January 19
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TRINITROTOLUENE
Health Advisory
Office of Drinking Water
U.S. Environmental Protection Agency
Washington, DC 20460
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Health Advisory on 2,4,6-Trinitrotoluene
Authors:
Loretta Cordon, M.S.
William R. Hartley, Sc.D.
Project Officer:
Krishan Khanna, Ph.D.
Office of Drinking Water
U.S. Environmental Protection Agency
Washington, DC 20460
January 1989
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P9IYACE
This report was prepared in ac ordanc,e th the emorandu of nderscandtng
Derveen the Departnent of the Ar -ny, Deputy for Environment Safety and
Occupational Health (OASA(I&L)) . and the U.S. Environmental Protection Agenc;
(EPA), Office of Drinking Water (ODW), Criteria and Standards Division, for
the purpose of developing drinking water Health Advisories (HAs) for selected
environmental contaminants, as requested by the Army.
Health Advisories provide specific advice on the levels of contaminants in
drinking water at which advers, health effects would not be anticipated and
which include a margin of safety so as to protect the most sens .tive members
of the population at risk. A Health Advisory provides health effects
guidelines, analytical methods and reco.nda treatment techniques on a
case—by—case basis. These advisories are normally prepared for One—day,
10—day, Longer—term and Lifetime exposure periods where availabi.
toxicological data permit. Thee. advisorie, do not condoms the presence of
contaminants in drinking water; nor are they legally enforceable standards.
They are not issued as official regi larione and they may or nay not lead o
the issuance of national standards or Maximum Contaminant Lavel. (MCLs).
This report is the product of the foregoing process. Available toxicological
data, as provided by the Army, on the munitions chemical 2,4,6—crinitrocoluene
(TNT) have been reviewed and relevant findings in, presented in this report in
a manner so as to allow for an evaluation of the dati without continued
reference to the primary documents. This report has been submitted to
critical internal and external review by the EPA.
A companion document, “Data Dificienciss/Probjem Areas and Recoendationa for
Additional Data Base Development for Trinitrotoluena” is included in this
report.
I would like to thank h. authors, Ms. Loretta Gordon and Dr. William Hartley,
who provided the extensive technical skills required for th. preparation of
this report. I am grateful to the members of ths EPA Tox—Revta’w Panel who
took time to review this report and to provide their invaluabl, input, and I
would like to thank Dr. Edward Oh.anja , Chief, Realth Effects Branch, and Dr.
Joseph Cotruvo, Director, Criteria and Standards Diviiion. for providing me
with c bs opportunity and encouragament to be a part of this project.
The preparation of this Advisory was funded in part by Interagency Agreement
(LAG) 85—PP5869 between the U.S. EPA and the U.S. Army Medical R.search and
Development Co and (USAI DC). This LAG v ia conducted with the technical
support of the U.S. Army Biomedical Research and Development Laboratory
(USABR.DL).
Kriahan anna, Project Officer
Office of Drinking Water
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:sT CF :A3L s
1. INTRODUCTION
TABLE OF CO .TE TS
• .
• .
• . v- i
• v—Il
• .
• V — 5
• .
• vt_I
• VI—7
• VI-7
Vt— i ’
t. — I
• L.
Vt—Is
• vt_Is
VI—24
Continued——
II. GENERAL INTORMATION AND PROPERTIES
III. OCCURRENCE
IV. ENVIRONMENTAL FATE
V. PHAR.MACOKINET:CS
A. Absorption
B. Distribution
C. Extretion
D. Metabolism
VI. HEALTH EFFECTS
A. Health Effects th
B. Health Effects in Anj.al Experiments
I. Short—Term Exposure
a. Skin and Eye Irritation, Dermal Seflgiti &tj .
b. Four—week Studies
2. LonZer—T.rm Expoaure
a. Thirteen—Week. Studies
b. Twenty—Six—Week Study
C. Lifetime Exposure . . VI—26
—1 .—
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:ABLE OF CONT!NTS (Conc ued)
\
3. Reproductive Effects
4. Developnental Effects
? AG
5. Carcinogenicity
6. Genotoxicity
7. Other Effects
V1. HEALTH ADVISORY DEVELOPMENT
A. Quantification of Toxicological Effects
1. One—Day Health Advisory
2. Ten—Day Health Advisory
3. Longer—Tez- Health Advisory
4. Lifetime Health Advisory
B. Quantification of Carcinogenic Potential
VIII. OTHER CRITERIA, GUIDANCE AND STANDARDS
IX. ANALYTICAL METHODS
X. TREATMINTTEC} OLOGIES
A. Red Weter
B.PjnkWater
C. Ocher Methods
XI. CONCLUSIONS AND PECO?* DATIONS
XII. REFER.E C s
Vu—)
v: : — s
—ii—
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.ABLE CF CO TS CcntLtue j
A2?EYDLX
I tnc .dence of tumors j Anjn.alg Fed TNT in the Diet • Ai—i
for 2 Years
2. Incidence of Xyelofibrosjs in Rats Fed TNT in the Diet . . . .
for 2 Years
3. Data Defjc1encjes/prob1e Areas and Reco zendations for
Additional. Data Base Deve1op ertt for Trinitrotojuen. A3—:
—iii—
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L:ST OF TA3LES
A3LE AC
tt-L GENERAL CH 1tCA.L AND PHYSICAL PROPERTIES OF
2, , 6-TRINITROTOLL’ENE
Itt-i 90th PERCENTILE CONCENTRATIONS AND RELATIVE CONCENTRATIONS
DETERMINED FOR CONDENSATE CO ONENTS tI:-3
PERCENTAGE OF ORALLY ADMINISTERED TNT
RECOVERED IN RATS IN 24 HOURS
V .-2 PERCENTAGE OF ORALLY ADMINISTERED TNT (100 MG/KG)
RECOVERED IN ALBINO CDI MICE IN 24 HOURS
V-3 PERCENTAGE OF ORALLY ADMINISTERED TNT (5 MG/KG)
RECOVERED IN RABBITS IN 26 HOURS V-5
V-4 PERCENTAGE OF ORALLY ADMINISTERED TNT (5 MG/KG)
RECOVERED IN DOGS IN 24 HOURS v-6
V-5 PERCENTAGE OF ORALLY ADMINISTERED TNT (50 MG/KG)
RECOVERED IN 24 HOURS v- 7
V-6 TOTAL RECOVERY OF 14 C-TNT A.FTER ORAL AND DERIIAL
ADMINISTRATION IN VARIOUS SPECIES v-9
V-7 PERCENTAGE OF ORALLY AND INTRATR .ACHEALLy ADMINISTERED 14 C—TNT
(50 MG/KG) RECOVERED IN SPRAGUE—DAWLEY RATS IN 4 HOURS . . . V- b
V—8 PERCENTAGE OF ADMINISTERED 14 C-TNT (50 KG/KG) RECOVER.ED
FROM VARIOUS ORGANS 24 HOURS AFTER ORAL ADMINISTRATION
IN FOUR SPECIES v-I:
V-9 TISSUE—TO—BLOOD CO ENTRATION RATIOS AT 24 HOURS AFTER ORAL
ADMINISTRATION OF C-TNT (50 MG/KG) TO MALE ANIMALS . . . . v- 3
V-jO PERCENTAGE OF OR.ALL.Y ADMINISTERED 14 C—TNT RECOVERED AND
TISSUE-TO—BLOOD CONCENTRATION RATIOS IN MALE DOGS V-15
VI—1 SUl tARY OF STUDIES: SHORT—TERM EXPOSURE OF ANIMALS TO TNT . Vt-B
Conc inued--
—iv-
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L:s: OF TA3LES (Con i .ued)
3E
C .TZ OP LD. (MG/KG) OF TNT ASTZWATER RESIDCE D
SYNTHETIC PRE tIQN IN MICE
VI-) SL A RY OF STUDIES: LONGER—TERN EXPOSURE OF IMALS TO TNT. V —
INCIDENCE OF URINARY BLADDER LESIONS IN F ALE PATS
FED TNT FOR ISP TO 24 MONTHS
A2—I. INCIDENCE OF BONE MARROW M ELOFIBROSIS IN F 1ALE
RATS FED TNT FOR UP TO 24 MONThS A2-
-v—
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e realch Advisory (HA) Program, sponsored by the Off ce of Driflking Aate
(C , provIdes rmar on on the health effects, analytical ethooo:o v a-:
treac:enc technology that would be useful in dealing with the c3n:a : aci3n
drinking water. Health Advisories describe nonregulatory concentrat ons of
drlr.king water contaminants at which adverse health effects would not be
antIcipated to occur over specific exposure durations. Health Advisories
contain a margin of safety to protect sensitive members of the population.
Health Advisories serve as informal technical guidance to a5sist Federal,
State and local officials responsible for protecting public health when
emergency spills or contamination situations occur. They are not to be
construed as legally enforceable Federal standards. The Advisories are
subject to change as new information becomes available.
Health Advisories are developed for One—day, ren—day, Longer—term
(approximately 7 years, or IOZ of an individual’s lifetime) and Lifetime
exposures based on data describing noncarcinogenic end points of toxicity.
Health Advisories do not quan ’titativ.ly incorporate any potential carcinogenic
risk from such exposure. For those substances that are known or probable
human carcinogens, according to the Agency classification scheme (Group A or
B) , Lifetime HAs are not recoended. The chemical concentration values for
Group A or S carcinogens are correlated with carcinogenic risk estimates .
employing a cancer potency (unit risk) value together with assumptions for
lifetime exposure and the consumption of drinking water. The cancer unit rtsk
is usually derived from the linear multistage model with 95Z upper confIdence
Limits. This provides a low—dos. estimat. of cancer risk to humans that is
considered unlikely to pos. a carcinogenic risk in excess of the stated
values. Excess cancer risk estimates may also be calculated using the
One—hit, Weibulj., Logic and Probjt models. There is no current understarding
of the biological mechanisms involved in cancer to suggest chat any one of
these models is able to predict risk more accurately than another. Because
each model is based upon differing assumptions, the estimates that are derived
can differ by several orders of magnicuds.
1— 1
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GENERAL : ,Fc y.A::JN AND ?RCPER::zs
:: oLuene (tN ’) or, nore specifically, —T’T s the common des nac’—
f ,- , — rinitroroluene, trte nost widely used military high—explos ve
(Castcr:na, :980). For purposes of this HA, the synonym, TNT, will, be used
chrou r.out to refer Co Z, 4 ,ô—crinitrotoluene. Along with TNT, the svmmeC ’ a
.somer, f ,ve meta or unsymmetrical trinitroto] .uene isomers are found in the
crude product resulting from the nitration of toluene with nitric acid in c-.e
presence of sulfuric acid. The nitration occurs in a Step—wise fashion by a
batch or continuous process.
The continuous process as employed at the Radford Army Auaitjon Plant
(RA ..P), a prototype for Army Aunition Plants (AAPs), ucili es 992 nitric
acid and Z oleum (1092 sulfuric acid, a solution of sulfur trioxide in
anhydrous sulfuric acid; Small and Rosenblatc, 1974) to nitrate toluerte in s.x
stages to crude TNT which is then subjected to purification with aqueous
sodium sulfite (sellite) (Ryon et al., 198I ). This process has been further
nodjfjed CO employ eight nitrator vessels fitted with dynamic (cent: fugal)
separators, thereby ensuring a greater degree of safety and efficiency. The
purification process consists of two acid washes, three sellite washes and two
post—selj ,ite washes.
The crude TNT contains approximately 52 of the meta—isomers. These are
reduced to about 0.62 by the sell.ite purification. Crude TNT also contains
approximately 1% of the six dinitroto],uene (DNT) isomers, which are not
removed during purification, and slightly more than 12 oxidation products,
which are reduced to <12 by purification, Three additional impurities,
anouncing to <1%, are introduced by the sellite process (Ryon et al., 1986).
total impur tjes constitute not more than 3.242 of the finished TNT (Pal and
Ryon, 1986).
The resulting monoclinic rhombohedric crystals, as described in Rosenbiact et
al. (1971), when very pure, melt at 80.99°C, although a melting point as high
as 81.6°c has been reported and 80.65°C is a coonly accepted figure (80.1 -
81.6°C). The color is usually pale yellow, but a chrom .atographicalj,y purified
sample has been described as faintly yellow to pure whit.. A boiling point of
21,0° to 212°C at 10 to 12 Mg has been determined. The specific gravity has
been variously reported over the range of 1.3 to 1.6 gm/cc. Although the
solubility of TNT in water at 20°C is only 0.0132 (130 mg/I..) 1 this is
significant for pollution and health issues. Its solubility in organic
solvents runs much higher, e.g., 109 gm/100 g of acetone at 20’C.
Two grades of TNT are used for military purposes and their purities are
measured by the solidification point (also termed freezing point or setting
point), which is considered more reproducible than a melting point. Grade
I tt, the more highly purified grad., has a solidification point of 80.4°C,
minimum, and exists as a fine crystalline form (Department of the Army, 196’
tt-. I
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General cne ical and physical characteristics of T T are presented tn Tate
Trinitrotoluene is among the least i pacc— and friction—sensitive of t e !-.t2n
explosives and the impurities for ed during its production (except for
cecrani.:ro echane) do not affect its sensitivity. It can be further
desensitized, however, by adding certain stabilizing substances in small
quantity (1Z to 2%) (Rosenblatt ec al., 1971).
The chemical stability of TNT is such that, even at 150 0 C, it undergoes no
great deco positioa in 40 hours. Molten TNT can be stored at 85°C for 2 years
without any decrease in purity. TNT has been found to withstand storage at
magazine temperatures for 20 years without any measurable deterioration.
Furthermore, oistura has no effect on the stability of TNT, which is
unaffected by iersion in sea water (Department of the Army, 1967).
11—2
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TABLE 1:—:
GENERAL C IE ICAL AND ?HYs:cAL ? QPERTES
or 2,4,6_TRi ,.t:ROTOL .:zNEaI
Color
Physical. state
Specific gravity
Liquid density
Vapor pressure
Solubility characteristics
Melting point
Boiling point
Freezing point
Flash point
Conversion factor
18—96—7
TNT, —crinicrocoluol, 1— echyl—2 . ,
6—crinitrobenzene, crotyl, tolice,
triton, tricol, trilice, —INT
227.13
C 7 H 5 N 3 0 6
0 N0 2
2
Yellow to white
Monoclinic rhonbohedra]. crystals
1 . 654
1.665 g/cn 3
0.053 (85°C); 0.106 ( [ 00°C)
Wacer 0.013 g/100 g (20°C)
Carbon tetrachlorids: 0.65 g/100 g
(20°C)
Tolusne: 55 g/100 g (20°C)
Acetone: 109 g/100 g (20°C)
80.1 — 8 1.6C
210°C (10 ) — 212’C (12 )
80.75 ± 0.OSC
240’C (explodes)
1 pp 9.28 g/ 3 (25°C; 760 1Ig)
I ng/ • 0.108 pp (25°C; 760 k(g)
CAS Nunber
sane s
Molecular weight
E p rical fornula
Structure
a/References: Clayton and Clayton (1981); Rosenblact cc al. (1973);
Departnent of the Army (1967); ndholz (1976); Zakhari and Villaume
(1978)
LL-3
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DCC L RRE .CE
rcroco1ue’ e .as produced arid used on an enormous scale during WorLd ar
and . orld .ar :i and may be considered the most Lmportari mjjjtary burs -
charge explosLve. tc has fourid wide application in shells, bombs, grenaces
demoli.zion explosives and propellant compositions (Department of the Ariy,
1967)
Trjnjtrocoluene is manufactured primarily by the Continuous process, as
described above, in Army Aunitjo Plants (AA.Ps). Production from 969—.97
was reported as 45 milLion pounds/month with a capacity of 85 m lij
poundslmonth (Ryan et al., 1984). tc has been reported that as much as ore
half million gallons of wastewacer have been generated per day by a single
plant involved in the roduccion of TNT (Hartley ec al., 1981).
Trinitrotoluene wastes have a unique terminology as described in Rosenbjatt et
al. (1973). “Nitrobodjes” include TNT, ocher TNT isomers, products from t’e
Sellite purification process and by—products from the production process. The
spent sellite washings are high in solids content and are called “red water”.
Ryon ec al. (1984) have reported chat “TNT is the largest single non—polar
component”. The na or organic components identified are 2 , 4 —dinicrotojuene...
3 —sulfonace and 2 , 4 dirlitrotoluen ,_5...5uifonite which make up approxi ateLv
one—third of the polar organic fraction. Such water is intensely red—co1 .ed
and either is sold to paper mills for sulfur content or is concentrated v
evaporation and incinerated. It is not amenable to purification and, because
it is classified by EPA as a hazardous waste, it cannot be discharged i.ntc
streams.
“Pink water” comes from both manufacturing plants and from load, assemble and
pack (LAP) facilities. that from manufacturing plants can aris, from arlon
fog filter effluents and nitrator fume scrubber discharges and is known to
consist of the DNTs. While not positivejy identified, these two sources of
“pink water” are also believed to contain all TNT isomer,, manonicrotolueres
(MNTs) and possibly dinjtro—m—cresol , arising from th. displacement of a tY:ro
group on TNT isomers. Additionally, “pink water” from manufacturing plants
arise, from “red water” distillate, (evaporator condensate from concencra rn
process) and consists of DNT ,, while thos. from finishing building hood
scrubber and wash—down effluents are also believed to contain primarily \Ts.
Spent acid recovery wastes may be an additional source of “pink water”
generated during the manufacturing process (Dacre and Rosenblact, 1974).
the ocher hand, “pink water” from LA.? facilities, resulting primarily f n
shell washout operations, contains essentially pure T NT, usually conta,i-a:e
with (P.DX) or ocher additives. s
pink color —— pale straw to brick red — arises under neutral or basic
condjtjo s, especially when the wastes are exposed to sunlight (Rosenb az: e:
al., 1973).
1 11—1
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A .unoer of phocOdegradat:on ?roducts of T riave been jdenc’..f:ed iri
sc: er.t extracts of “pLr ik water”. Those deg:adat on products that are ace:
so1ub e (but nor extractable by organic solvents) have not been fully
characterLzed; ho .,ever, as many as thirty components of condensate aste acer
(i.e. steam d stil.ates arising from the concentration of “red .racer ” by
evaporation) obtained from the Volunteer AAP have been identifIed and
quantified (Table LU—I). Other constituents not derived from TNT de3radat cn
include the toxicologically significant DNT isomers, particularly 2,4— arid
2,6—DNT (Dacre and Rosenblatt , 1974).
ttl—2
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ab,e 90th ?er:enc .e Cor .ce cra — 5 ar.d e1a:. .
Concentrations Deter .ned for Lo censata Co ponertsa
Reference: Spanggord et al., [ 978.
Determin.d by cluster analysis of data points from two studies via a
ispia1 developed computer program.
the 90th percentile concentration of thos. compounds appearing in at least
of the samp1es/et of the concentrations of all components.
Conpounds were riot present in [ 02 f the samples. Value given represents
the nean of the non—zero values.
90th ?ercentjle
Concencra tjon ’
Condensate Compor.ertc (ngiliter)
( )
:c
Toluene
0.200
2 —Nitrotoluene (NT)
0.030
0.590
6 —Nitrocoluene
3—N icrcbenzonicrile
4 —Nitrobenzor iitr le
0.100
0.013
0.009
0.089
0.293
0.035
2—Anino—4—NT ,
2—Anino—6—NT ,
3—Amino—4—Nt
0.033
0.010
0.027
o.c
0.097
0.030
3—Nethyl—Z—njcrophe nol
0.012
0,080
0.033
5—Mechyl—2—nicrophenol
0.032
0.094
l,3—Dinitrobertzerie (DNB)
4.000
2,3—Dinjtrotoluene (DNT)
0.400
1.180
2 ,4—DNT
14.700
2 ,5—DNT
0.400
2,6—DNT
7.300
21.541
3,4—DNT
0.500
1.475
3,3—ONT d/
3,5—Dinjeroaniline
0.520
0.058
1.534
1,5—Dinechyl—2,4 —DNB (DNX)
0.390
i. :s:
2—Amico—3,6—DNT
0.030
0.089
2—An ino—4,6—DNT
0.020
0.059
3—Anino—2,4—DNT
1.500
3—Amino—2,6—DNT
1.200
3.541
4 -Aj r io—2,6—DNT
0.600
1.770
4 —Ai ino—3,5—DNT
0.200
0.390
5—A ino—2,4—DNT
2, 4—Dinitro—5—methyLphenol 1
l ,3,5—trinjtrobenzen. 3 d/
2 ,3,6—Trinjtrotoluene ( tN T)
0.700
0.085
0.153
0.268
2.066
0.251
0.651
0.791
2,4,6—tNt
0.400
[ .180
111—3
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‘ .o’ :A: TATE
Several studies have been conducted to dete j e the environmentaL sate
g:ewacer. Sased on a rev:ew of the available Literature and the h•: -
anc c..enlcal properties of T>.T, Spanggord ec al. ( 1 980a) indicated that
sed1 en: adsorption and volatiliza .on were not likely to be significant fate
processes, but further investigation was recoended. Degradation via
phozolysis and biotransformation, but not hydrolysis, were considered
sigtiifi.cant.
Based on the weight of evidence in experiments conducted under a wide range cf
conditions, Tucker et al. (1985) reported that adsorption in soil would be an
important process affecting the migration of TNT, with th. cation exchange
capacity and organic carbon content most critical in determining the degree of
adsorption. Molecular diffusion was also considered an important factor and
was related largely to percolation rate while vapor phase diffusion woujo on!.v
be significant in arid climates where soil clay content was low.
tn contrast, Sikka ec al. (1980) showed that sorption of TNT to pond sedinent
is measurable but not extensive, is correlated to time arid concentrac on and
is stongly, perhaps irreversibly, bound. Sorption of TNT to sediment is also
directly related to pH and temperature. The degradation products of TNT also
appear to be adsorbed to sediment.
The phocolytic degradation of TNT wasrewater is well known, as indicated by
the formation of “pink water”, and numerous degradation products of TNT ‘ave
been identified. Laboratory studies have determined that photolysis is the
primary process involved in the Loss of TNT from the environment, arid chat tne
concentration of TNT in wastewater will decline rapidly within a short
distance of its discharge point. Furthermore, photolysis is accelerated by
the products formed during the photolytic process as well as by natural
substances. Using a computer model, Spanggord at al.. (1980b) estimated
probable concentrations of TNT arid its degradation products in water bodies.
Based on detailed kinetic investigations, an environmental half—life of 3 to
22 hours was calculated. Burlison (1980) determined that the major
transformation product in natural water was 1,3,5—crjnitrobenz,ne.
In contrast, Spanggord et al. (1980b) demonstrated a slow biocrarlsformat ’..3n
TNT in natural water, even in the presence of small quantities of organic
nutrients. The major process of transformation was reported to be via
nitro—group reduction with no ring cleavage detected. The 2 —amino and
‘—amjnodjnjtroto1uenes were the major biotransformation products (Burlison,
1980). Half—Lives of 8 to 25 days were experimentaily estimated for T T
Similarly, Spanggord cc al. (1981) determined that volatilization of : : -as
very slow and that actual races were consistently lover than those esc a:e:
by physical and chemical properties.
LV- I
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• ? .kR.’ .ACOK:\E::cs
- . c:ctoluene is absorbed by inhalation. ingestion or ski.n contact,
b:ocrar.sformed in the Liver, excreted in the ur.ne ano discr.buted tO Z e
organs; however, rapid clearance precludes extensive bioaccumuLat on. it
‘etaboi.ized primarily by reduction of the nitro group and, to a lesser excer.c,
by oxidation of the methyl group and ring hydroxylacion. Glucuronide
conjugates have been found and 4—amino—2,6—dinitrotoluene is the main
mecabolice identified (Ryon, et al., 1984). Available data indicate that TNT
is well absorbed (more than 50% of the administered dose in most experiner.ts)
in a variety of test species. The major route of excretion is via urine wicn
some (generally the balance of the recovered dose) in the feces and
gastrointestinal (GI) tract plus its contents. Distribution to other tissues
is usually less than 1%. Only negligible amounts (0. 1%) were recovered in
expired air. Several metabolites have been identified in the urine of various
species including hydroxylated. mono— and dinitro and mono— and diamino
derivatives.
A. Absorption
Lee ec a)... (1975) studied the absorption of TNT in Charles River CD female
rats (175 to 250 g) that were fasted overnight before being given a single
oral dose of approximately one tenth the LD 0 of the test compound (i.e.,
82 mg/kg), spiked with 10 Ci of the C—label ed compound. the test materIal
was suspended in peanut oil and administered via intragastric incubation at a
volume of I mi/tOO g body weight. After dosing, each rat was placed in a
“Roth—Delmar” metabolism cage where feces and urine were collected separately.
Ac the termination of each experiment, each rat was anesthetized, and aorcic
blood was collected in a heparinizsd syringe. Liver, kidneys, brain, Lung,
thigh muscle, C I tract plus contents and the feces were homogenized and
assayed for radioactivity at 24 hours after TN t administration.
Approximately 602 to 742 of the administered dose was absorbed in the 26—hour
period. Most of the absorbed radioactivity was excreted in the urine,
averaging 53.3% of the administered dose, with about 26% of the radioactivity
found in the GI tract and the feces. A negligibl, amount of radioactivity as
recovered in the expired air.
From the sama laboratories, Modgson et al. (1977) and El—hawari cc al. (1981)
reported on the absorption of TNT admini ered as a single oral dose at
100 mg/kg (spiked with 25 uCi/kg of the C—TNT) to male and female Charles
River CD or Sprague—Daw].ey rats (200 to 300 g) using the same procedure as
described by Lee at al. (1975). Table V—I compares the results of these three
studies. About 53% to 652 of th. administered radioactivity appeared in the
urine within 24 hours. Most of the remaining radioactivity was recovered in
the C I tract plus contents and in the feces, averaging 262 to 382 of the dose.
The urine was bright red in color indicating the presence of metabolic
products.
v—I
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abLe
?ercert:age c OraL].y Aarn .c..stered TNTa
Recovered in Rats in 24 Hours
Scrai.n C
Dose
harles River
82 mg/kg
CD
Charles
100
River CD
mg/kg
Sprague Dawj .ey CD
100 mg/kg
rota]. Recovery
Female(3)b
Male(4)
91.3
Female(4)
102.0
Male(4) Female(Z )
91.6 102.4
82.1
Feces
5.3
8.1
2.1
8.0 2.O
GI Tract
plus contents
20.7
29.7
33.9
29.8 33.9
erine
53.3
52.7
64.5
52.7
Expired Air
0.1
NAd ’
NA
NA NA
Other Tissues
2.5
0.8
1.5
1.1 2.0
Author Lee ec al. (1975)
Hodgson et
al. (1977)
El—havari et al. (1981)
a/ 14
C; standard deviations omitted.
of animals analyzed.
different (p
-------
; .. :ar studies were cortoucted by these sane aucnors ( odgson et al. . •,
E:— a ari ec al. , 961) in a1.Sino CD n .ce, .ew eajand raobics and eage
3;S. Results are conpared in Tables V—2, V—3 and V—4, respective1
As seen in Table V—, about 42% to 60% of the raaioacciv:ty adnjniscered Z0
nice appeared in the urine within 26 hours. A range of 16% to 55% of the dose
ren.ained in the GI tract plus contents and the feces. As in the rat, nouse
urine was bright red.
Ln rabbits, Table V—3, about 66% to 79% of the adninistered radioactivity
appeazed in the urine within 24 hours. The radioactivity in the C I tract plus
contents and in the feces averaged 6% to 13% of the dose. Unhikt that of the
rat and nouse, rabbit urine did not contain a red pignenc.
The results for dogs were sinilar to those for rabbits. As indicated .n Table
V—4, about 56% to 60% of the ad iru.scered radioactivity appeared in the urine
within 24 hours. The CI tract plus contents and the feces contained 15% tř
21% of the dose. Sini1.ar to the rabbit, the urine did not contain a red
pig enc.
El—hauarj ec al. (1981) also compared the 24—hour recovery of radiolabej.led
TNT in all four species following a single oral dose of 50 ag/kg to each. As
seen in table V—5, rats, nice and dogs excreted relatively equivalent anounts
in the urine whereas, in rabbits, the urinary excretion of radioactivity was
higher with a proportional decrease in the percent of radioactivity recovered
fron the Cl tract plus contents and feces. Even at this 10—fold higher cose,
red pignenc was not detected in the urine of the rabbit and dog. This red
pignent has been reported to occur in the urine of humans “poisoned” with TNT
(Hassnan, 1972 as cited in El—hawari cc al., 1981).
El—hawari cc al. (1981) studied species differences in the absorption and
excretion of TNT as a function of the route of ad inistratjon. Swiss albino
‘ice (20 to 30 g), Sprague—Dawj.ey rats (200 to 300 g), New Zealand rabbits (3
to 4 kg), and beagle dogs (8 to 14 kg) were used in parallel oral and der al
studies.
tn the oral stud1 s, test aninals were fasted overnight before receiving
single doiss of C—TNT dissolved in peanut oil. In the der al study, the fur
on the back of the anth.als was re ovs4 with electric clippers and, on the
following day, the C—TNT in peanut oil was spread over the depilated areas.
Rats and nice were dosed at 50 nglkg and dogs and rabbits were dosed at either
5 or 50 ng/kg. Ani .a1s were placed in individual netabolis: cages for the
separate collection of urine and feces. At 24 hours after dosing, aninals
were anesthetized and killed, and blood, liver, kidney., lung ., spleen, braLn.
skeletal muscle, fat (recropericoneal) and the CI tract plus contents were
analyzed for radioactivity.
V—3
-------
Table —2
?erce cage of Orally A Lnistered TNTa/ (100 gikg)
Recovered .n Albino CDI 1.ce in 2 Hours
Male ( 7 )b/ Fl (8) Male (7) Female S
rocal Recovery 101.6 86.8 80.0
Feces 42.6 18.2 22.0
GI Tract
plus contec ts 12.4 7.4 13.4
Urine 44.7 60.5 41.9 42.9
Other Tissues 1.7 0.7 2.7 1.
Author Hodgson et al. (1977) El—hauarj et al. (I981)
ring—ut—’ c; standard deviations omitted.
(n)—numbers of ani .als analyzed.
C/Si hi], different (pO.05) frog .ales.
V-6
-------
ab .e .—3
?ercer age oi Sral .y Ad .n .scared (5 m;, )
ecovered in Rabbits n nours
Male ())‘D ’ Female (3) Male (3) Fe aie (3 )
TotaL Recovery 78.1 94.7 77.6 88.9
Feces 1.8 1.8 1.8 1.3
C I ract
plus cor tents 7.5 10.8 7.3 4.?
Urine 66.3 78.9 66.3 78.3
Other tissues 2.5 3,2 2.0 3.6
Author iod.8son at a].. (1977) El—hawart et a].. (1981)
ring—UL— 14 C; standard deviations o itc.d.
(n)—nu bers of animaLs analyzed.
v-s
-------
Table
?e cencage of 0r .11y Ad .niscered
ecovered in Dogs in 2!. hours
‘.a1e (3) ’ Fe a1e (3) ale (3) Fe a1e (3 )
Total Recovery 79.6 87.5 77.4 88.2
Feces 5.4 16.7 5.4 16 . 8 c
C L Tract
plus contents 9.9 4.3 10.0 6.!.
LTrine 59.1 60.0 55.9 60.2
Other Tissues 5.2 6.5 6.1 6.8
Author Hodgson et al. (1977) El—hawari ec al. (1981)
ring—UL— 14 C; standard deviations omitted.
,(n).numbers of animals analyzed
‘Sign2.ficant].y different (p
-------
abLe V—S
?ercet ca e o raL].y Ad .n .stered (50 gi.q)
Recovered in 26 hours
Rats (3) Mice (8) Rabbics (2) (‘. )
Total Recovery 92.2(8t.3) 94.4 103.6 9L .1
Feces 10.7 (2.1) 24.1 5.1
GI Tract
plus contents 20.2(35.3) 10.2 22.7 i.7
Urine 59.5(4Z.5) 59.0 ’ 74.3 ÔLO
Other T .ssues 1.8 (1.4) 1.5
Author El—havari at al. (1981)
a/ 14
rthg—UL— C; specific activity 19.76 iCi/ g; single dose; standard
b/deviations onitted.
1 (n)•nu bers of animals analyzed.
1 iale(Fenale) values.
Bright red pigment in urine.
V —7
-------
of TNT as confir .ed by both the oral and der al r uzes of
aa 4 strac on. The authors repc rad that TST s read 1y absor5ed af:er oral
ad nis::acion with rabbits and dogs appearing to absorb nore T T than rats
and . e. the extent of oral absorption is reported as approx ate based on
the recovery data since the extent of biliary excretion and encerohepac.c
circulation was not evaluated. the dermal experiments confirmed the poreflcja.
absorption of TNT through the skin with absorption being highest in male
rabbits (57—68%) followed by male mice (42%), female rats (25%), male rats
(23%), and male dogs (16—17%). In all species, total elimination of the
administered radioactivity was reported to be lover after dermaj. application
s compared to oral administration. Table V—6 compares the total recovery as
a percent of the administered dose in rats, mice, rabbits and dogs by both
routes. As in the previously described oral studies, red pi ment was present
in the urine of rats and mice treated dermally with a single application of
4 C—TNT but not in the urine of dogs and rabbits.
In an effort to simulate inhalation exposure, El—hawari et a].. (1981)
instilled 50 mg/kg of TNT suspended in methyl cellulos, into the trachea of
anesthetized, tracheotomjz,d, male and female Sprague—Dawley rats. Concurrent
e perimertts were performed in rats treated orally with the same dose of
C—TNT under the same experimental conditions. After 4 hours, both groups of
rats were sacrificed and tissues and bladder urine were collected for analysis
of radioactivity.
After intratracheal maculations, the authors reported that the rate of
absorption was faster, and the extent of absorption greater and more uniform
with less individual variation as compared to orally treated rats. Urinary
excretion in intact male rats, 4 hours after intracrachea]. instillation.
averaged 19.3% while, 4 hours after oral administrations, th. urinary
excretion averaged 14.6%. Urinary excretion in female rats treated
intratracheally or orally was somewhat lower, averaging 13.2 and 10.0%,
respectively. The authors described the pharmacokinetic behavior of the TNT
after intratrachea]. instillation as “comparable to the behavior usually
observed after intravenous administration of other xenobjotjcs.” Direct
comparison between intratracheal and dermal routes was not studied 14 As in the
previous studies, the urine of rats treated with a single dos. of C—TNT by
incratracheal instillation contained the characteristic red pigment.
Since the GI tracts of the rats treated by intracracheal instillation
contained considerable amounts of radioactivity, some of the rats in this
experiment were bile—duct cannulated for collection of bile. Table V—7
compares the recovery of thu radioactivity in cannulateci an 4 non—cannu1aced
rats four hours after oral or incracrachea]. treatment with C—TNT. Excrec-
of radioactivity in the urine and bile was higher after intratracheal
administration but lover in the GI tract when compared to oral administrac c-
The authors reported that enterohepacic recycling of TNT and/or its
metabolites was suggested by a higher recovery of radioactivity in the ur e
V-8
-------
Table
tal Recover’, of C—TNT After Oral and 1 Der aj
Ad irtistracion in Various Speciesa
Dose Percent of Dose Recovered
pecies Sex _____ Oral Der al
Rat / 6 )b/ Male 50 92.2
Rat (3/6) Female 50 81.3 24.8
Mice (8/6) Male 50 94.4 41 . 7 c/
Rabbit (3,’4) Male 5 95.6 68.3 ’
(2/2) 50 103.6 569 d1
Dog (3/3) Male 5 994 158 C/
(i/i) 50 94.2 159 d/
Based on El—hawari et al. (1981)
Standard deviations omitted; fat and skin (including site of application)
blare not included in recovery estizates.
— number of ani .a1s evaluated oraj/der .a] ..
1 Significantly different (pO.O5) from oral. treat .nt.
Not statistically analyzed.
v-9
-------
Tabie V—
?erceritageaJ of Orally a’hd Incracracheally Ad .n1.scered • C—T .’T
(50 g/k ) Recovered in Sprague—Dawley Rats .n ‘. ours
Bile Duct—Cannulated
Oral ( 5 )b ’ trttracracheal (6) Oral (3) Intratracheal ( )
Total Recovery 95.6 429 c/ 93.4 46 .Oc ’
Urine 12.3 16.3 9.6 15.1 ”
GI Tract 76.4 66.2
Bile 10.6
Othet Tissues 6.9 11.4 7.0 11.4
Based on El—havari er a].. (1981)
a/Average recovery male and female; standard deviation! omitted; fat not
b/included in recovery estimates.
— number of animals analyzed.
Significantly different (p
-------
- e cact rats and a rad acr v : ’ Level :n C e b e : ra:s e:
zreacer char . that excreted in che urine.
3. D str burion
Studies on the recovery of radioactiv ;y from various tissues 2 hours after
treatment indicate that retention of C—TNT in the tissues of rats, nice,
rabbits and dogs is not extensive but that differences between species and
routes of administration did occur.
et al. (1975) reported on the distribution of radioactivIty from
C—labelled TNT in Charles River CD female rats. Ac 24 hours after dosing,
small but significant amounts (0.2 to 1.01) of radioactivity were found n tne
blood, liver, kidney, and skeletal muscle. Small amounts ( ‘0.11) were also
found in the lungs and brain. The tissue to plasma radioactivity rat o
suggested some retention of radioactivity in the liver and kidney.
In the same Study, another group of rats received a lethal dose of TNT in
order to determine how much of this compound was distributed to the brain. In
these animals, only 0.11 of the administered dose was found in the brain at 30
minutes. Small amounts (0.11 to 0.31 of the total administered dose) were
also found in the liver, urine, kidneys, and whole blood of chess animals.
Data on the distribution of 14 C—TNT in four animal species in the study by
Hodgson cc al. (1977) indicate that there were no major species differences in
the distribution of radioactivity in the tissues analyzed. In mice and rats,
small amounts (0.2 to 0.71) of radioactivity were found in the blood, liver
and kidneys. The other tissues contained only negligible amounts of C.
In rabbits, small amounts (0.3 to 1.01) of radioactivity were found in the
blood and liver, whil, in dogs small but aignjfican amounts of radioactivtty
were found in the blood, liver and muscle (1.2i o 2.21). The other tissues
contained only negligible ( ‘ 0.21) amounts of C.
In the study by El—hawati at al. (1981), at 24 hours, blood and tissue of dogs
contained a higher percentage of administered radioactivity than did the blood
and tissue of rate, mice, or rabbits. Table V—8 compares the radioactivity
recovered from various organs, as a percentage of administered dose, in all
fou 4 gp. j, 24 hours after t ie oral administration of a single 50 mg/kg dose
of C—TNT. Administration of different doses (100 mg/kg in rats and nice or
5 mg/kg in dogs and rabbits) produced generall 4 comparab 1e tissue levels 4
hours after oral administration. Recovery of C—TNT was greatest in liver,
skeletal muscle and blood.
Table V—9 compares th. tissue—to—blood concentration ratio (ug eq/g of tissue
per g eq/mi blood) in rats, mice, rabbits and dogs at the same 50 mg/kg orai
dose. Higher tissue—to—blood ratios (‘1.0) vera noted in the liver, kidneys
and lungs in rats, mice and rabbits and in the spleen of mice. Lower ratios
v—il
-------
table V—
?er:ancage of Ad n stered C—tNT (50 g/kg)aI Recovered fr Var:ces
Orgar.s 2. Hours Aicer Oral Ad iriiscration in Four Species
Rats ())bPC/ Mice ( 8 )dI Rabbits ( 2 )dI D (I)C
Liver 0.4 0.4 0.6 1.5
Kidneys 0.2 <0.1 <0.1 0.1
Spleen <0.1 <0.1 <0.1 0.3
<0.1 <0.1 <0.1 0.1
3ra n <0.1 <0.1 <0.1 <0.1
Skeletal Muscle 0.6 0.4 0.6 1.7
(as 40% body weight)
whole Blood 0.3 0.2 0.3 5.4
(as 7% body weight)
based on El—hawari. et al. (1981)
Single dose of ring—U1..— 14 C—TNT
1 (n)numbers of ar.ials analyzed
,,Average recovery, saL. and fe .a] .eg co biaed
Males only
V— 2
-------
a e .—
issue—co—3lood Co cer t c:on Raciosa/ at 2 iours Af:er
Oral Ad .r.iscration cf ‘C—N1 (50 gIkg) to Male Animals
Rats
Liver 4.1
Kidneys 3.3
Spleen 0.6
Lungs 1.2
Brain 0.3
Muscle 0.5
Fat 0.6
Blood
Based on El—hawart et al. (1981)
eq/g tissue per ug eqI J. blood
(n)ug eq/al
5.2
3.8
0.8
3.3
1.6
0.3
1.2
0.5
0.7
0.5
0.3
<0.1
0.8
0.8
0.2
1.0(0.93)
1.0(2.26)
LO(29 .22)
13
-------
were generally nocec in brain, muscle and ar of al spec:eS, Se
a s and rabbits, and in all tissues of the dog. The results of :- :s
cc oar.son seem to indicate chat the unusually high blood cortcencracons in
g eç!nl, ay account for the distribution pattern in dogs dosed at 50 :g. g.
when dosed at 5 mg/kg, the distribution pattern in various tissues of the dog
indicates a higher percentage of radioactivity in the liver (2.4:), but a
lower percentage (1.1%) in the blood. Comparison of the tissue—to—blood
concentration ratios at this lower dosage level indicates values similar to
those of the other species. Table V—IO compares these two dosing levels in
dogs.
Radioactivity remaining in most tissues was comparable after oral and dermaj.
adm .njstratjon, however, residual radioacciy cy was higher in the fat of a.l
species following dermal. application. The C content of the liver wa
generally higher after oral dosing.
In general, radioactivity in most tissues of the rat was higher four hours
after intratracheaj. instillation than after oral administration. The highest
amounts of recovered radioactivity (1—8%) were, found in muscle, blood and
liver. In this study, Levels in the muscle and blood of female rats were
about two times higher than in males for both routes.
C. Excretion
Lee et al. (1975) measured the excretion of 14 C—labelled TNT in Charles River
CD female rats. Ac ZC. hours after dosing, an average of 53% of the
administered dose was excreted in the urine while approximately 212 was found
in the CI tract plus contents and 6% in the feces. Only negligible amounts
were recovered in expired air.
et al. (1977) and El—hawari. et al. (1981) also studied the excretion
of 4 C—labelled TNT in mal. and female Charles River CD or Sprague—Dawley
rats. About 532 to 65% of the administered radioactivity was recovered in rre
urine in 26 hours while an average of 302 to 34% of the dose was found in the
GI tract plus contents. Males and females excreted approximately 82 and 2:,
respectively, in the feces.
In similar studies in albino CDI mice, New Zealand rabbits and beagle dogs.
Hodgson et al. (1977) and El—havati et *1. (1981) reported that excretion
radioactivity in mice was 422 to 602 itt the urine, 92 to 43% in the feces anc
72 to 13% in the CI tract plus contents (Table V—2).
In rabbits, these same authors reported the 24 hour excretion of the majort:v
of the radioactivity in the urine (66% to 192) with much smaller amounts
the Cl tract plus contentS (52 to 11%) and feces (22) (Table V—3).
Similar to rabbits, dogs excreted 562 to 60% of the radioactivity in the :.
V— [ 4
-------
able —i0
Pe ce a 3 e of Orally Ad inisCered L_C_T\T Recovered and
Ssue—co—Blcod Concencration Rac os in i a1e Doss
Z Recovery
Concentration Raciosa
50 ag/kg 5 mg/kg 5 O’g .
Liver 2.4 1.5 4.9 03
Kidneys 0.1 0.1 1.4 0.3
Spleen <0.1 0.3 0.8 0.7
Lungs 0.1 0.1 1.1 0.3
Stain <0.1 <0.1 0.2 <0.1
iusc1e (40Z of body Vt.) i.4 1.7 0.2 <0.1
Blood (7Z of body yr.) 1.1 5.4 10 ( 072 )b 1.0(9. 2)
Fat 0.2 C.2
Based on El—hawari et al. (1981)
a/ 8 eq/g tissue per i g eq/mi. blood
b/
(n)—ug eq/al
V— 5
-------
after oral dcsing while the GI tract plus conte’i co .:a ed •.
: :‘.e feces 5Z Z (Ta l V—.) .hodgson er al., i.97, EL— -a ar: cc
al., :9Sj,
‘ .hen l—hawari cc a.. (1981) conpared the 24 hour recovery of radiolabeije:
‘IT in all four species following a single oral, dose of 50 ng/kg to each,
rats, otice and dogs excreted relatively equivalent amounts in the urine,
averaging 60% of the dose while recovery from the urine of rabbits was
approximately 75% of the dose. The percentage recovered from the feces arid
the GI tract plus contents was somewhat more variable between the specLes as
shown in Table V—5.
El—hawari ec al. (1981) also compared the excretion of TNT in rats, mice,
rabbits and dogs as a function of route of administration, In all species,
total excretion 24 hours after administration was lower after derma].
application as compared to the oral route. when the oral, route was compared
to intratracheal instillation, 4 hour recovery was highest in the GI tract
plus contents of the orally treated rats (76%) while similar amounts
(approxl.marely 15%) were recovered in the urine and C I tract plus contents
after intracracheal instillation. A direct comparison between the dermal, and
intratracheal routes was not studied.
In this series of studies, El—hawari et al. (1981) reported the presence of a
bright red pigment in the urine of rats and mice treated by different routes
of administration while the urine of rabbits and dogs treated orally and
dermally contained no such colored pigment, even after doses up to SO mg/kg.
D. Metabolism
The presence of four functional groups on the TNT molecul, would permit a
variety of metabolic transformations to occur, to include oxidation of the
methyl group, oxidation of the benzerte nucleus, reduction of the nitro groups
and conjugation. Only minute quantities of un staboljz.d TNT have been
identified in the urine, and in vitro experiments suggest that the liver is
the major site of TNT biotransform acion.
In the study by Lee c c a],. (1975), a thin—layer chromatographic (ThC) analysis
of the radioactive compounds in the urin, and in brain of rats using a solvent
system of ethyl acetate;pscro leum ether indicated that all the radioactivity
in the )O—minuc, and 24—hour urine samples remained at the origin (point of
application), whereas TNT has an R value (ratio of movement from
ortgin:soj.vent front) of about O.7 in chj& system. The radioactivity from
the 30—minute brain extract had an R of 0.2, indicating cbs presenc, of one
mecabolite different from that in the urine. A 24—hour urine sample was also
developed in a bucanol:methamo l:wac ,r system. Ther, were one major and
several minor peaks of radioactivity. Almost no radioactivity was associated
with the I’NT or the dinitrocoluenes which have R values of about 0.95 in ch s
V— 16
-------
s . ;etit s; sCe . ‘ o actenpcs were nade : cnis study Co Loer.t fv
:e:a oLites.
-odgson et al. (197) stated that analysis of ur .nary netabolices recovered
c ated chat T T was metabolized extensively in all four species studLed ar.d
sinilarly ri rats, mice and dogs. The rabbi.c, on the ocher hand, had a
somewhat d fferent metabolic profile, Suggesting a species difference in the
netabolism of TNT. The presence of a red pigment in the urine of rats and
nice, but not rabbits and dogs, also suggests species differences in
mecabol .sm. Furthermore, glucuronide conjugation appears to play an important
role in the metabolism of TNT in the rat and dog, but not the rabb .t.
In this study, initial TLC analysis was carried out on rat urine that had been
extracted with CHCL 3 :Me014 (3:1, v/v) both before and after hydrolysis wLth 5
iCl (at 100C for 1 hour) and then evaporated and assayed for radioactivity.
Metabolices idenc fied in rat urine, with and without hydrolysis, included
trinitrobenayl. alcohol, 4—amino—2,6— dinitrotoluene, Z—amino—4,6—djnjero..
coluene, diamirtonitrotoluenes (not specified) and crtnicrobenzojc acid (in
hydrolyzed urine only). Unmetabolized TNT accounted for <12 of urinary
radioactivity. Urinary meta&oliteg from other species were noc presented.
tn the study by EL—hawari ec al. (1981), urinary metabolites of rats, nice,
dogs and rabbits were extracted with ethyl acetate under mildly acidic
conditions and separated by thin Layer chromatography using two solvent
syscems with different polarity. Tentative identification of metabolites was
carried out by comparing solubilicy characteristics, reactions with specific
spraying reagents, and Rf values of the netabolite, with those of standard
reference compounds. Quantitative determination of individual metabolites was
not feasible in this study.
The authors stated that TNT was metabolized extensively in all species
ex mjned, whither treatment was oral, derm.aJ. or intratracheaj.. Large portions
of the metabolic products wire conjugated with glucuronic acid, but no
conjugation with sulfate, was indicated by incubation with aryl suLfatase.
Most of the metabolic products were reduction derivative,, including the 2-
and 4—hydroxylamine ,, the 2— and 4 —monoaminodjnjcro and the 2,6— and
4 ,6—djamjnomononjtro derivatives. The trinitrobertzyl alcohol and the
tr riitrobenzoic acid seemed to be present, but confirmation was not possible.
The parent compound. t NT, was detected in the urine of some species in n.nuce
quantities only. Th. extraction procedure, used minimized the alterations of
the hydroxylamines to azoxytoluene, but some of the latter was present.
especially after fractionation of the urinary product, in the presence of
NaOH. Glucuronjde conjugation appeared to play an important role in TNT
metabolism, but while the amount of extractable radioactivity increased after
incubation with 3 —glucuronidase, the TLC profiles remained unchanged. Other
products of TNT metabolism remained unidentified. The metabolic profiles of
urine from rats, nice, and dogs differed only quantitatively.
V-U
-------
The r e of rats ConcaLned Large amounts of the ô—d amine (and, to a Lesser
excer.c, :ne 2 ,6—dianine) and Cflo mjnes (the 4—amino and/or 2 -anino). The
ana ‘— , droxvlamines and some azoxytoluene (probably formed during
fractionation) were present in small quantities. hetabolic profiles of urine
from na .e and female rats showed no significant differences. The amounts of
glucuronides in urine collected from bile duct cannulated rats were lower than
those collected from intact rats. In addition, the 4—hour urine from
caruiulated rats contained more of the polar metabolices and more parent : r.
Only minimal differences were apparent between orally and dermally treated
rats (more unchanged TNT eliminated after dermal application) and quanti:at ve
differences between orally and irttratracheaj.ly treated rats. The source of
the red pigment was not identified.
Compared with rat urine, mouse urine contained smaller quantitLes of the polar
mecabolices and the diamines and more of the monoamines and hydroxvlamjnes.
house urine also contained considerable amounts of the trinitrobenzy]. alcohol
and crinitrobenzcic acid. The presence of azoxytoluene was demonstrated after
fractionation by acid or base. Urine of mice contained the least glucuronide.
No major differences were evident after oral, or dermal treatment except for
the presence of larger quantities of unchanged TNT after dermal exposure.
The metabolic profiles of dog urine indicated the presence of appreciable
amounts of diamineg and monoamines. Only small amounts of the 4—hydroxyLamine
and 2—hydroxylamin., and minute amounts of azoxytoluen. (which seemed to e
formed during fractionation) were present. Smaller amounts of polar
metabolites and larger amounts of parent TN? were demonstrated in the urine of
dermaijy treated dogs when compared to those treated orally.
abbit urine showed a unique profile which differed quantitatively, and
probably qualitatively, from that of rats, mice, and dogs. The presence of
larger quantities of monoaines and hydroxylamines was demonstrated, in
addition to either or both of the diamines. The presence of trinitrobenzvl
alcohol and trinitrobenzojc acid was indicated by their R positions. TNT and
the azoxytoluene were absent from fresh urine, but some of ch. latter was
formed during fractionation with ether in the presence of NaOH. Urine from
dermally treated rabbits differed quantitatively from that of orally treated
animals with a sharp decrease in polar metabolices and some increases in the
monoamines, hydroxylamines and azoxytoluene in the dermal treatment group.
These studies by Łl—havarj et al. (1981) indicate that TNT is extensively
metabolized in all four species regardless of the route of exposure. The
majority of urinary metabo],jte, are of high polarity with very low
extractability in organic solvents and conjugation with glucuronic acid 1 .s
indicated. Most of the metabolic products are reduction derivatives and
include the hydroxylamines and mono/diamino—dinitro derivatives. The prese ’ ce
of b.nzyL alcohol and acid are indicated but not confirmed. Parent TNT is
present in the urine of only some of the species but only in minute
V—! 8
-------
ç ancitiesi although der al admin.scraciort seems C3 i ncrease the exc:et: f
. c anged T. The azoXVCOlueEe appears to be formed during tne exzract:
pr3cedure :n the presence of alkali. Identification of the source of tne red
p: nent in rat ar.d mouse urine was no: successful.
cst differences in the metabolic profiles were quantitative in nature &r.d
were demonstrated both within and between species. Quantitative differences
between oral and dermal treatment groups were minimal, being largely evidenced
by an increased amount of unchanged TNT present after dermal. administ;ac:ot .
Conversely, major quantitative differences were demonstr4ted between crally
and intracracheally treated animals.
Excretion patterns from rabbit urine presented a somewhat unique profi.e wL-ich
was considered by the authors to most closely approximate that reported ..n
literature for humans. Even this dissimilarity from other animal species
appeared mostly quantitative in nature, differing primarily in the amounts of
hydroxvlamines, which are present in the rabbit urine in larger quantic es
than in the other three animal species. Of significance may be the fact that
only the urine of rats and mice contained a red pigment similar to chat found
in human urine after exposure to TNT.
V-19
-------
F EZ s
affects data from human occupational exposure to TNT and from
a o;a: r: exper nents with animals adnin stered T T are Su arjzed in
secc on. Les .ons have been observed in many tissues and organ systems
including brain, liver, blood, reproductive organs, kidneys, urinary bladde—
and eyes. Evidence is presented that TNT is both mutagenic and carcinogen
in bactertal and animal tests, respectively.
A. Health Effects in Humans
With the advent of the large scale manufacture of TNT during World War 1, nar..
munitions workers reportedly died of TNT intoxication. During one 7 month
period, 45 deaths (2.8Z) occurred among 17,000 cases of t I poisoning. In
cr.e munitions plant alone, 105 fatalities (1.5Z) occurred among 7,000 cases of
TNT intoxication during a 20 month period (Zakhari and Villaume, 1978).
Overall, in the four year period between 1914 and 1918, 580 deaths (2.4Z) were
reported in the United States out of 24,000 cases o.f knovn TNT poisonings
(Rosenblatt, 1980). In 8ritish a uniCion plants, 125 deaths (26.3Z) over a
25 year period were reported among 475 cases of toxic jaundice resulting from
exposure to TNT (Zakharj and Villaume, 1978).
With the increased awareness of the hazards of TNT exposure, the number nf
fatalities significantly decreased during World War II, despite a dramatic
increase in the production of this explosive. Only 22 fatalities were
reported in the period between June, 1941 and September, 1945 among all
governmenc —o ed ordnance explosives plants. Eight (36Z) were due to toxic
hepatitis and 13 (59Z) were due to aplaseic anemia (Zak.hiri and Villaume,
1978). Only ne—thjrd of the 22 were exposed to TNT at average concentrations
over 1.5 mg/m , the existing workplace standard (OSHA, 1981). Among these
cases, hepatitis was reported to occur more frequently among younger persons
(average age, 30 years), with aplastic anemia being the cause of death among
older persons (average age, 45 years). Th. pathologic findings in the
clinical hepatitis cases invariably included degenerative damage to the liver.
usually accompanied by a great reduction in size and weight (NRC, 1982).
Since World War II, only occa5jo l deaths due to TNT exposure have been
reported and very few problems related to TNT use have been found in the
English_language literature (Morton et al., 1976).
In an extensive review of the Literature, Zakhari and Villaum. (1978) reported
on the various signs and symptoms of TNT toxicity and provided detailed
descriptions of the more specific effects of TNT on individual body systems.
Th. following is a su ary of this report.
Initial exposure to TNT in the atmosphere may result in mild irritation of : e
respiratory passages (nasal discomfort, sneezing, epistaxis and rhinitis
Vt—I
-------
pcss:5:. associated n.ch headac ’e and skin (erychena and papular erupc ns
prress: to desquamacion and exfoliation). Gascrointest .naj disorders, C:
i c . ,ude nausea, anorexia and constipation, somecL es associated with
tightening of the chest, are among the first signs of possible intoxication.
Epigastric path no: associated with food intake is a cardinal symptom.
Absorption of sufficient amounts of TNT through the skin or lungs can produce
signs of cyanosis (due to methemoglobin formation), toxic .laundjce (due to
severe liver damage), aplastic anemia (due to damage to the erythropoietjc
system), cataract formation (possibly a direct effect of TNT vapor or dust;
nay be first and only clinical manifestation), menstrual, disorders (hypo— or
hypermenorrhea), neurological manifestations (neurasthenia, nystagmus,
irregularities of tendon reflexes and adiadochokinesia; only 2.2% o the cases
in one study manifested diffuse brain lesions; 50% of the persons examined in
another study shoved irregularities in their thermoregulating reaction to heat
and cold (Kaganov et aX., 1970 as cited in Zakharj and Villaume, 1978)) and
nephrotoxicjty (as evidenced by a significant rise in glomeruj ,ar filtration
rate, sodium retention, urgency, frequent micturitiort and lumbar pain).
L’pon physical examination, the findings may include a yellow discoloration of
the skin, nails and hair. This is usually due solely to staining with TNT and
s not to be confused with the jaundice associated with liver toxicity. ore
bignificant would be a bluish discoloration of the mucosa indicative of
developing cyanosis. Other physical findings might include dermatitis with or
without rash (early appearing rashes may clear), eptgastrjc pain, tenderness
and/or spasm, enlarged and palpable liver and changes to the electrocardiogram
(bradycardia, decreased amplitude of QRS complex, flattened T—wave) and
electroencephalogram (decreased amplitude of biopotentiaj ,s, slowed activity,
poor reaction to stimuli), functional in nature, and apparently due to
vascular disturbances in the brain (Ermakov ec al., 1969 as cited in Zakhari
and Villaume, 1978).
Laboratory findings include an amber to deep red coloration of the urine and
various effects on the h .m.atologjcaj , parameters and blood chemistries. In
several cases where TNT exposure resulted in death, specific post—mortem
findings included fatty changes in the liver and kidneys. Foulerton (1918) as
cited in Zakh,rj and Vil] ,aum. (1978) reported that in 3 specific cases of
death due to TNT intoxication (exposure level, and duration not specified), the
liver shoved signs of advanced degeneration, disintegration of parenchyma,
fibrosis and advanced interlobular round—cell infiltration. Fat was
distributed in both parenchyma and fibrotic tissue. The kidney also shoved
signs of fat accumulation along with cloudy degeneration of the epithulium of
th. convoluted tubules. The glomeruli were, however, free of fat globules.
Numerous fat granules were scattered throughout the interalveolar tissues of
the lungs. Masses of brownish mactrial were found in all three organ systems
While there have been only limited reports in the English literature of
-------
cataract for .at .on resuLc.r. fron industr:a], exposure to TNT, :aKhar: a-:
(1978) descrioed seve’ al studies chac reported the f .nd:rg of
ca:a:ac:s a or.g European anc Russian dyna tite workers. The cataracts ‘ ave
seer. reporcec Co oZCen occur Without other toxic nanifestacions (Manc:.ova,
1968) . n .:e T;iu r a (1967) described changes in the crystalline lens as
occurri in four stages and being characteristic of TNT—induced opac t:es,
eas2 .l) d:stinguishab].e from those of different origins. Hass aa and .Juran
(1968) reported the occurrence of cataracts in 26,61 (42.6%) workers, avera2e
age of 44.5 years, exposed to TNT for an average of 8.4 years. The cataracts
were described as V—shaped or Lunar, white—grey in color and located tn the
area of the lens equator. In some cases, the opacities had merged to form an
irregular ring. While at ospherjc levels were not reported, the authors
indicated that cataract for .acjc was not associated with other toxic effects,
and that repeated exa jnacjons indicated no other health effects in 26.9% of
the workers with TNT—related cataracts. In 1978, Hass an at al. confirned the
occurrence of cataracts characteristic of TNT exposure in 87% of a group of 5’.
TNT workers with previously diagnosed or suspected TNT cataracts. Ccr.trol
subjects were not included in this study. Average exposure duration was
approxi.nate ly 14 years. Other TNT—related effects were ini a1, confirned in
on.i.y 9% of the exposed group and reported as chronic TNT intoxication.
More recently, Harkonen at al. (1983) reported on the occurrence of ecuacor:al
lens opacities in 6 of 12 occupationally exposed workers in Finland. he
opacities were described as bilateral and syetrical. They had no effect on
visual acuity or visual fields. They were detectable only in the periphery of
the lens, being either continuous or discontinuous. Exposure duration was *
approxinately 6.8 years with wor 9 oo air concentrations averaging 0.3 ngin
with a range of 0.14 to 0.58 g/ . Physjcaj. exa inacion as well as several
blood chemistry parameters were normal. The average age for the 12 workers
as 39.5 years with the subgroup having positive cataract findingo averaging
43.8 years vs 35.2 years in those without cataracts. In 1984, Makitie cc al.
reported that 18/21 (85) worker, exposed to TNT for a mean of 12.3 years in
the processing and packing of explosives had detectable equatorja lens
opacities, ‘oge frequently in the anterior cortex of the lens with decreasing
density toward central areas. The mean age of the exposed 3 worker , was 41.1
years while atmospheric levels ranged from 0.1 to 0.4 mg/m . Ten workers
showed varying degrees of central opacity, from minute spots to small.
rosettes, but these opacities were so slight that no effect was detectable on
visual acuity. In 50Z of chose with the peripheral lens opacici s, the
density was so slight that no shadow was seen in fundug reflex photography.
There have been no reports in the literature nor in occupational health
surveys on the occurrence of cataract, in munitions workers in the United
States.
The mechanism of TNT—cataract formation is not clearly defined. While note
recent studies (Harkonen at al. , 983) have investigated radical fornac tori,
based upon the vulnerability of t lt peripheral lens fibers to effects of
v i — 3
-------
per x aC1Ofl , as a 7O S2.ble cause of TNT—related cataracts, no def n c ’ie
C nC1’_S. .OnS could be drawn fron c’hts invesctgac .on. Several scudLes tnpi ca:e
d::ecc contact and local absorption as the probable cause (Kroll and
KolevatyKh, 1965; ‘anoilova, 1967 as cited in Zakharj and Villaune, 1.978)
basec upon the absence of syscenic effects in the ma)oricy of the exposed
individuals w .th the positive cataract findings. The weak polarity of TNT
also supports its ability to directly penetrate the lens.
It has also been found that individuals deficient in glucose—6—phosphate
dehydrogenase (G6PD) nay be particularly Su8CeptiblS to TNT intoxication. In
one report (Djerassi and Vitany, 1975 as cited in Zakhari and Villaune, 1978),
onset of henolytic episodes occurred in 3 individuals within 2 to 4 days after
initial exposure to TNT. Based on these and similar findings, it was
recoended chat determination of G6PD activity be made a pre—employmenc
requirement for TNT workers.
Effects on the white blood cells (WBCs), as evidenced by an increase in r-e
large mononuclear Leukocyte count, may also be an early indicator of TNT
poisoning. Hamilton (1946) reported chat increases in these cells usually
preceeded symptom .s of illness and Levels remained elevated for 2 to 3 months
following initial occurrence (cited in Zakhari and Villauze, 1978).
Toxic hepatitis and aplastic anemia have been reported as the principal cause
of death following TNT intoxication. Zakhari and Villaue (1978) reportee
chat several fatal cases of aplastic anemia were associated with earlier
episodes of non—fatal toxic jaundice or hepatitis. Thsy further indicacea
chat aplastic anemia can occur after a Latent period of several years
following an attack of toxic jaundice. Hyperplasia of the bone marrow is t e
first reaction of the hemapoietic tissues to TNT poisoning.
In a report prepared by the Department of the Army, as guidance standards n
industrial medicine and hygiene (DARCOM, 1976), gastrointestinaL symptoms were
reported as often the ftrst indication of toxicity. This report also
indicated the lack of a clear relationship between the occurrence of the
dermatitis often associated with exposure to TNT and the development of
systemic effects; either may exist in the absence of the other.
Cider reports on the advers, health effects associated with exposure to T’IT
generally did not. include information on workplace concentrations. In on.
uncontrolled study, Ermakov cc al. (1969) as cited in NRC (1982), reported
chac 3 l22 (212) of 574 employees exposed to an average T NT concentration of
mg/m were chronically poisoned; work exposures ranged from 6 to 25 years
Most of those affected had functional disorders of the central nervous svsce .
with 22% (27) having chronic anemia and leukopenia, 202 (24) with cataracts.
and 122 (15) with symptoms of hepatitis. No comparisons were made with
unexposed control populations.
VI-4
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Severa reports of cc .croLled scuaies nave provLded sone infor a cn on : e
• and subcl:ni.cal effects of TNT exposure (Stewart et al. , !9 5, EL
et al . , L97 , and Hathaway, 1974 as cited in NRC, 1 8 ; or:on et a _
A s: .if:canc finding in these ep .denioLogic studies s the occurren:e
of henaco1 .og .c and hepacic abnormalities at NT concentrations well below :-e
?erm ss .bLe Exposure Limit (PEL) of 1.5 mg/n (OSHA, 1981). Among the rnost
persistent frndings were mild reductions in hematocrit (Hct) , hemogLobin (‘ g5)
concentrations and red blood cell (RBC) counts of exposed persons. These
f.ndings have been attributed mostly to the destruction of red cells by
hemolysis due to exposure to TNT or to its metabol .ites (Voegtlin et a l. , l ::,
Cone, 1941k, as cited in NRC, 1982; Hathaway, 1917).
In one study cited by Zakhari and Villaune (1978), a group of 62 undergraduate
srudents 3 were exposed to atmospheric concentrations of TNT ranging from 0.3 Co
1.3 mg/n for approximately 33 days (Stewart et a)..., 1945). Observed changes
in 20 or more of the subjects included a decrease in Hgb and circulating
blood cells, art increas. in the number of reticulocytes, a small but
significant decrease in plasma proteins and a significant increase in
bilirubiri. The authors incicated that males were more susceptible to the
hemolycic effects of TNT than were females.
Goodwin (1972) reported that, in a 1951 study at the Lone Star Army A tur.t: ..cn
Plant (LSAAP) in Texarkana, te 9 s, mean atmospheric contaminant levels for T
(dust and fumes) were 2.38 mg/n , with no exhaust ventilation systems in ‘ se.
In a series of tests conducted under a Physical Recheck Examination Program,
the Thymol turbidity test, indicative of liver call irritation, was used to
evaluate liver impairment. From a total of 1,537 tests run during one
screening period, 87.5Z of th. workers were within the selected normal range
(to 2.9 MacL.ag.n units) with no signs of Liver toxicity. Of the remaining
workers with liver function teats above the normal range. from 2.9 to S
4 acLagen units, 36 (c2.51) showed classical symptoms of liver damage. Liver
function values in the affected workers, initially >5 MacLagen unfts, returned
to normal Limits within three .i .eks of their r. ova1. from the contaminated
environment.
In an occupational health study conducted by the U.S. Army EnvironmentaL
Hygiene Agsncy (USAEHA) at a TNT washout facility at Lerterkenny Army epot .rt
Pennsylvania, Fri.dlamder et al. (1974) reported that employees exposed for
months to TNT at various work locarions 3 in the facility and at atmospheric
Levels ranging from <0.02 to 3.00+ mg/rn displayed clinically and
statistically significant decreases in Hgb and Hct levels when compared to
pre—exposure values. Furthermore, a statistical comparison of these
post—exposure values with those of matched controls (non—exposed indivtdua .i
at the same facility indicated a higher rats of abnormalities in the exposed
individuals and mean value differences between the vwo groups.
In addition to significant differences in the 11gb and Hct values (0.005
vt—S
-------
0.Q ), s &n fjcant th.fferences vere also found in RBC count and b.ood urea
ger1 (3LN) (0.005 p 0.Olf’ and in reticulocytes, eos1noph ls and
ucose 0.0L p < 0.05). No si njficant differences could be demonsera- d
in severa]. ocher laboratory parameters including serum glutamic_oxajoace c
cransaminase (SGOT) , lactic dehydrogenase (LDH) , serum alkaline phosphacase
(SAP), cholesterol and total bilirubin, among others. It could not be
determined from this report if the positive clinical findings were dose
dependent.
In another occupational health survey (Morton and Ranadive, i 74) Conducted by
LSAEH.A at the Newport Army Aunitjon Plant (NAAP), Indiana, the d .scr buc±on
of abnormal values among workers correlated closely with both an increased
production rate (from 3 75% to )lOOZ 3 capacicy) and an increase in TNT dust
levej (from 0.3 mg/rn to 0.8 mg/rn ). Various parameters were tested
including Kgb, SGOT and LDH. Based on the measured values, 62.8% of the T
exposed individuals demonstrated abnormal findings. The detection race
(ability to identify abnormal results) ranged from approximately 26% when only
hgb values were evaluated to 100% when the values for all 3 parameters ( gb,
SCOT and LDH) were assessed. Recovery to normal lsvel occurred upon removal
of the individual from sources of exposure but the time required for recovery
could not be determined from the available data. No statistically significant
differences could be found in the incidence of abnormalities when results were
compared as to sex, age or race, but sampling size may not have been
sufficient.
Further statistical analysis of these clinical parameters as measures prior to
the time of increased TNT production (atmospheric levels of 0.3 mg/rn ) paired
with those one month after production was increased (atmospheric levels of 0.8
mg/n ) indicated a statistically significant increase in L.DM levels (P <0.005)
anc SGOT leveig (P <0.01) following th. increase in production race. No such
correlation was seen in hemoglobin levels (Morton cc al., 1976). This
increase in both the number of individuals with abnormal test results and the
degree of the abnormality were correlated with the higher atmospheric levels
of TNT, leading the aut ore to question the suitability of the Threshold Linit
Value (TLV) of 1.5 m$/m recoended at that time (ACCIM, 1971).
In a follow—up to the two previously cited occupational health surveys at Army
facilities, USAE A performed a cross—sectional epidemiologicai. stu y invg3 ving
626 employees exposed to one or more muniçion compounds (TN J RDX H2(X )
and 865 non—exposed employees from 5 Army Aunition Plants (Buck and
Wjlso , 1975). A.].]. individuals wer, evaluated for liver functjo (SAP, SCOT,
serum glucamjc—pyr vj transaminage (SGPT) and bilirubin) and hemacological
cyc1ocrjzechyjenetrjnitramjae (1 hexahydro_I,3 ,5_trjnocro_1,3,5_trjazjne)
(occahydro_ 1 ,3,5,7_cecrafljtroI ,3,5,7_
tetrazo line)
JoJ.iet, Iowa, Milan, Volunteer and Hoistort
V 1—6
-------
cc and retLculo re count) abrorma ities. N evidence of L .ver
: x c::y as dicacec by tne’ araneters studied. This result appears to
.n c .zrasc Co the positive f dings of liver toxicity in the NAAP SCU y•
cwe’ er, exposure levels in this cross—sectional study were generali> C.3
with on .y approxImately 1.2Z of the TNT workers exposed at lev 5 ls ‘0.5
g/m wr.iie at AAP, exposure levels rose to app 5 oxinacely 0.8 ng/n
Accordi . gly, the authors indicated that 0.5 mg/n nay be considered a
reasonable no effect level for hepatotuxicity.
On the other hand, a significant hemacological effect was observed among T’T
.crk rs exposed in this cross—sectional study to atmospheric levels of .0.5
This positive effect was evidenced by a dose response relacior.shlp for
all three parameters arid occurred more readily among males. Th se results
suggested to the authors that low level TNT exposure (<0.5 mg/n ) may induce a
low grade henolysis with a compensatory mild reticulocyrosis. It was not
possible o determine a no effect level for hematological effects from the
stucy. As a result of this study, LSAENA reCommended that th TLV for TNT n
the work)Place be lowered from the existing lev l of 1.5 mg/n to a level of
0.5 mg/n and that the U.S. Army adopt 0.5 mg/n as their airborne exposure
standard for TNT.
3. Health Effects in Animal Experiments
1.. Short—Term Exposure
As indicated by studies in rats, mice and dogs for periods up to four weeks,
dietary intake of TNT resulted in early but not persistent decreases in body
weight and food intake while the red pigmentation in the urine persisted
throughout. Sow. anemia was evident but somewhat inconsistent while
hemosjderosjs of the spleen was seen in all three species. Rats developed
testicular atrophy. Table VI—1 summarizes thee. toxicity studies.
Lee et al. (1975) determined the acute oral toxicity of TNT in Charles River
CD rats and albino Swig, mice. Rat, and nics were fasted for at least 16
hours prior to dosing by intragastric intubation with a 4.12Z saturated
solution of TNT in peanut oil. After treatment, the survivor, were observed
daily for 14 days for delayed mortality or toxic signs. The LD 5 A was
calculated by a computer program based on the method of maximum 1 ikelihood of
Finney (1971).
The acute LD values in ma].. and female rats were 1,010 and 820 mg/kg,
respectively; in male and female mice they were 1,014 and 1,009 mg/kg.
respectively. Symmetrical coordinated convulsions associated with respiracor
inhibition occurred within 5 to 1.5 minutes after dosing and continued for to
2 hours. Death, when it occurred, was usually due to respiratory paralysis
while survivors appeared cyanotic and exhibited ataxia. Recovery was complete
in 24 to 48 hours. No gross pathology attributable to treatment was noted,
Vt-7
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Table Vl—I Summary of Studies: Short—term Exposure of Alilulaib to TNT
rat, mouse
rabbit
guinea pig
rabbit
mouse
rat, mouse
dog
rat (male)
(female)
Dose
mg/kg/daya/
1.5, 1.0,
184.3
1.5, 7.7,
176.9
oral
dergial, ocular
derma I
dermul, ocular
oral
oral
oral
oral
0.3, 2.0, 14, 100, oral
700
Reference
Species
‘lout e
Lee et al. (1975)
Newell et al. (1976)
i ir it hii
a!
uceks
50x
4.
I 22
92
b/
Litiley et a!. (19)8, 1982)
/2 hours
0.2, 2.0,
1.8, 8.8,
190.4
Li, 8.5.
180.4
20.0
42.7,
41.2,
35.3,
35.9,
mouae (male)
(female)
up to 1 d.ays
I-
4
4
4
Levine et al. ( 1984a) mouse
Uiileui i otherw1t e stated.
A 1 ’pruxloiat.. 2; wastewatcr reilduc.
oral
4
-------
:- 0 CO O . uces after dosing, a br gnr red p &nenc vhic sraLred : e
fir ad bedcing aopeared in th e urine of both species and Continued : be
exc:e:ed for several hours after dosing.
D ::ev et aL. (97S, 1982) also conducted acute toxicity tests of TNT using
iature Sprague Dawley—derived rats and Swiss Webster mice. The acute oraL
LDs were determined in animals that were fasted for at least 16 hours bef3re
th y were dosed. Four or five dose levels with 10 males and 10 females per
dcse were used. The test material was administered as a suspension in corn
oil via oral—dosing needles at a volume of I nl/100 g body weight.
Cbservation of survivors continued for 2 weeks and any toxic signs were ncced.
All deaths were recorded and a gross examination was performed. The c as
calculated by the same method of Finney (1971).
The acute oral LD. 0 s of TNT were 660 mg/kg in both male and female nice and
1320 and 794 mg/kg in male and female rats, respectively. Initial toxic signs
included inactivity and tremors within the first I or 2 hours, followed by
petit na . convulsions. Red urine was noted in both species within 60 ninuces
after dosing. Death, when i; occurred, was within 4 hours. Animals that
survived the convulsions recovered and were still alive at 14 days after
treatment.
The acute toxicological effects of actual and synthetic TNT wastewaters were
reported by Newell cc al. (1976) a d Sasmore at al. (1977) before and after
irradiation and at different pH values. The oral LD values of the
].yopnilized, reconstituted residues were determined in fasted, adult male and
female Swiss—Webster m ce (15—20 g). Authentic TNT wascewaters were obtained
from the Joliec Army Aunitions Plant (JAAP) load and pack (LAP) operations
and initially contained 125.5 ppm T NT and 30 ppm RDX plus several minor.
unidentified components. The LAP vIstswats was Lyophilized and reconstituted
with water and TNT was added to give the expected 9% level (some TNT was lOSt
under conditions of large—scale lyophilization). The bulk of the lyophilized
residue (91:) was inorganic salts. The reconstituted solutions were adjusted
for specific pH levels, photoly sd as specified and either lyophilized to
solid residues or extracted with b.nzene and lyophilized. The residues here
administered as corn oil suspensians/solutions. Table VI—2 compares the
results of this toxicological evaluation. For the synthetic preparation, the
degree, if any, of irradiation could not be accurately determined from the
available data; pH via stated to be 7.0; lyophilized residues ware
administered in corn oil. The pure TNT samples were originally dissolved in
di rjljed water.
Toxic signs following oral administration included lassitude, cyanosis.
occasional muscular twitching, convulsions and red urine. Death occurred
the first 24 hours or not at all. Recovery was completa in 2—3 days. So
gross pathological lesions were reported for the surviving anim.als autops e
after the 14—day observation period.
Vl—9
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Table Vt—2
Acute Oral L 50 (nglkg) of TNT Wascewater esLdue
a d Synchecic Preparation n iicea
Percer age trradiacion
pH 50 100
JAAP residue 5.0 —— 1500 9OG
(pink water) 7.0 1300 2600 >5000
9.4 —— 1600 4200
JAA2 residue / 5.0 ——
(benzene fraction) t 7.0 500
9.4
JAAP residue 5.0 —— 4200 4700
(aqueous fraction) 7.0 2500 4700 ‘5000
9.4 —— 3900 4400
TNT
Syntheti Wascewater
7.0
7.0
830
zsc_ 2 sod/
a!
Reference: evell et al. (1976); residue ad jnjstered as corn oil
suspensions/soluc ona; LD 50 calculated using ethod of Litchfjejd and
b/ Wj oxon.
Phocolyzed until initial TNT concentrations decreased by SOZ and near y
i100z.
TNT reported to be major component of thie fraction.
Result of 2 separate determinations; percent irradiation, if any, not clear
for this evaluation.
vt— b
-------
: ese studies, the authors concluded that the toxic eor.ponertt o : e _.- ?
.astewacers obtained from J 2’was mostly extractable with benzene and thaz
Lrrac.ation a.so reduced cne wascewacer toxicity. They further postulated
chat ‘.T nay be crte principal toxic component as it constituted approxj ace:’.
9O. of tie organic material in the LAP water and was removed from wascewacer
both by irradiation and bertzene extraction.
Oral LD.. 0 studies in other species were not available in the literature. The
followi g lethal doses for TNT (oral LD, , lowe5t dose reported to have caused
death) were reported: rabbit, 500 mg/kg; rat, 700 mg/kg; ear, 1,850 mg/kg
(Wyon, 1921 as c ced in NRC, 1982).
a. Skin and Eye Irritation, Dermal Sensitization
The primary skin and eye irritation tests in rabbits, using the modified
Dra ze machod (Federal Register, 1574 as cited in Lee et at., 1975) indicated
that TNT was a mild irritant to the skin but did not irritate the eye. A red
stain, similar to that seen in the urine, appeared both art th. skin and around
the eye after application of a 50% TNT—peanut oil pasts (Lee et al., 975).
Topical application of a saturated 4.12% solution of TN T in peanut oil to the
clipped skin of guinea pigs produced a 40% response and was considered a
moderate sensitizing agent (method of Magnusson and Kligman, 1969 as cited .n
Lee et al., 1975).
Newell et at. (1976) also evaluated the skin and lye irritation potential of
the .JAAP LAP wastsvater dry, powdered residues using the Draize method in
adult male and female New Zealand white rabbits (2—3 kg). No skin irritacton
was observed when either the 0% or 100% irradiated neat (unextracted) residue
was applied to the prepared sits; however, considerable red skin staining
occurred particularly when the 100 irradiated residue was applied.
o eye irritation was observed when the nonirradiated lyophilized wastewater
residue was instilled and washed 30 seconds or 5 m.tnutss after instillation.
Irritation, including iritis and corneal opacity, was observed for up to 3
days when this residue remained in the eye for 24 hours. Irritation was
nearly absent 4 days after instillation and recovery was complete by 7 days
post—treatmant.
b. Four—Week Studies
In a four—week range finding study, Levine et at. (1984a) fed TNT ( 99Z pure)
mixed in ground Purina chow to groups of 10 36C3F1 mice/sex at levels of 0.0,
0.3, 2.0, 14, 100 or 700 mg/kg/day. Animals vets observed for clinical signs
of toxicity, and body weight an4 food coasu ption were measured. Prior to
sacrifice during Test Week 5. the animals were subjected to an extensive
hem.atological and clinical chemisty evaluation . Animals were sacrificed v
V t—I l
-------
car5on dioxide arteschesja, maor organs here weighed, and all organs vere
::xed or histological exam nac1or ; bone marrow smears were prepared.
5cac.sc ca]. analyses were accomplished using Analysis of Variance tests and
Dunne:z’s c—test when necessary.
No signif cant cl nical signs of toxicity nor mortality were observed. iice
receiving the 100 and 700 mg/kg/day doses had red—stained bedding. High—cosed
mice showed a reduction in weight gain and/or loss in body weight throughout
the study but food intake was increased in males receiving this dietary level.
At 100 mgikg/day, only occasional and slight decreascs in body weight gain
were recorded with no alteration in food intake.
The only significant changes in the hematological parameters were a decrease
in BC in males and an increase in platelets in females receiving 0 the high
dose. A dose—related increase in bilirubin was apparent in both sexes with
increases of 25Z and SOZ at the 100 and 700 mg/kg/day dose levels,
respectively. Weights of the kidneys and testes were significantly decreased
in male mice at the 700 mg/kg/day level.
A diffuse increase in the relative amounts of yellow—brown pigment, resembling
hemosiderin, was present in the red pulp of the spleen. The increase was c f
minimal severity at the 100 mg/kg/day level and of mild severity at the
high—dose level. Extramedullary hematopojegis did not differ in incidence
from controls. No treatment—related lesions of the testes were evident in
this study. No neoplastjc lesions were observed. This study identifies a
o —Observed—Adverse_Effect..Level (NOAIL) of 14 mg/kg/day for absence of
effects on body weight 1 serum bilirubth and the spleen.
In four—week oral toxicity studies conducted by Dilley et al. (1978, 1982) in
cogs, rats and mice, the appearance of red urine consistently occurred at the
highest dose level in dogs and at the two highest dose levels in rats and
mice. A decreas, in weight gain accompanied by a decreased food intake
occurred during the first week or two of treatment with recovery toward normal
levels often occurring thereafter. Mild to moderate anemia and increased
spleen weights, usually accompanied by hemřsiderosjs, at the high dose level
were also coon to all three species. Atrophy of the testes occurred in
rats, an increased cholesterol levels and decreased SGPT activity were
evident in dogs and rats.
In the dog study, 40 beagle dogs, approximately six months in ag. at the start
of the experiment, were divided into four groups of fiv, males and five
females each. The treatment group, received a TNT/lactose mixture equivalent
to 0.2. 2.0 or 20.0 mg/kg/day by capsule. The control group received lactose
only, also by capsule. All dogs were observed for signs of toxicity and
weighed weekly, and food intakes were recorded five days/week. Tests include
extensive hematology, clinical chemistry and urinalysis. At the end of four
weeks, one dog/sex/group was sacrificed and subjected to a complete
‘ I — 12
-------
s : a:hological exaai r.acion. An additIonal dOg/sexI oup was renoved
z e ::eatmenc routine and plac’ed on a recovery study. The remaLn .ng dogs we
c r.::nued on the treatment regimen, and results are discussed under l. 3 —wet 1 (
scuc es.
No toxic s ns were reported for the dogs treated at 0.2 or 2.0 mg/kg/day.
20 mg/kg/day, loose mucoid stools and diarrhea were frequently observed.
Orar.ge—cjnted urine was evident by day six of treatment and conc1n ued to
termination. Sporadic periods of inactivity occurred in males receiving the
high—dose level. Both treatment and control groups lost weight during tne
first week of the study with the high—doge group showing the greatest degree
of ioss and the slowest recovery rate, but no statistically significant
effects on body weight were detected. Food intakes were appreciably lower Ln
the high—dose group during Week 1 and slightly lower during the second and
third weeks. Organ weight analysis indicated an enlarged spleen in the male
and an enlarged liver in the female sacrificed after four weeks of treatment
with TNT at 20 mg/kg/day. Anemia, as evidenced by decreases in RBCs, Hgb, Mc:
and mean corpuscular hemoglobin concentration (MCHC) and an increase in mean
corpuscular volume (MCV), was pronounced at the high—dose level with Hgb and
hCHC values significantly decreased in both SiXiS. Clinical chemistry studies
revealed statistically significant increases in cholesterol and bilirubin and
decreases in SGPT and iron in dogs receiving 20 mg/kg/day but not all
parameters were significant in both sexes. After the four week recovery
period, cholesterol in males and bilirubin in females tended to remain
elevated while SGPT values returned toward normal. tn contrast, iron levels
were greatly increased in both sexes after this recovery period.
Histopachologjcaj examination revealed no clear cut treatmtnt —re lated effects
except possibly the hemosiderosis of the spleen, seemingly related to the
anemia, in the female dog receiving 20 mg/kg/day for four weeks. A NOAEL of 2
mg/kg/day for absence of anemia and effects on the liver and spleen is
indicated.
In the rat studies (Dilley et *1., 1978, 1982), five groups of Sprague—Dawley
rats, 2 O/Sex/group, received 0%, 0.002%, 0.01%, 0.05% or 0.25% TNT mixed in
powdered Purina Laboratory Chow (or approximately o, 1.8, 8.8, 42.7 and [ 90.4
mg/kg/day of TNT, respectively, for males and 0, 1.7, 8.5, 41.2 and 180.4
mg/kg/day, respectively, for females, based on the authors’ data for averaSe
intake of TNT over the four week period). AU animals were observed for toxic
signs; animal weight and food intake were determined weekly. Tests included
hematology and clinical chemistry. At the end of four weeks of treatment,
five rats/sex/level were fasted for 16 hours, anesthetized with chloroform,
and blood was wirhdra by heart puncture. The animals were sacrificed, major
organs were weighed and all. organs wire fixed for histological examination.
Five additional rats/sex/level were removed from the treatment regimen and
allowed to recover. Th. remaining rats VSVS continued on the treatment
regimen for 13 weeks, and results are described under “tonger—Te Z .xposure.
VI— 13
-------
Q< signs were apparenc. Those rats receiv ng the two ‘ighest dos 5
(O’.OfZ arid O.25) developed a red’coLor in the urine from Day ‘2 contLnuous1 .
hr gh : e day of sacrifice. Body weights were significantly reduced iri e
nigh—cose grouc, as were food intakes. In the high—dose male rats, a
si nif canc iricrease was observed in the .bsolute and relative liver and
spleen weights and a decrease in the weight of the testes. Only the spleens
of these females were similarly affected although the liver to body.wej .ght
ratios, but not the absolute Liver weights, were increased. There were no
statistically significant differences in hematologjcaj parameters after 4
weeks of treatment although RBC, Hgb, and Mct values were reduced in both
sexes receiving the high dose. Only cholesterol was significantly increased
in both sexes at the high TNT dos .. After four weeks of treatment, all males
at the high dose (Q.25Z) displayed testicular atrophy and hyperplagia of the
.nterstjtial cells. One male at the 0.05% TNT dose also had testicular
atrophy. Hemosiderosts of the spleen was present in all rats receiving the
high dose and in one female receiving 0.052 TNT. No m.icroscopjc lesions
associated with the gross hepatomegaly were found. No other lesions were
found to be related to TNT treatment although the rats receiving the highest
dose appeared to have greater susceptibility to respiratory disease, a
evidenced by an increased frequency of alveolar collapse and dilation in the
high—dose females.
During the four week recovery study, the red color disappeared from the uri’e
in 15 days, weights increased toward normal levels, and food consumption
increased in the high—doge groups. Tb. weight of th. testes from the
high—dose males remained low but the anemia was absent and cholesterol values
and SCPT activity were normal. Testicular atrophy and hyperplasia of the
inter!titial cells were present in all five high—dos. males along with
hemosiderosis of the spleen in four of the five female .. 3aa.d on the absence
of testicular effects and lesions in the spleen, a NOA.EL of 8.8 mg/kg/day Ls
indicated.
In the mouse studies (Dilley et a L., 1978, 1982), 20 male and 20 female Swiss
Webster mice per group received TNT in the diet at 0.0, 0.001, 0.005, 0.025 or
0.125% TNT by weight (equivalent to approximately 0 1.5, 7.0, 35.3 and 186.3
mg/kg/day, respectively, in males, and 0, 1.5, 7.7, 35.9 and 176.9 mgJkg/d .av,
respectively, in females). Weekly body weights and food consumption were
determined. Hsm.atologjcal parameters were measured. At the end of four weei s
of treatment, five mice/,ex/].ev.j. were sacrifj .d by anesthesia, major organs
were weighed, and all organs wer, prepared for histopathological examination
Five additional mice/sax/level were placed on a four week recovery study Th,
remaining 10 mice/sax/level were continued on the Study for a total of 13
weeks. R s ltg are reported under “Longer—term Exposure”.
No toxic signs related to TNT treatment ware apparent. The urine of mice
receiving the two highest dose levels (0.025% and 0.125%) became red in f ur
to six days after the start of treatment and remained red throughout the
Vt—14
-------
::eaz enc per od. the color dtsa peared fron the urtne LO days after Ze
“tre ?Lacea on the recovery st dy. Body veights vere signif:ca 1y Lo e: :-a-
n both sexes receiving O..257. TNT after one week, of treat erc 5u:
:ecover’ coward control levels occurrea over the next severa : eeks.
ecreased we gnt gain was also evident a: the O.C5Z level but not
signif .cart :1v so. Food consumption was decreased in both sexes at the two
h .ghesc nose levels during the first week but was not signif cancly different
from control n:ake; recovery to normal intakes occurred over the r ext two
weeks. During the recovery period, the mice at the high—dose level shghc . ’..
increased food consumption during the first week but body weights did no:
change to any notable degree. The only treatment—related effect on organ
.eighc was a significant increase in the spleen—co—body weight ratio in the
h gh—dosa males. “hile not statistically significant, the mice receiving the
high dose of TNT, particularly the females, showed signs of anemia, as
evidenced by decrease in RBC count, Hgb and Mc:, and smaller increases in
.CV, MCH and MCHC. After the four week recovery period, few signs of anemia
were evident. No treatment—related effects were observed upon histological
examinatLon , A NOAZL of 35.3 mg/kg/day is based on marginal effects a: the
higher dose level on the spleen and hemacopotetic system, both apparent target
organs for TNT toxicity.
2. Longer—Term Exposure
In studies conducted in rats, mice, dogs and monkeys for periods ranging frcn
L) weeks to 2 years, dose—related reductions in body weight and food intake as
well as anemia and red pigmented urin, were evidenc in most species. Organs
consistently affected by TNT intake include the Liver and spleen in dogs, rats
and mice as well as the testes in rats. No ophthalmic effects were ev .cerc.
Evidence of carcinogenicity was established. Table VI—3 suarizes these
toxicity studies.
a. Thirteen—Week Studies
In a continuation of th. four—week study described ur4.r “Short—term
Exposure”, DiiJ.ey cc al. (1978, 1982) exposed dogs, rats and mic, to TNT for a
total of 13 weeks. Groups of these animals were removed from the test d ec
and allowed to recover fot an additional four weeks. A red coloracjcjn a: the
urine was coon to all, three species at the higher dos. levels. Only rats
showed a persistent negative effect of TNT treatmanc on body weight gain.
A.nemia was apparent in afl. three species while hemosiderosis of the spleen -as
seen predominantly in rats and mice. Liver weights were increased in dogs rd
mice with some necrosis in the mice. Cho1. tsro1 levels were increased arc
SGPT activity was decreased in dogs and rats; these para .ters were not
measured in mice. Treatment with TNT for 13 weeks had no apparent effect
survival in any of these species.
In the dog study, three males and three females per group were adminisce-e
VI — IS
-------
Table V1—J Summary of Sti&d1e : long—term Expo 6 ui e ol AiiImal to TNT
Levine et al. (l981 , I984b) rat
MartIn (1974) monkey
hart (1974) dog
Levine (1983) dog
Furedi et at. (1 98 4a, b c) rat
( 1984d, e, 1) •ou
a Unleaa othei-vj e ataced.
0.2, 2.0. 20
1.4, 1.0, 34.7, 160
1.4, 7.4, 36.4, 164.4
1.6, 1.5. 35.7, 193
1.6, 8.0. 37.8, 184.2
I, 5, 25. 125, 300
0.02, 0.1, 1.0
0.02, 0.1, 1.0
0.5, 2, 8, 32
0.4, 2, 10. 50
1.5, 10, 70
oral ii
oral 13
oral 13
Reference
Dilley et al. (1978, 1982)
pec lea Dose
mgIkg/duya f
koute
dog
rat (male)
(female)
mouae (male)
(female)
oral I) —
oral 90 day
oral 90 day
—--------
oral 26
oral 104
oral 104
-------
:apsue 0.0. 0.2. 2.3 or 20 mg TNT/kg/day mixed with lactose for a rota:
ees at which z.ne two dcgs/ ex/1evei were tasted overnight. The an as
here anesthetized, and blood was witherawn for hematological and chemical
analvs.s. The aninals were sacr ficed, major organs were weighed, and a1
organs were fixed for histological examination. body weights and food Intakes
were measured weekly. The remaining dog/sex/level was removed from the rest
regimen and allowed to recover for an additional four weeks.
The only gross signs of toxicity in the dogs were loose mucoid stools and
diarrhea in the 20 mg/kg/day group. Periods of sporadic inactivity were
evident in these males throUghout the study, becoming persistent by Week 11
through termination of the treatment. On one occasion, one male displayed
s .gns of nystagmus. Red colored urine appeared in all dogs on the high—dose
level, beginning on Day 6 and persisting as long as TNT was administered. The
color returned to normal during the recovery period. During Week 12, one male
receiving 20 mg/kg/day became moribund and was sacrificed. Necropsy revealed
swelling of the left upper cerebral hemisphere, lung congestion, hemorrhagic
l’rmph nodes and enlarged kidneys, liver and spleen. Anemia was marked, and
cholesterol, and SAP were incrçased. The authors indicated that the presence
of a duodenal nemacode may have been a contributing factor in some of these
lesions. No other dogs succumbed to the treatment regimen.
No statistically significant effects on overall body weight were evident
although all groups lost some weight during the first week; high—dose females
lost appreciably more weight than controls. Of note is the observation that
the male and female placed in the recovery study after receiving TNT at 20
mg/kg/day for 13 weeks lost *eighc during this period, with the reversal in
weight beginning around treatment Week 10 and continuing thereafter. This
prompted the authors to speculate on a possible delayed onset of toxicity.
Food intake was appreciably lower in the high—dose group during the first week
and remained somewhat lower throughout. No other differences in intake could
be related to treatment.
Absolute and relative liver and spleen weights were increased in both sexes
receiving the high dose but both values were within normal. range in the
recover c group. Whil, anemia was pronounced in both sexes on the high—dose
level during the first four weeks of treatment (as indicated by significant
decreases in Hgb, Hct and MCHC, along with an apparent decreas, in RZC count).
by Week 13, only the MCHC remained significantly depressed. These parameters
were within normal range in the femal, dog on the four—week recovery regimen.
but they appeared depressed in the high—dos. male. Clinical chemistry tests
showed a significant increase in bilirubin and creatinine and a significant
decrease in SGPT and BUN in females receiving 20 ag/kg/day for 13 weeks. tn
the males, no significant differences occurred; however, bilirubin and
cholesterol appeared appreciably increased while SGPT and BUN appeared
similarly decreased. All parameters were within norme]. range in the recover:
animals except for an apparently high cholesterol and low SGPT in the
high—dose males.
Vt—17
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E.x:epc f3r the nocabie pathology in the high—dose nale sacrificed dur ng
(as preViously described), che only ocher lesions of possible S.& a .ce
were an eri.Largecenc and pigt’.encacion of the parenchvmal nacrophages in c e
liver c the renainir.g nale and one of the two females. This study
establishes a ‘0AZL of 2.0 ng/kg/day for the absence of anemia along with t e
absence of toxic effects on the liver and spleen.
In the tar study, essentially a continuation of the four—week study by Di.Jev
ec al.. (1978, 1982) previously described, groups of 10 male and 10 fem.ale
Sprague—Dawley rats were exposed to TNT in the diet at 0%, 0.002%, 0.OLz ,
0.05% or 0.25% for a total of 13 weeks. These doses were equivalent to
approximately 0, 1.4, 7.0, 34.7 and 160 mg/kg/day, respectively, for males and
0, 1.4, 7.4, 36.4 and 164.4 mg/kg/day, respectively, for females, based on an
average calculated by the authors. Body weights and food intake were recorded
weekly. At 13 weeks, five males and five females per dose level were
anesthetized with chloroform, and blood was withdrawn by heart puncture f3r
hematologicai. and chemical analyses. The animals were sacrificed, major
organs were weighed, and all organs were fixed for histological examination.
The five remaining rats/sex/dose were removed from the test diets and allowed
to recover for four weeks.
No deaths occurred before scheduled sacrifices and no signs of toxicity were
observed at any dose level. All rats on the two highest dose levels (0.3
anc 0.051) developed a red color in the urine beginning on the second day oi
treatment. A similar color appeared on Day 50 in the urine of rats receivir g
0.01%. This red color persisted through termination of treatment but
disappeared after 16 days on the recovery study. Sigaific -. findings in t e
rat study at the high—dose level (0.25%) included growth s _ sression ,
beginning during the first week and continuing throughout (possible “syscenic
errors” in the weighing procedure was indicated for the two love t dose levels
during i eek 9), accompanied by a decreased food intake, a significant decrease
in the absolute weight of the kidney and absolute and relative weight of the
testes in males and an increase in absolute and relative spleen weight in both
sexes. Anemia, &i evidenced by significant decreases in Hgb, Hct and MCHC, an
increase in MCH, and an accompanying but non—significant decrease in R3C
count, was present in both sexes receiving the high dose. Clinical chemistry
values included a significant increase in uric acid and cholesterol and a
decrease in glucos, in males and females while males also showed significant
decreases in SGPT, SAP and iron and an increase in crsacjnine.
At the next highest dose level (0.05%), aigt s of anemia were evidenced by
significant decreases in Hgb and Hct in both sexes and RBC count in females.
Female race receiving 0.01% TNT also shoved similar signs of anemia. Iron
levels were significantly decreased in males receiving TNT at 0.05% and O.O
but not at the loveit dose (0.002%). GLucose levels were significantly
decreased in males at the 0.05% test level and in females at the two lowesc
levels (0.01% and 0.002%). Hemosiderosis of the spleen and interstitial
VI — 18
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;rioc/:es .n cr e .dney were the only Lesions co on to both sexes a: : e
-:;- —dos .evel arid, to some d’čgree, at the next highest level (O.25 a
.O5 , respecciveL ) that were attributed to treatment wic TNT (a3.thouzh : c
ra:s sex at the control level also displayed some degree of hemosiceros .
This, however 1 became evident in controls only after four weeks on the
extended recovery scuQy.) Male rats on the high dose were reported to ha e
atrophy of the epidid)mis and testes accompanied by hyperplasia of the
interstitial cells. No hiscopathology was done on rats at the two lowest
levels after U—weeks of treatment, despite significant findings at the r.exc
highest level.
After four weeks of recovery, most of the above described parameters returr’ed
toward normal level; however, the Hgb, Hct and MCH in the high—dose males were
significantly increased while the kidney and testicular weights remair.ec
significantly decreased. The liver, spleen and brain weights were
significantly increased in females. Histological findings were similar to
chose after the 13 weeks of treatment. An unexplained finding at the three
highest dose levels (0.012, 0.052 and 0.252) allowed to recover for four weeks
was a significant decrease in albumin (A) to approximately one half of control
levels and a significant and marked increase in globulin (C) levels to 6 to 0
times the control levels. This resulted in a dramatic reversal of the A/C
ratio, 10 to 25 times higher than control levels. The authors discounted this
finding as “inconsistent”. A NOAEL of 1.4 mg/kg/day is indicated by the
occurrence of anemia at the next highest treatment level.
Dilley et al. (1978, 1982) also continued 10 Swiss—Webster mice/sex/level on
diets treated with TNT at levels of 0.02, 3.0012, 0.005, 0.0252 or 0.125: for
a total of [ 3 weeks. From intake and weight data, th. average intake of TNT
was calculated to correspond to approximately 0, 1.6, 7.5, 35.7 and 193.0
mg/kg/day, respectively, for males and 0, 1.6. 8.0, 37.8 and [ 84.2 mg/kg/day,
respectively, for females. Animals were observed for signs of toxicity; body
weight and food intake were recorded weekly. Ac the end of 13 weeks, the mice
were fasted for 16 hours, anesthetized with chloroform, and blood was
withdrawn by heart puncture for hematology. The mice were sacrificed, major
organs were weighed, and all organs were fixed for histological examirtacton.
No toxic signs attributed to treatment vets reported. Red color appeared ri
the urine of mice receiving the two highest doses (3.0252 and 0.1252) wichiri
6—6 days from the start of treatment and remained throughout the dosing
period. Premature ds.ths included one male per group from the three highest
dose levels (0.0052, 0.0252, 0.1252) during Week 2 and on. additional male
each at 0.0252 and 0.0052 during Weeks 6 and 13, respectively. No TNT created
females died during the study but one control died during Week 8. This
attrition rat. was not considered significant by the authors.
A significant decrease in body weight occurred in both sexes on the high-dose
level during Week 1, but quickly returned to normal Levels during the riexc : .‘
weeks. A slight but not significant decrease in food intake during this a:e
VI.— 19
-------
period was probably d cative of an initial aversion to the diet. It s cu::
:e c:ed that control females in “this study exhibited an ‘abnormally low
growth pattern” With low intake throughout. No significant effects were
present in hemacological parameters although males at the two highest levels
showed a slight aecrease in lict. Heart and heart—to—brain weight ratios were
significantly increased in males receiving 0.005% and 0.125% TNT but this
finding did not correlate to any pathology. A slight but significant Increase
in absolute and relative spleen weight was evident in females receiving the
high dose; all females at this level displayed a hemosiderosis of the spleen
as did 3/5 males. The authors felt that this was the only treatment—related
effect in mice receiving TNT for 13 weeks. Dilation and/or hyperplasia or
edema of the uterus occurred in some TNT treated females at [ 3 weeks or after
four weeks of recovery but was not statistically significant.
while chronic respiratory disease occurred in many mice at all levels in this
study, male mice on the two highest dose levels had a greater incidence of
complications, such as alveolar dilation and collapse. Significance of these
findings is not known but could indicate an increased sensitivity to this
condition.
In the mice treated for 13 weeks and allowed to recover for four additIonal
weeks, the only significant findings were an increase in absolute and relatIve
kidney and liver weight in high—dose males and in spleen weights of the
females. Two of these males also displayed necrosis of the liver.
iemosiderosjs of the spleen was evident in 80% to 100% of the mice on the
high—dose level (0.125%) and 80% of the females receiving the 0.025% TNT level
for 13 weeks with four weeks of recovery. A NOAEL of 1.6 mg/kg/day is
indicated by premature deaths (2/10) along with effects on the heart weight in
males at the next higher dose level.
Levine et al. (1981, 1984b) conducted a 13—week study in which Fischer 344
rats (lO/sex/dose level) were administered TN ( 99Z pure) in the diet at 1,
5, 25, 125, or 300 mg/kg/day. Thirty animals per sex were used as controls
and received the sama rodent chow used to prepare the test discs. Animals
were observed daily for pharinaco] .ogjc and/or toxic signs; test diets were
changed weekly and prepared on the basis of weight and intake for each sex:
clinical chemistry and hematology tests were performed following a 17—19 hour
fast on all surviving animals during Test Week 13. Ac sacrifice, the brain,
gonads, heart, kidneys, liver, and spleen were weighed, and all tissues were
collected and fixed for histological examination.
Sacrifice of the surviving animals was accomplished over Weeks 14 and 15 of
the study with one rat/sex/test group and three rats/sex/control group
sacrificed each day. Test diets were administered up to approximately one day
prior to the animals’ sacrifice by carbon dioxide anesthesia. Control and
high level test animals were subjected to a complete hiseopathological
examination with all other levels limited to examination of those major orgars
VI—20
-------
-a ere routinely we.ghed. The auc ors further noted that since : e a_e
a:s ere receivec on site tw weeks before the females, the study was
r.oucted n two 2hases witn the males always two weeks ahead of the fenaes.
C .injcal oDservacions included lethargy and/or ataxia which were seen duri
Leek 1 and to a lesser extent during subsequent test weeks for several art: a:s
receiving TNT. Ac Week 6, some of the males rece .ving TNT at 300 ng/kg.’dav
were noted by palpation to have smaller than normal size testes. Red—sca ec
bedding was seen at all TNT doss levels except for the lowest (1 ngIkg/day)
level.
Administration of various levels of TNT in the diet did not appear to have an
adverse effect on survival time. Wbile one male and one female at the 300
mg/kg/day level died during Weak 13 of the study, death occurred within days
following blood collection and was postulated to be due to a combination of
TNT induced anemia and the stress of a reduced blood volume.
Dose—related reductions in body weight gains were, in general, observed for
nala TNT—treated rats throughout the 13—week treatment period. These
reductions amount to approximately 5% at the I mg/kg/day level, 10% at 5 and
25 mg/kg/day, 24% at 125 mg/kg/day and 46% at the 300 mg/kg/day level at Test
Week 13. Although female body weight gains were largely unaffected at doses
up to 25 mg/kg/day at Test Week 13, an approximate 27% reduction in body
weight gain was observed at 125 mg/kg/day with approximately a 38 reduction
at the 300 mg/kg/day Level. These reductions in weight gain were significar.t
(p
-------
: cl ’icai chemistry measurements, a dose—dependent elevation of serum
c .o escerol levels was seen in TN t—creaced rats. Cholesterol values for bot .
sexes at L 5 and 300 ngikgiday were significantly increased (p
-------
::al to mild lr.creases in hemosiderin—li.ke pigment were found the
ac;;pnages of the splenic rea\ pulp and liver at the high—dose Level. These
ooservacions, along with the dose—related anemia, suggested to the authors
crtac TNT—induced anemia was hemolvt c in origin. This concept was
supported b . a lack of bone marrow cytocoxicicy and a slight ethenoglcb: e a
at the 300 ng/kg/day dose level.
Histologic examination of the testes revealed a dose—related degeneration of
the germinal epithelium lining the seminiferous tubules in one, six and :en
t ales at 25, 125 and 300 mg/kg/day, respectively. At the 300 mg/kg/oay dcse
level the testicular lesions were diffuse, bilateral and marked in seventy,
the minim.al to mild Lesions were characterized by a diminution of spermatozoa,
spermacids, and spermatocytes as a result of degeneration and necrosis.
Spermatocytic and spermatidic giant cells, present in the lumen of some
affected tubules, appeared to represent an early degenerative stage of ehts
lesion. those animals with lesions of moderate severity exhibited an absence
of spermatozoa and spermatids, with only a few spermatocytes remaining in the
degenerative tubule. The Sercoli cells and spermatogonia appeared unaffected.
Atrophic seminiferous tubules lined with a few Sertoll. cells and spermacogonia
characterized those lesions graded as marked in severity. All males at the
highest TNT dos. (300 mg/kg/day) also exhibited a mild to moderate diffuse
hyperplasia of interstitial (Leydig) cells in both testes along with
inrertubular edema. A NOA.EL of 5 mg/kg/day is indicated by the absence cf
testicular degeneration and effects on the spleen at this dose level.
tartin (1974) conducted a 90—day study to evaluate the toxicity of TNT
administered by gastric intubation as a suspension in a IZ aqueous solution of
methyl cellulos, to cynomolgus monkeys at daily dosages of 0.02, 0.1, or L.0
mg/kg/day. The animals ranged in age from 36 to 56 months and in weight from
2.0 to 4.2 kg for females and 2.6 to 4.6 kg for males, and groups consisted of
three males and thre. females each. Controls received the aqueous solution of
U methyl cellulose.
Daily observations were made to detect clinical signs of toxicity; body
weights were recorded weekly. Prior to the start of the study and during
Weeks S and 9 and at the termination of the test period, the authors conducted
hemato].ogical and clinical chemistry determinations, urinalysis,
bromosulfopLithajein (BSP) clearance tests and determinations of plasma TNT
Levels. Ophthalmoscopjc examinations were conducted prior to the study and
again at the close. At necropsy, histological examinations of thyroid, heart,
liver, kidneys, adrenal gland, stomach, small intestine, lung, spleen, bone
marrow and brain were made. Because only three animals of each sex were used
in each dosage group, statistical analysis of results was not conducted.
No clinical signs of toxicity were attributed to TNt administration, nor were
there any consistent abnormalitie, in any of the clinical laboratory tesc
conducted. Histologic examination shoved some increases in numbers of
Vi—23
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-.e : megakaryocytes in h ;h—dose females and determined that two of these
e a.es showed no normaL negakary cvtes in bone marrow sections. ThiS
concic on as described as a toxic manifestation, possibly related L’
cnrot.coc;toperu.a, but the lack of platelet counts resulted in an ir.abi1 .:y ;
confirm this suspicion. Increased amounts of iron—positive material in liver
cord cytoplasm was found at the highest dosage of TNT (1.0 mg/kg/cay). The
authors stated that the toxicologic importance of these two findings is
uncertain. A NOAZL or Louesc—Observed—Adverge —Effect..Level (LOAEL) could r.cc
be determined for this study due to the small numbers of animals evaluated
along with the lack of statistical evaluation.
Hart (1974) also conducted a 90—day toxicity study in purebred beagle dogs
administered TNT in the diet (consisting of ground dog chow supplemented w tn
coercial canned dog food) at dosage levels of 0.02, 0.1, or 1 mg/kg/day.
Three dogs per sex per dosage level were used. Controls received the normal
ground dog chow mixed with canned dog food. Daily observations of toxic and
pharmacologic effects were conducted, and animals wer, weighed weekly. Twice
prior to the start of this study and during the 4th, 8th, and terminal weeks
of the study, the authors conducted hematological and clinical biochemical
tests, and urinalysis. Examination by a veterinary ophthalmologist was done
prior to start and again during Week 13 of the study. Ac necropsy, gross and
histologic examinations of various organs were conducted.
o clinical signs of toxicity, body weight changes, diagnostic abnormalities
nor gross or microscopic lesions were noted at any dosage level.
Ophthalmoscopic examination revealed “some increased granularity and mild
hyper—reflecrivity of the fundu ” in the high—dosage group, possibly
inuicating a mild retinopathy. It was not considered toxicologically
important. No cataracts were reported. A slight increas, in hemosiderosis of
the bone marrow in th. high— dose group could not be properly assessed with
the group size in this study. The small number of animals evaluated precludes
the determination of a NOAEL or LOAEL for this study.
b. Twenty-Six-Week Study
Levine at c i. (1983 ) studied th. effects of TNT (approximately 99% pure)
administered daily, by meana of a gelatin capsule containing TNT mixed with
Purina Certified Rodent Chow to reduce th. hazards of explosion, to groups of
six beagle dogs per sex at 0.0, 0.5, 2, 8, or 32 mg/kg/day for 26 weeks.
(Study report indicates that dogs were administered TNT capsules for 25 weeks
with a blank capsule administered daily for one week prior to TNT
administration.) Animals were approximately six and one—half months old at
the start of the TNT dosing schedule and were maintained throughout on daily
rations of Purina Dog Chow. Animals were observed several times daily, before
and after dosing, for toxic signs and were examined weekly by palpation for
detectable masses. Body weight and food intakes were recorded weekly. Other
toxicologic endpoints included a comprehensive clinical chemistry and
VI—24
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e az . gical evaluacton, urinal’,ses. and per od c eecc:Qcard1cg..a .
a’d hainic exa.nacions. turing ..eek 27, a1 an majs, ioL1ow g a
r oLr fast, were sacrificed by injection of rtcraver.ous pentobarb al sodi ;
na or : ans were wei.ghed and all organs were collected and ftxed for
iicroscopic exa inacion. Statistical analyses were performed.
At the highest dose tested (32 mg/kg/day), TNT was found to be lethal, w it ’
one female sacrificed in a moribund condition during Test Week 14 and another
found dead during Test Week 16. Clinical signs in these two animals Lncluded
dehydration, emaciation, jaundice, hypothermja, weight loss, diarrhea and
ataxia. Clinical signs of toxicity observed in dogs surviving this lethal
dose Level included orange—brown urine and faces (also observed, to a lesser
extent, at 8 mg/kg/day), darkening of the tongue and/or gums, jaundice and
ataxia. in addition, body weights were reduced in all rNT—treated dcgs w:th
significant losses evident at 8 mg/kg/day (males only) and 32 mg/kg/day.
High—dosed dogs also showed significantly reduced food intake throughout osc
of the study with similar Losses evident at the 8 mg/kg/day level during Test
eek 1. Urine was a light to dark brown color in dogs at the 32 mg/kg/day
and, to a significantly lesse; extent, at the 8 mg/kg/day level throughout the
study. Additionally, during the final test week, urinary protein levels were
increacec in the two highest dose levels.
At the 32 mg/kg/day dcse level, the observed jaundice was accompanied by
elevated bilirubja levels in serum and urine and trace levels ot urobilinogen.
These findings were consistent with the observed anemic state (as evidenced by
significant reductions in Hct, Mgb, and R.BCs) for both sexes receiving either
8 or 32 mg/kg/day. Compensatory responses to anemia included increased
numbers of raticulocytes, macrocyrosis, and elevated numbers of nucleated
aBCs. Leukocytoaje with neutrophilia was evident in a dose—related manner at
the two highest doses along with methemoglobinemia. These observations, along
with evidence of a heaosjdsrjn—ljk. pigment in macrophages of the spleen and
liver and sinusoidal congestion of the aplenic red pulp with accompanying
increased spleen size, suggested to the authors that TNT—induced anemia was
hamolytic in origin. Reduced numbers of erythrocytes and their precursors in
bone marrow were also seen.
Other abnormalities in blood chemistry parameters included statistically
significant increases in ser globulin and LDH, dose dependent decreases in
SGPT, with females shcwj g significant reductions down to the 2 mg/kg/day
level, and decreased glucose at 32 and 8 mg/kg/day (males only). Cholesterol
levels were variable with slight increases at 8 mg/kg/day (males only) and
slight decreases at 32 mg/kg/day (females only).
No definitive treatment—related effect was reported upon ophthalmic
examination although some vitreal. stranding or haze was observed at all levels
(controls, 17%) with the two highest levels shoving a somewhat higher
incidence (62% to 50%). No ocular Lesions were observed. Likewise,
V 1—25
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elec:;ocardiograpny tracings did not reveal any fl T—relaeed effects.
$eve:al indications of liver injury were obEerved upon gross and histologic
exan .nacior.. Male (8 and 32 mg/kg/nay) and female (32 mg/kg/day) dogs had
significant increases in relative and/or absolute liver weight acconpanied y
moderate to nar ed hepatocytic cloudy swelling and hepacocytomegalia seen at
the high dose and, to a lesser degree of severity, at all, dose levels with
Lesions at the low dose (0.5 mg/kg/day) described as trace to mild. No such
lesions were seen in the control animals. Microscopic evidence of cirrhosis
was seen, primarily in males, at the 8 and 32 mg/kg/day dose levels and
hemosiderosts of the liver was seen in the majority of dogs at the two highest
levels as well as one female at the 2 mg/kg/day level. Non. of these
microscopic Lesions were seen in the two females necropsied prior to
termination of this study.
The absolute and relative weight of the spleen of both sexes was signlficant:y
increased at the high—dose level with a significant increase in the relative
weight in females at the 8 mg/kg/day level. This finding corresponded to a
marked co severe generalized congestion, primarily at the two high—dose
Levels. Hemosjd.rosjs of the spleen was evident at all dose levels and
extramedullary erythropoiesis was demonstrated primarLly in the high—dose
group.
Absolute heart weights were significantly decreased in males at the two
highest dose levels but no corresponding pathology was reported. Increases
occurred in the absolute weight of the thyroid of females at the high nose and
was accompanied by bilateral C—cell hyperplasia in all groups, including
controls, but with a greater degree of severity in the high—dose group. The
testes of male dogs receiving any level of TNT were unaffected.
Other notable pathological findings apparently related to TNT intake included
nembranous enteritis of the small intestine and .rythroid hypoplasia at all
TNT—treatment levels of both sexes, possibly due to administration of the TNT
as a bolus dose. Enlarged, pigmented lymph nodes with no apparent
histopathology were seen, primarily in the high—dose females. The 0.5
mg/kg/day test level appears to be a LOAEL for liver effects with
histopathology at this level indicated as trace to mild. No effects were seen
on the Liver enzymes and organ weight at this low dose level nor on the
spleen, another consistent target organ for toxicity. Therefore, a LOAEL of
0.5 mg/kg/day is considered to be appropriate.
c. Lifetime Exposure
Data on the toxic effects of lifetime exposure to TNT are available in an
extensive series of studies by Furedi et al . using Fischer 344 rats
( 1984a,b.c ) and B6C3FI hybrid mice (1984d,e,f).
VI—26
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: e rat stud’i, groups of 5 animals per sex (approx:nacely 6—7 wee s od)
rece:veo T T ( - 99Z pure) nl.xe’d in a diet of Purina rodent chow neal at cse
e els of 0.0, 0.4, 2, 10, or 50 mg/kg/day for 24 months. Diets were prepa
weekly, by sex, on the basis of projected body weight and intake data. All
ani.majs here observed daily for signs of toxicity; examination by palpation,
ana body weight and food intake determinations were evaluated weekly thrcugh
.eek 13 and bi—ueekly thereafter. Ten rats/sex/dose were sacrjfjceu at 6 a d
12 months of treatment with the remaining rats sacrificed during eek 5
105—106, Blood was collected via the orbital sinus; animals were fasted for
1.7—19 hours and weighed prior to their sacrifice by carbon dioxide euthanasia.
iajor organs were weighed, and all tissues were fixed for histological
examination. Clinical evaluations included hematology, clinical chemistry,
ophthalmology, and gross and tissue morphology; statistical analyses were
performed.
The chronic administration of TNT at doses up to 50 mg/kg/day did not alter
mean survival times. Dose—related reductions in food consumption and a
corresponding decrease in body weight gain were seen in both sexes at 10 and
50 ag/kg/day. At the high—dose level, thes. effects were statistically
significant in the first few weeks of the study and remained so throughout.
Ac 10 mg/kg/day, the changes developed somewhat later and tended to be more
sporadic. -
Anemia evidenced by a reduced Hct, Hgb and RBC count was seen at 10 and SC
mg/kg/day. The parameters were generally significant in both sexes receiving
the high dose, beginning at Week 14 and continuing through termination. Ac
the 10 mg/kg/day level, these same parameters were significant in males
through Week 52 but were inco si te t in females. Mechemoglobin values and/or
percent machemoglobin were significantly increased in males receiving the high
dose through their terminal sacrifice and in high—dose females at the Week 78
analysis. At 10 mg/kg/day, these parameters were sig jficant in males only
and only through the first year. Beginning at Week 52, a sporadic increase In
platelets, lymphocytes and/or WBC was significant in the high—doss females,
with an occasiomally significant increase in all, or some of these parameters
in the high —4 0 5 . males. In general, male rats appeared to be somewhat more
sensitive than females to the hematological effects of TNT. An increased
production of reciculocyces was seen as a compensatory response to the anemIc
state. Related lesions detected at terminal sacrifice and/or in rats found
dead or sacrificed moribund included focal to multifocal myelofibroejs of c e
bone marrow, mild to moderate in severity, and significantly increased in
incidence in the females receiving TNT at levels of 2.0 mg/kg/day and above
See Incidence table A2—1 in Appendix 2.). At the high—dos. level, this
lesion was seen in 17/54 females (31.5Z; p>0.01) but was not seen in males.
Spl ,enic lesions consisting of sinusoidal congestion, extramedullary
hemacopojesis, and increased quantities of a hemosjderjn—ljke pigment were
seen in both sexes, generally at c e two highest dose levels, with the
incidence and severity of the diffuse sinusoidal congestion also signif ar:
VI—27
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creased at the 2.0 mg/kg/ ay dose level in females. These lesions :n : e
spleer. vere fi.rst decec:ed during the six—month scheduled sacrifice and
remainec si nificanr through termination. The weight of the spleen was
significantly increased in males and females through most of the study (not
significant jn females at Week 104) at the high—dose level and was
sporadically increased in both sexes at 10 mg/kg/day. The authors suggested
Chat TNT appeared to induce anemia by a hemolytic process. This suggestion
was further supported by the observance, minimal in nature, of Howell—Jolly
and Heinz bodies at the 50 mg/kg/day level and the presence of methemogiobin
in the circulating blood, suggesting the oxidizing nature of TNT and/or its
metabolities.
Liver injury at 50 and, to a lesser extent, at 10 mg/kg/day was indicated upon
gross examination by focal and multifocal red to tan areas. Increases in
absolute and relative liver weight were evident in both sexes at the two
highest dose levels. Upon histological examination, a dose—related increase
in the incidence of foci and areas of hepacocel].ular hyp.rpl.asja with cystic
degeneration was observed for males but not females receiving the 10 and 50
mg/kg/day dose during the second year of the study. Hepatocoxicity was also
suggested by altered lipid and protein metabolism as evidenced by increased
serum cholesterol, total protein and alb .in levels and a more sporadic
increase in globulin Levels. Albumin/globulin ratios were significantly
decreased. These chemical alterations were most predominant at the high—dose
level but cholesterol levels in males were also significantly increased at the
2.0 and 10.0 mg/kg/day levels.
Blood urea nitrogen was slightly elevated at 50 mg/kg/day, particularly among
females. Renal injury was indicated grossly as spotted, granular and cystic
kidneys. Histological examination revealed a doss—related increase in
incidence and severity of mild to moderate pigmentation, beginning at the 2.0
mg/kg/day dose level in females and the 10 mg/kg/day level in males.
Infla.ation with lymphocytic infiltration was apparent in both sexes at the
high—dose level and in females at the 10 mg/kg/day level. Hyperplasia of the
renal pelvis was also significantly increased in the females receiving the 50
mg/kg/day dose level. Kidney weights were elevated for animals of both sexes
receiving 10 or 50 mg/kg/day. The brown mottled kidneys seen at necropsy for
high—dose animals contained iron—negative cytoplasmic bodies and, nuclear
hypertrophy of cortical proximal convoluted tubular cells was observed
microscopically in males at 2 mg/kg/day or greater. Additional toxic effects
on the urogenital system, primarily seen in high—dos. females, included
urinary bladder hyperplaija, papilloma, and carcinoma (discussed in section 5.
Carcinogenicity).
No effects, other than a significant increases in absolute weight, were seen
on the testes of males receiving the two high—dose levels. No other effects
related to TNT intake were reported.
V 1—28
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:. e c for an increased frequency of ocular d scharge n the nigh—dose :ar ,
o ::eac ent-r .aced a rtormalities were detected either by ophchal ic
exa .r ac.on or scological evaluation. A number of lesions were detected a:
a:. cse :evels, with increased frequency versus tine, but were considered
be r ..ated to ocular trauma or penetration at the time of orbital bleea g.
An apparent .‘OAEL of C.4 mg/kg/day is based on the absence of effects of m:
on the spleen, kidney, and bone marrow.
: the 24—month study conducted by Furedi et al. (1984d,.,f) in B6C3F hvbr d
mice, T T (>99% pure) was administered in a diet of ground Purina chow to
groups of 75 mice/sex/group at dosage levels of 0.3, 1.5, 10, or 70 gikgidav.
Animals’ were observed daily for signs of toxicity. Examination by palpacLon,
measurement for weight changes and food intakes and examination of bedding for
red stair.ing were conducted weekly through Week 13 and bi.—weekly thereafter.
Periodic ophthalmic examination as well as measurement of hemacological and
c..i.nical chemiscy parameters were conducted at defined intervals througnouc
the study. Ten mice/sex/dose were killed at 6 and 12 months with sur’iving
animals killed after 24 months of treatment. Euthanasia was accomplished by
carbon dioxide anesthesia following a 2 to 5 hour fast. Major organs were
weighed, and all organs were fixed for histological, evaluation. Tissues from
control and high—dose mice underwent a comprehensive examination with chose of
the remaining levels undergoing a more limited examination. Appropriate
statistical analyses were conducted.
The TNT did not cause deaths at the doses tested in this study; mean survivaL
times were similar among control and treatment groups. Clinical signs re .aced
to TNT administration were not readily apparent. Reductions in body weight
gains at the 70 mg/kg/day dose level were approximately 10% for both sexes up
through the first 6 to 8 months of the study, with further reductions of about
15% for females and 202 for males through the remaining test period. An
approxiz.ate 5% to 7% reduction in body weight gain for males but not females
at the 10 mg/kg/day dose level for the majority of the study was not
statistically significant. Body weights were not affected in mice given 1.5
mg/kg/day. Food intakes were variable with significant decreases apparent n
high—dos. males through approximately Week 19 and significant increases over
control intakes beginning at Week 25. In females receiving th. high aose,
dietary intakes were significantly increased through most of the study. wi.cr a
significant decrease in intake only during Week 1.. At other dose levels,
significant differences were sporadic in mature and varied from increases :
decreases throughout.
Hematologic observations included anemia in both sexes administered TN a:
mg/kg/day, as evidenced by gsnerally significant reductions in Mct, H b
RBC counts from Test Week 27 through Test Week 79. The effect was mi.:. .as
no longer apparent in males and was apparent but not significant in fence,
Test Week 105. Normal physiologic compensatory responses to the aaemt.
(e.g. reciculocytosis, macrocytosis, etc.) were not apparent. Lymphoc’t.s . -
VI—29
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.BC ’s s gnif:car. ’, increased during test weeks 2;’ and 52. ‘tales
rece:v:ig L0 mg/kg/day also .ad .z creased l ’mphocyee counts at test week 27.
No c r.sLstently significanc changes Lfl clinical chemistry parameters were
evident in this study.
Occasional elevations in relative Liver, kidneys, spleen and heart we.ghts
were seen in mice receiving 70 mg/kg/day. Although statistically signif±ca ,
these changes were small, and no pattern with respect to sex or time was
observed, nor were absolute organ weights significantly different from
controls.
Ophthalmic abnormalities were random and not considered to be related to
treatment with TNT. A high incidence of cataracts in all test groups was
significant in low—dosed females only, was not dose—related and was consLdered
“spurious” and related to aging changes.
No TNT-induced gross lesions nor microscopic abnormal.ities were observed at
the 6 and 12 month interim sacrifices. Increased extramadullary hematopojesis
of the spleen, cytoplasmic vacuolization of renal tubules (males), renal
lymphocytosig (females) and a variety of inflaacory dermal lesions were
considered spontaneous and were observed in control and treated animals alike.
Enlargement of the spleen and lymph nodes in females receiving 70 mg/kg/day
was observed at the 24 month necropsy as well as in mice that died or were
sacrificed moribund between 12 and 24 months. The study authors reported ct-ac
the incidence of combined leukemia/malignant lymphoma in the spleen of females
increased with dose. They reported that the iacreiss wag statistically
significant (p
-------
- r:-ter Exposure, Four—weeK Studies and :..onger—Ter Exposure,
5:.c:es.) These ef ec:s were not evident the Li.fet .ne Exposure st d.es,
‘cr :s :he signif.cartce, if any, of these effects on the reproducti ;e ca7ac::
of z-e :ac k o n.
eveLop ental Effects
No data were available in the literature concerning the developmenta. effec:s
of TNT.
5. Carcinogenicity
The carcinogenic potential of TNT was evaluated in 24—month studies in Fiscier
344 rats ( Furedi er a].., 1984a,b.c ) and in hybrid B6C3FI mice (Furedi et ak.,
1984 d,e,f).
In the study in rats, TNT was administered at 0.0, 0.4, 2, 10, or 50 /k iday
by diet to dosage groups of 75 rats per sex. Histopathologic lesions cbserved
in females dosed at 10 and 50 mg/kg/day during the 12 to 24 month TNT
treatment period included an increas, in the incidenc, and severity of
hyperplastic, preneoplastic and neoplascic lesions of the mucosal. epitheliuct
of the urinary bladder. Malignant neoplastic changes as well as benign
neoplastic changes were present in th. bladder spithelium. Based on these
observed changes, th. authors considered TNT a carcinogen to F344 rats under
conditions of the study. The incidence of urinary bladder hyperplasia among
females rats receiving the high dose was 12/55 (21.8Z; p 0 .O1), for urinary
bladder papilloma, 5/55 (9.IZ; p
-------
(:.e ?‘O.OS) using the Fisher—1ç’ jn Exact Test to compare dosage gr u; and
the C ciran—Armirage Test for Trend. Therefore, based upon the scac:s ai
analyses, this study s considered to be negative with no tunors related to
exoosure.
6. Genotoxtcltv
The strong mutagenic activity of TNT was reported by Ellis ec al. (1978). As
little as 10 ug/plate of TNT, dissolved in dimethylsulfoxide (DMSO), with or
without metabolic activation, was mucagenic in Salmonella typhimurium strains
TA—98, TA—1538, and TA—1537, indicators of frame—shift reverse mutations; at
30 ug/plate with or without metabolic activation, mucagenic effects were noted
in TA—tOO as well as in those three strains. Ac 300 ug/plate with metabolic
activation, TNT was positive in all five tester strains thc].udirtg TA—1535,
indicating that TNT is positive for both frame—shift reverse mutations and
base—pair substitutions.
Si oa ec al. (1977) conducted studies using bacteria (S. yphimurium , strains
TA—1535, TA—1537, TA—1538, TA—98 and TA—tOO), with and without S9 metabolic
activation, and yeast (S. cerevisjae ) to evaluate the mutagenic activity of
TNT before and after application of chlorination or ozonation disinfection
techniques. Results were negative both before and after either disinfecc on
technique except for two experiments in which TNT was reported as appearing
weakly mutagenic (<2—fold increase in revertants) in TA—tOO without metabolLc
activation. Using the same standard t,r mucagenicity as in the study by EJ .s
et al. (1978), i.e. a mutagenic ratio > 2.0 as positive, none of the
concentrations tested in the Sion study would be positive. It is important
to note, however, that the concentrations tested in these experiments were,
generally below 10 ug/plat. with 33.5 ug/plate being the highest concenrrat on
tested in a single experiment. The increases seen in this study, while
generally occurring at the highest concentration, were not always
“dose—related”.
Dilley cc al. (1978) reported that TNT dissolved in DMSO and incubated at 10
to 500 ug/place increased reverse mutation rates in a dose—related manner .n
S. tvphimurium strains TA—1537, TA—1538, TA—98, and TA—tOO, both in the
presence and absence of the 5—9 metabolic activation system. The toxicity and
nuragenicity of TNT were reduced upon metabolic activation.
Dilley et al. (1978) performed in vivo cycogenetic analyses on bone marro
cells from two groups of five young male Spragu.—Davley rats, each treated : r
28 days with TNT at 0.25Z (190.4 mg/kg/day) or 0.002Z (1.8 mg/kg/day) in the
number revercanes in test/number revertants in control
VI-32
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feec ar.d from two adc .:ionai groups of five rats, each s m larLv creared ar
a::c ed to reccver :or 23 days. The evaluation procedure used was a
oc:f.cation of tne method Outlined by NLchols et al. (1972) in wrtich tne
:reaced rats were ir ected, one and one—half hour 2r or to sacrtff.ce, tch
0. 3 of colcnic ne to obtain a high number of cells Lfl r etaphase. The
bone marrow cells .ere obtained by aspiration from the distal end of the
femur. Although the high dose was considered the highest tolerable dose, no
rats died prematurely. Mitotic indices were depressed in the rats exposed to
the high dose, but no cytogenecic abnormalities were observed. Following 3
nays for recovery, the. proliferative capacity of the bone marrow ce .s
returned to normal levels s indicated by the mitotic indices. The authors
concluded that, basec on their assay, genetic damage induced by TNT was not
evident.
In vitro measurement of unscheduled DNA synthesis (UDS) in human diploi
fibroo .ascs ( 1—38 cells) was also conducted by Dillay et al. (1978) in the
presence and absence of a metabolically active system obtained from the ..ver
of adult male nice. A dilution of TNT in DhSO was added to the culture medium
to yielc concentrations ranging from 0 to 1000 gIml. Results in this system,
without metabolic activation, indicated that L’DS was suggested at the higher
concentrations, A definitive evaluation, however, was obscured by
discoloration of the samples at the two highest concentrations, 500 and :000
..g/ml, thus interfering with the colorimetric determinatior. ot DNA cor.cenc.
in the presence of metabolic activation, at concentrations ranging from C to
6000 ug/mi, LDS was not observed, however, the sclubility of TNT was a
.mi:1.ng factor in this test (a precipitate was observed at all concentrations
of TNT).
Ashby et al. (1985) reported that TNT gave a negative response in a mouse bone
marrow micronucleus assay in which th. mice wire administered TNT by
intraperitonea]. injection at levels up to 80 mg/kg (described as 802 of the
Laximun Tolerated Dose, MTD) and evaluated at 24, 68 and 72 hours after 00 51mg
for an increase in the presence of micronuclei.
in an in vivo/in vitro rat liver assay for UDS, conducted by Aahby et a]..
(1985), ths hepatocytes of TNT treated rats were evaluated in an in vitro
system following administration of TNT to ths intact animal at dose levels uo
to 1000 mg/kg. A negative response was also observed in this system, wh Ich .S
used to msasure genotoxic—carcinogenic response.
7. Other Effects
Male and female adult Sprague—Dawley rats were fed TNT in the diet at 0. 5
for three weeks (Dilley it al., 1978) for the purpose of evaluating the
ability of TNT to stimulate the hepatic nicrosow.al enzyme system. Ani aLs
were killed by decapitation, and the livers were remřved and prepared r
V 1—33
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ar aiysis by standard e:hods. Substrates used represented three ecabo .:
N—de echvla on, O—de ’echylaci.on and aromatic ring hydroxy1at
sesu: s ir.dicated cnac TNT showed no stimulacory activities in two of t ’e
three systems, with a Li.mited positive response in the O—demechylacior. svsten
as indicated by a scimulacion of the metabolism of o—nitroanjsoj.e. However 1
metaboi sm of TNT tsalf is not altered by pre—creacment of the rat u ch
phenobarbical, TNT or RDX, indicating to the authors chat the decreasen
toxicological manifestations to repeated dosing of TNT cannot be explaLned cn
the basis of an increased metabolic disposition of TNT.
V1—34
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‘EALT t .:5O y
Ava:.acle toxicity studtes in var .ous ani.mal spec es, for perIods ranging
a sir.gie oral, dose (LD 50 ) to continuous 24—month feeding studies, along u:c
data on the health effects .n humans exposed to TNT :n the atmosphere have
been evaluated. Several toxicity endpoints considered relevant to a !{A for
TNT in drinking water have been identified. Numerous signs and symptoms of
TNT toxicity in humans exposed in the workplace have been reported (Zakhari
and Villaume, 1978), to include such relatively mild effects as respiratory
irritation, skin lesions, and gastrointestinal disorders and progressLng to
more severe symptoms such as methemoglobirtemia, jaundice, aplasric anemia,
cataract formation, menstrual disorders, neurologic dysfunction and
nephrotoxicity. Of these disorders, the most consistently reported effects of
TNT exposure in humans, including those which have been reported as the
principal cause of death when sucn exposure resulted in mortality, are
hepatitis and aplastic anemia (lakhari and Villaume, 1978).
Human exposure data gathered through occupational health surveys conducted at
various Army A unitiort Plants have irtdi 9 ted that atmospheric exposure to TNT
at levels ranging from <0.02 to ‘3.0 mg/n for periods generally up to 6
months consistently caused abnormalities in Kgb, Hct, and R3C count (estimated
absorbed dose could not be determined from the availabl, data). AbnormalitIes
in other hematological parameters and such clinical chemistry parameters as
BLN, SGOT, LDH and bilirubjn have also been reported (Friedlander et al.,
19 Ci; orton and Ranadive, 1974; Buck and Wilson, 975). tn almost all cases,
removal of the affected individuals from the source of exposure has resulted
in a return of these parameters to normal levels, although the time required
for recovery could not be determined. A consistent sign of TNT exposure has
been a red discoloration of the urine, apparently due to unidentified TNT
metabolites.
tn animals, significant and consistent findings following feeding of TNT in
the diet include hemolytic anemia with compensatory responses such as
reticulocytosis and macrocytosis; methenoglobinemia; increased spleen weight
usually associated with hemosiderosia and, in lifetime studies, congestion and
extramedullary hematopoiesis; and an increas, in Liver weight generally
associated with increased cholesterol, decreased SGPT, pigmentation,
hyperplasia and hepatocytomegaly (Dilley m c al., 1978, 1982; Levine et al.,
1981, 1983, 1984a,b; Fursdi at al., 1984a—f). Reported effects of TNT on the
testes (atrophy and hyperplasia) have been lass consistent as have effects on
other hemacological and clinical chemistry parameters. Consistent decreases
itt body weight gain at high dose levels seem to be associated with a
corresponding decrease in food intake. Red color appeared in the urine of
rats, mice and dogs fed TNT in the diet at levels of approximately 5 mg/kg/day
or greater, depending upon species.
Anemia in dogs, rats and mice fed TNT, evidenced by decreases in Kgb, Hct and
t I —
-------
? C c:’_ :, first appeared in dogs’after four weeks of feeding at 20 g day
(JiL2ey ec al., 198, 1982) and remained evident . en TNT was fed at dcses
S mg;kg, day or ‘ore for up to 26 weeks ( Levine er al., 1983) . .e aco1o Lca
signs of ar.emia also appeared in rats when TNT was fed at doses ranging fro
25 to 300 ng/kg/day for periods of 6 to 13 weeks (Dilley et al., 1978, 1982;
Levine ec al. , 1981, 1984b). In a two—year rat study ( Furedj et al., 198 4a) ,
the anemia was associated with a statistically signifjc increase in foc 1
to multifoca]. myelofibrosis of the bone marrow along with an enlarged spleen
and splenic lesions consisting of sinusoidal congestion, extramedullary
hematopoiesjg and hemosiderosis. These effects were significant in females at
2.0 mg/kg/day and above and in males at 10 and 50 mg/kg/day. Mice were less
sensitive to the e : : s of TNT on the hemacological parameters for anemia
with only slight or no effects at doses up to 700 mg/kg/day for periods up t
13 weeks (Dii.ley et al., 1978, 1982; Levine at a].., 1 98 4 a). When nice were
fed diets containing 70 mg/kg/day TNT for up to two years, anemia was
evidenced by decreases in the Hgb, Hct, and RBC counts by Week 79, but these
changes were evident but no Longer significant by Week 105 (Furedi et al.,
1984d).
The hepatomegaly reported in dogs and rats fed TNT for periods up to 13 weeks
was generally not associated with histological abnormalities except for a
muitifocal or diffuse hepatocelluj.ar hypererophy (hepatocycomegaly) in two
groups of rats fed TNT at 125 or 300 mg/kg/day for 13 weeks (Levine et al.
1981, 1984b). When TNT was fed to dogs at lower levels (0.5, 2, 8, or 32
mg/kg/day) for a longer duration (26 weeks), the hepatomegaly was accompanied
by moderate to marked hepacocytic cloudy swelling and hepatocycomegalia at all
creatmanc levels, but of only trace to mild severity at the lowest dose
( Levine at at., 1983) . In the two—year feeding study in rats ( Furedi et al. ,
I9 84 a,c) , the liver injury was further evidenced by a dose—related increase in
the incidence of foci and areas of hepacocellu].ar hyperplasia with cystic
degeneration in the males, but not females, fed TNT at 10 and 50 mg/kg/day.
Clinical chemistry parameters, while not always significant, were generally
indicative of altered liver function as indicated by increased cholesterol,
total protein, albumin and globulin levels. These alterations in liver
morphology were not evident in the mouse (Furedi ec a].., 1984d,f).
Testicular atrophy and hyparplasia, seen in high—dose rats (160 mg/kg/day)
receiving TNT in the diet for up to 13 weeks, were first detected in those
animals sacrificed after four weeka of treatment (Dilley at at., 1978, 1982)
Similar dog, related effects Vera seen in rats fed TNT at dos.s of 25, 125 or
300 mg/kg/day for 13 weeks (Levine cc at., 1981, 1984b). The testicular
atrophy in this study was first noted by palpation in the highest—dosed group
during Week 6. No testicular effects were evident at 5 mg/kg/day. Similar
testicular effects were not evident in rats fed TNT for up to two years.
contrast, significant findings in the two—year study ( Furedi et at., l98 .a,c
indicated an increas, in the absolute weight of the testes at the two highest
dose levels (10 and 50 mg/kg/day) with no treatment—related histopachology
VlI—2
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e a.e rats fed .T at evels p to 30 mg/kg/day for two years ‘ t reoi et a..
. - a,: ) showed a si&r.ificanc Increase in incidence and sever:ty of
- . erpLast , eneopLastjc and neopiastic (papilloma and carc:no a) .es.cr.s
cf :ne ucosai epithelium of the urinary bladder at the 50 mg/ kg/da> cose
Level (ab.e A’ —t, ApVendix 1). This carcinogenic effect was nor ev: enc n
nales and was nor reported in any other species.
whiLe the development of cataracts has been reported in humans exposed
chronically to TNT (Zakhari and Villauma, 1978; Hassman, 1968; Harkonen er
a).., 1953), no such Lesions were found in rats and mice exposed to TNT at
doses up to 50 and 70 mg/kg/day, respectively, for up to two years and
periodically subjected to a comprehensive ophchalmologjca l examination
throughout the two—year study ( Furedi ec a ) .., 1984b,.) . No lesions or
cataracts were found in dogs fed TNT at levels up to 32 mg/kg/day for 26 weeks
( Levine et a).., 1983 ) nor in dogs and monkeys treated with TNT at doses up to
I mg/kg/day for 90 days (Mart, 1974; Martin, 1974). Cataract development
observed n some human epidemiologica). studies does nor provide sufficient
data to estimate a health advisory.
Based on the foregoing data, the dog appears to be the species most sensitive
to the toxic effects of TNT in the diet/bojus dose. In this species, systemic
effects were associated with the liver, th. organ system most coortlv Linked
with TNT toxicity, and were apparent at a level of 0.5 mg/kg/day as evidenced
by hepatomegaj.y with hepatocytic cloudy swelling and hsparocyromegaiia of
trace to mild severity ( Levine er al., 1983) . The dog also appears similar to
nan in its metabolism of TNT, with differences being Largely quantitative n
nature, and is considered the most appropriate animal model for estimation of
a human health advisory.
A. Q antjfjcatjon of Toxicological Effects
Health Advisories are generally determined for One—day, Ten—day, Longer—cer
(approximately 7 years) and Lifetime exposures if adequate data are available
that identify a sensitive noncarcinogenic end point of toxicity. The HAS for
noncarcinogenic toxic& t. are derived using the following general formula:
( NOA.E1. or LOAEL) x (SW )
(UT) x (_ 1./day) — m g/L (_ ug/L)
where:
NOAEL or LOAEL • No— or LQwe5t-Obge ed..Adverg._Effect Level
(in mg/kg by/day).
BW — assumed body weight of a child (10 kg) or
an adult (70 kg).
v :1—3
-------
• uncertalrity’factor in accordance w ch NASIOD . .
guideli.nes.
L/dav • assumed daily water consumption of a child
(1 L/day) or an adult (2 Llday).
I.. One—Day Health Advisory
No data were located in the available Literature that were considered suLtable
for the calculation of a One—day HA. Short—term studies were Limited to
assessments of acute oral LDç 0 values. It is suggested that the D EL (20
-gIL) be used as a conservative estimate for the One—day HA.
2. Ten—Day Health Advisory
No appropriate data of less than 30 days duration were located in the
available literature for the calculation of a Ten—day HA. The four—week
studies of Dil.ey et al. (1978, 1982) in dogs, rats and mice evaluated only a
small numbers of the available animals with the remaining animals being
continued for a total of 13—weeks. A four—week range—finding study i.n mice
(Levine ec al., 1984a), th. least sensitive species, used doses that increased
by a factor of 7 and were not considered adequately sensitive for
toxicological evaluation. It is, therefore, suggested chat the DWEL (20 g/L)
be used as a conservative estimate for the Ten—day HA.
3. Longer—Term Health Advisory
Several studies were of appropriate duration to be considered for calculatIng
a Longer—Term HA. Thirteen—week studies were Conducted in dogs, rats and nice
(Dilley et al., 1978, 1982; Levine et al., 1981, 1984b) and a 26—week study
was conducted in dogs ( Levine et al., 1983) .
Mice were least sensitive to the toxic effects of TNT, shoving only minimal
but signifjca increases in spleen weight with hemosiderosis and slight but
not significant decreases in Met at the higher dose levels. No signifIcant
effects were evident on the liver. This species was not considered
appropriate for MA development.
Dogs Created with TNT for 13—weeks at doses up to 20 mg/kg/day displayed
evidence of anemia and liver toxicity indicated by changes in the clinical
parameters. However, no clear-cut NOAEL could be determined as only two
dogs/sex/Levei were available for analysis, with only one male surviving on
the high—dose level.
tn the 13—week study in rats, conducted by Dilley ec al. (1978, 1982), anen:a
was evident at the three higheat dose levels (7, 35 and 160 mg/kg/day) and as
VIt—4
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ac:orpanied by s nif:;ant eff ects on the spleen along with tox effec:s
c e . . r, kidney and testes. however, several L itations in thjs study,
icud g the small number of available animals, Possible “svste .ic’ errors
we.;’.1 g :oeedures and a lack of histological data at the two lowest dose
Levels .es ire s: nificanc findings at the next higher Level, precluded Its
use for calculating a Longer—term HA. The remaining 13—week study in rats
(Levine et al., 1981, 1984b) was generally well conducted with good
dose—response data but was considered to have too broad a dose range, with
doses decreasing by a factor of five, to provide adequate evaluation of the
lower levels. The lowest dose, 5 mg/kg/day, while apparently a YOAEL in th:s
study, was higher than doses producing positive effects in other strains and
species.
The 26—week study itt dogs ( Levine at al., 1983 ) remains as the most
appropriate from which to derive a Longer—term HA. This study produced
clearly toxic effects to the target organs for TNT, specifically the l:ver,
spleen and hemacopoietic system, at levels of 2 mg/kg/day and above, and trace
to mild effects on the liver, described as hepatocycomegalia with hepacocytic
clouding swelling, at the 0.5 mg/kg/day level (LOAEL). Based on NAS/ODW
guidelines, use of a study of appropriate duration with a LOAEL in animals
would require an uncertainty factor of 1000. Calculation of the Lifetime A
with these factors would produce a value equivalent to the calculated D EL
(Lifetime Health Advisory). It is, therefore, suggested that the DWEL (0
.g/L) be used as a conservative estimate for the Longer—term HA for both t te
10 kg child and the 70 kg adult.
4. Lifetime Health Advisory
The Lifetime HA represents chat portion of an individual’s total exposure :tat
is attributed to drinking water and is considered protective of
nortcarcinogenic adverse health effects over a lifetime exposure. The Lifeclne
HA is derived in a thre. step process. Step 1 determines the Reference Jose
(RfD), formerly called the Acceptable Daily Intake (ADI). The RID is an
estimate of a daily exposure to the human population that is likely to be
without appreciabl. risk of deleterious effects over a lifetime, and .s
dertved from the NOAEL (or LOA.EL), identified from a chronic (or subchrortc
study, divided by an uncertainty factor(s). FROM THE R.fD, A DRINKING -AE
EQUIVALENT LEVEL (DUEL) CAll BE DETERZiINED (STEP 2). A DWEL IS A
MEDIUM—SPECIFIC (I.E., DRINKING WATER) LIFETIME EXPOSURE LEVEL, ASSUMING ::
EXPOSURE FROM THAT MEDIUM, AT WUICH ADVERSE, NONCARCINOGDIIC HEALTH EFFECS
WOULD NOT BE EXPECTED TO OCCUR. The DWEL ii derived from the mu].tipltcac r.
of the RfD by the assumed body weight of an adult and divided by the assu .
daily water consumption of an adult. The Lifetime HA it determined in 5ce
by factoring in other sources of exposure, the relative source concr u:::
(RSC). THE RSC FROM DRINKING WATER IS BASED ON ACTUAL EXPOSURE DATA CR.
DATA ARE NO’r AVAILABLE, A VALUE OF 20 IS ASSU . If the contaminant i
V U-S
-------
JassL:i.ed as a GrouD A or 3 carcInogen, aCcord n to the Agency’s
c ss:: cacion SC eme of :arcinogenjc potent.al (U.S. EPA, 986), then cau :-
shc uld be exercised in assessing the risks associated ‘.‘ith lifet:ze exposure
Co cnis chemical.
the two—year studies in Fischer 344 rats ar.d BÔC3FI mice ( Furedi ec al.,
1 984a—f ) were well conducted lifetime exposure studies. The rat study clearl .
defined a NOAEL of 0.4 mg/kg/day for bone narrow, spleen, and kidney effects.
Ac this dose Level, there was no evidence of the splenic congestion, .ncreased
deposition of pigment in the kidneys or nyelofibrosjs of the bone narrow seer.,
primarily in females, at 2.0 mg/kg/day. Other effects seen at the higher
doses (10 and 50 mg/kg/day) and generally in both Sexes included anemia,
increased pigmentation and excranedullary henatopojesis of the spleen,
inflammation of the kidney ana hyperplasia of the renal pelvis, liver and
urinary bladder. Urinary bladder papillomas and carcinomas were present at, a
significant level in the high—dosed (50 mg/kg/day) females.
Similar effects reported in the two—year studies conducted in nice included
anemia and hepatomegaly without microscopic alterations at the high—dose level
(70 mg/kg/day), and a dose—related lymphocycosis at the 10 and 70 mg/kg/day
levels. Th. study authors reported that the incidence of combined
leukenia/mai.jgnant lymphona in the spleen of females increased with dose.
They reported that the increase was statistically significant (p’O.05) dC the
70 mg/kg/day dose level (high dose) and that the lesions were considered to be
treatment—related. This was an inappropriate conclusion based upon current
NIP guidelines (McConnell at al., 1986). These guidelines indicate that it Is
appropriate to combine all types of malignant lymphama and i.ymphocytic
Leukemia, but not in a single organ. These types of tumors occur throughout
the hematopoj.tjc system. Upon recounting these tumors, by each sex or both
sexes combined, in th. whole animal, the statistical significance is lost
(i.e., pcO .05) using the Fisher—Irvin Exact Test to compare dosage groups and
th. Cochran—Armjcage Test for Trend, Therefore, based upon the statistical
analyses, this study ii considered to be negative with no tumors related to
TNT exposure. The NOkEL for this study was 1.5 mg/kg/day.
The 26—week study in dogs ( Levine et al. , 1983 ) might also be considered for
development of a Lifetim. HA. This study resulted in clearly toxic effects to
the target organs for TNT, specifically the liver, spleen and hematopojetic
system, at levels of 2 mg/kg/day and above. At the 0.3 mg/kg/day level, this
study produced hepatocytomegajia with hepatocytic cloudy swelling of trace to
mild severity. No accompanying effects to the liver enzymes and organ weight
were seen at this do e. level. The 0.5 mg/kg/day Level may, therefore, be
considered a LOAEL for hepacic .ffsctg in the dog.
Based or. the foregoing studies, It appears chat the dog ( Levine at al. , 192J
is somewhat more sensitive to the hepacic effects of TNT than either rats or
V I 1—6
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;:ce and ay be ccnsLdered che rnosc appropr .ace species for ca.culat:-; a
:L:ec ’e HA. when c .e study .s viewed in relation to the two—year scud’, t-.
rats ( uredi er al. , 198 4 a—c ) with a NOAEL of 0.4 g/kgiday, it is felt ::-at
the dose of 0.5 gikg/day in dogs is close to the actual threshold dose ard
that an uncertainty factor of 1000 will provide a suffjcLenc nargin of safety
for hunans.
TNT is classified EPA Group C, possible human carcinogen based on ur narv
bladder papilloma and carcinoma in female Fischer 344 rats.
Step 1: Determination of Reference Dose (RfD)
RfD • ( 0.5 mg/kg/day ) • 0.0005 mg/kg/day (0.5 g/kg/da .)
.Th e r e:
0.5 mg/kg/day • LOA.EL’, based on trace to mild effects on the ].iver
of dogs exposed Co TNT in the diet for 26 weeks.
1000 • Uncertainty factor: It is based on the primary study
of Levine et al., 1983 and the strong supporting
study of Furedi cc al., l 9 84a—c . The LOAEL of 0.5
mg/kg/day in the Levine cc al., 1983 study appears to
be close to the actual threshold dose for liver
effectg. tt approximates the NOAEL of 0.4 mg/kg/day
in the Furedi et al., l 9 S 4 a— study in rats.
Reproductive toxicity data are not currently
available.
Step 2: Determination of a Drinking Water Equivalent Level (DWEL)
DWEL ( 0.0005 mg/kg/day) (70 kg ) s 0.0175 mg/L
(2 1./day) (rounded to 20 g/L)
Where:
0.0005 mg/kg/day — RiD
70 kg — assumed body weight of an adult
2 1./day assumed daily water cona ptjon of an adult
VII—7
-------
f:eo 3: Decer .nac.on of the L:fettne Health Advisory
The L:fet: e iA is derived from the DWEL by factoring in other sources of
exposure. The relative source contribution (RSC) is the percent of the tcta:
exposure to the chenical from drinking water. Actual data may be used vhen
available. Hence, RSC may potentially vary from 1 to 100 percent. A value of
20 for RSC from drinking water is assumed in the absence of actual exposure
data. Additionally, it is EPA policy that an additional uncertainty factor of
be used for Group C carcinogens. A valid quantitative cancer risk assessment
was developed from the Furedi ec al. (1984a, c) study and is provided in
Section VII B. This quantitative cancer risk assessment indicates that a
mitn.mal additional uncertainty factor is necessary to account for cancer rtsK.
Hence, the additional uncertainty factor of 2 is applied to the Lifetime .
For TNT the lifetime HA is as follows.
Lifetime HA — 0.0175 mg/L (0.2 ) — 0.00175 mg/L (rounded to 2 ugIL)
2
Jhere:
0.0175 mg/kg/day — Drinking Water Equivalent Level (DWEL)
0.2 • Relativ, source contribution (RSC) assumption of
2 • Uncertainty factor for Group C classification, C .
policy
B. Quantification of Carcinogenic Potential
TNT is classified EPA Group C based on urinary bladder papilloma and carcinoma
that were observed in female Fischer 344 rats. Mutagenic activity was
observed in the Ames test with and without metabolic activation. The risk
manager must balance assessment of carcinogenic potential. against the
likelihood of occurrence of health effects related to noncarcinogenic
endpoints of toxicity.
In order to assist the risk manager in this process, drinking water
concentrations associated with can er risks over th. range of one excess : mor
in po ulaciogs of ten chousaad(10 ) to one excess tumor in populations of o e
million (10 ) for the 70—kg adult, drinking 2 liters of water per day, are
provided.
In the lifetime feeding study conducted by Furedi cc al. (198 4 a,c) , the ht-
dosed female rats exhibited a statistically significant (p
-------
; ,dav, respecc.vely. The tui.tistage odel was used for h —co—Low dose
e<::a olacjon (Crunp and atson, 1979; Howe and Crump, 1982). CiobaLS3 was
sec to fit the data in the experimental dose range and tc obtain upper 95
confidence limits on the combined incidence of bladder papilloma and
carcinoma. The nultistage model conforms to a biological model of tumor
initiation and promotion (Crump ec al. , 1977) and provided an adequate fit to
the dose—response data for TNT. The relationship of the concentration (..g ’:)
of a chemical in drinking water to cancer risk is expressed as follows:
35000
x R—C
where:
q 1 * — (mg/kg/day
R — risk (i0 , 10 , 106, etc.)
C — concentration of chemical in ug/L
35000 • conversion factor for mg to ug and assumption that a 70
kg adult drinks ZL of water/day
The animal doses were converted to equivalent human exposures using a surface
area correction assumi 9 a 0.30 kg ray, and a 70 kg human. The human slope
factor (q 1 *) is 3 x 10 (mg/kg/day) for the Linearized multistage model.
The slope, q 1 *, is taken as an upper bound of potency of the chemical to
induce cancer at low doses below the experimental dose range. Assuming that a
70 kg human adult consumes 2 Liters of water a day over a 70 year Lifespan the
estimated cancer risk is as follows:
Level of Dos . in
Human Risk ug/L
100
10 10
io_6 1
For comparison purposes, rinking water concentrations associated with an
excess cancer risk of 10 were 0.7 .ag/L, 20 ug/L, 700 ug/L, 20 ug/L and LO
.g/L for the one—hit. multihit, probit, logit and Weibull models,
respectively. The parameter estimates for these models were calculated with
RISK8I (Kovar and Krevskj, 1981).
The estimated excess cancer risk associated with ].ifetim! exposure to dr .rk
water containing TNT at 20 wg/L is approximately 2 x 10 . The estimated
VIl—9
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exces5 cartCet risk assocja ed 1ifec e expos re :o dr nk .rtg a er
conca:rir g TNT at 2 ..g/L is approxi ate1y X 10 . represents the u;per
95Z con :dence 1i 1: on risk Eros extrapolation usl.ng the linearized
ultiscage model. The actual risk is unlikely to exceed this value.
v u—b
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OThER cR: E?IA, G ’ID CZ A D SLDA?DS
The ACGH (1936) 3—hour cine—w 9 ghted average threshold linit value (A—n.
for exposure t NT is 0.5 g/m . The [ 5—ninuce short—term exposure
t STEL) has 5een elimir.aced pendLng addic onal coxicolcgical 3 daca. The G$ A
( 981) Permissible Exposure Limit (PEL) remains a: 1.5 mg/n
As summarized by NRC (1982), the following workroom standards have been
adopted for TNT by various countri 5 s: Czechoslovakia, 0.1 ppm or I
es : Germany, 0.15 ppm or 1 5 mg/n ; East Germany, 0.15 ppm or 1.5 ng/m ; and
t.S.S.R., 0.1 ppm or I mg/n (Verschueren, 1977). The U.S. Navy 9ureau of
Medicine and Surgery (SITMED, 1980) as cited in NRC (1982) has established a
target interim maximum contaminant level (TIMCL) of 0.05 ng/L. (50 ugIL) for
TNT in drinking water. Dacre (1980) calculated an interim criterIa for TNT
for the protection of human health of 44.24 ug/L. Earlier, the U.S. Army had
established Limits of I mg/L (1000 .g/L) in drinking water and 5 mg/L (5000
g/L) in water used by fish and wildlife (Smock ec al., 1976, as cited in NRC,
1982). CSAMBR.DL (1980), as cited in NRC (1982), has recoended a TNT Limit
of 0.01. mg/L (10 g/L) in was,cewater, and the U.S.S.R. has set 1 ng/L (1000
ug/L) as the maximal permissible concentration in surface water (McKee and
wolf, 1963, as cited in NRC. 1982).
VItt—1
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A ALY::cAL z: - 1 0
\
Several nethods have been published for TNT analysis in water ( .T wastes)
s pie, guLck and reproduc .ble nechod using high performance liquid
chro acography (‘ 1PLC) is described.
The following methods for the determination of TNT in TNT wastes have been
listed by Zakhari and Villaume (1978):
1. Colorimetric or spectrophocometric determination at 500 nm following
crea menc of wastes with sodium sulfite and sodium hydroxide solutions.
2. Colorimetric measurement of a Meisertheimer complex at 440 nm
following treatment with 15% potassium hydroxide solution, usable to 80 ppm
a—tNT (2,4,6TNT).
3. Gas chromatographic detection of ppb to ppt in sea water using a
nickel—63 electron capture detector.
4. Liquid chromatographic characterization after adsorption of nicro
compounds on a styrene—divinylbenzerte copolymer type resin.
The HPLC method described by Brueggeman (1983) utilizes a trace enrichment
sample preparation technique to keep preparation tima to a minimum and affords
analysis to be conducted at ambient temperatur. without loss of efficiency of
separation or speed of analysis. The detection limits were reported as 0.2
g/ml. Several military explosives were analyzed by this method.
In the HPLC method, separation of the explosives is achieved by using a
reverse phase column (C 18 ) and a mobile phas. of methanol and water. A linear
gradient elution program is used in which the elusnt is changed from 95% Pump
A (25% MeOH/H 0) to 50 Pump 8 (80% M.OH/H 2 0) in 30 minutes at a flow rate oi
1.7 mL/min an a pressure of 500 psi. The column effluent is monitored at 240
nm. Separation was accomplished in <28 minutss.
Prior to analysis of th. aqueous samples. the C cartridge is activated with
two to four milliliters of methanol followed by m .I. of glass—distilled
water. Th1.a procedure removes organic impurities, displaces the methanol and
activates the cartridge. Concentration of the sampl. is achieved by passing
20 ml of each sample, containing 200 ppm of the internal standard
(1,3—dinitrobenzene), through the SEP—PAX cartridge at approximately
10 mI/mm. followed by the sequential injection of 10 ml of air, 4 ml of a
aceronitril./vater solution and an additional 10 ml of air. The combined
elements are collected in a screw—cap test tube and centrifuged at 2000 rpn
for 15 minutes. the supernacanc is analyzed by H.PLC as described.
The ratio of peak area of TNT to 7 ak area of the internal standard was
LX-1
-------
pc::ed versus cOflCentra ion of TNT. By exa i.ning the R’ correlation
: e.e’.cs, it was denonscrated chat TNT showed a Linear relation over :. e
concenc:ac:on range analyzed (R - 0.998) over a 4 —day period. The lower
dececc:on l it was 100 ng.
Recovery studies done with Laboratory—spiked wastewater indicated a recover’,•
range of 70% o 76% for TNT. The peak areas of the SEP—PAK—crapped nacer:als
were cc pared with standards having identical concentrations of the compound.
Recovery of the internal standard over a 5—day period was considered
reproducible (S.D. 2Z). Separation of a synthetic vastewacer influent and
effluent showed good resolution for all compounds tested. This method, as
described, is considered by the author to be suitably precise, accurate,
sensitive, and selective for the determination of TNT and various other
explosives in wascewater.
The analysis of actual wascewater samples were not reported by the Brueggmann
procedure; however, Spanggord et al. (1978) reported on the analysis of
several AAP wastewater samples (LA.? discharges) using .similar HPLC
procedure. esing a C 18 Bondpak Reverse—Phase column, a 60:40 methanoj:wacer
solvent at a flow rate o 1.6 mi/mm. and uv detection at 254 nm, the
retenc on time for TNT was 209 seconds in LAP discharge samples. Peak areas
were determined by digital integration and quantitation was by the internal
standard method using beazophenone. The lower limits of detection were
0.1 ppm and these huts could be lowered by a factor of two at 210 nu. This
procedure did not utilize any special sample preparations or concentration
techniques.
IX-2
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: EAi z : EC:-NCLccL s
\
‘ ‘ec ds for wascewater treatment for the removal of TNT and j proth c:s are
:scLssed. The rrent treatnents oi choice for both ‘red” and “pink” water
are descr 5ed n greater deta j.
.asce water discharges from AAP’s involved in the production of TNT are of two
unique types:
1. “Red water” is the spent salute waste solution formed durIng t ie
I T purifioarjon process. It contains TNT and its isomers, sodium carbonate,
sodium sulfate, and sodium sulfite, along with complex chemicals resulting
from the degradation of the TNT isomers. Many of the components of red water
are toxic and/or carcinogenic and it has been classified as a “hazardous
substance” by the EPA (Ryon et al. , 1984).
2. “Pink water” is the aqueous effluent generated during TNT
anuiaccure from plant clean—up and scrubbing processes, LAP operat .ons and as
a condensate from the evaporative concentration and incineration of red water.
It contains varying amounts o’f TNT and it mata—jiomers and photodegradac ion
products of TNT to include water—soluble, organic —insojubj e anions, orgar .c
solvent—extractable products and DNT isomers (Ryon at *1., 1984).
Treatment methods for these two major types of TNT vast, require different
procedures which are treated separately below.
A. Red Water
Disposal of the wastes generated during the s.llic. purification process is
the one of most serious concern. During the period between World War II and
the Korean War, red water wastes were dumped into screams as a means of
disposal. Later, after evaporative concentration of the red water at the AAP
facility, the concentrates were either sold to paper mills for incinerac:on
and recovery of the sodium sulfat, needed for th. pulping process or
incinerated at the AAP, followed by landfilling of the ash. Transportat. .on of
the concencrats is no longer feasible due to its hazardous classification ar.d
stricter pollution regulations have decreased the secondary market for t’e
recoverable sodium sulfate. Incineration of the concentrat, is not Ot v
expensive but, more importantly, adds to the existing air and solid vast.
pollution problem. Current techniques are aimed at the recovery of the su. f
and sodium present in the red water. Several. companies and their processes
are listed below:
L. Molten salt bath reduction process — Atomics International
2. Carbonate process — Tampella Smelt
3. Pyro1ysj reduction process — SCA Sillerud
6. SuLfite recovery process — SONOCO
-------
Of c-iese, the SCNOCC process appears to be the nost techn .cally and
eccr.cn caijy feasible. Rvon et a’l. (1984) described the sulfite reco’:er.
process (SRP) as adapted for use at the Radford AAP as follows: red water :s
nixed with aluminum hydroxide and reduced in a furnace to sulfur dLoxide and
soluble sodium aluminace. The sulfur dioxide is scrubbed with sodium
carbonate to form sodiuz sulfite, which is reused to purify TNT. The sodiur
aluminace is converted to aluminum hydroxide, which is recycled for the
treatment of the red water feedscock. In sum ary, the Radford adaptation cE
the SRP, essentially a closed—loop process, converts red water to sellite
which is reconverted to red water when used in the TNT purification process.
Impurities from TNT itself are converted to water, nitrogen and carbon dioxide
and released to the atmosphere. A more detailed description along with a flow
diagram of this process is contained in Pal and Ryon (1986). Problems artsing
from this adaptation are currently under active investigation.
B. Pink Water
Under condition of full production of TNT, it has been estimated (Forsten,
1980 as cited in Ryon ec al. , 1984) that the amount of pink water generated
could be as high as 100,000 gal/day/line and may contain 140—160 mg TNT/L
along with other contaminants and explosives. The current method of choice
for pink water abatement is adsorption by activated carbon (Ryan it al.,
1984). W1 ile this method has been long and widely used, it is limited by a
24Z drop in efficiency of the carbon with the first regeneration with
successively lower losses at each subsequent regeneration (Castorina, 1980).
To increase the efficiency and reduce the cost of carbon regeneration, a new
thermal carbon regeneration process using a rotary kiln has been developed and
put into use at the Iowa AAP. Ryon et al. (1984) described the process as
follows: the explosive—laden carbon is devatered and calcined at 110°C. In
the second step, spent carbon is pyrolyzed at 300C to remove the adsorbed
explosives. Steam at 20 kg/hr and carbon dioxide at 25 t/min are fed at this
stage to maintain a reducing atmosphere. In the third step, the carbon is
subjected to a reactivation temperatur, of 861C. An average regeneration
efficiency of 92Z, decreasing significantly vith time, has been achieved
(Forgte , 1980 c i cited in Ryan it ci., 1984). Th. carbon can be regenerated
four times using this process after which it is discarded by open burning (Pal
and Ryon, 1986).
A study conducted at the Holston A&P (Burrows, 1982) indicated that while
adsorption on granular activated carbon (CAC) is a viable treatment method for
removal of TNT from deionized water when present alone, this would not be a
method of choice for mixtures of munition compounds as competition for
adsorption sites and reduced overall efficiency has been demonstrated. Other
munitions are progressively displaced in favor of TNT adsorption.
A hydroperm microfiltration system has been proposed as an alternate means cf
treating pink water (Sundaram it al. , 1981 as cited in Pal and Ryan, 1986’,
x-2
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Th:s nethod iS based on cross—flow f:ltracion with thick—wa1.led, por:_s
asz r cubes (hyaropern tubes which nave been demonstrated :o :encve
si; icanc Levels of hi h—molecu1ar—weight dissolved solios and, i c e case
of water, the color associated with these conscituent . The eEfue :
fron : e svsce can be recycled in LAP operations or d:scharged co iatura.
screams after creat enc with carbon. The feasibility of this system is vet to
be determined.
C. Other Methods
While open burning of waste munitions is still practiced at AAP’s,
incineration is becoming the preferred method. In the treatment of TNT
wastes, open burning or incineration of the red water concentrate anc the
spent carbon from pink water treatment has been practiced at various A.AP’s.
In many cases the incineration process, despite its potential for the release
of new and un onitored chemicals Co the atmosphere, is preferable to storage
in landfills or elsewhere. New hazardous waste disposal furnaces are being
developed and tested at various facilities. Pal and Ryon (1986) have
described the following:
1. Rotary Kiln Incinerator — propellant slurries are incinerated by a
continuous process at 1000C in refractory lined cylinders rotating at a slow
speed. Capital and maintenance costs are high in this system.
2. Fluidized Bed Incinerator — incorporates a slurry feed system (for
the aqueous explosive slurries), a cyclon, particulate collector and a stack
gas analyzer. The addition of 6Z (by weight) of nickel oxide to the aluminum
bed has produced a drastic reduction in the emissions making this method safe,
efficient and economical.
3. Pyrolytic Incineration — a two step, continuous process system
combining pyrolysis and combustion in a conventional incinerator in one step
with a pyrotherm system (pyrolytic system with heat recovery) in the second
step. This system has many advantages for the treatment of sludges or wet
solids including the fact that it is totally enclosed thereby eliminating
fugitive emissions.
4. Simplified Incinerator Technology for Pollution Abatement (SITPA
‘II) System — consists of an unlined rotary kiln, a combustion chamber heated
by oil burners and a cyclone particulate collector, bag house filter and wec
scrubber. Whils this system is simple and low in cost, it is basically a dry
feed system, making it mors hazardous for disposal of explosives.
Biodegradation has also been considered as a means of disposing of munitions
wastes including those encountered in the TNT manufacturing process. T e
microbial transformation of TNT has been extensively studied with conflicting
reports concerning the degradation of the aromatic ring (Pal and Ryon. 98 )
X- 3
-------
T e c xic:t’i of the ring ccnpounds that m:ghc Earn during :h:s pr3cess 3
:ge_i kriovn Sec sini..ar compo nds are known to be toxic (Castorina, C
F -:-er research has been contracted to Atlantic Research Corporation to stud.
the effec veries5 cf compasting TNT waste. (Renard. 1984 as cited i ?al and
or., L986)
The primary physical method for the breakdown of N1 involves phocolysis.
Exposure to sunlight or any other source of UV light results in a rapid
breakdown of TNT arid its by—products, including 2—4— and 2,6—DNT, to a pInk
residual. Ring cleavage to CO 2 and volatile organics has been shown (Andrews
and Osmon, 1976 as cited in Ryon at al., 1984). Treatment of T NT and SDK In
an aqueous solution by exposure of a 1000 gaL/day pilot test sample to LV
light in the presence of ozone resulted in a reduction of dissolved TNT and
RDX to <1 mg/L. 3y converting the TNT rapidly to CO , HNO 3 and i 2 O. no
by—products requiring disposal are formed (Roth and urphy , 1978 as cited In
Castorina, 1980). Further study of this process awaits the development cf
more cost—effective equipment.
Several bench—scale models have undergone testing at the Holaton AAP
Industrial Liquid waste treatment Facility for their ability to degrade TNT ,
aLone or in combination with ocher munition chemicals, in aqueous solutions.
These nodels ir..:. . de:
1. Corona Oxidation (Inriova Process) — uses electrolysis arid graphite
fiber particles; results in degradation, both singly arid in mixtures;
degradation products not identified; both oxidation and reduction processes
seem to occur ( obylinski and Burrows, 1983).
2. Combined UV Radiation—Ozone Treatment — apparently useful for reso’. a
of TNT from relatively small, arid clean process screams (Burrows, 1983).
3. UV Radiation with Hydrogen Peroxide — resulce in an increased rate of
destruction of TNT when 0.O1Z hydrogen peroxide is added to mixed munition
wastes (Noss and Chyrek, 1984).
Pilot scale testing of authentic wastevacers would be th. next step in
evaluating those mode].s considered most promising as tertiary treatment
methods.
X- ’4
-------
v:. cm’:C: s:c :s
3ased on the availaole ani.nal toxj ty and human ep de iolog cal data, the A
for One—day, Ten—days and onger—te exposures is 20 _g/L. The D T
:s 0 _g/ for ltfeci e exposure and the Lifetine HA, assuming 20% relative
source contribution, is 2 gIL. TNT is classifIed EPA Group C, possibe human
carcinogen. The classification of TNT in EPA Group C Is based upon l::±ted
aninal data. A quantitative cancer risk assessment, based on the united
data, is provided to support selection of uncertainty factors for the
recoended lifetime HA.
As indicated in the companion report, “Data Deficiencies/Problem Areas and
Recommendations for Additional Data Base Development for Trinitrotoluene’
(Appendix 3), standard reproductive and developmental toxicity studies were
not available and should be considered in future medical research plans.
Also, ophthalmological evaluation of workers in occupational settings should
be routinely performed.
XI— ’
-------
x::. __________
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Levine BS, Rust JH, Burns iN, Li h PM. 1983. Determination of the chronic
na aLjan toxicological effects of TNT. Twenty—six week subchronic oral
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DA. 17—79—C—9120., AD—Aj57 082.
M.agrtusson B and Kligman AN. 1969. The identification of contact allergens by
animal assay. The guinea pig maximization test. J. Invest. Derm. 52:268—276,
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Makicie J, Harkonen H, Som.roja S, Ahonen R. [ 984. Trjnicroco].uene induced
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Manoilova 1K. 1968. [ Pathogenesis of the crystalline lens involvement
following crinitrotoluen, action on th. human organism.] Vegt . Oftalmol.
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NR 108—985. AD—A044 650/0.
McConnell EE, Solleveld HA, Suenb.rg JA end Boorman GA. 1986. Guidelines f r
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Cart. Ins. 76:283—289.
I I—
-------
“ cKee JE. Wolf eds. 1963. (Repr , 9 ) . Water Qual tv Criter:a, ::
o. ?jbllcatLon NO. 3 . CA.. The Resources AgC C of California, St
ac esources Control Board. 5. 8 pp., as cited tn NRC, 196:.
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Aberdeen Proving Ground, Mp., D923285L ,.
Morton AR 1 Ranadive MV, and H.athaway JA.. 1976. Biological effects of
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Newell GW. Dilley JV, Spanggord RJ. 1976. Maalian toxicological
evaluations of TNT wasrevater (“pink water”). Final Report. Stanford Resedrch
Lnsc tute, Menlo Park, CA. DA1 17—74—C—4115. AD—A044 785.
Nichols WW, Moorhead P. Breven C. 1972. Chromosome Methodologies in Xutdtion
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Bioengineerjng Research and Development Laboratory, Technical Report 8308, ‘t.
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XI—7
-------
R se iarc DH, Lauce:bach GE, Dav s GT. i9 . ater poi:ur on probens
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(Abstract)
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XII—8
-------
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creac en: of
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Development
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ae cr. Phase
-- — AO
‘ I —, 1
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XII—9
-------
m n ott the toxic effects of cr.rutrctcj.ue e
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studies . _c toxic che ica1s. Vo1u e 3 of 8.
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- .c— 3A. 1921. I I.
aria..s. Ln 1ed .ca].
: - sate of —
Scac.ortery Off:-
Prob1e definit cn
Occupational health and
Report, Science
DAZW 17—77—C—7O2o,
XII—1O
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APPENDIX 1
tncidertce of Tumors in Ani a1s
Fed TNT th the Diet for 2 Years
At-I
-------
rable Ai- .i. Thc’der.ce of Lr..nary 3.adder Les ons
n FemaLe Rats Fed T for up o .
Lesion
0.0
0.4
Dose ( g,kg/day)
2.0
10.0
5C.
iucosal
1/54
0/54
0/55
2/55
L S
Hyperplasia
Transfticnal cell
0/54
0/54
0/55
1/55
‘
5/55’
?apillo .a
T:artsit1onal—squa ous
0/54
0/54
0/55
0/55
1 I’35 ’
C 1I Carc .no as
Combined ?api1lo a
0/54
0/54
0/55
1/55
Car c i now .a
Reference: Furedi et
al.
(1984a,c)
a!
Number with 1esion/nu ber necropsied. Includes chose rats sacrificed at
the end of the study along with spontaneous deaths and ortbunc
sacr ices (12—24 months)
p< 0 . 05
p< 0 . 0 l
A1-2
-------
APPENDLX 2
Incidence of Myelofibrosis in Rats
Fed TNT in the Diet for 2 Years
A2-I
-------
-ab .e A2—L c .dence of 3cr e arro e1cfi ços:s
th E’e ae Racs Fed T for up :0 24 OflChSa
L.esLor
0.0
0.4
Dose (mg/kg/day)
2.0 10.0
50.0
XyeLofibrosis of
5/54
6/53
13155 b/
12/54
L i s 4
Sterna]. Sone Marrow
References; Furedj
et
a]..
(1964
a,c)
ai
Number wi: 1esion/nu ber necropsied. Intludes those rats sacrificed ac
the end of the study along with spontaneous deaths and moribund
sacr .f ces (12—24 months)
b/
c/
A2—2
-------
APPZNDIX 3
Data Deficte r c es/?rob1e Areas and R. oendations For
Additional Data 8a . Develop enc For trinitrocoluene
A)- 1
-------
: ‘
‘e off_ce of Dr -tr. . ater (OD ) Environmental ?rocect on Agenc: ?A) ,
ccr. u:1c:i3n witri tie Department of the Army, has reviewed :he availaD le data
on 2 , ,ô—cr:n::rozcluene (TNT) for the purpose of developing a Health Advisor,
C tA) useful in dealing with contamination of drinking water, to include
“state—of—the—art” information on health effects, analytical methodology and
treatment technology.
CBJECT:VES
The objective of this appendix is to provide an evaluation of data
deficiencies and/or problem areas encountered in the review process for TNT
and to make recoendations, as appropriate, for additional data base
development. This document is presented as an independent analysis of the
current status of TNT toxicology, as relates to its possible presence in
drinking water, and includes a suary of the background information used in
the development of the HA. For greater detail on the toxicology of TNT, the
Pealch Advisory on Trinitrotoluene should be consulted.
B ACKCROLND
trtnitrotoluene, a pale yellow to white crystalline substance, is the nost
widely used military high—explosive with applications in shells, bombs,
grenades, demolition explosives and propellant compositions (Department c,i the
Army, 1967). It is produced at certain selected Army Ammunition Plants
(AAP’s) and loaded at other A.A.P’s. Production in the United States between
1969—1971 was approximately 45 million pounds per month with a total capacity
of 85 million pounds per month (Patterson et ii., 1976 as cited in Ryon ec
al. , 1984)
It has been reported that as much as one half million gallons of wastewacer
have been generated per day at a single plant involved in TNT production
(Hartley et a ]., 98L). The pollutants of greatest concern are in wastewater
discharges from manufacturing, purification and load and pack (LAP) processes
at the AAPs, and are primarily designated “red water” and “pink water” (Ryon
ec al., 1984). These two major types of wastes differ substantially both in
composition and treatment techniques.
Red water arises from the sellits purification process for crude TNT, is high
in solids and, in one recovery study, contained approximately O.6Z TNT by
weight as well as other substances, largely as suspended solids (Ryon ec al.
1984). Pink water results from plant clean—up and acrubbers processes. LAP
operations and red water treatment procedures and contains, in addition to
approximately IZ 2,4,6—TNT, the other TNT isomers and numerous degradation
products (Spanggord et al., 1978). Continuous flow raw wastewater from o e
AAP, prior to its final treatment by neutralization and sedimentation, was
AJ—2
-------
est aced tO COntain an average of 20 ng/L of -—TNT (Nay ec al. • 1974 as c tec
;n Rvon ec al., 198 ). These values varied from <0.05 to 2 0 ng/L deper.dj
on c e site of sanp].lng, production Levels and stage of production and/or
creac enc (Spanggord cc a].., 1973).
Treatment technology for red and pink waters differs considerably. while tne
volume of pink water generated is considerably higher, the handling of red
waters poses the greatest health risk. A sulfit. recovery process (SONOCO
Process; Ryon cc a].., 1’984) appears most promising for treatment of red water
via recovery of sellite and conversion of impurities to natural products.
Current treatment of pink water involves adsorption on activated carbon. A
process to regenerate the spent carbon, if successful, could increase the
efficiency and efficacy of this procsss.
Various laboratory studies (Jerger et a].., 1976; Spanggord et al., 1980)
indicate that the persistence of TNT in the aquatic environment, while varying
somewhat due to physical and biological conditions, is considered low due to
degradation both by photolysis in the water column and bacterial species in
sediments. Persistence in soil and groundwater is Lengthier with one study
(Sanocki et a].., 1976 as cited in Ryon cc a].., 1984) indicating a level over
3000 mg/kg in a former waste lagoon now filled with sediment and coal wastes,
20 years after its active use as a disposal site. Bioaccumulatjon in
organisms is not considered a significant problem (Ryon et al. 1984).
The pharmacokinetic properties of TNT have been studied in dog., rats, mice
and rabbits. Available data indicate chat it is will absorbed by inhalation,
ingestion or skin contact, detoxified by cbs liver with a low distribution to
other tissues, and is excreted primarily in the urine. It is metabolized
largely by reduction of the nitro group, extensively in all four species and
similarly in rats, mice and dogs (Hodgson et al., 1977).
Mecabolices include the hydroxylamines, the monoamjnodinjtro and
diaminomononitro derivatives. Little unchanged T NT was found. Quantitative
differences in metabolite. were evident when different treatment routes were
used. Rabbits appeared to metabolize TNT at least quantitatively different
than ocher species. A red pigment in th. urine, believed to result from a
netaboljte of TNT and evident in humans exposed to TNT in the workplace, was
also found in rats and mice but its source could not be identified. (El—hawari
et a].., 1981).
While numerous effects of TNT in humans exposed during the manufacturing
process have been reported, the most persistent effect. involve the
hematopoietic system and the liver (Zakharj and Vii.laumg, 1978). Occupational
health surveys (Priedlander it a].., 1974; Morton and R.anadive, 1974; Buck and
wilson , 1975) indicate that thes. effects are generally detectable by changes
in the hemacological and chemical parameters of the blood and are readily
reversible upon removal of the individual from the source of exposure.
A3-3
-------
Cataract development in occupationally exposed workers has seen reported
j . assnan and Juran, 1958; Hassmdn et al.. 1978; {arkonen ec a.., 1933; ak::.e
ec a:., .984), but not among U.S. munitions workers.
Acute coxtc tv studies were conducted in rats and nice with ranging f:c
approximately 800 Co 1320 mg/kg in male and female rats and from 660 to 1015
mg/kg in male and female mice (Lee at al., 1975; Dilley et al., 1972, 1982),
Toxic signs included inactivity with tremors, proceeding to syetrical
coordinated convulsic- ith death usually due to respiratory paralysis.
Survivors showed signs ataxia and cyanosis. Red urine was noted in both
species within 10—60 minutes of dosing.
In skin and eye irritation tests, TNT produced no to mild irritation of rabbit
skin but did not ir: .:ate the eye when washed within five minutes. Longer
periods of contact resulted in iritis and cornea]. opacity. In the guinea pig,
TNT was a moderate sensitizing agent (Lee et a].., 1975; Newell et al., 1976).
Red staining of the skin and tissues surrounding the eye was evident.
Four—week feeding stunies conducted by Levine at a].. (1984a as Appendix IV in
Furedi et a].., 1984d) in B6C3F I mice at levels up to 700 mg/kg/day produced no
mortality and few toxic signs. At the high dose, body weight was low,
biiirubin was increased, kidneys and testes of males were significantly
decreased in weight and white blood cell counts were low. Females had
increased platelet le els. The only significant pathological lesion found i
this study was a diffuse increase in the amount of hemosiderin—].jke pigmenc n
the red pulp of the spleen. At 100 mg/kg/day, body weights ware only
occasionally and slightly decreased, bilirubin was increased and hemosiderosis
of the spleen was of minima], severity. Red urine was evident at high dose
levels.
Dilley et a].. (1978, 1982) conducted four—week feeding studies in dogs, rats
and mice at levels up to 20, 100 and 185 mg/kg/day, respectively. Red urine
appeared in dogs at the high does and in rats and mice at the two highest
levels. Significant findings in all three species included a decrease in body
weight with an accompanying decrease in food intake, mild to moderate anemia,
and increased spleen weights with hemosid.rosis. Rats also shoved signs of
testicular atrophy and increased liver weights while dogs and rats had
increased cholesterol Levels and decreased sertz glutamic pyruvic—acid
transaminass (SGPT) activity. In the one dog/sex/level allowed to recover fir
four weeks, iron levels were greatly increased in both sexes. In the rat
study signs of testicular atrophy and hemosideroets of the spleen were aso
seen at the next highest treatment level, approximately 42 mg/kg/day. Effects
of TNT on mice were minimal.
This four—week feeding study was continued in surviving animals for an
additional nine weeks (Dilley at a].., 1978, 1982). Rats shoved a p.rsis . :
negative effect on body weight while anemia remained evident in all three
species. Rats and mic. had hemosiderosis of the spleen and dogs and mice
A3—4
-------
elevated Liver weight with sone necrosis evident in nice. Cholesterol Leve:s
were Lncr ased and SGFT acciv y was decreased in dogs and rats but were no
studied in nice. Only onedeach occurred in these studies with one acg dytr.
during . .eek 12. Signs of anemia were m ninal in dogs. La the rats, ane :a
and henosiderosLs of the spleen were also evident at the 3 S/mg/kg/da level.
Iron levels were decreased in males at all but the lowest dose and glucose was
decreased in the two lowest dose levels. High—dosed male rats fed TNT for [ 3
weeks were reported to have testicular atrophy and hyperplasia of the
interstitial cells. The only effect reported as treatment—related for mice
fed TNT for 13 weeks was hemosiderosis of the spleen. Some necrosis of the
liver was seen in the mice allowed to recover for an additional four weeks.
A [ 3—week feeding study conducted by Levine Sc a].. (1981, 1984b) in Fischer
344 rats fed TNT at levels up to 300 mg/kg/day produced similar results.
Lethargy and ataxia were observed early in the study. Small testicular size
was noted by palpation during Week 6 in the high—dosed males. Reductions in
body weight were seen at most dose levels in males and in females at 125 and
300 mg/kg/day. Other dose—related findings included anemia, elevated serum
cholesterol levels, hepatomegaly, testicular atrophy, and increased spleen and
kidney weights. Histopathology revealed brain lesions, hepatocytomegaly,
increased pigmentation of the spleen and the kidney, splenic congestion and
testicular lesions.
In a 90—day feeding study La monkeys (Martin, 1974) and dogs (Hart, 1974), the
only effects reported as possibly trsatment—rejaced were necrotic or abnormal
tnagak.aryocytes in high—dosed female monkeys (1.0 mg/kg/day) with increased
iron—positive material in the liver cord cytoplasm and a slight increase in
hemosiderogis of the bone marrow in high—dosed (1.0 mg/kg/day) dogs.
Levine cc al. (1983) also studied the effects of TNT on dogs dosed over a 26
week period. Two deaths occurred at the highest dose level (32 mg/kg/day).
Other effects at this level included dehydration, weight lois, jaundice,
hypothermia, diarrhea, etazia, anemia, elevations in bilirubin, serum globulin
and lactic dehydrogena,. (LDH), and decreases in SGPT and glucose.
HistoLogical. sxamiuatioit revealed hemosid.rosjs of the spleen and liver,
splenic congestion, increased spleen and liver weight., hepatocytomegaJ.y with
hepatocytjc cloudy swelling and cirrhosis. Effects on the liver were evident
at all dose levels (0.5 to 32 mg/kg/day), although minimally so at the lowest
doses.
Lifetime feeding studies were conducted by Puredi et aX. (1984a—f) in Fischer
344 rats and B6C3FI. mice at levels up to 50 and 70 mg/kg/day, respectively.
Signific*nt adverse effects in rats fed Tht for two years included decreases
in weight gain and food consumption, anemia, methemoglobinemia, myelofibrosis
of the bone marrow, congestion, extrazedullary hemacopoie.is and hemosiderosis
of the spleen, increases in liver, kidney and spleen weights, hepatocslluiar
hyperplasia with cystic degeneration, alterations in lipid and protein
A3-5
-------
ecaoo_ stn, spotted ana cystic kidneys with pigmentation, nf1anr ation anc
.n: ltrat .on, hyperpl sja of the renal pelvis ar .d urinary ba der
H.perpias:a, papillo and carcinoma. Several effects were s .gnificant at e
2 ngik icav dose level.
In the study in rice, effects related to TNT intake included mild decreases
weight gain, anemia, occasional elevations in relative weights of the liver,
kidneys, spleen and heart, extramedu].].ary hematopolesig of the spleen,
cytoplasmic vacuolization of the renal tubules (males), renal lymphocycosis
and enlarged spleen and lymph nodes (females). Effects in mice were seen in
animals receiving 10 mg/kg/day or more.
Trinitrotoluene was reported to be strongly mutagenic in Salmonella
yph murium in all test strains at various dose levels (Ellis et al., 1973)
In vivo cytogenic analysis on bone marrow cells from rats revealed no evtdence
of genetic dan.age induced by TNT while an in vitro measurement of unscheduled
DNA synthesis (TJDS) in human diploid fibroblasts suggested a positive response
at hIgher dose levels (Dilley et al., 1978).
Ashby et al. (1985) reported a negative response in a mouse bone marrow
nicronuclaus assay while an in vivó/in vitro rat liver assay for IDS was also
negative.
Intake of TNT ifl the diet for two years at levels up to 50 mg/kg/day in rats
was reported to cause cancer in the female animals. Female rats fed TNT at 1C
and 50 mg/kg/day showed an increase in the incidence and severity of
hyperplastic, prerteoplastic and neoplastic lesions of the mucosal epithelium
of the urinary bladder (Furedi et al., 1984a—f).
No studies on the possible reproductive or developmental effects of TNT were
found. However, potential reproductive effects were nocsd in 13—week feeding
studies (Levine St c i., 1981, 1984b).
Several methods of chemical analysis for TNT in water have been reported with
a high performance Liquid chromatography (HPLC) method apparently suitable for
detection of TNT in LAP discharge wastewater samples. The lower Limit of
detection was reported to be 0.1 ppm (Spanggord et *1., 1978).
Treatment of “red water” and “pink water” wastes is accomplished differently.
A sulfite recovery (SONOCO) process appears most feasibl. for treating red
water waste and has been adapted for use at the Radford AAP (Ryon it al.,
1981.).
Granular activated carbon (GAC) adsorption Is the current method for treating
pink water wastes. A method to regenerate the spent carbon is under
development (Pal and Ryort, 1986) but the presence of other munitions in the
wascevacer places limitations on the GAC method (Burrows, 1982). An alternate
A3-6
-------
ydroperm nicrof rat.on system is under deveLop enc (Sundara ec a].., ; .
as cited in ?al and Ryort, 1986),.
Based on the sign .ficanc flndLrlgs of the foregoing studies; HA values for
One—day, Ten—days and Lortger—Ter were established at 20 ..g/L, the Drk
acer Equivalent Level (D 1EL). A DWEL is defined as the ediu —specifjc (:n
this case, drinking water) exposure which is interpreted to be protective for
non—carcinogenic endpoints of toxicity over a Lifet .tie of exposure. This D tL
is calculated for a 70 kg adult consuming 2 liters of water per day. The
Lifetime HA is 2 ugIL and assumes 20% relative source contribution. TNT is
classified as EPA Group C, possible human carcinogen.
The estimated excess cancer risks associated with lifetime exposure to
drirtkirt water conta rting TNT at 20 wg/L and 2 wg/L. have been calculated cc be
2 x 10 and 2 x 10 , respectively.
DISCT.SSION
Available data on the pharacokinetics, health •ffect,, analysis and treatment
of TNT have been reviewed.
The pharm .acokjnetjc properties of TNT have been studied in various species and
results indicate that it is easily absorbed, and that metabolism is
qualitatively similar, if quantitatively different, in all three species.
Little data is available on the metabolism in humans; however, humans as well
as rats, mice and dogs (at high—dose levels) produce a metabolic., as yet
unidentified, that causes a red color to appear in the urine. Identification
of this color—producing metabolite could be significant to the metabolic
profile of TNT but does not necessarily impact on the development of a HA.
The available studies on the toxicity of TNT include 5O in rats and mice,
short—term (four—week... in dogs, rats and mice) and longer—term (13—weeks in
dogs, rats, :1cc and monkeys; 26—weeks in dogs; and 24—months in rats and
mice) studies including assessments for carcinogenic potential. Results of
these studies produced similar results in most species with effects on the
hematopojetic system (anemia with related affects in the spleen) apparent from
the shortest term through lifetim, studies. Effects on the hepatic and renal
systeme becam. apparent with increasing length of exposure. Available data
following human exposure to TNT in the workplace indicate similar effects in
the various human systems. The only seeming inconsistency in thes. studies
was the testicular atrophy apparent in the several studies in rats fed TNT for
up to 13—weeks but not evident in a lifetime study in this species. Data
suitable for One—day, Ten—day and Longer—term HA.. are unavailabl, but the
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20 g,L is corsicered to be a conservative est aee fcr safe exocs
es for non—carc.ncSenjc er.dpo nts of toxicity over a L :et ne of exposure
ese caca are supported by consistent effects Lr. the variouo nger—t rz
S CUO $.
:n view of the testicular effects in rats 1 the need for a chree—generac.or
reproduction s - .:ttg with developmental studies, both of which are
currently Lao .s of primary importance.
while the various mutagertic assays differed somewhat in their results, nest
negative data occurred at low doses with positive results for mutagenic
act .v.ty predominating at the higher doses. The results of in vivo and in
vitro assays for cycogenetic effects were somewhat uncertain, Largely due to
limitaC3.OrtS based on solubility of TNT in the various systems and to
interference from discoloracions in the samples. Nevertheless, mutagenic t
studies are adequate.
Evidence has been presented that NT is carcinogenic to rats. The carc .nomas
of the bladder in female rats fed TNT for 2 years were not previously
.ndicated by short—term toxicities (but identification of the red
color—producing netabolite of TNT becomes of possible significance). In the
mouse study, TNT was administered in the diet for up to 24 months. GrQups of
75 nice per sex received TNT at doses of 0, 1.5, 10, or 70 mg/kkg/day. Ten
mice per sex per dose were killed folLowing 6 and 12 months on test wLch
surviving animals Itilled after 24 months of treatment. The major systemic
effects observ.-: the high (70 mg/kg/day) dose group included anemia with
hepacocoxicity. this indicates the MTD vas achieved. The study authors
reported that the incidence of combined leukemia/malignant lymphoma in the
spleen of females increased with dose. They reported that the increase was
statistically significant (pcO.05) at the 70 mg/kg/day dose level (high dose)
and that the l.siofll vere considered to be treatment——related. This was an
inappropriate conclusion based upon current NTP guidelines (McConnell et al.,
1986). Thesi guidelines indicate that it is appropriate to combine all types
of m.alignant lymphoma and lymphocytic leukemia, but not in a single organ.
These types of tumors occur throughout the hematcpoietic system. Upon
recounting these tumors, by each sex or both sexes combined, in the whole
animal, the atatistical significance islose (i.e., p0.05) using the
Fisher—Irvin Exact Test to compare dosage groups and the Cochran—Armitage Test
for Trend. Therefore, based upon the statistical analyse., this study is
considered to be negative with no tumors related to TNT exposure.
It has been reported in the literature that TNT may be associated with
cataract formation in humans (Hasam.an and Juran, 1968; Missman cc al., [ 978;
Zakhari and Villaum., 1978; Harkonen ec al., 1983; Makicie cc al., 1984).
Similar findings have not been reported among munitions workers in the Lni’e
A 3-8
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States no has this f.ndirtg been supported by data in rats and mice subTecte
to extensive ophthalmic examitiations at 1/artoug periods throughout the 2—:ear
seeding studies.
$everal methods of analysis for TNT in vastevater have been reported. One
method utiliz .ng api_C appears to be capable of being adapted to a sensitivity
level suitable for detecting those levels of TNT that may be considered
hazardous to health. The lover Limits of detection for this method were
reported as 0.1 ppm, lowerable by a factor of 2 at 210 rim (equivalent to 50
g/L for this Lower limit of detectability (Spanggord et al., 1978)).
Methods for treating both “red” and “pink” waters an, available. Since red
water has been declared a “hazardous waste” by the EPA (Ryan et al., 1984),
its disposal by concentration, incineration and Landfilling of the ash is rio
longer considered suitable. Alternate methods of treatment are under active
investigation (Ryan et at., 1984) and an adaptation of a eulfits recovery
process ( 0N0CO) currently being used at the Radford AAP appears to be a safe
and effective treatment method for this toxic waste.
Treatment of pink water, vhil. not presenting as hazardous a waste disposal
problem, does present problems of economic feasibility due to the quantities
of pink water wastes generated during the various production processes.
Adsorption by activated carbon has been and remains a viable treatment method.
Methods to regenerate the spent carbon are undergoing active inveatigaton
(Forsten, 1980 as cited in Ryan et al., 1984) and their developmertt should
relieve some of the economic burden of this waits treatment method. An
hydroperm microfiltration system is also undergoing active study and may
provide an alternate treatment method (Sundarem et al., 1981. as cited in Pal
and Ryan, 1986). Both analysis and treatment methods for TNT in wast wacers
are adequate.
C0NCLUSIONS/REC0 1 ’ lzNDATI0NS
Based on the abov, discussion, the following conci .u.ton./reco endations are
made:
1. The available studie. on th. toxicity of flIT are generally
considered adequate for development of a BA useful in dealing with
the potential contamination of drinking water.
2. No data ar, availabl, on the reproductive and developmental effects
of TNT. La view of the testicular effects seen in rats fed T T for
periods up to 1)—weeks, a three—generation reproduction study and a
study to determine possible developmental effects utilizing
currently accepted protocols, is recoended.
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3. Available data on the occurence of cataracts in European yorkers
clearly ind:cace that TN’T does produce this effect. Ho .rever,
air levels, absorbed dose, and echanL.- s of toxicity are not clear.
these d fficulcies are compounded by the fact chat cataracts have r.oc
been noted in L’.S. TNT— orkerg. Cataract developnenc is art effect
that nust be clarified by further studies to decer ine the dose and
echanis producing this effect. Additional studies in individuals
exposed to TNT in occupational settings should be considered. :
particular . thorough ophthal.wological exa inacions should be
routinely performed.
4. Aside frog the aforementioned data gaps, no further studies on TNT,
as relates to its possible presence in drinking water, are deemed
necessary at this time.
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