April 1992
HEALTH ADVISORY FOR
2,4- AND 2,6-DINITROTOLUENE (DNT)
3EPA
Health and Ecological Criteria Division
Office of Science and Technology
Office of Hater
U.S. Environmental Protection Agency
Washington, DC 20460
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April 1992
HEALTH ADVISORY FOR
2,4- AND 2,6-DINITROTOLUENE (DNT)
AUTHORS:
Margaret E. Brower, Ph.D.
Welford C. Roberts, Ph.D.
William R. Hartley, Sc.D.
PROJECT OFFICER:
Krishan Khanna, Ph.D.
Office of Science and Technology
Office of Water
U.S. Environmental Protection Agency
Washington, DC 20460
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AUTHORS AND CONTRIBUTORS
The following Dynamac Corporation personnel were involved in the
preparation of this document: Margaret E. Brower, Ph.D. (Task
Manager/Principal Author); Nicolas P. Hajjar, Ph.D. (Project
Manager/Senior Reviewer); William L. McLellan, Ph.D., Mary E. Cerny,
M.S., Robert Platz, Ph.D., Jess Rowland, M.S., Patricia Turck, M.S.,
Dawn Webb, B.S. (Contributing Authors); and Karen Swetlow (Technical
Editor).
This document was prepared by Dynamac Corporation under contract No.
68-03-3417 to the Office of Water, Washington, DC; Krishan Khanna,
Ph.D., Project Officer; Wei ford C. Roberts, Ph.D., Task Manager.
This document was revised under subcontract to Environmental
Management Support, Inc., Contract Number 68-CO-0006.
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PREFACE
This report was prepared in accordance with the Memorandum of
Understanding between the Department of the Army, Deputy for Environment
Safety and Occupational Health (OASA(ILiE)), and the U.S. Environmental
Protection Agency (EPA), Office of Water (OW), Office of Science and
Technology 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 adverse health effects would not be anticipated
and which include a margin of safety so as to protect the most sensitive
members of the population at risk. A Health Advisory provides health
effects guidelines and analytical methods, and recommends treatment
techniques on a case-by-case basis. These advisories are normally prepared
for One-day, Ten-day, Longer-term, and Lifetime exposure periods where
available toxicological data permit. These advisories do not condone the
presence of contaminants in drinking water, nor are they legally enforceable
standards. They are not issued as official regulations and they may or may
not lead to the issuance of national standards or Maximum Contaminant levels
(MCLs).
i
This report 1s the product of the foregoing process. Available
toxicological data, as provided by the Army and as found in open literature
sources, on the munitions chemicals 2,4- and 2,6-dinitrotoluene (DNT) have
been reviewed, and relevant findings are presented in this report in a
manner so as to allow for an evaluation of the data without continued
reference to the primary documents. This report has been submitted for
critical internal and external review by the EPA.
I would like to thank the authors who provided the extensive technical
skills required' for the preparation of this report. I am grateful to the
members of the EPA Toxicology-Review Panel who took time to review this
report and to provide their invaluable input, and I would like to thank
Dr. Edward Ohanian, Chief, Human Risk Assessment Branch, and Ms. Margaret
Stasikowski, Director, Health and Ecological Criteria Division, for
providing me with the opportunity and encouragement to be a part of this
project.
The preparation of this Health Advisory was funded in part by
Interagency Agreement (IAG) 85-PP5869 between the U.S. EPA and the U.S. Army
Medical Research and Development Command (USAMRDC). This IAG was conducted
with the technical support of the U.S. Army Biomedlcal Research and
Development Laboratory (USABRDL), Or. Howard T. Bausum, Project Manager.
Krishan Khanna, Ph.D.
Project Officer
Office of Water
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TABLE OF CONTENTS
Pace
LIST OF FIGURES ..... ix
LIST OF TABLES .........'... . x
EXECUTIVE SUMMARY xiii
I. INTRODUCTION . . . . 1-1
II. GENERAL INFORMATION II-l
III. SOURCES OF EXPOSURE . . . . III-l
A. Environmental Exposure . III-l
B. Occupational Exposure . III-3
IV. ENVIRONMENTAL FATE ..... IV-1
A. Biodegradatlon . . . IV-1
B. Photolysis -....'.' IV-3
C. Volatilization IV-4
0. Sorption on Soils and Sediments IV-4
E. Hydrolysis IV-5
F. Other Fate Routes IV-5
V. TOXICOKINETICS . V-l
A. ABSORPTION AND EXCRETION . ........ V-l
1. Humans ....'.. V-l
2. Animals ....... V-3
a. 2,4-DNT . , V-3
b. 2,6-DNT .................... V-6
c. tg-DNT V-7
B. DISTRIBUTION V-7
1. Humans V-7
2. Animals . . . . . V-8
a. 2,4-DNT V-8
b. 2,6-ONT . V-12
c. tg-ONT V-13
3. Covalent Binding of ONT Isomers . V-13
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TABLE OF CONTENTS (cqnt.)
Page
C. METABOLISM V-15
1. Humans ......... V-16
2. Animals V-17
a. 2,4-DNT . V-17
b. 2,6-DNT V-25
c. tg-ONT . V-26
3. Microbial and in vitro Metabolism of 2,4- and
2,6-DNT .. V-26
VI. HEALTH EFFECTS VI-1
A. Humans . VI-1
B. Animal Experiments VI-9
1. Short-term Exposure (<4-week studies) VI-9
a. Acute VI-9
b. Primary Eye and Skin Irritation and
Skin Sensitization ........ VI-11
c. Subacute VI-12
1) 2,4-DNT . VI-12
2) 2,6-DNT VI-16
3) tg-DNT . VI-20
2. Longer-term Exposure VI-21
a. 13-Week Studies ........ 1 . VI-21
1) 2,4-DNT . VI-21
2) 2,6-DNT VI-25
3) tg-DNT VI-29
b. 6-Month Study . VI-29
c.- Lifetime/chronic Studies . . . VI-31
1) 2,4-DNT VI-31
2) 2,6-DNT VI-38
3) tg-DNT VI-40
3. Reproductive Effects VI-42
a. 2,4-DNT VI-42
b. 2,6-DNT . VI-47
c. tg-DNT VI-47
4. Developmental Toxicity VI-47
a. 2,4-DNT VI-47
b. 2,6-DNT . VI-4«
c. tg-DNT VI-4«
5. Carcinogenicity .'.'...' VI-52
a. 2,4-DNT VI-52
b. 2,6-DNT VI-sa
c, tg-DNT . . , ,....,.. . . . . VI-S«
d. DNT Isomers VI 61
vi
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TABLE OF CONTENTS (cont.)
6. Genotoxicity .............. ..... VI-61
a. Microblal Systems ............... VI-61
b. Mammalian Cells in vitro .... ....... VI-64
c. In vivo Assays ................ VI-64
d. Unscheduled DNA Synthesis (UDS) ........ VI-65
e. Initiation, Promotion, and DNA Binding .... VI-66
7. Neurotoxicity ................... VI-68
VII. HEALTH ADVISORY DEVELOPMENT ..... . .......... VII-1
A. Summary of Health Effects Data ..... ....... VII-1
B. Quantification of Toxicological Effects ....... . VII-4
1. 2,4-DNT ... ........ ..... ...... VII-5
a. One-day Health Advisory ....... ..... VII-5
b. ten-day Health Advisory ............ VII-5
c. Longer-term Health Advisory ..'...• ..... VI I -6
d. Lifetime Health Advisory ...... ...... VII-8
2. 2,6-DNT ....... : ......... . .... VII-12
a. One-day Health Advisory ....... ..... VII-12
b. Ten-day Health Advisory . . ........... VII-12
c. Longer-term Health Advisory ..... ..... VI I -13
d. Lifetime Health Advisory .... ........ VII-15
C. Quantification of Carcinogenic Potential ....... VII-18
1. Dose-Response Data (Carcinogenicity, Oral
Exposure) . . .................. VII-19
2. Summary of Risk Estimates ...... ....... VI I -20
3. Drinking Water Concentrations at
Specific Risk Levels ...... ......... VII-20
4. Discussion of Confidence (Carcinogenicity,
Oral Exposure) ............ . ..... VII-21
VIII. OTHER CRITERIA, GUIDANCE, AND STANDARDS . ......... VIII-1
IX. ANALYTICAL METHODS ....... ........... . . IX-1
A. GC Analysis . . ...... .... ..... ..... IX-1
B. HPLC Analysis .... ........... ..... . IX-3
C. Other Methods . ... .............. ..... IX-4
X. TREATMENT TECHNOLOGIES .................. X-l
XI. CONCLUSIONS AND RECOMMENDATIONS ....... ....... XI -1
vii
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TABLE OF CONTENTS (conl.j
Page
XII.-REFERENCES . . . XII-1
APPENDIX: Data Deficiencies/Problem Areas and Recommendations for
Additional Data Base Development for 2,4- and 2,6-DNT
viii
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LIST OF HGURtS
Figure No.
V-l
V-2
V-3
Distribution of Free and Conjugated Metabolites of
2,4-ONT in the Urine of Orally Dosed Animals. ....
Proposed Pathway for the Anaerobic Metabolism of
2,4-DNT by Rat Intestinal Microflora
Proposed Metabolic Pathway for the Hepatic
Metabolism of 2,4-DNT . .
Pace
V-24
V-29
V-32
IX
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LIST OF TABLES
Table No. Page
II-l Physical and Chemical Properties of 2,4-DNT
and 2,6-DNT . . II-2
II-2 U.S. Manufacturers of 2,4-.and 2,6-DNT II-4
V-l Distribution and Excretion of Radioactivity 24 Hours
After Oral Administration of a Single Dose of
[r1ng-UL-l4C]2,4-DNT to Various Species ....... V-2
V-2 Tissue/Plasma Radioactivity Ratios in Various
Species of Animals 24 Hours After Administration of
a Single Oral Dose of [ring-UL-r4C]2,4-DNT V-9
V-3 Urinary Metabolites Excreted by Humans Exposed
Occupationally to Technical Grade DNT V-18
V-4 Radiolabeled Metabolites Found in the Urine of Various
Species 24 Hours After Administration of a
Single Oral Dose of r4C-2,4-DNT
.... V-20
V-5 Radiolabeled Metabolites Found in the Urine of
Various Species 24 Hours After Administration
of a Single Oral Dose of 14C-2,4-DNT V-21
V-6 Urinary Metabolites of 2,4-DNT Excreted by Orally
Dosed Fischer Rats . . . V-22
VI-1 Symptoms Exhibited by 2,4-ONT-Exposed Workers .... VI-3
VI-2 Clinical Signs in 2,4-DNT-Exposed Workers VI-4
VI-3 Acute LD,n Values for 2,4- and 2,6-DNT in Mice
and Rats VI-10
VI-4 Adverse Effect Levels of 2,4-DNT Following Subacute
Exposure VI-17
VI-5 Adverse Effect Levels of 2,6-DNT Following Subacute
Exposure VI-U
VI-6 Summary of Subchronlc Toxicity of 2,4-DNT in Mice . . VI-22
VI-7 Summary of Subchronlc Toxicity of 2,4-DNT 1n Rats . . VI-24
VI-8 Adverse Effect Levels of 2,4-ONT Following Subchronlc
Exposure ; VI-26
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LIST OF TABLES
Table No.
VI-9
VI-10
VI-11
VI-12
VI-13
VI-14
VI-15
VI-16
VI-17
VI-18
VI-19
.
VIM
VII-2
VII-3
VIII-1
XI-1
Adverse Effect Levels for 2,6-DNT Following
Subchronic Exposure . . .
Incidence of Histopathological Lesions in
CD Rats Fed 2,4-ONT for 24 Months
Incidence of Histopathological Lesions in Mice
Fed 2,4-DNT for 12 or 24 Months . .
Adverse Effect Levels for 2,6-DNT Following
Chronic or Lifetime Exposure
Effects on the Fertility and Gestation Length of
Parental Rats Fed 2,4-DNT in the Diet for Three
Generations . . .
Summary of Reproductive Effects in Rats Fed
2,4-DNT in the Diet for Three Generations
Tumor Incidence in F344 Rats Fed 2,4-DNT for
78 Weeks . . . ... . . ... . . . . . . . . . .
Tumor Incidence in CO Rats Fed 2,4-DNT for
24 Months
Tumor Incidence in Male CD-I Mice Fed 2,4-DNT
for 24 Months ...........
Incidence of Liver Tumors in CDF Rats Fed
tg-DNT for up to 2 Years ...-...-
Comparative Mutagenicity of 2,4-DNT and Its
Metabolites
Summary of Candidate Studies for Derivation
of the Drinking Water Equivalent Level (DWEL)
for 2,4-DNT
Summary of Candidate Studies for Derivation of the
Drinking Water Equivalent Level (DWEL) for
2,6-DNT .
Cancer Risk Data for Exposure to 2,4-DNT, 2,6-DNT,
and tg-DNT .
Regulations and Guidelines Applicable to 2,4- and
2,6-DNT
Drinking Water Advisory Values for 2,4- and
2,6-Dinitrotoluene . .........
Page
VI-30
VI-35
VI-37
VI-39
VI-44
VI-45
VI-53
VI-55
VI-57
VI-60
. ' VI-63
VII-10
VII-17
VII-23
VIII-2
XI-2
xi
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EXECUTIVE SUMMARY
, Dinitrotoluene, commonly known as DNT, is a white- to buff-colored solid
at room temperature and exists as a mixture of two or more of its six isomers;
the 2,4- and 2,6-DNT isomers are the most predominant and have been used in
military munitions, dye manufacture, and'the synthesis of toluenediamine (the
organic intermediate used in the production of polyurethane). The toxicity of
2,4-DNT, 2,6-DNT, and technical grade DNT (tg-DNT) is reviewed in this HA.
Where appropriate, data for each pure chemical isomer (-98%) or tg-DNT is
discussed separately in subsections. The technical grade DNT is a mixture
composed of approximately 76.5% 2,4-DNT, 18.8% 2,6-DNT, and 5% other DNT
isomers (2.4% 3,4-DNT,-1.5% 2,3-DNT, 0.7% 2,5-DNT, and 0.4% 3,5-DNT).
The toxicokinetics of primarily the 2,4-DNT Isomer has been studied
extensively in laboratory animals. Data indicate that approximately 70 to 90%
of orally administered [UC]2,4- or [UC]2,6-DNT is eliminated in the urine of
CD (Sprague-Dawley) and Fischer rats, New Zealand white rabbits, beagle dogs,
rhesus monkeys, and A/J mice. Fecal excretion generally accounts for less
than 10%. In contrast, urinary excretion of orally administered 2,4-DNT by
two common strains of mice (CD-I and B6C3F,) accounted for only 8 to 12% of
the dose and fecal excretion accounted for 81 to 84%. Results from one study
indicated that gastrointestinal absorption of 2,6-DNT in the rat 1s somewhat
slower than that of 2,4-DNT. Percent absorption of either Isomer 1s difficult
to estimate since biliary excretion and enterohepatic cycling are significant
biological processes in most animals. Metabolites that are eliminated in the
bile and into the gut are hydrolyzed and reduced by Intestinal microflora.
Many of these compounds, in turn, are reabsorbed from the gut into the
systemic circulation and then oxidized In the liver. Biliary excretion of
these metabolites Into the gut results in additional reduction by intestinal
bacteria. Male rats usually excrete less of the administered dose of 2,4-DNT
in the urine and more in the feces than females, a difference most likely due
to higher biliary excretion in males. Similar sex-related differences in the
absorption or elimination of 2,6-DNT have not been observed in animals or
humans. Elimination of either compound via the lungs 1s minimal (<0.35%).
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I
Workers exposed to tg-DNT absorb the munitions chemicals via the inhalation
and dermal routes, but the extent of absorption has not been determined
conclusively.
The data on distribution of 2,4- and 2,6-DNT suggest that accumulation of
these compounds in the body following a single exposure 1s, at most, minimal.
Repeated oral exposure in CO rats Indicates preferential uptake of 2,4-ONT
and/or Us metabolites by the liver, kidneys, brain, lungs, and skeletal
muscle, although total retention in the body is low. Limited data suggest
that placental transfer of 2,4-DNT occurs in rats.
Significant portions of 2,4-DNT administered orally or intraperitoneally
to most animals (A/J mice, CD and Fischer 344 rats, New Zealand rabbits,
beagle dogs, and rhesus monkeys), and in humans exposed occupationally by .
inhalation and dermal routes, are converted to 2,4-dinitrobenzyl alcohol
(2,4-DNBalc) and/or its glueuronide (i.e., 2,4-DNBalcG). Other primary
metabolites excreted by these animals include 2-am1no-4-n1trobenzyl alcohol
(2A4NBa1c) and 4-am1no-2-n1trobenzy1 alcohol (4A2NBalc); humans excrete only
the former in the urine in large quantities. For Fischer rats and humans,
another major product of 2,4-ONT metabolism is 2,4-dinitrobenzoic acid (2,4-
DNBadd). A significant portion (4 to 9%) of a single oral dose of 2,4-ONT is
reduced in vivo to 2.4-diaminotoluene (2,4-DAT) by rats, rabbits, dogs, and
monkeys, but none is found in the urine of occupationally exposed humans. In
contrast, metabolism of 2,4-ONT by CD-I mice and Wistar rats 1s not extensive,
and only small amounts of an oral dose of 2,4-ONT given to these animals are
oxidized to one of the benzyl alcohol metabolites; reduction of 2,4-ONT to
2,4-OAT is negligible in mice. Two mutagenic metabolites of 2,4-DNT (I.e., 2-
nitroso-4-nitrotoluene and 2-am1no-4-n1troto1uerie) have been recovered
IQ vitro fro* cultures containing cecal or ileal material from male Swiss-
Webster mice, male and female Fischer 344 rats, and male humans. It has been
.suggested that these Intermediates may bind covalently to hepatic
macromolecules. 1
Sex-related differences In the metabolism of 2,4-DNT have been reported
only in Fischer rats and humans. Exposure in rats was by the oral route,
xiv
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whereas human exposure was via the inhalation and dermal routes. For both
species, females produce up to three times more 2,4-DNBalc and/or 2,4-DNBalcG
than males, and in humans only, men excrete nearly twice as much 2,,4-DNBacid
as females.
The metabolism of 2,6-DNT has not been studied extensively. As with
2,4-DNT, Fischer rats convert the 2,6-DNT isomer to the corresponding
dinitrobenzyl alcohol glucuronide and dinitrobenzoic acid; however, only one
metabolite (2-amino-6-nitrobenzo1c add, 2A6NBacid) results from the in vivo
reduction of 2,6-DNT. Similarly, only three major metabolites of 2,6-DNT
(2,6-DNBalc, 2,6-DNBalcG, and 2,6-DNBacid) have been recovered from the urine
of men and women; women appear to excrete more 2,6-DNBalcG than men. No other
sex-related differences in the metabolism of 2,6-DNT have been reported.
Occupational studies of DNT characterized concurrent exposures to 2,4-DNT
and tg-DNT; 2,6-DNT has not been studied epidemiologically. In humans, the
toxic effects of DNT are on the heart, the circulatory system, and the central
nervous system. Chronic DNT exposure, primarily via the inhalation route
(although percutaneous absorption and ingestion are considered to be secondary
and tertiary routes of exposure), has been characterized in munitions workers
by nausea, vertigo, methemoglobinemia, cyanosis, pain or paresthesia in
extremities, trenwfrs, paralysis, chest pain, and unconsciousness. A
retrospective epidemiology study suggested that following a latency period of
15 years, workers exposed to 2,4-DNT and tg-DNT exhibited excess mortality
from ischemic heart disease and residual diseases of the circulatory system.
However, most of the workers had taken other jobs during the 15-year latency
period and were not continually exposed to either 2,4-DNT or tg-ONT. A more
recent study failed to demonstrate excess mortality from cardiovascular
disease in DNT-exposed workers and suggested a possible increase in mortality
from liver and biliary tract cancer. Limited evidence suggests that DNT does
not result in adverse effects on human reproductive performance.
Acute oral toxicity studies Indicate that rats are more susceptible to
2,4-DNT than mice. The LD50 values ranged from 1,340 to 1,954 mg/kg in mice
and from 270 to 650 mg/kg in rats. Both species exhibited ataxia and
xv
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cyanosis. When rats were administered doses of 2,4-DNT in the diet ranging
from 45 to 143 mg/kg/day for 14 days, body weight gains and food consumption
were decreased and testicular lesions were found in a dose-related manner.
When mice (dose range, 47 to 468 mg/kg/day), rats (dose range, 34 to
266 mg/kg/day), and dogs (dose range, 1 to 25 mg/kg/day) were administered
2,4-DNT in the diet for 4 weeks, dogs were found to be most sensitive,
exhibiting incoordlnation and paralysis at doses of 25 mg/kg/day; central
nervous system (CNS) effects were not observed in rodents. Similar CNS
effects were seen when dogs were orally administered 20 mg 2,6-ONT/kg/day.
Methemoglobinemia, reticulocytosis, and Heinz body formation resulted from
oral administration of 37.5 mg tg-ONT/kg/day to F344 rats for 4 weeks.
Subchronic toxicity was assessed in mice and rats administered 2,4-DNT
via the diet and in dogs administered 2,4-DNT via capsules for 13 weeks (same
doses tested as reported previously for 4-week studies). In mice of both
sexes, anemia and reticulocytosis were observed at dose levels of 413 (males)
and 468 (females) mg/kg/day. In rats, mortality occurred prior to study
termination in all females fed 145 mg/kg/day and 75% of males fed
266 mg/kg/day. Anemia and reticulocytosis were observed at dose levels as low
as 93 (males) and 108 (females) mg/kg/day; splenic hemosiderosis and depressed
spermatogenesis were found In these animals as well as those dosed at 266
(males) and 145 (females) mg/kg/day. In .dogs, neuromuscular incoordlnation
and paralysis, methemoglobinemla, aspermatogenesis, hemosiderosis of the
spleen and liver, cloudy swelling of the kidneys, and lesions of the brain
were observed in males and females at a dose level of 25 mg 2,4-DNT/kg/day.
Similar effects were seen when mice (dose range, 11 to 299 mg/kg/day), rats
(dose range, 7 to 155 mg/kg/day), and dogs (dose range, 4 to 100 mg/kg/day)
were orally administered 2,6-DNT for 13 weeks. All species exhibited
methemoglobinemla, anemia, bile duct hyperplasia sometimes accompanied by
hepatic degeneration, and depressed spermatogenesis. Incoordlnation, rigid "
paralysis, and renal degeneration occurred in dogs at a dose level of 20 «g
2,6-DNT/kg/day.
xv i
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In a lifetime feeding study in male and female CD (Sprague-Oawley) rats
fed 2,4-DNT In the diet at a level giving a daily intake of 34 and
45 mg/kg/day, respectively, lifespan was shortened, and the incidence of
hepatocellular carcinomas was increased (6/30 and 19/35 high-dose males and
females, respectively, as compared with 1/25 and 0/23 concurrent controls).
Seminiferous tubules atrophied resulting*1n almost complete cessation of
spermatogenesls, and excessive hemos1der1n accumulated in the spleen. An
increased Incidence of benign mammary gland tumors (33/35 as compared with
10/23 1n controls) was exhibited in females. Anemia (partially compensated)
and testicular atrophy were evident at 3.9 mg/kg/day, and hepatocellular
alterations were observed at 0.57 mg/kg/day, the lowest dose tested. In a
study with CD-I mice, all the animals fed 898 mg/kg/day died by month 18
(males) or month 21 (females). Effects at 14 mg/kg/day, the lowest dose
tested, Included testicular atrophy, decreased body weight in males, and
hemosiderosls of many organs, primarily the liver and spleen. The Incidence
of malignant renal tumors was elevated in males fed 95 mg/kg/day (15/17 as (
compared with 0/20 concurrent controls). In a 2-year oral toxldty study 1n
dogs, 2,4-DNT administered dally in capsules caused neurotoxicity
(incoordlnation and paralysis, often leading to death) in all dogs at
10 mg/kg/day, and anemia (partially compensated) and biliary tract hyperplasia
at 1.5 mg/kg/day. No adverse effects were observed at 0.2 mg/kg/day.
Hale and female CDF (F344) rats fed tg-DNT in the diet at a dally intake
of 35 mg/kg/day were sacrificed in extremis after 55 weeks. At 14 mg/kg/day,
96% of the males and 59% of the females developed hepatocellular carcinomas.
After 78 weeks, decreased body weight, Increased liver weight, hepatotoxicity,
increased incidence of hepatocellular carcinomas in males (19/20, as compared
to 0/20 in concurrent controls), renal toxicity (chronic Interstitial
nephritis), and parathyroid hyperplasia were reported at 3.5 mg/kg/day, the
lowest dose tested.
In a 1-year study, 47% of male CDF (F344) rats given tg-DNT at dose
levels of 35 mg/kg/day (the only dose tested) developed hepatocellular
carcinomas (0/20 control, 9/19 treated). Under similar conditions, 2,6-DNT
induced hepatocellular tumors in 100% of the high-dose (14 mg/kg/day) and 85%
xvii
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of the low-dose (7 mg/kg/day) males; no lesions were found in the control
group. Rats exposed to 2,4-DNT (35 mg/kg/day) did not develop liver tumors.
Although limited in duration and in the number of animals tested, the data may
indicate that the 2,6-DNT isomer caused much of the carcinogenic activity in
the previous tg-DNT bioassays.
The 2,4- and 2,6-DNT isomers are weak mutagens in Salmonella test
systems. However, metabolites of 2,4-DNT (in particular the 2,4-nitrobenzyl
alcohol and the 2-amino- and 2-n1troso-4-n1trotoluenes) are mutagenic without
metabolic activation. The dlnltrotoluenes are nongenotoxic in mammalian cells
In vitro, in the dominant lethal test in mice and rats, and in Drosophila
systems. Technical grade ONT gave negative responses for unscheduled DNA
synthesis (DOS) except when an in vivo/in vitro testing system was used. It
has been concluded that biliary excretion, metabolism by gut mlcroflora, and
resorption from the Intestine are prerequisites for genotoxic activity.
Metabolites of 2,4-DNT can bind to liver DNA; 2,4-DNT appears to act as a
promoter of hepatocarcinogenesis, inducing gamma glutamyl transferase positive
foci in the livers of rats initiated with dimethylnltrosamine. On the other
hand, 2,6-DNT had both Initiating and promoting activity in the GGT* focus
assay.
The 2,4-dinitrotoluene Isomer has been shown to cause severe reproductive
effects in both sexes of rats, mice, and dogs. Oral exposure to 2,4-DNT
results in testicular atrophy and degeneration, reductions in spermatogenesis,
cessation of follicular function/and reduction in the number of corpora
lutea. Consequently, fertility Is reduced in both sexes. 2,4-DNT has also
been shown to cause reduced viability as well as decreases In the body weight
of offspring at birth and weaning. No data on the reproductive effects of
2,6-DNT or tg-DNT were found in the available literature.
Limited data suggest that 2,4-DNT is not teratogenic in mice following
ingestion. Technical grade-DMT was not teratogenic to rats administered oral
doses, although embryotoxicity was observed at maternally toxic levels. In
addition, the administration of tg-DNT to pregnant rats also resulted in
changes in relative organ weights and hematologic parameters in their fetuses.
, xviii
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No data on the developmental effects of 2,6-DNT were found 1n the available
literature.
Based on decreased body weight gain, decreased food consumption, changes
in serum chemistry levels in male and female Sprague-Oawley rats, and
testicular lesions in male rats administered 2,4-ONT In the diet for 14 days,
the Ten-Day Health Advisory (HA) for 2,4-DNT for a 10-kg child 1s 0.5 mg/L
(500 M9/L)- In the absence of available animal data to determine a One-day
HA, the Ten-day HA 1s considered acceptable (0.5 mg/L or 500 M9/L). Based on
dose-related decreases in body weight gain and food consumption in rats
administered 2,4-ONT in the diet for 13 weeks, the Longer-term HA for exposure
in a 10-kg child 1s 0.3 mg/L (300 M9/L); the Longer-term HA for exposure in
an adult is 1.0 mg/L (1,000 Mg/L). The Drinking Water Equivalent Level
(DUEL) for 2,4-DNT 1s 0.1 mg/L (100 Mg/L), derived from a Reference Dose
(RfD) of 0.002 mg/kg/day, which was based on neurotoxicity, Heinz bodies, and
biliary tract hyperplasia in dogs dosed orally with 2,4-DNT for 2 years.
All HAs for 2,6-DNT are based on a 13-week study with dogs administered
2,6-DNT orally. The critical effects were neurotoxicity, Heinz bodies, bile
duct hyperplasia, liver and kidney histopathology, and death. The Longer-term
HA for exposure in a 10-kg child is 0.4 mg/L. In the absence of available
animal data to determine One- and Ten-day HAs, the Longer-term HA for a 10-kg
child is considered acceptable (0.4 mg/L or 400 M9/L). The Longer-term HA
for exposure in an adult 1s 1.0 mg/L (1,000 ng/l). The Drinking Water
Equivalent Level (DWEL) for 2,6-DNT is 0.04 mg/L (40 M9/L), derived from a
Reference Dose (RfD) of 0.001 mg/kg/day.
' . ' [ -
DNT 1s classified 62: Probable Human Carcinogen; thus a Lifetime HA is
not recommended. The estimated excess cancer risk associated with lifetime
exposure to drinking water containing 2,4-DNT at the level of the DWEL
(100 M9/L) 1s 2 x 10"3. The estimated excess cancer risk associated with
lifetime exposure to drinking water containing 2,6-DNT at the level of the
DWEL (40 M9/L) 1s 1 x 10~3. The cancer risk assessment 1s for the
2,4-/2,6rONT mixture. The data for 2,6-ONT are limited, and 2,6-DNT is
usually found in the presence of 2,4-DNT with 2,4-DNT being the more
xix
-------
predominant component of the mixture by volume. Therefore, the cancer risk
assessment for the 2,4-/2,6-DNT mixture is used for the cancer risk assessment
for both 2,4- and 2,6-DNT. Derived by the linearized multistage extrapolation
method, the oral slope factor (q^) for the mixture is 6.8 x 10"1 (mg/kg/day)'1
and the drinking-water unit risk is 2 x 10"5 jug/L- At drinking water
concentrations of 5 M9/U 0.5 M9/U and 0.05 /ig DNT/l, excess cancer risks
are estimated to be 10"4, 10"5 and 10"6, respectively. For comparison,
drinking water concentrations associated with a 10"6 cancer risk are 8 x 10'8
Mg/L, 5 x 10"2 M9/L, 2 x 101 M9/L, 2 x 10"1 M9/U and 2 x 10"5 ng/l for the -
one-hit, multihit, probit, logit, and Wei bull models, respectively.
xx
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I. INTRODUCTION
The Health Advisory (HA) Program, sponsored by the Office of Water (OW),
provides information on the health effects, analytical methodology, and
treatment technology that would be useful in dealing with the contamination of
drinking water. Health Advisories describe nonregulatory concentrations of
drinking 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 assist Federal,
State, and local officials responsible for protecting public health when
emergency spills or contamination situations occur. They are not to be
construed as legally enforceable Federal standards. Health Advisories are
subject to change as new information becomes available.
Health Advisories are developed for One-day, Ten-day, Longer-term
(approximately 7 years, or 10% of an individual's lifetime), and Lifetime
exposures based on data describing noncarcinogenlc endpoints of toxicity. For
those substances that are known or probable human carcinogens, according to
the Agency classification scheme (Group A or B), Lifetime Health Advisories
are not recommended. The chemical concentration values for Group A or B
carcinogens are correlated with carcinogenic risk estimates by employing a
cancer potency (unit risk) value together with assumptions for lifetime
exposure and the consumption of drinking water. The cancer unit risk is
usually derived from the upper 95% confidence limits of the linear mult1sUg«
model. This provides a low-dose estimate of cancer risk to humans that is
considered unlikely to pose a carcinogenic risk in excess of the stated
values. Excess cancer risk estimates may also be calculated using the ont-
hit, Weibull, logit, and probit models. There is no current understanding of
the biological mechanisms involved in cancer to suggest that any one of th«st
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 magnitude.
1-1
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II. GENERAL INFORMATION
Dinitrotoluene (DNT) appears as a white- to buff-colored solid at room
temperature and exists as a mixture of two or more of its six isomers; the
2,4- and 2,6-ONT isomers are the most significant by volume (Beard and Noe,
1981; Small and Rosenblatt, 1974). The isomers 2,4-DNT (CAS No. 121-14-2) and
2,6-DNT (CAS No. 606-20-2) are combustible, nitroaromatic compounds that
compose technical grade ONT (tg-ONT) (ATSDR, 1989; NIOSH, 1985). When heated,
DNT forms an oily liquid that turns yellow when exposed to sunlight. The
general physical and chemical properties of 2,4- and 2,6-ONT are presented in
Table II-l.
Oinitrotoluene is used in the manufacture of dyes, in munitions (as a
smokeless propel1 ant powder), and as a gelatinizing and plasticizing agent in
both commercial and military explosive compositions, However, its major use
(99%) is in the synthesis of toluenediamine, the organic intermediate used in
the production of polyurethane (ATSDR, 1989; Beard and Noe, 1981; NIOSH/OSHA,
1985; Santodonato et al., 1985; Small and Rosenblatt, 1974).
Both 2,4- and 2,6-DNT are produced through the dinitration of toluene
with nitric acid in the presence of concentrated sulfuric acid. This reaction
produces a mixture that consists of approximately 75% 2,4-DNT isomer, 20%
2,6-DNT isomer, and small quantities of other DNT isomers. If the single
2,4-DNT isomer is required, the nitration is stopped at the mono- stage, and
pure p-nitrotoluene is obtained by crystallization. Subsequent nitration of
the p-nitrotoluene yields only 2,4-DNT. Small concentrations of DNT isomers
also occur as byproducts in the production of trinitrotoluene (TNT) (ATSDR,
1989; Santodonato et al., 1985).
The military requirement for DNT specifies a minimum melting point of
65.5'C, which corresponds to a 2,4-DNT purity of 92%. The current approach is
to purchase commercial DNT and to purify it by a process known as sweating.
The commercial material is about 75% 2,4-DNT isomer, 20% 2,6-DNT isomer, and
5% other isomers. The mixture is melted and subjected to a controlled
cooling-heating process. In the cooling step, DNT containing mostly the
Il-i
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Table II-l. Physical and Chemical Properties of 2,4- and 2,6-DNT
Property
2,4-DNT
2,6-DNT
Reference
CAS No.
Synonyms
Chemical formula
Chemical structure
121-14-2
2,4-01nitrotoluol;
1-methyl-2,4-
d1nitrobenzene
CH,
Molecular weight
Melting point
Boiling point
Density
Vapor pressure,
mmHg (20'C)
Solubility
Water, mg/L
Organic
solvents
Partition
coefficients
Log octanol/
water (K^)
Log organic
carbon-soil I
Conversion
factors
ppm (v/y)
to mg/m in air
(20*C)
NO,
182.14 .
70.5'C
Decomposes (300*C)
1.521 015*C
0.0051
270 (22'C)
Soluble in alcohol,
ether, acetone,
benzene
2.0
1.65-364.0
606-20-2
2,6-Dinitrotoluol;
1-methyl-2,6-
dinitrobenzene
182.14
66'C
Decomposes (260*C)
i.28 enrc
0.018
180 (20*C)
Soluble in alcohol
2.3
1.96-77.6
1 ppm » 7.5 mg/m3 1 ppm a 7.5 mg/m3
mg/m3 to ppm 1 mg/m3 « 0.13 ppm
(v/v)
1 mg/n a 0.13 ppm
NIOSH/OSHA (1985)
NIOSH/OSHA (1985)
ATSDR (1989)
NIOSH/OSHA (1985)
NIOSH/OSHA (1985)
NIOSH/OSHA (1985)
Sax (1975); Small
and Rosenblatt
(1974)
ATSDR (1989)
ATSDR (1989);
Verschueren (1977)
ATSDR (1989);
Hawley (1981)
ATSOR (1989)
ATSDR (1989)
Spanggord et al. (1985)
Burrows (1989)
Mabey et al. (1982)
ATSDR (1989)
ATSDR (1939)
II-2
-------
2,4-ONT isomer crystallizes, while the liquid impurity-rich fraction is
drained off (or sweated) (Small and Rosenblatt, 1974). One procurement
specification requires that the DNT used in the production of military
munitions be composed of at least 98.5% of the 2,4-isomer (Department of the
Army, 1990). The composition of the supplied product is approximately 99.5%
2,4-DNT and 0.5% 2,6-DNT.
As of 1986, a small number of domestic chemical companies were
manufacturing DNT; these are listed in Table II-2. Du Pont and Air Products
and Chemicals are the major U.S. producers of DNT. In 1982, U.S. production
of tg-DNT (2,4- and 2,6-DNT isomer mixture) was approximately 720 million
pounds. No data describing import or export activities for 2,4- or 2,6-DNT
were found in the available literature (ATSDR, 1989; Santodonato et al.,
1985). , ,
II-3
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Table 11-2. U.S. Manufacturers of 2,4- and 2,6-DNT
Isomer
Manufacturer
Location
2,4-DNT
2,6-DNT
Air Products and Chemicals, Inc.
E.I. Du Pont de Nemours
ICI Americas (Rubicon)
Mobay Chemicals
Allied Chemical Corporation
Uniroyal, Inc.
Allied Chemical Corporation
Air Products and Chemicals, Inc.
E.I. Du Pont de Nemours
Pasadena, TX
Deepwater, NJ
Geismar, LA
Cedar Bayou, TX
New Martinsvllie, WV
Moundsville, WV
Joliet, IL
Moundsville, WV
Pasadena, TX
Deepwater, NJ
Source: Santodonato et al. (1985); U.S. EPA (1986).
II-4
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III. SOURCES OF EXPOSURE
According to McGee et al . (1942), the body may be exposed to DNT via four
routes: (1) Inhalation of DNT vapors; (2) Inhalation of DNT dust particles;
(3) ingestion of DNT-contaminated food; and (4) absorption through the skin.
Exposure to DNT may occur in the environment, or more likely, in occupational
settings.
A. ENVIRONMENTAL EXPOSURE
Information concerning environmental levels of DNT Is limited.
Significant amounts of DNT-containing wastewaters arise from the preparation
and production of smokeless powder and trinitrotoluene (TNT) at U.S. Army
ammunition plants (AAPs) (Small and Rosenblatt, 1974). The major consistent
discharges of DNT occur during dry solvent and water recovery processes.
Total estimated rates of DNT discharged Into local waters by AAPs In Virginia
and Tennessee were 40 and 550 pounds per day, respectively.
STORET (Storage and Retrieval computerized database maintained by EPA)
data show that five raw water observations of 2,4-DNT, each at 5 /xg/L, and
seven raw water observations of 2,6-DNT, from 5 to 10 ng/L with a mean of
6.4 M9/L, were made. The Chemical Manufacturers Association (CMA, 1990), on
behalf of most U.S. manufacturers of DNT (Table II-2), reported median
concentrations of 2,4- and 2,6-DNT in surface and groundwater in the vicinity
of plant production sites. The 2,4-DNT isomer ranged from 360 ng/L to
20,000 ng/L (median 3,500 M9/L) 1n groundwater and up to 32 M9/L (median
9.1 M9/L) in surface water. The 2,6-DNT isomer ranged from 7 ng/L to
*
20,000 ng/l (median 3,700 ng/L) in groundwater and up to 49 pg/L (median
14.9 Mg/L) 1n surface water.
During a study of 17 military installations, DNT was found to leach to
groundwater at six sites where open burning directly on the ground was used as
a method for munitions disposal (Murnyak, 1991). The concentrations detected
in monitoring wells near five of the sites were 1 to 265 jig/L 'fo.r 2,4-DNT and
III-l
-------
2 M9/L for 2,6-DNT. Concentrations were higher at the sixth site, ranging
from 32 to 1,788 ng/L for 2,4-DNT and from 11 to 651 Mg/L for 2,6-DNT.
In a review of analytical methods for the determination of groundwater
contamination, Gar-man et al. (1987) gave Practical Quantification Limits
(PQLs) for a number of priority pollutants. These limits are estimates of the
lowest concentrations that may be expected to be routinely measured. The PQLs
for 2,4- and 2,6-DNT ranged from 0.1 to 10 nq/L depending upon the analytical
method used.
Hashimoto et al. (1979) reported the occurrence of 2,4- and 2,6-DNT in
the seawater of Dokai Bay, Japan. Concentrations of the isomers ranged from
0.890 to 206 M9/L for the 2,4-DNT isomer and from 0.072 to 14.8 M9/L for the
2,6-DNT isomer. This area was found to be heavily polluted by Industrial
waste. Hashimoto et al . (1982) estimated that the main source of waste
discharged an average of 76 kg of DNT (mixed Isomers) per day with a maximum
of 150 kg discharged per day.
The 2,4-DNT Isomer was detected in the soil of 2.2X of 800 to 900
hazardous waste sites at a geometric mean concentration of 1 mg/kg. 2,6-DNT
was detected at 1.3% of the sites at a mean concentration of 0.140 mg/kg
(140 /ig/kg) (ATSDB, 1989).
Oinitrotoluene volatilizes from water at a relatively insignificant
level. Environmental exposure to DNT in the air may occur if contaminated
surface soils are eroded and re-entrained. DNT may also be released or
transported in the air as dusts, aerosols, or other suspended solids.
Matsushita and lida (1986) collected and analyzed airborne samples (from
unspecified sources) for the presence of nitroaromatics. Sampling was
performed using a quartz filter, and analyses Involved gas chromatography and
flame thermolonlc detection (GC/FTO). Detected concentrations of 2,4- and
2,6-DNT were 0.024 and 0.006,.ng/m3, respectively.
Toxic Release Inventory (a computerized database maintained by U.S. EPA)
reports for the year 1989 show that 169,291 Ibs of 2,4-DNT were released: 52%
III-2
-------
to air, 41% to underground injection, 7% (12,657 Ibs) through surface water,
and 0.2% to landfills. In the same year, 102,977 Ibs of 2,6-ONT were
released: SIX to air, 18% to underground injection, and 1% (1,083 Ibs)
through surface water discharge.
B. OCCUPATIONAL EXPOSURE
It is estimated that approximately 500 workers are potentially exposed to
2,4- and 2,6-ONT in the production of munitions and explosives. Highest
exposures occur in DNT unloading procedures, as a result of fugitive emissions
from leaky pumps and loose pipe connections, during certain maintenance
operations, and during cleanup operations (Santodonato et al., 1985).
Hamill et al. (1982) conducted occupational sampling at two plants of the
Olin Corporation, a toluene diamine/DNT production facility. Air
concentrations (not reported) at the plants (Lake Charles, LA, and Bradenburg-,
KY) were reported to be well within acceptable OSHA, ACGIH, or Olin internal
standard levels.
Levine et al. (1985a) evaluated worker exposure at a tg-ONT manufacturing
plant. Sampling included breathing zone air, skin, environmental surfaces,
and urine to determine absorption. Concentrations obtained from personal air
sampling ranged from 0.1 to 4.4 mg/m3 for pure 2,4-DNT and from 0.6 to
5.9 mg/m3 for tg-ONT. The lowest levels were found in stripper operators and
maintenance mechanics, and the highest levels were found in "operators" (type
of operator not specified) and loaders. Skin testing revealed contamination
ranging from <2 to 179.5 g. The lowest concentration was found on the hands
of an operator performing ONT analysis, and the highest concentration was
found on the hands and face of an operator following sample collection and
analysis. Environmental surface samples ranged from <2 (door handles,
desktop, knobs, and switches) to 433.2 g (handrails and valve handles).
Results of the wipe survey and urinalyses showed that frequent skin
contamination with small quantities could add up to considerable exposure.
III-3
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Two biological monitoring studies among workers exposed to tg-DNT in an
explosives factory were carried out by Woollen et al. (1985). Routine
personal sampling revealed atmospheric levels ranging from undetectable to
0.1 mg/m3. Static samples positioned near potentially dusty areas revealed
atmospheric concentrations ranging from 0.02 to 2.68 mg/m3 (mean, 0.40 mg/m3).
Urinalyses were also performed. Concentrations of DNT metabolites as well as
unmetabolized DNT in the urine were much higher than routine environmental
sampling concentrations.
Difficulties in detecting metabolites and frequent occurrence of samples
with values near the detection limits hampered interpretation of the actual
exposure and disposition of DNT in both the Levine et al. (1985a) and Woollen
et al. (1985) studies. The results indicated that dermal contact and
inadvertent ingestion were contributing routes of exposure for workers.
III-4
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IV. ENVIRONMENTAL FATE
Spanggord et al. (1980a) conducted a literature review to identify and
estimate the magnitude of the transport and transformation processes believed
to be important in the environmental fate of 11 munitions wastewater
constituents (including 2,4- and 2,6-DNT.) in the aquatic environment.
Possible fate routes of ONT are biodegradation, photolysis, volatilization,
sorption on soils and sediments, and hydrolysis.
A. BIODEGRADATION
Oinitrotoluene degradation occurs under aerobic and anaerobic conditions.
Biotransformation occurs mainly through the reduction of the nitro group
(Spanggord et al., 1981). Local (Searsville Lake in Uoodside, CA, and Coyote
Creek in San Jose, CA) eutrophic waters did not contain microorganisms that
degrade these compounds, but they did contain microorganisms that transformed
DNT when other metabolizable organic compounds were present for their growth
(co-metabolism). However, Spanggord et al. (1980b) found that natural surface
water microorganisms could use 2,4-DNT as a sole carbon source. After a 2- to
3-day lag period, >90% of the added 2,4-DNT (10 mg/L) was transformed in
aerated water in 6 days of incubation. Bausum et al. (1990) found that both
2,4- and 2,6-DNT served as sole carbon sources for microorganisms in water
samples taken downstream from wastewater outflows from the Radford (VA) Amy
Ammunition Plant. Up to 60% of substrate carbon appeared as CO,. Compared to
the 2,6-isomer, the 2,4-isomer was degraded at a higher rate, had a shorttr
lag time, and had a larger population of degrading organisms. In contrast,
degradation was not observed In samples taken from Maryland surface freshwater
sources. Using respiratory techniques, Chambers et al. (1963, as cited in
Spanggord et al., 1980a) reported evidence of 2,4-DNT (100 mg/L) degradation
by phenol-adapted bacteria.
Spanggord et al. (1985V-estimated the environmental persistence of
2,4-DNT in Vfaconda Bay, TN. They determined that photolysis
(ks - 0.017 days"1), biotransformation (k, - 0.113 days"'), and volatilization
(kv => 0.004 hours"') control the loss and movement of 2,4-DNT in Waconda Bay.
IV-1
-------
The estimated half-life was 5.2 days. Based on the data, persistence is
water-body dependent and the length of time any particular nitroaromatic
compound will reside in the environment will ultimately depend on the
compound's unique interaction with the natural organics and biota in its
surroundings. Since microorganisms readily metabolize ONT to CO, as the final
product, it is not expected to persist in the environment.
Tabak et al. (1981) determined the acclimation periods and extent of
microbial degradation of 2,4- and 2,6-DNT via the static-screening-flask test.
A significant bio-oxidative activity with gradual adaptation and induced
enzyme formation was noted initially. This was followed, however, by a
deadaptive process from the loss of the metabolically efficient microbial
population and the gradual buildup of toxicity of either the parent substrate
or the metabolic products to the microbiota.
Davis et al. (1981) found that 2,4-DNT (50 mg/L) degraded rapidly in
2 days in a batch culture containing glucose and inoculated with an industrial
seed containing four bacterial genera (Acinobacter. Alcalioenes.
Flavobacterium. and Pseudomonas) and one yeast (Rhodotorulal. Municipal seed
(activated sludge organisms), however, was inhibited by concentrations as low
as 10 mg 2,4-DNT/L. Under the same conditions, 2,6-DNT inhibited municipal
seed at >50 mg/L and appeared to react differently in wastewater treatment
operations.
Parrish (1977) screened 190 fungi, in shake cultures of basal medium with
/
glucose, for the ability to transform 2,4-DNT. Only five organisms were found
to effectively transform at an initial concentration of 100 mg 2,4-DNT/L.
McCormick et al. (1978, as cited in Spanggord et al., 1980a) identified the
products of transformation by a fungus Hicrosoorum species grown in DNT,
glucose, and basal medium as 2-aminb-4-nitrotoluene, 4-amino-2-nitrotoluene,
2,2'-dinitro-4,4'-azoxytoluene, 4,4'-dinitro-2,2'-azoxytoluene, and 4-
acetamido-2-nitrotoluene. --
A study to identify and quantify the routes.taken by 2,6-DNT in a model
waste stabilization pond (12-hour detention time) was conducted by Davis
IV-2
-------
et al. (1983). The percentages of 2,6-DNT lost by degradation,
volatilization, sedimentation, water column residuals, and effluent were 92.2,
0.3, 3.6, 1.2, and 2.7, respectively, while the half-life was 8.3 days and the
bioconcentration factor was 5225.
Other sources report transformation'occurring only under anaerobic
conditions. Liu et al. (1984) conducted a study to identify the intermediates
of the microbial transformation of 2,4-DNT (5-25 mg/L) by a mixed bacterial
culture derived from activated sludge. The biotransformation took place only
under anaerobic conditions with an exogenous carbon source and appeared to be
energy dependent. Intermediates identified were the aminonitrotoluenes, and
2-nitroso-4-nitrotoluene and 4-nitroso-2-nitrotoluene. The two nitroso
\
compounds were very unstable and could be found only at early stages of
biotransformation.
Hallas and Alexander (1983) studied the microbial transformation of
2,6-DNT in sewage effluent. Under anaerobiosis, 2,6-DNT disappeared slowly,
but no loss was evident in aerated sewage. The transformation product
detected via gas chromatography-mass spectrometry was aminotoluene.
B. PHOTOLYSIS
Because nitroaromatic compounds absorb sunlight strongly in the
ultraviolet and blue spectral region, they are generally susceptible to
photochemical transformation in aquatic systems. Photolysis of DNT isomers
was rapid in natural waters with the 2,6-lsomer photolyzing more rapidly than
the 2,4-isbmer. Zepp et al. (1984) calculated a half-life of 17 minutes for
2,6-DNT in river water exposed to sunlight; the photoreactlon was determined
to be indirect. However, data suggest that direct photolysis is a major
environmental fate of 2,4 DNT. Spanggord et al. (1978, as cited in Spanggord
et al., 1980a) reported that 50% of the 2,4-ONT added (amount not specified)
was lost after exposure to sunlight for about 5»days. A control solution
\
maintained in the shade showed that the same amount of material was lost in
IV-3
-------
about 11 days. Spanggord et al. (1980b) studied sunlight photolysis of 2,4-
DNT in distilled and natural waters. Half-lives were 43, 2.7, 9.6, and 3.7
hours in distilled, river, bay, and pond water, respectively.
Burlinson and Glover (1977, as cited in Spanggord et al., 1980a) reported
that 2,4-DNT photolyzes more rapidly at a higher pH, while Wetter-mark et al.
(1965, as cited in Spanggord et al., 1980a) reported that the first-order rate
constant for decay was steady from pH 8.4 to pH 13 (1.0 sec"); below pH 5,
the decay rate increased with lowered pH. Permanent visible absorptions
appeared in the range 380 to 600 nm with the extent of coloration depending on
pH, buffer concentrations, and the presence of oxygen.
Simmons and Zepp (1986) studied the influence of various humic substances
on the photoreactions of 19 nitroaromatic substances including 2,4- and
2,6-ONT. The results observed indicate that dissolved humic substances in
natural waters enhance the sunlight-induced photodegradation rates compared
with rates observed in distilled water and were, by far,.most pronounced in
six compounds including 2,4- and 2,6-ONT. Additional experiments with 2,6-ONT
to determine the effects of varying concentrations of humic substances on the
sunlight photolysis rate constant showed a linear relationship between the
photolysis rate constant and the total organic carbon concentration.
C. VOLATILIZATION
An estimated value of Henry's Law constant (He, 3.4 torr M*') suggests
that the volatilization rate of 2,4-ONT is limited by gas-phase mass transport
resistance. The estimated environmental half-life (using laboratory half-life
information) is 410 days. Consequently/volatilization would not be an
important environmental fate owing to the rapid biodegradation and photolysis
of the 2,4- and 2,6- isomers (Spanggord et al., 1980a).
i
0. SORPTION ON SOILS AND SEDIMENTS
Spanggord et a.1. (1985) measured the organic carbon soil ()<<*)> octanol-
water (log K,,.), and microorganism (K,) partition coefficients for 2,4-DNT and
IV-4
-------
found them to be 364, 2.0, and 64, respectively. Since the partitioning of
organics to the sediment from the aqueous phase does not become a major loss
until the <„,. values exceed 104, the relatively low K^ values for 2,4-DNT
indicate that this compound would have only a slight tendency to sorb to
sediments, suspended solids, and biota. In general, nitroaromatics in soil
are reduced to ami no compounds and covalently bound to soil organic matter
(ATSDR, 1989). It is concluded that adsorption on soils and sediments is not
a significant environmental fate.
E. HYDROLYSIS ,
No hydrolysis is expected under environmental conditions (Spanggord
et al., 1980a). •
/
F. OTHER FATE ROUTES ,
Other fate routes to be considered are transport, partitioning, and plant
uptake. Since the water solubilities of ONTs are moderate and
octanol-water partition coefficients are low, there 1s potential for transport
of DNT by surface or groundwater (ATSDR, 1989).
Sediment-water partitioning coefficients calculated for 2,4- and 2,6-DNT
were 45 and 92, respectively. Depending on the nature of the sediment load,
the total concentration of DNT carried in the soil and sediment water column
could be high. However, no data are available to give support to this theory
(ATSDR, 1989).
Limited information 1s available on the direct measurement of plant
uptake of DNT. The structural analogy with 1,3-dinitrobenzene and
paranitrobenzene suggests that 2,4- and 2,6-DNT would be readily taken up by
plants (ATSDR, 1989). This suggestion is consistent with findings by several
investigators who have shown that plants grown in media containing
nitrotoluenes have a tendency to take up and translocate the chemical (Cataldo
et al., 1989; Folsom et al., 1988, 1985; Pennington, 1988; Palazzo and
Leggett, 1986a,b; Pallazzo et al., 1984).
IV-5
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V. TOXICOKINETICS
A. ABSORPTION AND EXCRETION
/
Absorption of DNT and excretion from the body occur rapidly and are
usually complete within 24 to 72 hours postdosing. The urine is the primary
route of elimination for both DNT isomers in most animals, including rodents,
rabbits, dogs, and monkeys; in CD-I and B6C3F1 mice and VHstar rats, however,
the major route of elimination of 2,4-DNT is the feces. Percent absorption of
the DNT isomers is difficult to estimate since biliary excretion is
significant in most animals; males excrete more of the 2,4-DNT isomer and its
metabolites in the bile and less in the urine than females. The amount of DNT
reabsorbed also varies among species. Humans absorb both DNT isomers
following dermal or inhalation exposure; the urine provides at least one route
of elimination. Elimination half-lives of about 1 to 5 hours have been
reported for occupationally exposed individuals; one study indicates that
urinary elimination of DNT may be biphasic. The comparative percentages of
excretion and distribution of radioactivity 24 hours following administration
of single oral doses of [UC]2,4-DNT to mice, rats, rabbits, dogs, and monkeys
are presented in Table V-l. Comparable data were not available for 2,6-DNT.
1. Humans
Absorption of DNT following oral exposure in humans has not been
determined. However, occupational studies indicate that technical grade DNT
(tg-DNT) is absorbed by humans after inhalation or dermal exposure and
excreted in the urine. Elimination via the feces or lungs has not been
examined in humans.
Woollen et al. (1985) conducted two studies in which a total of 33
workers in an explosives factory were exposed to tg-DNT (76% 2,4-DNT,
20% 2,6-DNT). Routine atmospheric sampling indicated DNT levels of 0.03 to
0.1 mg/m3 (Threshold Limit Value (TLV) 1.5 mg/m3); static air in dusty areas
of the plant contained 0.02 to 2'.68 mg/m3 (mean 0.40 mg/m3). The authors
suggested that the skin was probably the primary route of exposure for these
V-l
-------
Table V-l. Distribution and Excretion of Radioactivity 24 Hours After Oral Administration of a Single .Dose of [r1ng-UL-"C]2.4-DNT to Various Species'
IV)
Fraction
Urine
Feces
Spleen •
Liver
Kidney
Brain
Lung
Skeletal muscle"
Gastrointestinal tract
plus contents
Whole blood1
Total recovery
CD-I
11. 313. 7*
81.014.3
<0.1
0.2±0.0
<0.1
<0.1
<0.1
O.llO.O
2.111.3
O.llO.O
,94.812.3
•House
B6C3F,
7.211.9'
84.012.9
<0.1
O.llO.O
<0.1
<0.1
<0.1
<0.1
0.610.2
<0.1
92.0±3.2
' X of Administered Dose
A/J Rat
66 75.9+2.6*
2.1 9.113.0
i
0. 310.01
<0.1
<0.l
<0.1
0.310.1
2:811.5
O.llO.O
88.512.6
1
Rabbit
75.2*
3.
<0.
<0.
<0.
<0.
<0.
0.2
10.4
0.1
89.8
Dog
76.3"
8.7
<0.1
1.1
0.2
<0.1
<0.1
1.6
8.6
0.6
97.3
Monkey
81.3" •
4.8
<0.1
0.7
<0.1
<0.1
<0.1
1.2
•i.7
11.3
9:i.O
*CD-1 and B6C3F, nice. rats, rabbits, dogs, and monkeys received 1/10 of the acute LOU (see text for additional details) (Lee et al., 1978); male A/J mice
were given 100 mg 2,4-ONT/kg (Schut et al., 1985). Values are for 24 hours postdosing except those of A/J mice, which Mere measured 8 hours postdosing
'Based on 40X of the body weight.
'Based on 7X of the body weight.
"Mean 1 SE of four female mice.
"Mean 1 SE of five female mice.
'Data not provided.
*Nean 1 SE of three female CD rats.
"Average of two animals (female New Zealand white rabbits, female beagle dogs, and female rhesus monkeys).
SOURCE: Lee et al. (1978); Schut et al. (1985).
-------
workers, and the lungs constituted the secondary route because of low
atmospheric levels of DNT. In the first group of individuals (20 males,
8 females), urine was collected during 2 consecutive workweeks; in the second
group (2 males, 3 females), 24-hour urine samples were collected during a
2-day exposure period and the subsequent 2-day nonworking period. Excretion
of 2,4-dinitrobenzoic acid (2,4-DNBacid),' a metabolite of DNT, was highest at
the end of the work shift and generally was much greater at the end of each
workweek than at the beginning of the week (I.e., mean urinary levels of 3.4
to 41 mg/mL versus <1 mg/mL); urinary concentrations of 2,4-DNBacid were
comparable between the 2 weeks. Blood levels of ONT (2,4- and 2,6- combined)
were low before the workday began (<10 ng/mL), gradually increased during the
exposure period (20 to-90 ng/mL), and peaked at the end of the work shift (70
to 250 ng/mL). These data suggest that DNT is absorbed, is readily cleared
from the body, and does not tend to accumulate. However, the extent of DNT
absorption by humans cannot be determined from this study.
Elimination half-lives for all five DNT metabolites excreted 1n the urine
of three workers exposed to 0.05 to 0.59 mg tg-DNT/m3 were between 0.88 and
2.76 hours (Turner, 1986; Turner et al., 1985). Values for Individual
metabolites were between 0.80 and 4.26 hours. In the study by Woollen et al.
(1985), a half-life of 2 to 5 hours was calculated for the urinary elimination
of 2,4-DNT by workers exposed to 0.02 to 2.68 mg tg-DNT/m3. These authors
suggested that since low but detectable levels of 2,4-DNBacid were found in
the urine 3 days after exposure was terminated, urinary elimination of DNT is
biphasic. •
2. Animals .
a. 2.4-DNT
Lee et al. (1978) reported that approximately 81 to 84% of a single oral
dose of [ring-UL-uC]2,4-DNT (80 mg/kg) given to female CD-I and female B6C3F,
mice was excreted in the feces within 24 hours postdpsing; only 7 to 11% was
recovered from the urine during the same period (Table V-l). In contrast, A/J
mice given single oral doses of 100 mg [3-3H]2,4-DNT/kg eliminated 66% of the
V-3
-------
dose in the urine and 2.1% in the feces within the first 8 hours postdosing
(Schut et al., 1985). Similarly, male A/J mice given a single intraperitpneal
(ip) dose of 1, 10, or 100 mg/kg of the 3H-labeled compound excreted about
52.5, 60.1, and 70% of the administered radioactivity, respectively, in the
urine within 4 hours (Schut et al., 1982, 1985). Fecal levels of
radioactivity were not measured, but the* concentrations of 3H in the large
intestine at the corresponding doses were 10.1, 3.9, and 2.1% at 4 hours.
Elimination of radioactivity via the lungs did not exceed 0.20%. These data
suggest that A/J mice either absorb DNT more readily from the gastrointestinal
tract or preferentially eliminate absorbed DNT in the urine rather than in the
bile (see below).
Following oral administration of [ring-UL-uC]2,4-DNT to male and female
F344 and CO rats, most of the radioactivity was eliminated in the urine
(Rickert, 1984; Rickert et al., 1981; Ellis et al., 1985, 1979; Lee et al.,
1975, 1978). Rats receiving less than 100 mg/kg excreted between 60 and 90%
of the radioactivity in the urine and between 3 and 32% in the feces within 72
hours postdosing. Urinary excretion generally was higher in females (61 to
90%) than in males (55 to 80%), and 14C levels in the feces usually were lower
in females (7.5 to 20%) than in males (3 to 32%). At 100 mg/kg, elimination
via the urine and feces was comparable between the sexes, accounting for about
52 and 16%, respectively, for both males and females. This dose therefore may
indicate a saturation point in the elimination of 2,4-DNT. In a study by Mori
et al. (1977), male Wistar rats given a single oral dose of [3-3H]2,4-DNT
(50 mg/kg) excreted only 16 and 30% of the administered 3H in the .urine and
feces, respectively, at 7 days postdosing. Similarly, only 12.5% of a
75-mg/kg oral dose of 3H-labeled test material was recovered from the urine of
male. Wistar rats within 7 days (Shoji et al., 1985). Low total recovery of 3H
may have been a problem with the latter two studies, however.
; Approximately 75, 76, and 81% of a single oral dose of [ring-UL-14C]-
2,4-DNT was eliminated in the urine by female New Zealand rabbits, female
beagle dogs, and female rhesus monkeys, respectively, within 24 hours after
compound administration (Lee et al., 1978). Between 3 and 9% was excreted in
the feces (see Table V-l). The authors stated that each animal received
V-4
-------
approximately 1/10 of the LD,0, but the dose administered to these animals,was
reported only as 10 jiCi [ring-UL-14C]2,4-DNT (specific activity 3.55 mCi/mM).
Elimination of 2,4-DNT following repeated oral dosing also has been
examined. Consumption of unlabeled 2,4-DNT in the diet for 1, 3, 9, or 20
months caused only a slight reduction in the total and urinary elimination of
2,4-DNT in male and female F344 (Rickert et al., 1981) or CD (Ellis et al.,
1979) rats when compared with animals administered DNT only once. In the
study by Rickert et al. (1981), animals were fed unlabeled 2,4-DNT at a level
of 35 mg/kg/day in the diet for 30 days before receiving a single oral dose of
[14C]2,4-DNT (35 mg/kg). Ellis et al. (1979) gave male and female CD rats a
single oral dose of 57 or 65 mg [ring-UL-uC]2,4-DNT/kg, respectively, after
animals had consumed a diet containing 0.01 or 0.07% unlabeled 2,4-DNT for 3,
9, or 20 months. In a study by Mori et al. (1980), male Wistar rats were
given a single oral dose of [3-'H]2<4-DNT (50 mg/kg) after being fed a diet
containing 0.5% unlabeled 2,4-DNT for 4 months. A total of 59% of the
administered 'H was excreted within 7 days postdosing; approximately 33% was
found in the urine, and about 26% was found in the feces.
Biliary excretion of 2,4-DNT appears to be significant, particularly in
male rats, and most likely accounts for sex-related differences in the rate of
fecal elimination of 2,4-DNT and/or its metabolites. Medinsky and Dent (1983)
demonstrated that biliary excretion of radioactivity following a single oral
dose of [ring-UL-'4C]2,4-DNT (35, 63, or 100 mg/kg for males; 35 mg/kg for
females) was higher in male than in female Fischer 344 rats. About 25 and 18%
of the administered dose was eliminated in the bile of males and females,
respectively, at 36 hours postdosing. Biliary excretion of radiocarbon was
essentially complete within 24 hours for males (excretion half-times 4.1 to
5.3 hours) and 12 hours for females (half-time 3.3 hours). Medinsky and Dtut
(1983) also collected more radioactivity (i.e., 60 to 90% versus 20 to 60% of
the administered dose) in the urine of rats from which bile was not colltcttd
than in bile-duct cannulated rats from which bile was collected. These data
suggest that biliary metabolites were absorbed from the intestines via
enterohepatic cycling. In all cases, females excreted more "C in the urliw
than males. In another study, biliary excretion of "C in female CD rats
*
V-5
-------
given a single oral dose of [ring-UL-KC]2,4-DNT accounted for about 10% of
the administered radioactivity at 24 hours postdosing (Ellis et al., 1980; Lee
et al., 1978). The rate of excretion in the bile peaked at 2 hours. Bond et
al. (1981) attributed the sex-related differences in elimination of 2,4-DNT in
Fischer 344 rats to three factors: a faster rate of transport of
2,4-dinitrobenzyl alcohol glucuronide (2,'4-DNBalcG) from the liver to the
bile, a larger amount of 2,4-DNBalcG excreted by the liver into the bile, and
a lower concentration of 2,4-DNT required to saturate the rates of metabolism
of 2,4-DNT by the liver in males when compared with females.
Metabolism by gut microflora may be required before reabsorption and
elimination of ONT can-occur. One group of investigators (Rickert et al.,
1981) demonstrated that axenic male F344 rats (i.e., those devoid of
intestinal bacteria) eliminated significantly less (p <0.05) 14C in the urine
and feces than did conventional male rats; corresponding values for the
bacteria-free animals were 33.8 and 15.7%, and levels for normal rats were
54.7 and 24.2%. Recovery of 14C from the urine of axenic female rats was also
lower than that from conventional females (39.8 versus 60.5%), but fecal
levels were comparable (17.3 and 13.8%). Sex-related differences in
elimination of 2,4-DNT following oral exposure in axenic rats were not
remarkable.
b. 2.6-DNT ,
Similar to the 2,4-DNT isomer, orally administered 2,6-DNT was primarily
eliminated via the urine. Male A/J mice excreted about 54, 54, and 49% of a
single oral dose of 1, 10, or 100 mg [3-3H]2,6-DNT/kg, respectively, In,the
urine within 8 hours postdosing (Schut et al., 1983). Feces contained no more
than 2.1% of the 3H dose, and levels in the small intestine accounted for
about 3 to 3.6X. Elimination via the lungs was negligible (<0.35%).
Twenty-four hours following administration of a single oral dose of
[ring-UL-14C]2,6-DNT (80 mg/kg) to female CD rats, about 60% of the UC was
recovered from the urine and 40% was recovered from the feces and
gastrointestinal contents (Ellis et al., 1980; Lee et al., 1975). Long and
V-6
-------
Rickert (1982) reported that male and female Fischer 344 rats eliminated about
54% of a single oral dose of 14C-labeled 2,6-DNT (10 mg/kg, 2 mCi/mmol) in the
urine within, 72 hours after dosing; feces contained about 20%. No sex-related
differences were noted. The authors reported that urinary excretion of 14C
was complete within 24 hours, but that fecal elimination was still evident at
72 hours. '
As with 2,4-DNT, a major route of elimination of 2,6-DNT 1s biliary
excretion. For example, female CO rats excreted about 25X of a single oral
dose of [ring-UL-14C]2,6-DNT 1n the bile 24 hours after dosing (Ellis et al.,
1980; Lee et al., 1978). The rate of biliary excretion of 14C peaked at
6 hours after dosing as compared with 2 hours in female GO rats administered
2,4-ONT (Lee et al., 1978). Long and Rickert (1982) reported that total
recovery of 14C-labeled 2,6-DNBalcG from liver perfusate and bile was
comparable for both male and female CO rats when livers were Incubated with
20 M [ring-UL-14C]2,6-DNT. At 70 /iM, however, total recovery of this
metabolite in the bile was significantly less (p <0.05) in females than in
males. These data indicate a possible saturation point in the metabolism and
excretion of 2,6-DNT in female rats. Biliary flow rates were similar for both
sexes, and disappearance of parent compound (20 or 70 fM) from the perfusate
was biphasic: half-times of elimination were 7.5 and 8.4 minutes for males and
females, respectively, for the initial phase, and 53.9 and 52.7 minutes,
respectively, for the second phase.
c. to-DNT
No information on the absorption and excretion of tg-DNT was found in the
available literature.
B. DISTRIBUTION .
1. Humans ' "
No Information was found on the distribution of DNT in human tissues,
although Woollen et al. (1985) detected trace amounts of 2,4- and 2,6-DNT in
the blood of workers exposed to this mixture.
V-7
-------
2. Animals
a. 2.4-DNT
As shown in Table V-l, very little radioactivity was retained by mice,
rats, rabbits, and monkeys 24 hours after administration of a single oral dose
of [ring-UL-14C]2,4-DNT (10 jid; dose equivalent to 1/10 the acute LD,0) (Lee
et al., 1978). For each animal, essentially all of the "C was recovered.(in
the urine, feces, and gastrointestinal tract. Most tissues contained no more
than 0.3% of the radioactive dose, although the whole blood of dogs and the
liver and skeletal muscle of both dogs and monkeys contained 0.6 to 1.6%.
Tissue/plasma radioactivity ratios showed a preferential initial uptake of "C
by the liver and kidneys of all animals within 24 hours postdosing; the ratio
for lung tissue in rats was also relatively high (Table V-2) (Lee et al.,
1978). After 5 days of dosing, tissue/plasma ratios in rats were 30.3, 19.9,
5.1, 12.5, and 5.4 for the liver, kidneys, brain, lungs, and skeletal muscle,
respectively (Lee et al., 1975). These data suggest that small amounts of
2,4-DNT and/or its metabolites may accumulate in animal tissue, particularly
the liver, kidneys, and lungs of rats.
/ '
Additional studies on the distribution of 2,4-DNT in mice and rats are
described below. Schut et al. (1981, 1982) examined the distribution of a
single intraperitoneal (ip) dose (1, 10, or 100 mg/kg) of [3-3H]2,4-DNT
(10 |iCi/mL) in male A/J mice.. Total recovery of JH in body fluids, and tissues
was 69.4, 74.8, and 81.7% for the low, middle, and high doses, respectively.
Concentrations of 3H in the blood were proportional to dose. Levels peaked at
2 to 3 and 15 nmol equivalents/mL blood for the 1- and 10-mg/kg groups,
respectively, within 30 minutes postdosing; for the 100-mg/kg group, blood
.levels of radioactivity (150 nmol equivalent/mL) peaked at 1 hour. Levels of
3H in the liver were initially about two to three times higher than levels in
the plasma, but after 2 hours, the two values were comparable. The authors
V-8
-------
Table V-2. Tissue/Plasma Radioactivity Ratios in Various Species of Animals
24 Hours After Administration of a Single Oral Dose of frinq-UL-
UC]2,4-DNT' - -.
Tissue
Mouse
Rat
Rabbit
Dog Monkey
Spleen
Liver
Kidneys
Brain
Lungs
Skeletal muscle
1.710.3"
6.3t0.8
3.4±0.4
0.6±0.1
1.9t0.3
o.6±o.r
c
18.1±0.7"
7.4±0,5.
l.StO.l
6.1±3.2
1.8±0.2
1.3'
8.7
7.0
0.6
2.2
0.5
i.r
6.9
4.9
0.5
1.7
0.5
2.6e
17.8
6.4
1.3
1.9
1.5
"Ratios represent the amount of radioactivity in 1 g of wet tissue divided
by the amount of radioactivity in 1 ml of plasma.
"Mean t SE of four mice.
cNot reported.
"Mean ± SE of three rats.
'Mean ± SE of two animals.
SOURCE: Lee et al. (1978).
V-9
-------
described similar results for the kidney, but they did not present supporting
data. Terminal half-lives for :H in the liver and kidney were less than 2
hours. In all cases, the lungs contained no more than 0.2% of the radioactive
dose. Maximum levels of 3H in the adipose tissue (3.9 to 4.7%) were reached
within 30 minutes but dropped to <0.1% within 4 hours. Uptake of
radioactivity by the small intestine was1 highest (9 to 14%) between 0.75 and
2 hours. Levels in the brain, heart, and spleen were negligible.
Preferential uptake or retention of radioactivity was hot apparent for any
tissue.
In male F344 rats, maximum UC levels in the liver and kidneys were
proportional to dose (}0, 35, or 100 mg [ring-UL-uC]2,4-DNT/kg) and were
about 5 to 10 times higher than levels in the plasma and red blood cells
(Rickert and Long, 1980). No sex-related differences were observed in the
concentration of UC in plasma or in the elimination of UC from the plasma
(2 to 2.5 hours), liver (terminal half-lives of 36 and 40 hours for males and
females, respectively), or kidney (30 and 41 hours, respectively). In
contrast, hepatic levels of radioactivity were about two times greater in
males than in females during the 72-hour postdosing period. Elimination of
14C from red blood cells was significantly (p <0.05) slower in female than in
male rats (half-lives of 123 versus 27 hours). No parent compound was
detected in the plasma at 12 hours postdosing.
Mori et al. (1978) gave male Wistar rats a single oral dose of 22 mg
3H-labeled compound/kg. Blood levels of radioactivity peaked at 6 hours and
dropped steadily during the subsequent 9 hours. Hepatic 3H levels also peaktd
at 6 hours and correlated with levels in the blood. The highest
concentrations of radioactivity in the stomach and small intestine were
detected before 6 hours. A whole-body half-life of 22 hours was reported.
In another study, low levels of radioactivity were recovered from 16
tissues 7 days after male Wistar rats were given 50 mg [3H]2,4-DNT/kg
(2.14 nCi/mg) (Mori et al., 1977). All tissues combined (including the
stomach and small and large intestine) contained only 2.95% of the
administered 3H. The highest levels of radioactivity were found in the
V-10
-------
adipose tissue (1.6%), skin (0.60%), and liver (0.40%). Total recovery of 3H
in this study, however, accounted for only 49% of the radioactive dose.
Irons and,Gross (1981) examined autoradiographs of female F344 rats
administered up to 100 jiCi [ring-UL-'4C]2,4-DNT (35 mg/kg). For same-sized
tissue sections of animals sacrificed 3 'hours after dosing, I4C concentrations
increased in the following order; muscle (lowest), lung, blood, liver, and
kidney. Brain, heart, and adipose tissue contained negligible amounts of
radioactivity.
Radioactivity was detected in the placenta and amniotic fluid of rats
(strain not given) administered a single oral dose of 35 mg [ring-UL-'4CJ-
2,4-ONT/kg on gestation day 20 (Rickert et al., 1980). (The time of sacrifice
was not reported.) Approximately 10 to 50% of the UC dose was recovered from
these two fractions. Clearance of radioactivity from the plasma was similar
for fetuses and dams (half-lives, 8.8 and 9.5 hours, respectively) but was
slower from the liver (39 and 17 hours) and kidney (27 and 13 hours) of dams.
The authors stated that concentrations of "C in fetal tissues were similar to
those in maternal tissues, but supporting data were not provided.
The effects of repeated dosing on the tissue uptake of 2,4-DNT have been
examined. As discussed above, the tissue/plasma "C ratios for the liver,
kidney, brain, lungs, and skeletal muscle of rats given a single oral dose of
radiolabeled 2,4-DNT for 5 consecutive days were about two to four times
greater than ratios of single-dose animals, indicating that 2,4-DNT and/or its
metabolites can accumulate in the body (Lee et al., 1978). In another study,
Ellis et al. (1979) gave male and female CD rats a single oral dose of 57 or
65 mg [ring-UL-'4C]2,4-DNT/kg, respectively, after animals had consumed a diet
.containing 0.01% or 0.07% unlabeled 2,4-DNT for 3, 9, or 20 months. Rats in
the 20-month experiment had relatively high levels of radioactivity (i.e., 4.4
and 5.7%) in the muscle tissue 24 hours after administration of the
"C-labeled compound. However, the level of radioactivity in the muscle
tissue of nonpretreated female controls also was high and contained about 2.3%
of the 14C dose. No other trends-were noted in any other group. Mori et al.
(1980) found that tissue levels of radioactivity generally were lower in male
V-ll
-------
Wistar rats fed unlabeled 2,4-DNT in the diet for 4 months before receiving a
single oral dose of 50 mg [3H]2,4-DNT/kg (47.6 /iCi/kg) than in those fed a
standard diet. At 7 days after dosing, the highest 3H residues were in the
liver, and with the exception of the kidney, heart, and small intestine,
levels of radioactivity 1n 12 other tissues were lowest 1n the pretreated
animals when both groups were compared.
b. 2.6-DNT
Schut et al. (1983) examined the disposition of a single oral or 1p dose
of [3-3H]2,6-DNT in male A/J mice. Animals were given 1-, 10-, or 100-mg/kg
doses of the radlolabeled compound (2.5 /iCi/mouse). Blood and liver levels
of 3H were similar in orally dosed mice and remained fairly constant for the
first 8 hours after dosing. In contrast, hepatic concentrations of
radioactivity In ip-dosed mice peaked during the first hour postdosing and
decreased steadily thereafter. The amount of 3H in the blood was two to four
times lower than that in the liver through the first 2 hours after compound
administration. Kidney 3H residue levels were reportedly equivalent to those
in the liver and showed no treatment-related differences (data not provided).
Uptake of 2,6-ONt In the small Intestine peaked between 1 and 3 hours, but the
maximum concentration was higher in orally dosed mice (9.5 to 16.9% of the
administered 3H) than in 1p-dosed animals (5.2 to 8.9%). Lungs contained no
more than 0.35% of the administered dose for either group, and the levels of
radioactivity in the brain, heart, and spleen remained low throughout the
experiment. Preferential tissue uptake was not apparent, but total recovery
data suggest that the 100-mg/kg dose may have been saturating for both routes
of exposure.
In a study by Ellis et al. (1980), only a small amount (<5%) of the
radioactivity administered to female CO rats was recovered from the carcass
24 hours after dosing. Each animal received, by gavage, 10 j*C1 of
[ring-UL-14C]2,6-DNT at a dose equivalent to 1/10 of the L050 (about 80 mg/kg).
No other studies on the distribution of 2,6-DNT were found.
V-12
-------
c. tq-DNT
No information on the distribution of tg-DNT was found in the available
literature.
3. Covalent Binding of DNT Isomers
Covalent binding of DNT to hepatic macromolecules has been examined
extensively and may be particularly important in light of data showing a
higher incidence of hepatic carcinomas and hepatic neoplastic nodules in
DNT-exposed male rats when compared with treated female rats. Results of
several studies indicate that conjugation, biliary excretion, microbial
metabolism in the gut, and intestinal reabsorption may be prerequisites to
hepatic binding of DNT. Hepatic binding may be greater for 2,6-ONT than for
2,4-DNT, and binding of DNT Isomers appears to be lower in females than in
males. Diet (I.e., as it affects microbial activity and number) may also
influence the degree to which binding of DNT metabolites occurs.
Medinsky and Dent (1983) demonstrated differences In the binding of 14C
to hepatic macromolecules following oral administration of [ring-UL-14C]-
2,4-DNT to male and female Fischer 344 rats. Binding was lower in females
than in males. I FT addition, covalent binding in bile duct-cannulated rats
from which bile was collected was lower than in animals from which bile was
not collected.
Swenberg et al. (1983) reported sex-related differences in the covalent
binding of [3H]2,6-DNT fpllowing oral dosing in Fischer 344 rats. DNA from
hepatocytes of males given a single oral dose of the compound contained at
least 15 to 20% more radioactivity than DNA from hepatocytes of females.
Binding of 2,6-DNT to DNA in liver sinusoidal lining cells was considerably
lower than binding to hepatocyte DNA through the first 4 days after compound
administration, particularly 1n cells isolated from female rats. The amount
of binding to hepatocyte DNA dropped throughout the entire 7-day postdosing
period, while binding in sinusoidal cells declined during the first 4 days and
then increased during the next 3 days. In another experiment, essentially all
V-13
-------
covalently bound radioactivity was recovered from the hepatocellular DNA of
male Fischer 344 rats given weekly doses of [3H]2,6-DNT for 1, 2, 3, or 4
weeks. These data indicate that covalent binding of 2,6-DNT is specific for
hepatocytes.
Covalent binding of 14C to hepatic DNA, RNA, and protein was evident in
male Fischer 344 rats at 12 through 96 hours after animals were given a single
oral dose of [r1ng-UL-l4C]2,4- or 2,6-DNT (Rlckert et al., 1983). The authors
reported that hepatic binding of 2,6-DNT was about two to five times greater
than that of 2,4-DNT. Neither isomer had an affinity for a particular
macromolecule.
Kedderis et al. (1984) used sulfotransferase inhibitors to determine the
effect of sulfate ester formation on the in vivo b1oact1vat1on of 2,4- and
2,6-DNT to electrophilic species, which in turn bind covalently to DNA. In
this study, male Fischer 344 rats were given an oral gavage dose (28 ing/kg) of
either isomer after administration of pentachlorophenol (PCP) or 2,6-dichloro-
4-nitrophenol (DCNP). Total hepatic macromolecular binding of 2,4- and
2,6-DNT were reduced by 33 and 69%, respectively, following treatment with the
inhibitors. Each inhibitor caused binding of 2,6-DNT to hepatic DNA to
decrease by more than 95%. In contrast, DNA binding of 2,4-DNT was reduced by
about 84 and 33% after administration of DCNP or PCP, respectively. These
data suggest that sulfation may be involved in the hepatic covalent binding of
reactive metabolites of DNT isomers.
As mentioned above, metabolism by intestinal bacteria appears to play a
role in hepatic binding of DNT. In a study by Rlckert et al. (1981), hepatic
covalent binding was significantly greater (p <0.05) in conventional male
Fischer 344 rats than In axenic male rats following oral administration of
14C-labeled 2,4-DNT. The amount of 14C bound to hepatic material also was
higher (p £0.05) In both conventional and axenic males than in corresponding
females. In contrast, hepatic binding was comparable in both groups of female
rats. Swenberg et al. (1983) reported that at 12 hours postdosing, the level
of 2,6-DNT bound to hepatocyte DNA in axenic male rats was about 10% of that
V-14
-------
bound to the ONA of conventional rats; female axehic rats were not included in
the experiment.
The effects of dietary pectin on intestinal microflora and on the hepatic
binding of 2,6-DNT were investigated by DeBethizy et al. (1983) and Rickert
et al. (1986b). Male F-344 rats in these studies were fed a purified diet
containing 0, 5, or 10% pectin or one of two cereal-based diets containing <1
or 8.4% pectin. No pectin-related changes in the hepatic covalent binding of
2,6-DNT were observed in rats given a single low dose (10 mg/kg) of
[3H]2,6-DNT on day 28. In animals given a 75-mg/kg dose of the
tritium-labeled 2,6-DNT, however, total covalent binding was increased by 40
and 90% after consumption of 5 or 10% pectin, respectively, when compared with
animals given pectin-free diets. Binding in hepatic tissue of rats fed the
cereal-based diets and dosed with 75 mg [3H]2,6-DNT/kg was 135 and 150% higher
than in controls. Activities of cecal glucuronidase and nitroreductase were
about two to three times, greater in pectin-fed rats than in controls; the
total number of anaerobic microbes in the cecum of treated animals was
similarly Increased. The authors suggested that through the changes described
above, dietary pectin could increase the potential toxicity of high doses of
2,6-DNT.
C. METABOLISM •
Following gastrointestinal absorption, DNT isomers undergo oxidation in
the liver. Metabolites generated from this reaction are often conjugated with
sulfate or glucuronate and subsequently excreted in the urine or Into the bllt
(section V.A.I). Metabolites that are transported from the bile to the gut
are hydrolyzed and reduced by intestinal microflora (section V.C.3). Many of
these compounds, in turn, are reabsorbed from the gut into the systemic
circulation and then oxidized in the liver. Urinary elimination may occur
next, but biliary excretion of these metabolites Into the gut results in
additional reduction by Intestinal bacteria prior to elimination from the
body.
V-15
-------
For most animals (i.e., A/J mice, CO and Fischer 344 rats, New Zealand
rabbits, beagle dogs, and rhesus monkeys) and humans, significant portions of
orally or intraperitoneally administered 2,4-DNT are converted to 2,4-DNBalc
and/or its glucuronide (i.e., 2,4-DNBalcG). Other primary urinary metabolites
excreted by these animals include 2-amino-4-nitrobenzyl alcohol (2A4NBalc) and
4-amino-2-nitrobenzyl alcohol (4A2NBalc);. humans excrete only the former in
large quantities. For Fischer rats and humans, another major product of
2,4-ONT metabolism 1s 2,4-DNBacid. A significant portion (4 to 9%) of a
single oral dose of 2,4-ONT Is reduced in vivo to 2,4-diarainotoluene (2,4-OAT)
and excreted in the urine by rats, rabbits, dogs, and monkeys, but none 1s
found In the urine of occupationally exposed humans. In contrast, metabolism
of 2,4-DNT by CD-I mice and Wistar rats 1s not extensive, and only small
amounts of an oral dose of 2,4-ONT given to these animals are oxidized to one
of the benzyl alcohol metabolites; reduction of 2,4-ONT to 2,4-OAT Is
negligible in mice. Sex-related differences in the metabolism of 2,4-ONT have
been observed in only Fischer rats and humans. For both species, females
produce up to three times more 2,4-DNBalc and/or 2,4-DNBalcG than males, and
in humans only, men excrete nearly twice as much 2,4-DNBacid as females.
The metabolism of 2,6-ONT has not been studied extensively. As with
2,4-DNT, Fischer rats convert the 2,6-ONT isomer to the corresponding
dinitrobenzyl alcohol glucuronide and dinitrobenzoic acid; however, only one
metabolite (2-amino-6-n1trobenzoic acid, 2A6NBacid) results from the in vivo
reduction of 2,6-ONT. Similarly, only three major metabolites of 2,6-ONT
(2,6-DNBalc, 2,6-ONBalcG, and 2,6-ONBacid) have been recovered from the urine
of men and women; women appear to excrete more 2,6-DNBalcG than men. No other
sex-related differences in the metabolism of 2,6-ONT have been observed.
1. Humans /
Metabolism of DNT in humans 1s similar to that in Fischer rats. The
primary urinary metabolites of 2,4- and 2,6-ONT excreted by Individuals
(14 men, 3 women) exposed occupationally to tg-DNT (0.05 to 0.59 mg/m3; 76%
2,4-DNT, 19% 2,6-DNT) were the corresponding dinitrobenzyl alcohols and their
glucuronides, the corresponding dinitrobenzoic acids, and 2A4NBacid
V-16
-------
(Table V-3) (Levine et al., 1985a; Turner, 1986; Turner et al., 1985). Males
excreted more DNBacid (2,4- and 2,6-DNT combined) than females (52.5 versus
28.8% of all urinary metabolites, respectively); most of the DNBacid
eliminated was 2,4-DNBacid (50.5% for men and 28.9% for women). In contrast,
men excreted less of the combined DNBalcG isomers than females (9.5 versus
33.3%, respectively). Approximately 29.1 and 73.9% of the urinary DNBalcG
metabolites in females were the 2,4- and 2,6- isomeric forms, respectively;
corresponding values for males were 5.0 and 35.6%. Urinary excretion of
2,6-DNBacid was slightly higher in males (2.2 to 14.3% of all metabolites)
than in females (2.5%), and levels of 2,6-dinitrobenzyl alcohol were
comparable between the sexes (4.8 to 6.6%). Concentrations of 2A4NBacid also
were similar for males and females (37.2 to 37.6%). Small amounts (0.3 to
0.8%) of the metabolites in the urine of men and women were 2Ac4NBacid.
Unchanged parent compounds (i.e., 2,4- and 2,6-ONT) were also recovered from
the urine, and the hydrolyzed urine of one individual contained trace amounts
of 4A2NBacid and 4Ac2NBacid. Reduction of both nitro groups was not evident.
Wide variations in urinary metabolite profiles were attributed to differences
in exposure and in the way an individual may metabolize ONT.
Woollen et al. (1985) found that the primary urinary metabolite of
workers exposed to tg-ONT was 2,4-DNBacid. Other urinary metabolites isolated
were 2-amino-4-nitro-, 4-amino-2-nitro-, and 2-amino-6-nitrobenzo1c acids and
4Ac2NBacid. The urine of these workers contained no 2,4- or 2,6-DNBalc.
2. Animals
a. 2.4-DNT
The primary metabolites identified in the urine of male A/J mice after
oral or ip dosing with [3-3H]2,4-DNT were 2,4-DNBalc and 2,4-DNBalcG (Schut
et al., 1985). In ip-dosed mice, the unconjugated form accounted for about
1.5 to 6% of the administered 3H; the urine of these mice; was not analyzed for
conjugates. Between 5.45 and 16% of the oral dose given to mice was
2,4-DNBalcG; less than 0.6% was unconjugated. Small amounts (<1.5%) of the
V-17
-------
Table V-3. Urinary Metabolites Excreted by Humans Exposed Occupationally
to Technical Grade DNTa'b
Percent of all urinarv
Study:
Metabolite
2,4-Dinitrotoluene
2,6-Dinitrotoluene
2-Amino-4-nitrobenzoic acid
4-Amino-2-nitrobenzoic acid
2,4-Oinitrobenzyl alcohol
2,6-Oinitrobenzyl alcohol
Levine et a.
Men
D
37.2
trd
. ..
...
1., 1985a)
Women
D
0
37.6
tr
--
-•-
metabolites
(Turrter et
Men
0
0
31.1
tr •
4.9e
5.3"
excreted by
al., 1985)
Women
0
D
34.3
tr
27. 3*
6.6e
Oinitrobenzyl alcohol
glucuronide (DNBalcG) 9.5 33.3
2,4-ONBalcG 5.09 29.I9
2,6-DNBalcG 35.63 73.9s,
Oinitrobenzoic acid (DNBacid)f 52.5 28.8
2,4-DNBac1d -- -- 50.5* 28.9e
2,6-DNBac1d .... 7.4- 2.5e
2-Acetylamino-4-nitrobenzoic
acid 0.8 0.3
4-Acetylamino-2-nitrobenzoic
acid .. .. 2.1 0.4
'76% 2,4-DNT, 19% 2,6-DNT.
Values are averages for 14 men or 3 women, except where noted (see
footnote e).
Detected but concentration not quantified.
Trace amount detected.
^Average of three men or value for one woman.
2,4- and 2,6- isomers combined.
3As percent of the total isomeric forms.
SOURCE: Levine et al. (1985a); Turner et al. (1985).
V-18
-------
following\metabolites were also identified in both orally and intraperi-
toneally dosed mice: 2,4-DAT, 4-amino-2-nitrotoluene (4A2NT), and
2-amino-4-nitrotoluene (2A4NT). Trace amounts of parent compound were
detected in the urine of both groups of mice. Unknown metabolites accounted
for approximately 0.2 to 5% of either dose. Only orally treated mice excreted
2A4NBalc and 2-acetylamino-4-nitrotoluene (2AcA4NT). Female CD-I mice
administered a single oral dose of [ring-UL-14C]2,4-DNT eliminated only 11.3%
of the total radioactive dose in the urine, with the largest amounts (2.6 and
2.8%, respectively) recovered as 2,4-DNBalc and 2-amino-4-nitrobenzyl- and
4-amino-2-nitrobenzyl alcohol combined (Table V-4) (Leeetal., 1978). These
metabolites accounted for about 23 and 25% of the total urinary radioactivity
recovered 24 hours after dosing, respectively (Table V-5); a third metabolite,
4A2NT, represented only 1.4% of the 14C dose but 12.5% of the urinary
radioactivity.
Shoji et al. (1985) detected several metabolites in the urine of male
Wistar rats given a single oral dose of [3H]2,4-DNT. The most abundant
urinary metabolites were 2,4-DNBacid, 2,4-ONBalcG, and 2-amino-4-acetylami no-
benzole add (2A4AcABacid), which accounted for 5.91, 3.15, and 1.85%,
respectively, of the 3H administered. The other metabolites identified were
2,4-DNBalc (0.83%), 4A2NBalcG and 2A4NBalcG combined (0.45%), 4A2NBalc and
2A4NBalc combined .(0.16%), and 4A2NT (0.03%). Repeated oral dosing (i.e.,
administration of a single oral dose of 3H-labeled material for 6 consecutive
days) in male Wistar rats appeared to cause an increase in the reductive
metabolism of 2,4-DNT (Mori et al., 1981b). For example, although no
2,4-DNBacid or 2,4-DNBalcG was recovered from the urine of these animals
during the 7 days after the first dose was given, 2A4NT, 2,4-DAT, and 2-nitro-
4-acetylamino toluene (2N4AcAT) were excreted. (Percent-recovery data were
not provided.) Formation of 2N4AcAT, 2A4AcAT, and 2A4AcABacid may be unique
to Wistar rats.
Male and female Fischer rats convert 2,4-DNT to two major oxidative
metabolites (2,4-DNBalcG and 2,4-DNBacid) and two major reductive products
(4-acety1amino-2-n1trobenzo1c acid (4Ac2NBacid) and 2-amino-4-nitrobenzoic
acid (2A4NBacid)) (Table V-6) (Medinsky and Dent, 1983; Rickert et al., 1981;
V-19
-------
Table V-4. Radiolabeled Metabolites Found in the Urine of Various Species 24 Hours After Administration of a Single Oral Dose of "C-2.4-DNT
Metabolite
2,4-Dinitrotoluene
4-Amino-2-nitrotoluene ,
2-Amino-4-nttrotoluene
2.4-Oiaminotoluene
2.4-Dinitrobenzyl alcohol
2-Amino-4-nttrobenzyl alcohol
^ and 4-amino-2-nitrobenzyl
alcohol
< 2,4-Diaminobenzyl alcohol
0 2.4-D1nitrobenzoic acid
Unidentified
Total
Mouse
0"
1,4"
1-1
0.6
2.6
2.8
0.2
0-4
2.2
11.3
Percent
Rat
Oc
1.9
0.6
8.8
25.2
19.2
4.6
6.2
9.5
76JD
of Administered Dose*
Rabbit
0.2*
6.6
5.0 .
4.2
32.9
7.6
5.6
7.2
5.9
75.2
Dog
0.6"
6.3
2.7
4.0
26.6
14.2
2.6
5.1
14.2
76~3
Monkey .
2.1"
1.8
. 7.1
3.8
20.2
14.8
2.5
'4.0
25.0
81.3
'Includes free compound and glucuronide and sulfate conjugates.
"Mean of four CD-I female animals.
'Mean of three CD female animals.
"Mean of two females (New Zealand white rabbits, beagle dogs, and rhesus monkeys).
SOURCE: Adapted from Lee et al. (1978).
-------
Table V-5. Radiolabeled Metabolites Found in the Urine of Various Species 24 Hours After Administration of a Single Oral Dose of "C-2.4-ONT
ro
Percent of all Urinary Metabolites*
Metabolite .
2,4-Dinitrotoluene
4-Amino-2-nitrotoluene
2-Ami no-4-ni t rotol uene
2 , 4 -01 ami notol uene
2.4-Dinltrobenzyl alcohol
2-Anino-4-nitrobenzy1 alcohol
and 4-amtno-2-nttrobenzyl
alcohol
2.4-Diamtnobenzyl alcohol
2.4-Olnltrobenzpic acid
Unidentified
Mouse
0s
12.5(10.3*)
9.5
5.2
22.6(19.6')
25.1(24.5')
1.6
3.9
19.6
Rat
Oc
4.8
0,7
11.1
30.3(27.4')
31.7(22.5')
3.1
4.2
12.5
Rabbit
0"
8.8(7.9*)
6.6
5.6
43.5(40.3')
10.2(9.4')
7.5
9.5
7.9
Dog
0.7"
8.4(4.6';3.7*)
3.5
5.4
34.8(33.1')
18.5(17.9')
3.4
6.7
18.6
Monkey
2.6'
2.1
8.7
4.8
25.3(21.5')
18.1(17.9')
3.2
4.9
30.7
'Includes free compound and glucuronide.and sulfate conjugates.
"Mean of four female CD-I animals.
'Mean of three female CO animals. Values are based on available data and do not add up to 100X in all cases.
*Mean of two female animals (New Zealand white rabbits, beagle dogs, and rhesus nonkeys).
'Sulfate conjugate.
'Glucuronide conjugate. ~
SOURCE: Adapted from Lee et al. (1978).
-------
Table V-6. Urinary Metabolites of 2,4-DNT Excreted by Orally Dosed
Fischer Rats
Metabolite
2,4-D1n1troto1uene
2-Amino-4-nitrotoluene
2-Am1no-4-n1trobenzo1c add
2-Am1no-2-n1trobenzyl alcohol
2,4-D1nitrobenzyl alcohol
glucuronlde
2,4-Dinitrobenzoic acid
4-Acetylamino-2-nitrobenzoic add
4-Acetylamino-2-nitrotoluene
F344
Males
__•
<4b-c :
2.2-4.2b
<4b'c
11. 4-15. 8b
6.3-18.8b
8.4-13.56
<4b,c
Rat
Females
/
--
<4b,c
1.8-4b
<4b'c
15. 9-36. 6b
6.9-21.3b
9.9-14.0b
<4b.e
"Not detected.
As percent of dose.
cExact amount not reported.
SOURCE: Medinsky and Dent (1983); Rickert et al. (1981); Rickert and
Long (1981).
V-22
-------
Rickert and Long, .1981). . These compounds accounted for about 11 to 37, 6 to
21, 8 to 14, and 2 to 4% of a single oral dose of [ring-UL-14C]2,4-DNT,
respectively. Other metabolites of 2,4-DNT recovered from the urine of
Fischer rats Included 2-amino-4-nitrotoluene (2A4NT), 2:am1no-4-nitrobenzyl
alcohol (2A4NBa1c), and 4-acetylamino-2-nitrotoluene (4Ac2NT) (Medlnsky and
Dent, 1983). Females eliminated up to about two times more 2,4-ONBalcG in the
urine than males; no other sex-related differences in the metabolism of
2,4-DNT were observed.
Lee et al. (1978) demonstrated that in female CO rats, female New Zealand
white rabbits, female beagle dogs, and female rhesus monkeys, the two major
urinary metabolites of orally administered [ring-UL-14C]2,4-ONT were the
glucuronide conjugates of 2,4-DNBalc (20 to 33% of the dose at 24 hours after
compound administration) and 2A4NBalc (8 to 19%) (values for free and
conjugated forms are given in Tables V-4 and V-5). Smaller amounts (about 2
to 7% of the 14C dose) of 2,4-DAT, 2,4-DABalc, 2A4NT, 4A2NT, and 2,4-DNBactd '
were also recovered from each species. The percent distribution of urinary
metabolites was fairly comparable to the amount of 14C dose metabolized by
these animals. In contrast, CD-I mice excreted disproportionate amounts of
2,4-DNBalc, 2A4NBa1c plus 4-amino-2-nitrobenzyl alcohol (4A2NBalc) combined,
and 4A2NT in the urine (22.6, 25,1, and 12.5% of all metabolites,
respectively) when compared with the corresponding amounts of administered
2,4-DNT metabolized (2.6, 2.8, and 1.4%). Unchanged parent compound was found
in the urine of rabbits (0.2% of the administered radiocarbon), dogs (0.6%),
and monkeys (2.1%); most 2,4-DNT in the urine of monkeys was present as a
glucuronide conjugate. No 2,4-DNT was found in the urine of mice or rats.
Between 7.9 and 30.7% of the urinary 14C (2.2 to 25% of the dose) was
unidentified. Figure V-l shows the relative proportions of free and
glucuronide- and sulfate-conjugated urinary metabolites excreted by the
various anlnals in this study.
The placenta and amniotic fluid of pregnant Fischer 344 rats contained
only unchanged parent compound after administration of a single oral dose of
[ring-UL-14C]2,4-DNT on gestation day 20 (Rickert et al., 1980). Small
V-23
-------
100
% 50
U
Mouse
Rat
Rabbit
Monkey
Figure V-l. Distribution of free and conjugated metabolites of 2,4-ONT in the
urine of orally dosed animals. Urinary products of an oral dose
of 2,4-ONT given,to mice, rats, rabbits, dogs, and monkeys and
expressed as free, glucuronide (GLUC) conjugates, sulfate (SULF)
conjugates, and unknown (?) metabolites.
SOURCE: Ellis et al. (1980).
V-24
-------
amounts of 2A4NT, 2,4-DNBacid, and 2A4NBacid were recovered from maternal
plasma. No other data were provided.
The metabolism of 2,4-DNT in CO and Fischer 344 rats 1s minimally
affected following repeated oral dosing. For example, male and female rats
fed 2,4-DNT in the diet for 30 days and then given a single oral dose of UC-
labeled 2,4-DNT excreted amounts of 4Ac2NBacid, 2,4-DNBacid, 2A4-NBacid, and
2,4-DNBalcG similar to those of nonpretreated animals (Rickert et al., 1981).
Males excreted less 2,4-DNBacid than females (9.8 versus 15.8% of the 14C
dose), and females excreted slightly more 2,4-DNBalcG than males (24.3 versus
19.1%), but these differences were not statistically significant. In a study
by Ellis et al. (1979)-, male and female CD rats were given a single oral dose
of [ring-UL-14C]2,4-DNT after consuming unlabeled test material in the diet
(at a level of 0.01 or 0.07%) for 3, 9, or 20 months. Metabolism of
2,4-DNT generally was minimally affected by dosing regimen, and nonpretreated
and pretreated males and females appeared to metabolize the test compound in a
similar fashion. As discussed earlier, repeated oral dosing over a 6-day
period appeared to cause a shift toward reductive metabolism in male Vlistar
rats (Mori et al., 1981b).
b. 2.6-DNT
Very limited data on the metabolism of 2,6-DNT in mice were available.
In a study by Schut et al. (1983), male A/J mice were given a single oral or
ip dose (1, 10, or 100 mg/kg) of 3H-labeled compound. No unchanged 2,6-DNT
was recovered from the blood, liver, lungs, or small intestine of animals
given a 1-mg/kg dose by either route, and less than 2% of the 3H recovered
from the urine of these animals during the 8-hour postdosing period was
unchanged parent compound. In contrast, unchanged 2,6-DNT was isolated in the
tissues of animals in all other groups, with the highest levels in high-dose
mice and the slowest rate of disappearance of unchanged 2,6-DNT in orally
dosed animals. No more than11% of the 3H in the liver or lungs of animals
given a 10-mg/kg ip dose was unchanged parent compound; for animals
administered 100 mg/kg ip, 2,6-DNT accounted for up to 20% of the total liver
or lung radioactivity. Levels of unchanged parent compound in the small
V-25
-------
intestine of ip-dosed mice initially were high (>50% of the total intestinal
3H) but dropped to low or trace amounts within 2 hours after dosing.
Unchanged 2,6-DNT in the liver and lungs of mid- and high-dose mice accounted
for up to 15% of the total corresponding tissue radioactivity through 4 hours
postdosing. Concentrations of 2,6-DNT in the small intestine of orally dosed
animals were high, representing approximately 23 and 50% of the total
intestinal 3H within 4 hours after animals were given 10 or 100 mg/kg,
respectively. Eight hours after oral administration of the high dose to mice,
about 35% of the tritium In the small Intestine was recovered as unchanged
parent compound. The large Intestine of orally dosed mice contained small
amounts of 2,6-DNT (I.e., <2% of total tissue 3H). About 50 to 90% of the 3H
administered to animals was excreted in the urine within 8 hours after dosing,
but less than 2% was recovered as unchanged 2,6-DNT. The data indicate that
2,6-DNT is rapidly and extensively metabolized by mice following oral or 1p
dosing. The liver and Intestines appear to be the primary sites for the
metabolism of 2,6-DNT in mice.
Three metabolites accounted for about 95% of the urinary 14C excreted by
male and female Fischer 344 rats given a single oral dose of [ring-UL-14C]-
2,6-DNT: 2,6-dinitrobenzoic add (2,6-ONBacld), 2,6-dinitrobenzyl alcohol
glucuronide (2,6-DNBalcG), and 2-amino-6-nitrobenzoic acid (2A6NBacid) (Long
and Rickert, 1982). In males, these metabolites accounted for about 21, 22,
and 14% of the 14C dose, respectively; in females, the corresponding values
were 20, 19, and 11%.
c. tg-DNT
No information on the metabolism of tg-DNT was found In the available
literature.
3. Microbial and in vitro Metabolism of 2.4- and 2.6-DNT
The role of gut nricroflora in the metabolism of 2,4- and 2,6-DNT has been
examined in vivo and in vitro, in a study by Rickert et al. (1981), urinary
concentrations of oxidized metabolites of orally administered 2,4-DNT (I.e.,
V-26
-------
2,4-DNBacid and 2,4-DNBalcG) were comparable between conventional and axenic
rats. In contrast, axenic rats excreted significantly smaller amounts (i.e.,
about 80 to 90% less) of metabolites requiring both oxidation and reduction
(i.e., 4Ac2NBac1d and 2A4NBacid) than conventional rats.
Guest et al. (1982) investigated the. metabolism of 2,4-DNT incubated with
cecal or ileal material from male Swiss-Webster mice, male and female Fischer
344 rats, and male humans. No remarkable sex- or species-related differences
in the metabolism of 2,4-DNT were observed, although the rate of disappearance
of test material was highest for mouse samples, and no 2,4-diaminotoluene
(2,4-DAT) was produced in human samples for at least 3 hours. Anaerobic
conversion of parent compound was complete within 20 minutes for cecal
material from rats. In contrast, no 2,4-DNT was metabolized aerobically
within 3 hours. The first metabolites recovered from all Incubations were
2-nitro-4-nitrosotoluene and 4-nitro-2-nitrosotoluene; decreases in the
concentration of these compounds were accompanied by Increased amounts of
2A4NT and 4A2NT. Thus, both nitro groups were reduced to ami no groups via
nitroso Intermediates. The authors suggested that these Intermediates may
bind covalently to microflora or hepatic macromolecules.
In a study by Mori et al. (1985), 2,4-DNT incubated with cecal microflora
from male Wistar rats was reduced anaerobically to 2A4NT, 4A2NT, and 2,4-DAT
via 2-hydroxylamino-4-nitrotoluene and 4-hydroxy1amino-2-nitrotoluene.
Similarly, Escherichia coll isolated from human intestines metabolized 2,4-ONT
to 2A4NT and 4A2NT and converted 2,6-DNT to 2A6NT via the corresponding
hydroxylaminonitrotoluenes (Mori et al., 1984b). The amount of parent
compound converted by human bacteria was 10.2, 18.8, and 45.7%, respectively.
The hydroxy1 ami no Intermediates were detected in vitro within 8 hours, and
production of the final metabolites peaked within 24 hours. Nitrosotoluenes
were not recovered In any human samples (Mori et al., 1984b). Microflora in
the cecal contents of male A/J mice and male Fischer 344 rats anaerobically
converted [3H]2,6-DNT (240 M, 20 Ci) to 2A6NT (6.4 to 9.6% of the total 3Hh
2-acetylam1no-6-nitrotoluene (2Ac6NT) (1.3 to 4.6%), and 2,6-diaminotoluene
(2,6-DAT) (0.4 to 0,7%) (Dixit et al., 1986). About 5.3 to 6.5% of the total
metabolites in mouse preparations were not identified; only trace amounts
V-27
-------
(0.7% or less) in"rat samples remained unidentified. Most parent compound
(82.1 to 88.7%) was recovered as unchanged 2,6-DNT at the end of the 30-minute
incubation period. Schut et al. (1985) reported that under aerobic
conditions, most 2,4-DNT incubated with intestinal explants or cecal material
from mice was metabolized to 2,4-DNBalc. Overall, the data suggest that gut
microflora are responsible for reductive •metabolism of DNT.
Figure V-2 shows the proposed pathway for the anaerobic metabolism of
2,4-DNT in rat Intestinal microflora. The primary metabolites of anaerobic
degradation of 2,4-DNT by rat hepatocytes are 2A4NT and 4A2NT (Lee et al.,
1978; Kozuka et al., 1978; Mori et al., 1980, 1981a; Decad et al., 1982);
Lee et al. (1978) reported that concentrations of these aminonitrotoluenes
were highest in male liver tissue. Little or no 2,4-DNBalc was recovered from
anaerobic liver homogenate preparations containing 2,4-DNT (Lee at al., 1978;
Decad et al., 1982), and no 2,4-DAT was isolated following prolonged anaerobic
incubation of rat hepatocytes with 2A4NT or 4A2NT (Bond and Rlckert, 1981;
Mori et al., 1984a). Kozuka et al. (1978) demonstrated that the 4-nitro group
of 2,4-DNT 1s more readily reduced than the 2-nitro group.
Aerobic metabolism of 2,4-DNT by liver tissue of male and female mice,
rats, rabbits, dogs, and monkeys leads to the formation of 2,4-DNBalc, the
major oxidative product of this munitions chemical (Lee et al., 1978; Bond and
Rickert, 1981; Decad et al., 1982; Schut et al., 1985). Similarly, the
primary metabolite of 2,6-DNT produced by hepatocytes from male A/J mice and
male Fischer 344 rats was 2,6-DNBalc, which constituted 57.5 to 83.8% of all
metabolites; most (61.3 to 70.9%) was conjugated (D1x1t et al., 1986). Small
amounts (1.2 to 5.3%) of unconjugated 2-amino-6-n1trotoluene (2A6NT) also were
recovered. The amount of unchanged parent compound varied considerably, from
4.8 to 28.8% of all metabolites. In another study, three metabolites of
2,6-DNT (2,6-ONBacid, 2,6-ONBalcG, and 2A6NBadd) were recovered from the bile
and liver perfusate of male and female Fischer 344 rats (Long and Rlckert,
1982). Although equivalent amounts of each metabolite were found in the urine
of orally dosed males and females (I.e., 11 to 22% of the 14C administered),
approximately 41 to 48% and 8.5 to 11% of the radioactivity in the bile of
males and females, respectively, were associated with 2,6-DNBalcG. Amounts of
V-28
-------
CH, CH, CH, CHj
NH
f CH3
CH,
CH,
NO.
NH
NH
NH
NO,
CH.
NH,
2
CH,
NH
CH.
NH,
CH3
NHj
Figure V-2. Proposed pathway for the anaerobic metabolism of 2,4-DNT by
rat intestinal microflora.
SOURCE: ATSOR (1989).
V-29
-------
other metabolites In the bile were not quantitated, and the bile and liver
were not further analyzed. Shoji et al. (1985) proposed that
2,4-dinitrobenzylaldehyde might be an intermediate in the conversion of
2,4-DNBalc to 2,4-DNBadd In vivo; however, the aldehyde was not isolated in
the urine of dosed male Wistar rats. A subsequent study by Shoji et al.
(1987) showed that 2,4-DNBalc can be converted in vitro to 2,4-dinitrobenzal-
dehyde in the presence of microsomal and cytosolic preparations.
1
Bond et al. (1981) tracked the oxidation, conjugation, and acetylatlon of
2,4-DNT in perfused rat liver and found that the primary route of metabolism
of 2,4-ONT in both male and female tissue was through oxidation to 2,4-DNBalc
and subsequent glucuronidation to 2,4-ONBalcG. Reduction of 2,4-ONT was
minimal. Bond and Rickert (1981) and Lee et al. (1978) reported that as the
concentration of oxygen in hepatocyte incubations was reduced, the levels of
oxidized metabolites of 2,4-DNT (e.g., 2,4-DNBalc) decreased, and the amount
of reduced metabolites (e.g., 2A4NT and 4A2NT) Increased but remained low.
For all five species studied by Lee et al. (1978), reductive hepatic
metabolism of 2,4-ONT was greater In males than in females. However, the data
indicate that hepatic reduction of 2,4-ONT most likely represents a minor
metabolic pathway.
In a study by.Mori et al. (1981a), the microsomal metabolism of 2,4-ONT
was blocked by carbon monoxide and primary amines, indicating an Involvement
of cytochrome P-450. In contrast, cytosolic fractions metabolized 2,4-DNT to
2,4-DAT through the formation of aminonitrotoluenes. It was suggested that
since allopurinol blocked this reaction, cytosolic xanthine oxidase most
likely mediated the conversion of 2,4-ONT to 2,4-DAT. Bond and Rickert (1981)
\
reported that administration of Aroclor 1254, phenobarbital, or
3-methylcholanthrene to rats Induced the production of 2,4-DNBalc In
hepatocytes, but that In vitro addition of SKF 525-A or 7,8-benzoflavone
inhibited Its production. These data together Indicate that hydroxylation of
the methyl group of 2,4-DNT requires both cytochrome P-450 and P-448
monoxygenases..
V-30
-------
Sayama et al. (1989), using HPLC, showed that 2,4 DNT is metabolized to
2,4-dinitrobenzaldehyde, a potent mutagen, in the male Wistar rat. This
metabolite constituted 35% of the total metabolites of 2,4-DNT;
2,4-dinitrobenzyl alcohol constituted 29%. A hypothesis was developed to
explain the enterohepatic circulation of 2,4-dinitrobenzaldehyde. The 2,4-DNT
is metabolized by the liver to 2,4-dinitrobenzyl alcohol glucuronlde and to
2,4-dinitrobenzaldehyde and other minor metabolites, the former metabolite 1s
excreted either in the urine or in the bile; in the bile, it is metabolized by
the intestinal flora to 2,4-dinitrobenzaldehyde through the enterohepatic
circulation. The 2,4-dinitrobenzladehyde formed by direct hepatic metabolism
of 2,4-DNT is then excreted in the bile. The authors cautioned that the
formation of 2,4-dinitrobenzaldehyde is strain dependent and, therefore, may
not be found in other rat strains. ,
Mori et al. (1989) supported the finding of Sayama et al. (1989) that
dinitrobenzaldehyde 1s a metabolite of ONT and that the metabolism of DNT
isomers Is strain dependent. .Using two rat strains, Wistar and Sprague-
Dawley, the authors Investigated the metabolism of 2,4- and 2,6-DNT, 2,4- and
2,6-dinitrobenzyl alcohol, and 2,4- and 2,6-dinitrobenzaldehyde In the
microsomal and cytosol fractions of the corresponding livers. It was
concluded that dinitrobenzaldehydes are intermediate metabolites formed from
the oxidation of dinitrobenzyl alcohols to dinitrobenzoic adds, and that the
conversion of dinitrobenzaldehydes to dinitrobenzyl alcohols and vice versa is
a reversible process. The oxidation of DNT to dinitrobenzyl alcohol occurred
in the presence of microsomal cytochrome P-450, and the oxidation of the
latter to the dinitrobenzaldehydes was mediated by P-450 and NAO-dependent
alcohol dehydrogenase; oxidation of dinitrobenzaldehyde to benzole add was
mediated by NAD-dependent aldehyde oxidase. Differences in oxidation and
reduction rates were clearly seen between the two rat strains.
Figure V-3 provides a summary of the proposed metabolic pathway for the
hepatic metabolism of 2,4-DNT-.
V-31
-------
CH3
CH
Y( minor)
NO2
2-amno-4-
nttrotoiu«n«
!
CH3
NH ^
fY ^
*^J> o
NO2
2-dinitrotolu«n« 2.
1
CH3
CHj ItfOa
Nr^
4-amino-2- 2.
nitrotoiu«n« alee
1
CH3
1
CH3
amno-
2-nitrotokMn*
Nq
2.4-o1nitrob«nzy«
aiconot
N0
NQ
2.4.dinitrocenzaidenyce
alcohol glucurondt
.4-dinitrobenzoic
acid
Figure V-3. Proposed metabolic pathway for the hepatic metabolism of
2,4-ONT.
SOURCE: ATSDR (1989).
V-32
-------
VI. HEALTH EFFECTS
A. HUMANS
Since data on isomeric composition, purity and, in many cases,
concentration of DNT exposures were not reported for the epidemiologic
studies, these reviews will not be subdivided by DNT isomer. The studies of
occupational exposure to DNT are limited by the small groups of workers
studied, by exposure to more than one chemical, and by the lack of individual
exposure monitoring. As a result, many of the conclusions found in these
human studies are not definitive and require further investigation.
Chronic DNT exposure, primarily via the inhalation route (particulate or
vapor), has been characterized in munitions workers by cyanosis, vertigo,
headache, metallic taste, dyspnea, weakness and lassitude, loss of appetite,
and nausea and vomiting (Etnier, 1987; Ellis et al., 1979; McGee et al., 1942;
Perkins, 1919). Other symptoms have included pain or paresthesia in
extremities, abdominal discomfort, tremors, paralysis, chest pain, and
unconsciousness (U.S. EPA, 1986). Many of these symptoms may indicate
neurotoxicity. Recovery from moderate intoxication generally has occurred 2
to 3 days following exposure; pain in extremities has persisted several months
in some cases.
The initial clinical sign of DNT toxicity is methemoglobinemia (U.S. EPA,
1980, 1986). The presence of methemoglobin in erythrocytes leads to the
formation of aggregates of hemoglobin degradation products, called Heinz
bodies, which are readily detected and have been found following exposure to
2,4-DNT and technical grade DNT (tg-DNT) (Ellis et al., 1979; Hughes and
Treon, 1954). A hemolytlc hypochromic anemia may result, depending on the
severity of exposure. When the methemoglobin concentration of the blood is
greater than 15%, cyanosis occurs. Headache increases in intensity as the
methemoglobinemia increases In severity. Weakness and dizziness occur when
the concentration reaches 40%; at 70%, ataxia, dyspnea, tachycardia, nausea,
vomiting, and drowsiness may occur (U.S. EPA, 1980, 1986).
VI-1
-------
Percutaneous absorption is considered to be the second most likely route
of exposure to DNT for the industrial worker (U.S. EPA, 1980). Skin
contamination may result from exposure to airborne vapors and particulates as
well as from splashes and direct contact with contaminated surfaces (Levine
et al., 1985a). /
The U.S. Department of Health and Human Services (1978) Occupational
Health Guideline for DNT indicates that hematological (e.g., methemoglobin)
and liver function testing should be conducted annually for Industrial workers
exposed to DNT. In addition, the level of DNT or 2,4-d1n1trobenzo1c add
(2,4-DNBA, a principal metabolite of DNT) in the urine of Industrial workers
should be measured on a routine basis (U.S. Department of Health and Human
Services, 1978; Woollen et al., 1985; Levine et al., 1985a). However, since
it is hot currently possible to estimate the absorbed dose of DNT from
2,4-DNBA excretion, It had been suggested that a human dosing study provide
;=-
these data. However, such a dosing study would be difficult to complete.
Ellis et al. (1979) indicated that the most objective hematologic parameters
for detecting exposure to toxic doses of 2,4-DNT consist of Heinz body
formation and retlculocyte counts. Heinz bodies persist and are easily
•\ .
detected, and Increased reticulocytes indicate a compensatory response to
erythrocyte destruction prior to frank anemia. Ellis et al. (1979) considered
the measurement of methemogloblnemia to be too transient, especially for low
levels of exposure.
^
McGee et al. (1942) reported 36 (23% of the study cohort) cases of anemia
and 52 and 55 cases (34 and 36% of the study cohort, respectively) of cyanosis
and pallor among 154 employees of a munitions manufacturing facility
processing primarily 2,4-DNT (Tables VI-1 and VI-2). Levels of exposure and
purity of the 2,4-DNT were not reported. After removal from exposure,
recovery fro* anemia required more than 2 months, although cyanosis and pallor
disappeared with 2 days. Two Individuals developed jaundice, acute liver and
kidney damage, and hyperchromlc macrocytic anemia within 3 to 4 weeks
following exposure; no instances of permanent physical impairment were
identified. Workers were exposed to DNT dust, and absorption was reported
VI-2
-------
Table VI-1. Symptoms Exhibited by 2,4-DNT-Exposed Workers'
Symptom
Unpleasant taste in mouth
Weakness
Headache
Loss of appetite
Dizziness
Nausea
Insomnia
Pain in extremities
Vomiting
Loss of weight
(5 Ibs or more)
Diarrhea
Number
96
78
76
72
68
57
57
40
29
10
8
Percentage
62
51
49
47
44
37
37
26
19 .
6.5
5.2
'Data based on 154 exposed workers.
SOURCE: McGee et al. (1942).
VI -3
-------
Table VI-2. Clinical Signs in 2,4-DNT-Exposed Workers1
Symptom
Pallor
Cyanosis
Anemia
Leukocytosis
Hypotension
c
Dermatitis
Leukopenia
Acute toxic hepatitis/
jaundice
Number
55
52
36
19
9
6
5
2
Percentage
36
34
23
12
5.8
3.9
3.2
1.4
"Data based on 154 exposed workers.
SOURCE: McGee et al. (1942).
VI-4
-------
to be primarily via inhalation; however, six cases of compound-related
dermatitis were reported. When hygienic measures were followed, the
incidence of DNT intoxication decreased.
Levine et al. (1986a,b) reported excessive mortality from ischemic heart
disease and residual diseases of the circulatory system. Ischemic heart
disease was characterized by myocardial infarction, coronary thrombosis, or
occlusion or acute coronary or myocardial insufficiency. Residual circulatory
system diseases included congestive heart failure, cardiac arrest, and
arteriosclerosis. A Standard Mortality Ratio (SMR) of 141 (p <0.01) was
reported for workers exposed to 2,4-DNT (90% pure) and tg-DNT (76% 2,4-DNT,
19% 2,6-DNT, 5% other isomers) at two separate manufacturing facilities
following a 15-year latency period. Evidence of a relationship between
duration (>5 months) and intensity of exposure to DNT and increased mortality
from ischemic heart disease was suggested. Mortality from Ischemic heart
disease, reported to be related to coronary atherosclerosis, was less than
expected prior to the 15-year latency period. Since the heart disease
mortality risk of workers is usually lower than that of the general population
because of the "healthy worker" effect, it is considered unlikely that risk
factors independent of DNT exposure could explain the observed excess of heart
disease. Although worker exposure included other chemical agents (nitric and
sulfuric acid, toluene, mononitrotoluene), DNT was the only exposure common to
both plant cohorts. In addition, duration and concentration of DNT exposure,
although estimated, correlated with the extent of mortality from ischemic
heart disease. Levine et al. (1986b) stated that compound-related
cardiovascular lesions were not reported in bioassays with rats or mice;
however, these species were considered to be resistant to naturally occurring
or experimentally induced atherosclerosis. This study was conducted to
determine whether the carcinogenicity of DNT in rodent bioassays had relevance
for humans. No evidence of a carcinogenic effect was exhibited in these
cohorts. However, the adequacy of DNT exposure and latency for the
development/ of carcinogenic Tesions was questioned by the study author; it was
concluded that while the results do not exclude the possibility that DNT may
cause human cancer, the risk of th.is occurrence is small.
VI-5
-------
A more recent retrospective cohort mortality study of ammunition plant
workers exposed to ONT was not consistent with the finding of Levine et al.
(1986a,b). Stayner et al. (1989) found that mortality from ischemic heart
disease (SMR - 0.95) and cerebrovascular diseases (SMR - 0.98) in DNT-exposed
workers was similar to that of nonexposed populations. The study site was one
of the facilities included in the Levine et al. studies; however, the workers
were from different time periods. Levine et al. (1986a,b) studied workers
employed during 1940 to 1950, and Stayner et al. (1989) studied employees who
were exposed during 1949 to 1980. Stayner et al. (1989) also analyzed workers
employed during a period time that overlapped Levine's studies and did not
,. - . . v -
observe an excess of ischemic heart disease. During the same study, and also
contrary to the results reported by Levine et al. (1986a,b), Stayner (1989)
detected a greater than twofold increase (SMR -2.67) in cancer of the liver
and biliary passages in ONT-exposed workers. Even though the excess cancers
were based only on six cases and .were not statistically significant, they were
•
nearly significant (p -0.054) and consistent with increased liver and bile
duct cancers In DNT-exposed rodents.
Woollen et al. (1985) indicated that 61111s et al. had conducted an
epidemiologic study of the carcinogenic potential of explosives workers using
a cohort that included persons exposed to ONT (unpublished). The results did
not indicate an excess of cancer at any site in the body; no further data were
provided.
Alcohol consumption has a synergistic effect on the toxicity of 2,4-DNT,
increasing susceptibility to ONT-induced cyanosis. Exposed workers who
ingested alcohol shortly after exposure experienced chest pain, palpitations,
head "fullness," and severe, acute illness (U.S. EPA, 1980).
Floret (1929, as cited in U.S. EPA, 1986) reported a case of 2,4-DNT
intoxication in which an Industrial worker exhibited tremors of the
extremities, head, and tongue; visual disturbances; and impaired reflexes. No
further information was reported.
VI-6
-------
Ahrenholz (1980)^studied the reproductive effects of exposure to mixed
isomers of DNT and toluene diamine (IDA) in 30 male workers employed at a
chemical manufacturing facility. Individuals were divided into three groups:
those with the highest current potential exposure to DNT (n - 9, Group 1);
those with intermittent exposure over several years but none in the preceding
2 years (n - 12, Group 2); and the control group with no known exposure to ONT
(n =• 9, Group 3). The DNT and TDA concentrations in workroom air ranged from
0.013 to 0.42 mg/m3 and from 0.008 to 0.39mg/m3, respectively, during the
initial survey, and from undetectable levels to 0.10 mg/m3 and from
undetectable levels to 0.38 mg/m3, respectively, during the followup survey.
No differences were detected between groups with respect to renal and hepatic
profiles or selected endocrine assays. Results indicated a significant "
(p <0.05) reduction in the sperm counts of exposed workers (Group 1)
(55.9 x 106) when compared with controls (156.8 x 10*); however, this
significant reduction in sperm counts has been questioned by Hamill et al.
(1982), who considered the sperm counts of the control group to be abnormally
high. Control sperm counts would be expected to be In the range of 80-100 x
106. A slight Increase in the number of spontaneous abortions was also
observed among the wives of exposed workers (Groups 1 and 2). However,
Ahrenholz (1980) did hot consider this effect to be due to chemical exposure
alone.
In response to the Ahrenholz (1980) study, Hamill et al. (1982) conducted
a more extensive survey (84 exposed workers, 119 controls) of male workers
exposed to DNT and TDA in a similar chemical manufacturing facility. Specific
exposure concentrations were not reported, although the authors indicated that
exposure levels were below the Occupational Safety and Health Administration
(OSHA) threshold limit value for DNT (1.5 mg/m3). Individuals were divided
into groups according to intensity, frequency, and recency of chemical
exposure. No?differences in sperm counts or morphology, serum follicle-
stimulating hormone (FSH) concentration, testicular volume, or fertility wtrt
observed among control or exposed workers of any group.
Ahrenholz and Meyer (1982) conducted a followup study of the reproductive
effects of exposure to ONT and TDA in 52 male workers employed at a chemical
VI-7
-------
factory. Exposure categories were based on quantisation and duration of
exposure. Area sampling of DNT and IDA ranged from 0.026 to 0.890 mg/mj
(mean, 0.207 mg/m1) and below detection limits to 0.687 mg (mean,
0.221 mg/m1), respectively; personal sampling data were found to be invalid.
No significant differences in sperm counts or morphology, liver function, or
renal function tests were observed among, control or exposed DNT workers of any
category. These results disagree with those reported by Ahrenholz (1980).
No significant reductions in fertility were observed in workers from
three separate factories exposed to ONT, TDA, sulfuric add, nitric add, and
nitrobenzene (Levine et al., 1985b). Exposure concentrations, duration of
exposure, and chemical purity were not reported. In general, small exposure
populations and lack of individual exposure monitoring limit the power of
these studies to detect adverse effects on human reproduction.
Urinary metabolites of dinitrotoluenes have been monitored in
occupationally exposed workers (Woollen et al., 1985; Levine et al., 1985a;
Turner et al., 1985; Turner, 1986). (A more complete review is found in
Chapter V, Pharmacoklnetics.)
Woollen et al. (1985) monitored urinary levels of 2,4-DNBA in 33
explosives factory, workers exposed to tg-DNT. Workers were generally exposed
to atmospheric levels of 0.03 to 0.1 mg/m1, although levels between 0.2 and
2.68 (0.04 ± 0.65) mg/m1 were measured in more concentrated processing areas.
Exposure levels were below the OSHA 8-hour Time-Weighted Average (TWA)
exposure of 1.5 mg/m1. These atmospheric levels of DNT were not considered to
be sufficient to account for the observed excretion of 2,4-DNBA. Dermal
absorption of DNT was considered to be a major route of uptake secondary to
that of inhalation. A concurrent study of five Individuals Indicated that th«
highest urinary 2,4-DNBA levels were measured in end-of-shift specimens.
Blood levels of DNT peaked at the end of the work shift at 250 ng/mL.
Although Woollen et al. (198S) reported that 2,4-dinitrobenzyl alcohol, a
major urinary metabolite and precursor of the proximal carcinogen in the rat.
could not be detected in the urine of exposed workers, this urinary metabolttt
VI-8
-------
was dctectsd and documented in the studies cunuucLeu by Levine et al. (i965a),
Turner et al. (1985), and Turner (1986) in DNT manufacturing plant workers
(see Chapter V, Pharmacokinetics). These latter studies also indicated that
the metabolic pathway for bioactivation of 2,6-DNT occurs to a much lesser
extent in humans as compared with rats. The presence of unmetabo11 zed 2,6-DNT
in the urine of these workers indicates that not all absorbed 2,6-DNT is
available for bioactivation. Since 2,6-DNT is genotoxic and active as a
carcinogenic initiator, the absence of evidence of reduced metabolites of
2,6-DNT in the urine of exposed humans may be an important difference
concerning the risk of DNT-exposed humans.
Levine et al. (1985a) estimated the atmospheric concentration of 2,4- and
2,6-DNT combined to be'between 0.05 and 0.59 mg/m1 in a DNT manufacturing
plant. Seventy-six percent of the DNT in the breathing zone was the 2,4-DNT
isomer. Wipe samples of surfaces in the work area contained up to 433 jig 2,4-
DNT, and the largest quantity of DNT recovered from the skin was 180 \ig
(isomer not specified). Clinical effects of exposure for the 17 persons (13
workers, 4 office employees) undergoing analysis of urinary metabolites were
not reported. Urinary excretion of DNT metabolites appeared to increase
during the workday and to decline to nearly undetectable levels by the start
of the following workday, suggesting rapid absorption of DNT following
inhalation (Turner, 1986). The proportion of metabolites excreted varied
considerably between all workers and within individuals on any one day (Levine
et al., 1985a). This was attributed to differences in exposure or to
technical error or differences in absorption, metabolic transformation, and
excretion.
B. ANIMAL EXPERIMENTS
1. Short-term Exposure (<4-week studies)
a. Acute
The acute toxicity data are summarized In Table VI-3. When administered
via the oral route, 2,4-DNT was moderately toxic to mice and highly toxic to
VI-9
-------
Table VI-3. Acute LDSO Values for 2,4- and 2,6-DNT in Mice and Rats
Species Strain Sex
. House Swiss . Male
Fe*ale
Mouse CF-1 r-'
Rat CD Male
Female
Rat Spranue-Dawley Nat*
Route Vehicle
Oral Peanut oi I
Oral Peanut oil .
Oral
Oral Peanut oil
- Oral Peanut oil
Oral
LDTff
2,4 DMT
1.954 t 68
(1.848 - 2,178)*
1.340 t 67
(1,205 - 1.50Q)
1.630
(1,180 • 2.240)
568 t 59
(434 • 705)
650 t 49
(520 - 743)
270
(180 - 400)
(ma/kg)
2.6 DNT
621 t 51
(488 - 721)
807 t 35
(735 - 693)
1.000
(590 - 1.700)
535 t 58
(397 - 646)
795 * 22
(744 - 844)
180
(130 • 240)
Reference
v_
Lee et al. (1975)
Lee et al. (1975)
Vernot et al. (1977)
Lee et al. (1975)
Lee et al. (1975)
Vernot et al. (1977)
' lite values In parentheses are 95X confidence limits.
* Data not reported.
-------
rats. The oral LD,a values for 2,4-DNT range from 1,340 to 1,954 mg/kg for
mice and from 270 to 650 mg/kg for rats. Toxic signs observed in both species,
were ataxia and cyanosis; death usually occurred within the first 24 hours or
not at all. Surviving animals recovered completely within 48 hours. No
treatment-related gross pathology was seen in animals found dead (Lee et al.,
1975; Vernot et al., 1977).
The acute toxicity of 2,6-ONT appears to be less species-specific, with
oral L0,0 values ranging from 621 to 1,000 mg/kg in mice and from 180 to
795 mg/kg in rats; male rats were slightly less tolerant than females. Times
to death, recovery, and gross pathology were similar for 2,6-ONT and 2,4-DNT
(Lee et al., 1975; Vernot et al., 1977). ' ,
b. Primary eve and skin irritation and skin sensitization
Neither 2,4- nor 2,6-ONT produced ocular irritation when instilled as a
50% paste in peanut oil (volume not reported) into the eyes of groups of six
New Zealand white rabbits (Lee et al., 1975).
In a primary skin Irritation test, groups of six New Zealand White
rabbits (sex not reported) received a single dermal application of 2,4-DNT
(98% pure) or 2,6-ONT (>99% pure) as a 50% paste with peanut oil. Test sites
were scored at 24 and 72 hours. Both isomers were classified as very mild
skin irritants (mean primary irritation score was 0.21 and 0.25 for 2,4- and
2,6-DNT, respectively) (Lee et al., 1975).
In a skin sensitization test, groups of 10 guinea pigs (sex and strain
not reported) were exposed to either 2,4- or 2,6-ONT as a 5% solution; 2,4-DNT
was not a skin sensitizer, and 2,6-DNT was a mild sensitizer producing a
response in two animals (Lee et al., 1975).
VI-11
-------
c. Subacute
1) 2.4-DNT
Lane et al. (1985)-orally administered to groups of 10 male Sprague-
Dawley rats, by gavage, 2,4-DNT (purity-not specified) 1n corn oil at doses of
0, 60, 180, or 240 mg/kg/day for 5 consecutive days. Because of high
mortality at the high dose, another group of 15 rats was started at
240 mg/kg/day on the same schedule; 8 of the 15 animals In this group died
within 2 weeks after receiving the first dose. No other deaths were seen.
Rats at the mid- and high-dose levels exhibited cyanosis; body weight loss was
seen at the high dose. This study is discussed in Section VLB.3,
Reproductive Effects.
Groups of 10 female CD-I mice were orally administered, by gavage,
2,4-DNT (purity not specified) In corn oil dally for 8 consecutive days,at 0,
310, 525, 1,250, 2,500, or 3,500 mg/kg body weight (bw). No treatment-related
mortality was seen at the low dose. Mortality occurred In six animals dosed
at 525 mg/kg. All mice dosed at the higher levels (1,250 to 3,500 mg/kg) died
by study day 3. The mean body weight of the surviving rats dosed at 525 mg/kg
was significantly (p <0.007) lower on day 8 of treatment and also on day 8
posttreatment. Toxic signs observed were lethargy, dyspnea, rough hair coat,
hunched posture, tilted head, tremors, ataxia, and prostration. The NOAEL was
310 mg/kg (Smith, 1983).
McGown et al. (1983) fed groups of five male and five female Sprague-
Dawley rats diets containing 0, 900, 1,200, 1,900, or 3,000 mg 2,4-DNT/kg (97%
2-4 DNT, 2% 2,6-DNT, IX unspecified) for 14 days. This represents an intake
of 0, 45, 60, 94, or 143 mg/kg/day, based on an average body weight of 0.25 kg
and an average total dietary Intake of 0.0175 kg/day (U.S. EPA, 1986).
Hematology, clinical chemistry, urinary parameters, and histopathology were
*
evaluated^ No treatment-related clinical signs of toxlcity were observed.
Both the control and treated groups gained weight consistently during the
dosing period; however, the rate of weight gain was decreased in a dose-
related manner in males and females fed 2,4-DNT. Food consumption was also
VI-12
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depressed in the same manner. No treatment-related effects were seen in
hematology or urinary parameters. Serum cholesterol levels were significantly
(p <0.05) higher than in the controls in all dosed males and females. Serum
glucose levels Increased in a dose-related manner in both sexes fed DNT;
however, the increase was significant (p <0.05) only in females fed the
143-mg/kg/day diet. The albumin/globulin ratio was significantly (p <0.05)
higher than controls in females fed diets containing 60 or 143 mg/kg/day.
Alanine aminotransferase levels were significantly (p <0.05) higher than
control groups at all levels in males; no significant differences were seen in
females. Histopathology revealed hyaline droplet formation (not
dose-dependent) in the epithelium of the proximal convoluted tubules of the
kidneys in both sexes, with males being more susceptible than females.
Oligospermatism with degenerative changes in the testes of male rats occurred
in a dose-dependent manner. Based on decreased body weight gain, decreased
food consumption, and changes in serum chemistry levels in males and females
and testicular lesions in males, the LOAEL was 45 mg/kg/day, the lowest dose
tested.
Groups of 10 male Sprague-Oawley rats were maintained on a diet
containing 0, 0.1, or 0.2% 2,4-DNT (50 or 100 mg/kg/day, based on Lehman,
1959) for 3 weeks. The final body weights were significantly (p <0.01) lower
in treated animals.at both dietary levels when compared with controls. No
systemic toxicity was seen. This study is discussed in Section VLB.3,
Reproductive Effects (Bloch et al., 1988).
Lee et al. (1978) studied the toxic effects of the subacute (4-week)
dietary administration of 2,4-ONT in Swiss mice, CD rats, and beagle dogs.
The study in rats was also published by Lee et al. (1985), and the study in
dogs was also published by Ellis et al. (1985).
In the study with mice and rats, groups of eight animals/sex/species were
fed diets containing 0, 0.07, 0.20, or 0.70% 2,4-ONT (98% pure) for 4 weeks.
This represents a corresponding dally intake of 0, 47, 137, or 413 mg/kg/day
for male mice; 0, 52, 147, or 468 mg/kg/day for female mice; 0, 34, 93, or 266
mg/kg/day for male rats; and 0, 38, 108, or 145 mg/kg/day for female rats
VI-13
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(U.S. EPA, 1986). At the end of 4 weeks, four animals/group/species were
sacrificed. .Treatment was discontinued and the surviving animals were
observed for 4 more weeks and then sacrificed to study the reversibility of
the adverse effects. Hematology and clinical chemistry tests were conducted
at 0, 4, and 8 weeks. At necropsy, terminal body weights were measured; the
liver, spleen, heart, and kidneys were weighed and organ-to-body weight ratios
were calculated. Selected organs were examined histologically. No mortality
occurred in mice. The low- and mid-dose levels were nontoxic, and mice at the
high-dose level showed slight body weight loss. No abnormalities were seen in
blood parameters, organ weights, or gross pathology. Histopathology revealed
a mild depression of spermatogenesls in two males at the high dose. After 4
weeks, the mice recovered. The NOAEL was 137 mg/kg/day in males and 147
mg/kg/day in females. Based on body weight loss in males and females and
depression of spermatogenesls in males, the LOAEL was 413 mg/kg/day in males
and 468 mg/kg/day in females.
2,4-DNT was toxic to both sexes of rats at all levels. At the high dose,
two males and two females died; the surviving rats showed body weight loss and
decreased food consumption. Rats dosed at the lower levels exhibited a slight
depression in body weight gain and food consumption. No consistent changes
were observed in hematology or clinical chemistry parameters. A significant
(p <0.05) increase was seen in both the absolute and relative liver weights of
males fed 93 mg/kg/day and females fed 38 or 145 mg/kg/day. Histopathology
revealed mild to moderate hemosiderosis In the spleen of males fed 93 or 266
mg/kg/day and females fed 108 or 145 mg/kg/day. Males at the high dose (266
mg/kg/day) showed aspermatogenesis and testicular atrophy. After 4 weeks,
there was only partial recovery; rats regained the weight lost, but the
hemosiderosis and testicular lesions were not reversible. Based on body
weight loss and decreased food consumption in both sexes, the LOAEL was 34
mg/kg/day In male rats and 38 mg/kg/day in female rats; a NOAEL was not
established (Lee et al., 1978).
In the study with dogs, groups of two males and two females were given
2,4-DNT in capsules at doses of 0, 1, 5, or 25 mg/kg/day for 4 weeks. Oogs
were observed daily, and body weight and food consumption were measured
VI-14
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weekly; blood samples collected prior to initiation and at termination were
analyzed for hematology and clinical chemistry parameters. After 4 weeks of
exposure, one male and one female from each group were sacrificed; the
remaining animals were maintained on a standard diet and sacrificed following
an additional 4 weeks. At termination, the liver, spleen, kidneys, adrenals,
and gonads were weighed; organ-to-body weight ratios were calculated; and a
bromosulfophthalein (BSP) retention test was performed. All major tissues
underwent histopathologlcal examination. No treatment-related toxicity was
observed in dogs given 1 or 5 mg 2,4-DNT/kg/day. At the high dose
(25 mg/kg/day), dogs showed signs of toxicity after 12 days of treatment.
Toxic signs included decreased food consumption, weight loss, yellow stain on
and near hind legs, pale gums, neuromuscular incoordlnation, and paralysis.
No significant changes were observed 1n the hematology or clinical chemistry
parameters, and 2,4-ONT did not cause any retention of BSP. Gross necropsy
showed that the high-dose dogs were in fair to poor nutritional condition with
little or no body fat. Treatment had no effect on organ weights.
Histopathology of the high-dose dogs revealed hemoslderosls in the liver,
cloudy swelling and tubular degeneration of the kidneys, and lesions of the
brain (gliosis, edema, and demyelination of the cerebellum, brain stem, and
spinal cord) in both sexes and aspermatogenesis in males. After 4 weeks, dogs
showed partial recovery. Two dogs kept for 8 months completely recovered.
Based on decreased .weight gain, decreased food consumption, neurotoxic signs,
and histopathology, the LOAEL was 25 mg/kg/day for males and females, and the
NOAEL was 5 mg/kg/day (Lee et al., 1978).
In summary, dogs were the most sensitive species tested; a dose of
5 mg/kg/day had no effect, and a dose of 25 mg/kg/day was toxic. In rats, -
dietary administration of 34 mg/kg/day to males and 38 mg/kg/day to females,
the lowest dose tested, was slightly toxic. Mice were much less affected,
since administration of 137 mg/kg/day to males and 147 mg/kg/day to females
had no adverse effects, and administration of high doses (413 mg/kg/day to
males and 468 mg/kg/day to females) was not as toxic as much lower doses given
to rats or dogs. Typical toxic effects observed in all species were decreased
body weight gain and food consumption in both sexes and decreased
spermatogenesis in males. Dogs showed neuromuscular symptoms of
VI-15
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incoordination and paralysis. After 4 weeks, mice recovered completely, and
rats and dogs recovered only partially. Based on the results of this study,
the NOAELs and LOAELS shown In Table VI-4 can be established for 2,4-DNT
following subacute exposure.
2) 2.6-DNT '
Lee et al. (1976) assessed the subacute (4-week) toxiclty of 2,6-DNT
(>99% pure) in Swiss mice, CD rats, and beagle dogs. The basic design and
procedure for these experiments were similar to those for studies conducted
with 2,4-DNT (discussed above). 2,6-DNT was administered In the diet to mice
and rats at levels of .0.01, 0.05, or 0.25% for 4 weeks. This represents a
corresponding daily intake of 0, 11, 51, or 289 mg/kg/day for male mice; 0,
11, 55, or 299 mg/kg/day for female mice; 0, 7, 35, or 145 mg/kg/day for male
rats; and 0, 7, 37, or 155 mg/kg/day for female rats. Dogs were given doses
of 0, 4, 20, or 100 mg/kg/day in capsules for 4 weeks.
; • ^ ' .
No treatment-related effects were observed in mice (elght/sex/dose) fed
the 11-mg/kg/day diet. No treatment-related deaths occurred; however, all
male mice dosed at 51 mg/kg/day died by week 3 as a result of fighting. The
mid- and high-dose levels caused decreased body weight gain and food
consumption. Extramedul1ary hematopoiesis in the spleen and the liver was
increased in both sexes at the high-dose level; males fed the high dose showed
aspermatogenesis and testicular atrophy. After a 4-week recovery period
following discontinuation of treatment, the testicular lesions were not seen;
however, the extramedul1ary hematopoiesis in the liver or spleen continued to
appear in treated mice. The NOAEL was 11 mg/kg/day for males and females.
Based on decreased body weight gain and food consumption, the LOAEL was
51 mg/kg/day 1n males and 55 mg/kg/day in females.
No treatment-related deaths were observed in rats (elght/sex/dose) fed
2,6-DNT. Treated and control rats gained weight during the dosing period.
However, the rate of weight gain was decreased in a dose-related manner in
treated males and females. Food consumption was also depressed in a dose-
related manner. Histopathology revealed hematopoiesis in the spleen and liver
VI-16 ,
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Table VI-4. Adverse Effect Levels for. 2,4-ONT Following Subacute Exposure
Parameter fmq/ko/dav)
NOAEL LQAEL
Species Males Females Males Fema-les Basis for LOAEL
Mouse 137 147 413 468 Weightless.
Rat -- -- 34 38 Decreased body weight gain and
food consumption.
Dog 5 5 25 25 Decreased body weight gain and
food consumption; neurotoxic
signs; and lesions in the
brain, liver, kidneys, and
testes.
VI-17
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of both sexes and degeneration of spermatogenesis in males. Allowed to
recover for 4 weeks, most rats regained their weight, but only partial
recovery of the spleen, liver, and testicular lesions was seen. Based on
decreased body weight gain and decreased food consumption at the lowest dose
tested, the LOAEL was 7 mg/kg/day for males and females; a NOAEL was not
established. •
Dogs given 4 mg 2,6-DNT/kg/day showed no signs of toxicity. At doses of
20 or 100 mg/kg/day, 2,6-ONT Induced body weight loss and reduced food
consumption. Dogs exhibited listlessness, incoordination, lack of balance,
pale gums, dark urine, and weakness (particularly of the hind limbs).
Laboratory tests showed anemia in these dogs with decreased hematocrit,
hemoglobin concentration, and compensatory reticulocytosis. Histopathology
revealed extramedul1ary hematopoiesis in the liver and spleen and bile duct
hyperplasia in both sexes, and degeneration of spermatogenesis in males.
After 4 weeks of recovery, dogs showed some improvement, with lesser amounts
of extramedul1ary hematopoiesis and testicular lesions. Two dogs given 100 mg
0 , i
2,6-DNT/kg/day and allowed to recover for 19 weeks showed complete recovery.
The NOAEL was 4 mg/kg/day. Based on decreased body weight gain and food
consumption, the LOAEL was 20 mg/kg/day for both males and females.
In summary, the effects produced by 2,6-DNT were very similar to those
seen in parallel studies with the 2,4-DNT isomer. Dogs appeared to be the
most sensitive, and mice were the least affected; rats were somewhat less
sensitive to 2,6-DNT with a NOAEL of 7 mg/kg/day. Treatment-related effects
observed in all species Included decreased body weight gain and food
consumption, hematopoiesis in the spleen and liver, and degeneration of
spermatogenesis. Dogs exhibited clinical signs of central nervous system
toxicity that were not seen in rodents. Based on the results of this stud/.
the NOAELs and LOAELs shown in Table VI-5 can be established for 2,6-DNT
following subacute exposure.
The Chemical Industry Institute of Toxicology (CIIT, 1977) conducted a
30-day toxicity study in Fischer .344 rats with technical grade DNT (76.4%
VI-18
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Table VI-5. Adverse Effect Levels for 2,6-DNT Following Subacute Exposure
Parameter (mq/ko/dav) .
NOAEL LQAEL
Species Males Females Males Females Basis for LOAEL
Mouse 11 11 51 55 Decreased body weight gain and
food consumption.
Rat -- ,. -- 7 7 Decreased body weight gain and
food consumption.
Dog 4 4 20 20 Clinical signs, weight loss,
and lesions in the spleen,
liver, and testes.
VI-19
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2,4-DNT, 18.83% 2,6-DNT, 4.6% other isomers). Groups of 10 male rats and
10 female rats were fed diets containing 0, 37.5, 75, or 150 mg ONT/kg/day for
30 days. Animals were observed dally, and body weight and food consumption
were measured weekly. Blood samples collected from females on day 27 and
from males on day 28 were analyzed for methemoglobin, reticulocytosls, and
Heinz body formation. At termination, gross pathology was evaluated, but no
histopathology was conducted. All animals survived the 30 days of treatment.
Urine stains on the fur were seen In six rats in the high-dose group. Two
females in the mid-dose group developed alopecia around the eyes; no clinical
signs were observed at the low dose. The mean body weights of females in the
high-dose group and of males in all groups were significantly (p <0.05) lower
than those of the controls. Suppression of body weights occurred earliest and
to the greatest extent in rats fed the high-dose diet. The mean food
consumption was slightly lower for rats in the low-dose group, moderately
lower for rats in the mid-dose group, and markedly lower for rats in the
high-dose group. A significant (p <0.05), dose-related Increase in mean
values was observed for methemoglobin, retlculocyte counts, and Heinz body
formation, with the exception of mean percent methemoglobin for females in the
mid-dose group. Methemoglobin values for females in the low-dose group and
for males and females in the high-dose group were significantly (p <0.05)
higher than the control values. Treatment-related gross pathological
alterations seen in rats in the high-dose group included discoloration,
enlargement, and irregular surfaces of the spleen in both sexes, and
"discoloration of the kidneys in males. In addition, males at all dietary
levels had livers with discolorations and/or surface irregularities. Based on
decreased body weight gain and food consumption, blood effects, and gross
pathological changes In male rats, the LOAEL was 37.5 mg/kg/day, the lowest
dose tested.
3. ta-DNT
No subacute studies on tg-DNT were found in the available literature.
VI-20
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2. Longer-term Exposure
a. 13-Week studies
1) 2.4-DNT
The subchronic toxicity of 2,4-DNT was evaluated in 13-week studies with
mice (Hong et al., 1985), rats (Lee et al., 1985), and dogs (Ellis et al.,
1985). These studies also were reported by Lee et al. (1978).
Hong et al. (1985) fed groups of 16 male and 16 female CD-I mice diets
containing 0, 0.07,'0.20, or 0.70% 2,4-DNT (98% pure) for 13 weeks. This
represents a corresponding daily intake of 0, 47, 137, or 413 mg/kg/day for
males and 0, 52, 147, or 468 mg/kg/day for females. Mice were observed daily,
and body weight and food consumption were recorded weekly. At the end of 13
weeks, four males and four females were sacrificed; treatment was discontinued
for the remaining animals. These animals (recovery group) were observed for
an additional 4 weeks and were then sacrificed. At termination, blood was
collected for a standard battery of hematology and clinical chemistry tests.
The liver, spleen, kidneys, heart, and brain were weighed, and organ-to-body
weight ratios were calculated. Major tissues from all animals underwent
histopathological examination. The results of this study are summarized in
Table VI-6. A total of five mice died during the study. A dose-dependent
decrease in body weight gain was observed for males, and decreased food
consumption was seen in males in the high-dose group. Females in the
high-dose group exhibited decreased body weight gain and food consumption.
Both sexes of mice fed the high-dose diet showed anemia characterized by a
decrease in erythrocyte count, hematocrit, and hemoglobin concentrations,
concurrent with reticulocytosis. Histopathology revealed mild hepatocellular
dysplasia and pigmentation in Kupffer cells of .the liver in both males and
females, and mild degeneration of seminiferous tubules in males in the
high-dose group. Mild hepatocellular dysplasia was also seen in some males
(number not specified) in the mid-dose group. After 4 weeks, mice recovered
completely. The LOAEL was 47 mg/kg/day, based on body weight loss in males,
and 52 mg/kg/day in females; a NOAEL was not established.
VI-21
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Table VI-6. Summary of Subchronic Toxicity of 2,4-DNT in Mice
.Hales
Parameter - 2.4-ONT In feed (X): 0 0.07 0.2 0.7
(mg/kg per day) : 0 47 137 413
Average feed intake (g/day/mouse) - 2.3 2.2 2.2 1.9
Total weight gain (g/mouse) 5.0 3.3 1.9 -2.9
Number of deaths/total number of mice 0/8 1/8 0/8 2/8
Hematology
Anemia • +
Lesions
Testlcular degeneration* +
Hepatocellular dysplasla1 • + +
Pigment deposition +
Females .
0 0.07 0.2 0.7
0 52 147 468
2.2 2.1 2.1 1.7
2.9 4.1 3.5 -0.3
0/8 0/8 0/8 -2/8
+•
•*•
+
'+ indicates positive finding of hematologic change or lesion;.these changes were not exhibited in control or
low-dose males or control, low-, or mid-dose females.
SOURCE: Hongetal. (1985).
VI-22
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Lee et al. (1985) reported the results of a subchronic toxicity study
with CD rats fed diets containing 0, 0.07, 0.20, or 0.70% 2,4-DNT (98% pure)
for 13 weeks. This represents a corresponding daily intake of 0, 34, 93, or
266 mg/kg/day for males and 0, 38, 108, or 145 mg/kg/day for females. The
basic design and procedure for these experiments with rats were similar to
those described for mice (Hong et al., 1985). The results of the study with
rats are summarized in Table VI-7. 2,4-DNT administered at lower levels
.produced minimal adverse effects; administration at higher levels caused
severe adverse effects. All high-dose females died within 3 weeks; one male
at the mid-dose level and six at the high-dose level died between weeks 4 and
13. Dose-dependent decreases in body weight gain and food consumption were
observed in both sexes. Orange to yellow colored urine stains were observed
on the fur of high-dose rats, and one male rat had widespread and stiff hind
legs. Hematologic Indices (erythrocyte count, hematocrit, hemoglobin)
indicated anemia with concurrent reticulocytosis in mid- and high-dose males
and females. Absolute liver and kidney weights were slightly increased in
mid-dose males; relative liver, kidney, and brain weights of these animals
were significantly Increased statistically. Relative increases in organ
weights of other dosed animals were consistent with body weight reduction in
these animals. Hlstopathology revealed splenic hemosiderosis in mid- and
high-dose males and females. Spermatogenesis was decreased in mid-dose males
and was completely, arrested in high-dose males. One high-dose male showed
some signs of neuromuscular effects with demyelination in the cerebellum and
brain stem. The LOAEL was 34 mg/kg/day in males and 38 mg/kg/day in females
based on decreased body weight gain and food consumption; a NOAEL was not
established. Hemosiderosis and testicular lesions of mid- and high-dose
animals were not reversible following the 4-week recovery period. Gliosis and
demyelination of the cerebellum of one mid- and one high-dose male were also
found in these recovery animals.
El11s et al. (1985) gave 2,4-DNT in capsules to groups of two male and
two female dogs at doses of 0.,. 1, 5, or 25 mg/kg/day for 13 weeks. At the end
of 13 weeks, one pair from each group was sacrificed. The remaining pair was
maintained on a control diet and observed for an additional 4 weeks, and was
VI-23
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Table VI-7. Summary of Subchronic Toxicity of.2,4-DNT in Rats
Hales
Parameter
2.4-ONT In feed (X): 0 0,.07 0.2 0.7
(mg/kg per day): 0 34 93 266
Females
o o.or 0.2 0.7
0 38 108 145
Average feed intake (g/day per rat)
Total weight gain (g/rat)
Number of deaths/total number of rats
Hematology
Reticulocytosis*
Anemia*
Lesions
Organ welght changes*
Abnormal pigments in spleen*
Spermatogenesis*
Oemyelination (cerebellum, brain stem)*
24.3 21.6 19.6 . 10.6
138 35 -11 -157
0/8 0/8 1/8 6/8
17.0 14.3 14.4 5.0
35 2 -10
0/8 0/8 0/8 8/8
a+ indicates positive finding of hematologic change or lesion; these changes were not exhibited in control
or low-dose males or females.
"+ indicates decreased spermatogenesis; ++ indicates aspermatogenesls. '
SOURCE: Lee et al. (1985). . .
VI-24
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than sacrificed. Dogs were observed daily, and body weight was measured
weekly. Hematology and clinical chemistry tests were conducted prior to
treatment and at termination. A BSP retention test was conducted at
termination. 2,4-DNT did not induce toxicity in the low- and mid-dose groups.
In the high-dose groups, 2,4-DNT was toxic after 12 to 22 days and was lethal
after 22 or more days. The primary target organs were the neuromuscular
system, erythrocytes, and testes. Dogs showed great variation in individual
susceptibility. All affected dogs exhibited decreased food consumption,
weight loss, urine stains on the fur, pale gums, neuromuscular incoordination,
and paralysis. Hematologic indices indicated the presence of
methemoglobinemia, anemia, and Heinz bodies. Treatment did not cause any
retention of BSP. Gross pathology Indicated that the dogs were 1n fair to
poor nutritional condition with little or no body fat. Histopathology
revealed hemosiderosis in the liver and spleen and cloudy swelling of the
kidneys of males and females and aspermatogenesls in males. Lesions of the
brain observed in high-dose dogs sacrificed during weeks 6 and 7 were gliosis,
edema, and demyelination of the cerebellum, spinal cord, and brain stem.
After 4 weeks, dogs recovered partially from the various effects. Based on
mortality, body weight loss, hematological abnormalities, neurological signs,
and histopathological alterations, the LOAEL was 25 mg/kg/day and the NOAEL
was 5 mg/kg/day.
Based on the results of these studies, the NOAELs and LOAELs shown in
Table VI-8 can be established for 2,4-DNT following subchronic (13 weeks)
exposure.
2) 2.6-DNT
Lee et al. (1976) conducted subchronic toxicity studies of 2,6-DNT (>99%
pure) in CD-I mice, CO rats, and beagle dogs. The basic experimental design
and procedures were similar to those described above in studies conducted with
2,4-DNT. Mice and rats were fed diets containing 0, 0.01, 0.05, or 0.25%
2,6-DNT for 13 weeks. This represents a corresponding dally intake of 0, 11,
51, or 289 mg/kg/day for male mice; 0, 11, 55, or 299 mg/kg/day for female
VI-25
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Table VI-8. Adverse Effect Levels for 2,4-DNT Following Subchronic Exposure
Parameter (mq/ko/dav)
NOAEL
LOAEL
Species Males Females Males Females Basis for LOAEL Reference
Mouse
Rat
Dog
47
34
25
52
38
25
Decreased body Hong et al
weight gain. (1985)
Decreased body Lee et al.
weight gain and (1985)
food consump-
tion.
Weight loss,
clinical signs,
hematological
effects, and
lesions 1n the
spleen, liver,
kidneys,
testes, and
brain.
Ellis et al.
(1985)
VI-26
-------
mice; 0, 7, 35, or 145 mg/kg/day for male rats; and 0, 7, 37, or 155 mg/kg/day.
for female rats. Dogs were given 0, 4, 20, or 100 mg 2,6-ONT/kg/day in
capsules for 13 weeks.
No treatment-related effects were observed in mice in the low-dose group,
and only a few mice in the mid-dose group showed weight loss. 2,6-DNT
administered at high levels produced decreased body weight gain and food
consumption, hematopoiesis in the liver and/or spleen, bile duct hyperplasia
in both sexes, and depression of spermatogenesis and atrophy of the testes in
males. After 4 weeks, mice showed partial recovery as Indicated by weight
gain and the disappearance of testicular lesions; however, hematopoiesis
continued in the liver or spleen. Based on decreased body weight and food
consumption and histopathologic changes in the spleen, liver, bile duct, and
testes observed at the mid-dose level, the LOAEL was 51 mg/kg/day in males and
55 mg/kg/day in females. The NOAEL was 11 mg/kg/day for both males and
females (Lee et al., 1976).
No adverse effects were seen in rats in the low-dose groups. Mid-dose
males and females exhibited decreased body weight gain and food consumption,
extramedullary hematopoietic activity in the liver and spleen, and bile duct
hyperplasia; males also exhibited depressed spermatogenesis and testicular
atrophy. Rats in the high-dose groups were severely affected. The effects
included decreased body weight, body weight gain, and food consumption,
methemoglobinemia, Heinz body formation, anemia, compensatory reticulocytosls,
severe extramedullary hematopoiesis in the spleen and liver, bile duct
hyperplasia, and renal degeneration in both sexes. In males, by the end of
13 weeks, the testicular lesions had progressed to encompass virtually all
connective tissues. After 4 weeks, only partial recovery was observed; rats
regained the weight lost, but lesions continued to occur in the spleen, livtr,
and testes of treated rats. Based on weight loss, blood effects, and
.histopathology, the LOAEL was 35 mg/kg/day in male rats and 37 mg/kg/day in
female rats. The NOAEL was 7_.mg/kg/day for both sexes (Lee et al., 1976).
In the study with dogs, Lee et al. (1976) did not observe adverse efftcts
in the low-dose group (4 mg/kg/day); however, 2,6-DNT was toxic at higher
t
VI-27
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levels. All high-dose (100 mg/kg/day) males (4/4) and females (4/4) died
between study weeks 2 and 8; 2 of 4 mid-dose (20 mg/kg/day) females died
during study week 9. Toxic effects included decreased food consumption
leading to weight loss; listlessness; incoordination leading to rigid
paralysis with occasional tremors; elevation in serum alkaline phosphatase,
serum glutamic-pyruvic transaminase (SGPT, currently referred to as "alanine
aminotransferase" or "ALT"), and/or blood-urea-nitrogen (BUN);
methemogloblnemia leading to Heinz body formation; anemia with compensatory
reticulocytosis and extramedullary hematopoiesis; and lymphold depression
leading to peripheral lymphocytopenia. Histopathology revealed bile duct
hyperplasia and degenerative and Inflammatory changes in the liver and kidneys
in both sexes, and degeneration and atrophy of the spermatogenic cells of the
testes. Effects in the high-dose animals were more pronounced and appeared
earlier than those in the mid-dose animals. A great variation in the onset of
symptoms was seen among dogs given the same dose. The effects were partially
reversed in 4 weeks and completely reversed in 19 weeks following cessation of
treatment. The NOAEL was 4 mg/kg/day. Based on mortality, weight loss, blood
effects, neurological signs, and histopathology, the LOAEL was 20 mg/kg/day.
In summary, dogs were the most sensitive species; doses of 4 mg/kg/day
had no effect, and doses of 20 mg/kg/day were toxic. Rats were somewhat less
sensitive, since 7 mg/kg/day was nontoxic. Higher doses (35 mg/kg/day in
males and 37 mg/kg/day in females) produced a variety of toxic'effects; more
severe, earlier effects were seen 1n the high-dose group. The effects
observed in rats and dogs exposed to 2,6-DNT were quantitatively and
qualitatively similar to those reported for 2,4-DNT. The main effects on th«
erythrocytes and testes were virtual!/ identical. In addition, 2,6-DNT
induced bile duct hyperplasia. Effects in mice were similar to those in rats;
the NOAEL was 11 mg/kg/day, and 51 mg/kg/day in males and 55 mg/kg/day in
females caused dose-related toxicity. In all three species, the primary
effect was methemogloblnemia; numerous sequelae .included Heinz body formation,
reticulocytosis, anemia, and-extramedullary hematopoiesis. 81le duct
hyperplasia, sometimes accompanied by hepatic degeneration or elevated senia
enzymes, was seen in all species, Males of all species had depressed
spermatogenesis and, in some cases, the effect was severe leading to
VI-28
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testicular atrophy. The principal effects seen in dogs, but not in mice or
rats, were incoordination leading to rigid paralysis. Dogs also showed renal
degeneration and elevated BUN levels. Based on the results of this study, the
NOAELs and LOAELs shown in Table VI-9 can be established for 2,6-DNT following
subchronic (13 weeks) exposure.
3) tq-DNT
No 13-week studies on tg-DNT were found in the available literature.
b. 6-Month study
Kozuka et al. (1979) fed a group of 20 male Wistar rats diets containing
0.5% 2,4-ONT (purity not reported) for a period of 6 months. The estimated
ingestion of 2,4-ONT was 66 mg/day for the first 3 months and 75 mg/day for
the last 3 months. Based on an average weight of 0.35 kg for ah adult rat,
this represents a daily Intake of 190 and 214 mg/kg/day for the first and the
last 3 months, respectively. A group of 23 rats served as controls. Rats
were observed dally, and body weight and food consumption were measured
weekly. Blood samples were collected for the determination of methemoglobin
and for biochemical .tests. Mortality occurred in two treated rats during week
2; 10 rats died between weeks 14 and 22, and 1 control rat died at week 14.
Toxic signs observed in treated rats were piloerection, whitened skin color,
humpback, jerky movements, decreased spontaneous movement, and general
weakness. Treated animals showed a slower growth rate, and body weights were
lower during the study. At the end of study, treated animal body weights were
approximately 58% lower than controls. A significant (p <0.01) Increase was
observed for relative liver, spleen, and kidney weights, and relative
testicular weights were significantly lower. A significant (p <0.01)
sevenfold Increase was seen in methemoglobin formation after 6 months based on
measurements 1n five animals. After 1 month of treatment, significant
(p <0.01) differences were observed in serum components (triglycerides,
glucose, albumin, and albumin/globulin ratios) and in serum enzymes (serum
VI-29
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Table VI-9. Adverse Effect Levels for 2,6-DNT Following Subchronic Exposure
Parameter fmq/ko/davl
NOAEL
LOAEL
Species Males Females Males Females
Basis for LOAEL
Mouse 11
Rat 7
Dog 4
11
7
51
35
20
55 Body weight loss; lesions in
spleen, liver, and testes.
37 Body weight loss;
hematological effects; lesions
in the liver, spleen, and
kidneys.
20 Body weight loss;
hematological effects;
neuromuscular Incoordination;
lesions in the liver, spleen,
kidneys, and testes.
VI-30
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glutamic-oxaloacetic transaminase [SGOT, now referred to as "aspartate
aminotransaminase" or "AST"], serum glutamate pyruvate transaminase [SGPT, now
referred to as "alanine aminotransferase" or "ALT"], alkaline phosphatase
[A1P], and acid-phosphatase) of treated rats as compared with controls. Gross
pathology revealed the formation of purulold matter and hypertrophy 1n the
liver, and atrophy of the testes. No hlstopathology was conducted.
c. Lifetime/chronic studies
i' •
1) 2.4-DNT
Leonard et al. (1987) observed significant reductions in body weight
gain (p <0.05) and significant increases in liver weight (approximately 150%
of control) of 24 male CDF (F344)/CrlBR rats following administration of
purified 2,4-DNT (>99.4% pure) 1n the diet for 12 months (6-month Interim
sacrifice of 4 rats/group) at concentrations that resulted in doses of
27 mg/kg/day. Hepatocytlc degeneration and vacuolation were apparent in the
majority of treated animals. Addophilic foci were present In 70% of the
animals, whereas basophilic foci were encountered in only 10% of the livers.
Neither type of focus was apparent in the controls. Bile duct hyperplasia was
noted in the majority of treated animals, and a highly variable incidence of
cholangiofibrosis was observed. Based on changes in the liver and biliary
tract in male rats, the LOAEL 1s 27 mg/kg/day, the only dose tested.
Ellis et al. (1979) evaluated the chronic toxldty of 2,4-DNT (£98.5%
2,4-DNT; £1.5% 2,6-DNT) 1n dogs, rats, and mice. The study in dogs was also
reported by Ellis et al. (1985); the study in rats was also reported by Lee
et al. (1985); and the study in mice was also reported.by Hong et al. (1985).
Groups of six male and six female beagle dogs were fed 2,4-DNT daily in
gelatin capsules containing 0, 0.2, 1.5, or 10 mg/kg. Thorough clinical and
histopathological examinations* were performed on each dog following sacrifice.
According to the protocol, one pair of dogs was to be killed for necropsy
after 1 year and another pair was allowed a 4-week recovery period to assess
reversibility. A similar procedure was to be used for the remaining four
VI-31
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pairs of dogs at 24 months. However, three of the six high-dose males were
killed for necropsy (at weeks 8, 18, and 19) following the development of
progressive paralysis starting in the hind legs and progressing to the
forelimbs, head, and neck. Initial symptoms included rapid weight loss,
intermittent tremors or convulsions, and vomiting. The death of another
high-dose dog (week 5) was unrelated to treatment. The two remaining males in
the high-dose group were continued to the end of 24 months, when one was
necropsied and the other was placed on the recovery study.
One high-dose female dog showed uncoordinated movements especially in
the hind limbs during week 10. Symptoms were most apparent 1 to 2 hours after
dosing; none were apparent by week 13. Intermittent symptoms indicative of
neurotoxicity observed in the surviving high-dose dogs lasted a few days or,
at most, weeks, and recurred weeks or months later. Recovery was facilitated
by supplying affected dogs with a soft diet when incoordination prevented them
from consuming the standard food.
In the raid-dose group (1.5 mg/kg/day), the first toxic signs were
observed in one male dog during week 66. Symptoms, Including loss of muscular
control of the hindquarters and occasional convulsive tremors, lasted about a
week and recurred occasionally during the remainder of the study. No other
mid-dose dog displayed such signs. One mid-dose female dog was found dead
during week 98, but the cause of death was not apparent.
The primary hematological effects were on erythrocytes; female dogs in
the high-dose group developed anemia with the erythrocyte count depressed by
20% after 3 months of treatment. Adaptive compensation was Indicated by
increases in the mean cell volume (MCV) and mean cell hemoglobin (MCHB) and
decreases in the total hemoglobin as the proportion of reticulocytes
increased. The cause of anemia was reflected in the small, inconsistent
methemogloblnenria and the more considerable, more consistent presence of Heinz
bodies. After 18 or 24 months of dosing, only a slight anemia or none at all
was evident with reticulocyte levels near normal, no Heinz bodies, and minimal
methemoglobln. Minimal amounts of methemoglobin were found in most of the
high-dose dogs, in half of the mid-dose dogs, and in very few low-dose dogs.
VI-32
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Although not statistically significant, these findings appear to be
toxicologically significant.
Histopathologic examination after 24 months revealed several lesions
that were related to treatment with 2,4-ONT. Central nervous system lesions
in the three high-dose dogs that developed paralysis and were killed for
necropsy during weeks 8, 18, and 19 Included vacuollzatlon, endothelial
proliferation, gliosis of the cerebellum, and peri vascular hemorrhage in the
cerebellum and the brain stem. A mild bile duct hyperplasla was seen, and
clusters of brown pigment-laden Kupffer cells were found In the livers of all
surviving high-dose dogs and 1n one low-dose dog. Cystic hyperplasla of the
epithelium occurred in the gallbladder of all high-dose dogs,,one mid-dose
dog, and one control dog. Brown epithelial pigmentation was seen in the
gallbladder, kidneys, and spleen of two high-dose dogs, and in the gallbladder
of three mid-dose dogs.
In summary, the primary targets affected by chronic oral dietary
administration of 2,4-ONT to dogs were the nervous system, erythrocytes, and
biliary tract (Ellis et al., 1979, 1985). The major adverse effect was
neurotoxicity, characterized by incoordination and paralysis. Effects were
observed within 2 months in all dogs given 25 mg/kg/day (see Section VLB.2.a,
Subchronic Toxicity), within 6 months in all dogs receiving 10 mg/kg/day, and
during month 16 in one dog given 1.5 mg/kg/day. A wide range of variability
was observed among individuals as to the onset and severity of the neuropathy.
Central nervous system lesions Including vacuollzatlon, endothelial
proliferation, and gliosis of the cerebellum were present in three of the
high-dose dogs. The primary erythrocyte effect, methemoglob1nem1a, was rarely
seen in the chronic study, but sequelae, including the presence of Heinz
bodies and retlculocytosis, were evident in the mid- and high-dose groups
within 3 months. During the second year, these erythrocyte effects were
minimal, presumably because of an adaptive response by the dogs.
Methemoglobineraia and Its sequelae were the only, reversible effects in dogs
allowed a 4-week recovery period. M1ld biliary hyperplasla was seen in most
dogs receiving 10 mg/kg/day and in some dogs given 1.5 mg/kg/day. Based on
neurotoxicity, hematologic changes, and effects on biliary tract
VI-33
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histopathology in dogs, the LOAEL is 1.5 mg/kg/day and the NOAEL is
0.2 mg/kg/day.
In a chronic study with rats, diets containing 0, 0.0015, 0.01, or 0.07%
2,4-ONT (0, 15, 100, or 700 ppm) were administered to groups of 38 male and
38 female CO (Sprague-Dawley) rats, yielding an average Intake of 0.57, 3.9,
or 34 mg/kg/day, respectively, for males and 0.71, 5.1, or 45 mg/kg/day,
respectively, for females (Ellis et al., 1979; Lee et al., 1985). After
12 months, eight males and eight females from each group were killed for
necropsy; the remaining rats were sacrificed after 24 months. Cumulative
deaths in high-dose males and females were significantly higher than in
controls; 50% mortality occurred in high-dose rats by month 20 and in controls
by month 23. Weight gains in high-dose animals were markedly reduced,
reaching a plateau considerably below control levels after 2 months in females
and after 4 months in males. Growth in mid-dose males and females began to
decrease during month 9, while low-dose rats exhibited growth rates comparable
to those of controls. Anemia, partially compensated as evidenced by Increased
reticulocyte counts, was prominent in mid- and.high-dose males and high-dose
females after 12 months. Histopathological examination revealed the
progressive development of hyperplastlc foci in the liver, the severity of
which was Increased In high-dose males and mid-dose females (Table VI-10).
There was marked atrophy of the testes with severe atrophy of the seminiferous
tubules and almost complete lack of spermatogenesis in 86% (6/7) high-dose
males at 12 months. Beyond 12 months, severe atrophy of the seminiferous
tubules occurred in 16% of the control males and in 25% of the low-dose, 32%
of the mid-dose, and 83% of the high-dose males. Based on the Incidence of
changes in the seminiferous tubules of male rats and in the liver of female
rats, the LOAEL 1s 0.57 mg/kg/day, the lowest dose tested.
In a chronic study with mice, diets containing 0, 0.01, 0.07, or 0.5%
2,4-ONT (0, 100, 700, or 5,000 ppm) were administered to groups of 38 male and
38 female CD-I mice, yielding an average Intake of 0, 14, 95, or 898 mg/kg/day
(Ell 1s et al., 1979; Hong et al., 1985) using procedures similar to those
described for rats by Lee et al. (1985). Unscheduled deaths occurred
considerably sooner in the high-dose mice; all males and females in this group
died by months 18 and 21, respectively. Weight gain was drastically reduced
VI-34
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Table VI-10. Incidence of Hlstopathological Lesions in CD Rats Fed 2,4-DNT for 24 Months
Males (ma/ka/dav)
Lesion
Testicular atrophy
Splenic henosiderosts
Liver hyperplastlc foci or
cellular alteration
0 0.57
/ 4/25 (16)* 7/28 (25)
4/25 (16) 5/28 (18)
9/25 (36) 10/28 (36)
3.9 34
6/19 (32) 25/30 (83)
1/19 (5.3) 5/30 (17)
9/19 (47) 16/29 (55)
Females (ma/ka/dav)
0 0.71 5.1 46
3/23 (13) 5/35 (14) 3/27 (11) 7/3!, (20)
7/23 (30) 18/35 (51) 19/27 (70) 13/34 (3d)
'Hunter of rats with lesions/number of rats examined for lesions (percent incidence).
SOURCE: Ellis at al. (1979).
en
-------
in all high-dose mice with effects more pronounced in males than in females.
Males consistently weighed less than controls after months 11 (low-dose) and 3
(mid-dose), whereas females in these groups weighed about the same as
controls. Toxic anemia and concurrent reticulocytosis occurred in high-dose
males and females after 12 months. In addition, all high-dose mice displayed
increases in relative spleen and liver weights after 12 months of treatment.
Mice at all dietary levels exhibited an increased, dose-related Incidence of
an unidentified, brown pigment in many tissues; the intensity increased with
length of treatment. This pigment was most prominent in the liver and spleen
but was also present in the lungs, kidneys, adrenals, bone marrow, heart,
gonads, and central nervous system (CNS). HepatocelTular dysplasia,
characterized by metabolic, degenerative, and hyperplastic alterations of
cells, was observed in all treated males (17/25, 11/20, and 31/32 low-, mid-,
and high-dose males, respectively) and in high-dose females (24/25)
(Table VI-llj. Renal tumors in males and testicular or ovarian atrophy, in
mid- and high-dose males and high-dose females, respectively, were also
prominent lesions (see Cardnogenicity, Section VLB.5, and Reproductive
Effects, Section VLB.3). Based on liver changes in male mice, the LOAEL is
14 mg/kg/day, the lowest dose tested.
In an NCI (1978) bioassay, groups of 50 male and 50 female Fischer 344
rats were fed diets containing 0.008 or 0.02% 2,4-DNT (80 or 200 ppm; >95%
purity) for 78 weeks and observed for another 26 weeks. Assuming that rats
consume approximately 5% of their body weight in food each day, the diets were
equivalent to 4 and 1.0 mg/kg/day of DNT, respectively (Lehman, 1959).
Corresponding control groups containing 25 (low-dose control) or 50 (high-dose
control) rats of each sex were assigned because the low- and high-dose groups
were tested at different times. Necropsies were performed on each rat that
died, that was killed when moribund, or that was sacrificed at the end of the
bioassay. Gross and microscopic histopathological examinations were conducted
on major tissues. The only significant clinical observation was a mean body
weight depression that was evident as early as week 16 1n high-dose males. At
the end of the assay, this group exhibited a mean average body weight about
25% less than controls, and high-dose females exhibited an 18% decrease in
mean body weight. The incidence and variety of nonneoplastic lesions in the
major organs were similar in control and treated rats.
VI-36
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Table VI-11. Incidence of Histopathological Lesions in Mice Fed 2,4-DNT
for 12 or 24 Months
Finding
Dosage (mq/kq/dav)
Males
14 95
898
Females
14 95
898
Generalized abnormal
pigmentation
12 months
24 months
Hepatocellular
dysplasia
12 months
24 months
Testicular/ovarian
atrophy
12 months
24 months
0/8 0/8 0/8 0/8
0/25 19/25 6/21 32/32
1/8 1/8 1/8 8/8
1/25 17/25 11/20 31/32
0/8 1/8 1/8 7/8
8/24 3/25 13/20 26/30
0/8 0/8 0/8 8/8
0/23 13/21 12/23 23/25
0/8 1/8 0/8 5/8
5/23 3/21 5/23 24/25
1/8 0/8 0/8 6/8
1/29 2/23 0/27 15/24
SOURCE: Hong et al. (1985).
VI-37
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A similar protocol was used for B6C3F, mice (NCI, 1978). Fifty mice of
each sex were exposed to 2,4-DNT at dietary concentrations of 0, 0.008, or
0.04% (0, 80, or 400 ppm, respectively) for 78 weeks followed by 13 weeks
without treatment. Because the low- and high-dose groups were tested at
different times, each was assigned its own control group. Mean body weight
depression was observed in all treated groups by week 30. At study
termination, the approximate weight gain for low-dose males was 91% of
control; for high-dose males, 82%; for low-dose females, 89%; and for
high-dose females, 76%. No clinical observations were reported, and no
increase was reported for the Incidence of nonneoplastic lesions in treated
mice as compared with controls. .
Based on studies by Leonard et al. (1987), Ellis et al. (1979), and NCI
(1978), the LOAELs and NOAELs shown in Table VI-12 can be established
following chronic or lifetime exposure.
2) 2.6-DNT .
Leonard et al. (1987) observed significant reductions in body weight gain
and significant Increases in liver weight following dietary administration of
purified 2,6-DNT (>99.4% pure) to 24 male CDF (F344)/CrlBR rats for 12 months
(6-month interim sacrifice of.4 rats/group) at concentrations that resulted in
doses of 7 or 14 mg/kg/day. Serum alanine aminotransferase was elevated
following administration of both doses of 2,6-DNT for 1 year, and serum gaou-
glutamyl transferase was Increased after 26 and 52 weeks at the high dose.
Hepatocytic degeneration and vacuolization were apparent in the majority of
treated animals, although these effects were not shown to be dose-dependent.
Acidophilic and basophilic foci were observed in over 90% of the animals ftd
2,6-DNT. Neither type of focus was apparent in the controls. Bile duct
hyperplasia was noted In the majority of treated animals, and a highly
variable incidence of cholangiofibrosis was observed. Based on changes in th«
serum enzyme levels,, the liver, and the biliary tract Mstopathology in IM!«
rats, the LOAEL is 7 mg/kg/day, the lowest dose tested.
VI-38
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Table VI-12. Adverse Effect Levels for 2,4-DNT Following Chronic or Lifetime Exposure
I some r
Species (X Purity)
fififl
Beagle 2.4-DNT
(98XJ*
M
SO 2.4-DNT
(9«X)
F344 2.4-DNT
(»99X)
F344 2.4-ONT
< (>95X)
i House
«o
C01 Z.4-DHI
(98*)
Exposure
Duration/
Route
' '
24 months/
diet
24 months/
diet
12 months/
diet
78 weeks/
diet
' 24 months/
diet
NOAEL LOAEL
Effects (mg/kg/day) Reference(s)
. Biliary hyperplasia; anemia; Heinz bodies; 0.2
paralysis; death
.
Anemia; hepatic foci; testicular atrophy " 3.9
' Decreased body weight; Increased liver weight; None
hepatocyttc degeneration; bile duct hyperplasia
Decreased body weight None
Decreased body weight; Increased liver and None
spleen weights; organ and tissue pigmentation;
testicular and ovarian atrophy
1.5 Ellis et al.. 1985;
Ellis et al.. 1979
/
34 Lee et al.. 1985;
Ellis et al.. 1979
27* Leonard et al.. 1987
200 ppm NCI. 1978
14 Hong et al . , 1985;
Ellis et al.. 1979
'Other 2X consists of other isoners. but primarily 2.6-DNT.
'Only one dose tested.
-------
Oinitrotoluene-induced hepatotoxicity was investigated in relationship to
structure-activity parameters using six DNT isomers (Spanggord et al., 1990).
Sprague-Dawley rat hepatocyte suspensions were used as the biological test
system. All Isomers produced a concentration-dependent inhibition of protein
synthesis and lactate dehydrogenase (LDH) release during 4-hour incubations.
This finding suggested that ONT isomers as well as their metabolites are
directly hepatotoxic without the need for metabolic activation. Protein
synthesis Inhibition occurred at lower ONT concentrations than those affecting
LDH release; therefore, protein synthesis inhibition was the most sensitive
indicator for hepatotoxicity. None of the Isomers affected lipid
peroxidation. The results also showed that the ortho- and the para-isomers
were more toxic at the same concentration than the meta-Isomers regardless of
the indicator. The authors concluded that based on the present metabolite
analysis (HPLC) and other reports, the reduction of the nitro groups of the
parent compounds plays the primary role in determining the potential of
hepatotoxicity of these compounds.
3) to-DNT
Leonard et al. (1987) administered a technical grade mixture of
dinitrotoluenes In the diet to 24 male COF (F344)/CrlBR rats for 12 months
(6-month interim sacrifice of 4 rats/group) at concentrations that resulted in
a dose of 35 mg/kg/day. The tg-DNT was prepared by mixing purified DNT
isomers in a ratio representative of a standard technical grade, with a final
composition of 76.5% 2,4-DNT, 18.8% 2,6-DNT, 2.43% 3,4-DNT, 1.54% 2,3-DNT,
0.69% 2,5-DNT, and 0.04% 3,5-DNT. Feeding tg-DNT for 6 months or 1 year
significantly reduced body weight gain and significantly Increased liver
weights at 1 year. No effects were seen on serum alanine aminotransferase or
serum gamma-glutarayl transferase activities. Hepatocytic degeneration and
vacuollzation were apparent In the majority of treated animals, although these
effects were not shown to be dose-dependent. AcidophlUc and basophilic foci
were observed In over 90% of the animals fed tg-DNT. Neither type of focus
was apparent in the controls. Bile duct hyperplasia was noted in the majority
of treated animals, and a highly variable incidence of cholangiof1brosIs was
VI-40
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observed. Based on changes in the body weight, liver, and biliary tract in
male rats, the LOAEL for tg-DNT is 35 mg/kg/day, the only dose tested.
The toxicologic effects of tg-DNT (composition not reported) in Charles
River COF (F344) rats were evaluated following administration at dietary
levels of 0, 3.5, 14.0 and 35.0 mg/kg/day (130 rats/sex/group) in a study
conducted by CUT (1982). Sacrifice and necropsy of 10 rats/sex/treatment
group occurred at weeks 26 and 52; 20 rats/sex/group were sacrificed at week
78, all surviving high-dose rats at week 55, and all surviving animals at
termination after 104 weeks. A significant dose-related decrease in body
weight gain was seen in all treated groups throughout the first year of the
study. After 54 weeks, a dose-related decrease in mean body weight was
observed at all dose levels in both sexes. Analysis of hematological data
suggested,a low-grade regenerative anemia in high-dose males and females.
Anemia at this dose level was also indicated by the increased hemosiderin and
extramedul1ary hematopoiesis in the spleen, which was indicative of enhanced
red cell turnover. Analysis of organ weight data revealed alterations in
several organs. Consistent significant increases were observed in relative
liver and brain weights in all treated rats, relative kidney weights in all
but low-dose males, and relative ovarian weights in low- and mid-dose females.
At 104 weeks, absolute organ weight increases were reported for liver and
kidney in both sexes at the mid-dose level and for liver in both sexes at the
low-dose level. Absolute testicular Weight was increased in low-dose males at
104 weeks. Gross necropsy findings revealed alterations in the liver
characterized by white or yellow foci in all treated males and females as well
as nodules and tissue masses in mid-dose males and females.
At the end of 1 year, histopathological examination showed hepatotoxicity
in all mid- and high-dose animals and in low-dose males, characterized by
areas of cell alteration, hyperbasophilia and megalocytosis of hepatocytes,
and vacuolizatlon and necrosis of individual hepatocytes. Other treatment-
related lesions in the high-dose males were observed in the kidney, pancreas,
testes, spleen, and bone marrow. Following 78 weeks of treatment, primary
histologlc findings pertained to the increased incidence of neoplasms in the
liver (see Section VLB.5, Carcinogenicity). Hepatotoxicity was observed in
VI-41
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mid-dose males,'as was an exacerbation of chronic interstitial nephritis in
mid-dose males and females. At the end of 2 years, low- and mid-dose animals
exhibited a high incidence of hepatoxicity as well as chronic interstitial
nephritis accompanied by parathyroid hyperplasia and adenoma. In addition, an
enhancement of degenerative changes in the adrenal glands was noted in mid-
dose rats. In general, treatment-related histologic tissue alterations;were
characterized by the presence of hepatic neoplasms in all treatment groups, an
increased incidence of mammary fibroadenomas in all mid-dose rats, and an
increased incidence of subcutaneous fibromas and sarcomas primarily,in mid-
dose males (see Section VLB.5, Carcinogenicity). Based on histopathological
changes in the liver, kidney, and parathyroid gland 1n rats, the LOAEL for
tg-DNT is 3.5 mg/kg/day, the lowest dose tested.
3. Reproductive Effects
a. 2.4-DNT
i
The dominant lethal effects of 2,4-DNT in rats were studied by Lane
et al. (1985). Groups of 10 male Sprague-Oawley rats were administered, by
oral gavage, 2,4-ONT (purity not reported) in corn oil at 0, 60, 180, or
240 mg/kg/day for 5 days. Significant reductions in the mating index were
observed in rats administered 240 mg/kg/day; a sharp decrease in sperm-
positive and pregnant females was observed at this dose level. Because of
this finding, statistical evaluation of the results was difficult. However,
no dominant lethal effects, characterized by early fetal deaths, were
observed. Dose levels at or below 180 mg/kg/day did not result in changes in
fertility or fetal death. The authors concluded that exposure to 2,4-DNT did
not result in dominant lethal mutations but did adversely affect reproductive
performance. ,
In a study by Bloch et al. (1988), groups of 9 to 10 male Sprague-Dawley
rats were fed 2,4-DNT (purity~97%) at dietary levels of 0, 0.1, or 0.2% (0,
1,000, or 2,000 ppm, respectively) for 3 weeks, and the effects of 2,4-DNT on
the testes were evaluated. Body weights were significantly reduced (p <0.05)
at 2,000 ppm when compared with controls. Significant increases (p <0.05) in
VI-42
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serum follicle stimulating hormone (FSH) and luteinizing hormone (LH)
concentrations were observed in rats ingesting 2,000 ppm. Total sperm count
was also significantly reduced (p <0.01) at 2,000 ppm. No significant
reductions were found at 1,000 ppm. Rats fed 2,000 ppm demonstrated extensive
disruption of spermatogenesis with vacuolization and other cytoplasmic
alterations of Sertoll cells. These Included varying sized vesicles, swollen
mitochondria* and distended endoplasmic reticulum. Degeneration of
spermatocytes and spermatlds was present, and Irregularity in the basal lamina
and distortion of the peritubular tissue were observed. No microscopic
changes were observed in rats fed 1,000 ppm when compared with controls. The
NOAEL is 1,000 ppm, which corresponds to a dally intake of approximately
80 mg/kg/day (assuming .that rats consume approximately 5% of their body weight
in food daily [Lehman, 1959], and body weights were approximately 325 g).
Ellis et al. (1979) evaluated the reproductive effects of 2,4-DNT in rats
k
following dietary exposure for three generations. Groups of 10 to 24 Sprague-
Dawley rats of each sex were fed diets containing 0, 15, 100, or 700 ppm
(approximately 0, 0.75, 5, or 35 mg/kg/day) 2,4-DNT (purity 98%) for up to
6 months prior to mating. Each parental generation produced two sets of
offspring (F. and Fb Utters); the study was terminated during the third
generation after weaning of the second Utter (Fb).
Parental body weights were significantly reduced (p <0.05) at the
high-dose level during the first two generations. Fertility was low for all
animals from the control and test groups during the first (F0) generation
(Table VI-13). This probably was due to the age of the animals at mating
(8 months). The survival of high-dose pups (F,b) from the first generation
was greatly reduced (Table VI-14). Four of five Utters died, and
subsequently, only three males and three females (F,) were available for
mating during the second generation. The fertility of the high-dose
F, animals was reduced, and consequently, no litter data were available from
the second mating of this generation, and a third generation was not produced.
Mean litter size and pup weight at weaning were slightly, but not
significantly, lower for high-dose pups when compared with controls from the
VI-43
-------
Table VI-13. Effects on the Fertility and Gestation Length of Parental
Rats Fed 2,4-DNT in the Diet for Three Generations
Dietary
level (ppm)
F- aeneration
0
15
100
700
F, aeneration
0
15
100
700
F, aeneration
0
15
100
:700
Mating
index (%)
75
84
76 .
87
100
91
97
67
97
97
95
a
Pregnancy
rate (%)
57
62
59
48
100
83
92
75
'
97
97
100
, "" "
Gestation length
(days)
23
23
23
23
•
22 '.
22
22
22
22
-22
22
• "
"No pups produced.
SOURCE: Ellis et al. (1979).
VI-44
-------
Table VI-14.
Summary of Reproductive Effects in Rats Fed 2,4-ONT
in the Diet for Three Generations
Litter Dietary
generation level (ppn)
F 0
. 15
100
700
F,. 0
15
100
700
Fj» 0
15
100
700
F36 0
15
100
700
Fj. 0
. 15
100
700
F» 0
15
100
700
Mean
litter
size
6.9*1.1
6.8*1.0
4.9*1.1
4.5*1.1
12.7*1.8
3.2*1.9
9.7*1.1
7,4*1.2
12.7*0.6
13.9*0.4
13.8*0.4
11.0*1.0
14.0*0.3
15.4*0.6
14.2*0.8
i
11.3*0.4
12.2*0.6
12.6*0.5
™"
12.2*0.6
13.2*0.4
13.6*0.5
Live
birth
Index (X)
85
83
89
94
.91
100
88
90
98
97
99
100
96
95
97
—
97
95 .
98
..
99
98
98
Mean
pup
birth
weight (g)
7.2*0.3
7.1*0.2
7.4*0.1
7.5*0.4
7.0*0.5
7.2*0.8
7.5*0.4
7.0*0.3
7.0*0.2
6.6*0.1
6.9*0.2
6.4*0.2
7.2*0.1
6.8*0.3
7.1*0.2
—
7.1*0.1
7.1*0.2
6.9*0.1
. '•'"
6.4*0.1.
6.4*0.2
6.5*0.1
Viability
Index
(X)
70
69
60
64
100
99
83
20*
98
99
94
89
98
96
94
~
96
96
94
""•
94
95
97
Lactation
Index
(X)
61
91
61
90
91
70
83
78
96
95
95
100
92
83
9'
— •
95
93
87
"•"
94
87
91
Mean
pup
weight
at weaning (g)
45*8
53*2
38*10
53*6
55*7
45*9
44*13
44
43*2
41*1
42*1
35*1
42*1
41*2
41*2
39*1
36*1
35*1
~ —
40*1
35*1*
35*2*
•
Sex
ratio
60
50
63
48
37
50
54
57
50
52
48 •
66'
48
41
45
—
49
38
43
--
43
38
46
aNo pups produced.
'Significantly different from controls (p <0.05).
SOURCE: Ellis et aT. (1979).
VI-45
-------
first generation. Slight reductions in body weight at weaning were also
observed in low- and mid-dose pups from the first and third generations.
However, parental fertility and offspring viability were not affected at the
low- and mid-dose levels. The parental NOAEL and LOAEL for this study were
100 and 700 ppm, respectively, based on consistent significant reductions in
body weight at 700 ppm. The NOAEL and LOAEL for reproductive effects were 100
and 700 ppm (approximately 5 and 35 mg/kg/day), respectively, based on severe
reductions 1n fertility at 700 ppm.
Although testicular weights were not recorded in the reproduction study
discussed above, significant reductions (p <0.05) in relative (to brain)
testicular weight were observed in Sprague-Dawley rats (38 animals/group) from
a concurrent study fed 700 ppm (approximately 35 mg/kg/day) 2,4-DNT for
12 months or longer (Lee et al., 1985). These rats developed severe
testicular atrophy and reduced spermatogenesis. However, no testicular
effects were observed in rats similarly fed approximately 100 ppm
(3.9 mg/kg/day).
In a 13-week subchronic study in which male CD-I mice (16 animals/group)
were fed 2,4-DNT, mild testicular degeneration was observed at approximately
1,000 ppm (137 mg/kg/day) but not at 350 ppm (47 mg/kg/day) (Hong et al.,
1985). Testicular degeneration and atrophy and decreased spermatogenesis were
observed in male mice (58 mice/sex/group were initially used) fed 700 ppm
(approximately 95 mg DNT/kg/day) for up to 24 months (Ellis et al., 1979). In
the same study, a lack of follicular function and reductions in the number of
corpora lutea were observed in female mice fed 5,000 ppm (898 mg/kg/day) for
24 months. Similar effects were observed in male beagles (six dogs/group)
administered 2,4-DNT in gelatin capsules at 25 mg/kg/day for up to 24 months;
the dogs exhibited mlId to severe testicular degeneration and reduced
spermatogenesis (Ellis et al., 1985). No testicular effects were observed in
dogs administered 10 mg/kg/day. (See Sections VLB.2.a, 13-Week Studies, and
VI.B.2.C, Lifetime Studies, for further discussion.) No data on the
reproductive toxicity of 2,6-DNT or tg-ONT were found in the available
literature.
VI-46
-------
b. 2..6-DNT
No studies of reproductive toxicity were found in the available
literature.
c. ta-DNT
No studies of reproductive toxicity of the technical grade mixture of
dinitrotoluene were found in the available literature.
4. Developmental Toxicitv
a. 2.4-DNT
In a series of screening tests, 60 chemicals were evaluated for
developmental toxicity. potential in CD-I mice (Hardin et al., 1987). Fifty
time-mated females were administered, by oral gavage, 2,4-DNT in corn oil at
390 mg/kg/day on gestation days 6-13. The dams were observed twice dally
during treatment and once dally before and after treatment for mortality and
clinical signs of toxicity. The dams were allowed to deliver their pups, and
on postnatal day 1, the number of live pups and litter weight were recorded.
On postnatal day 31, the number of pups, litter weight, and maternal body
weight were recorded. External, visceral, and skeletal examinations of pups
were not conducted.
Maternal toxicity as evidenced by a 30% mortality rate (15/50 dams
during the study) was observed. However, 2,4-DNT had no effect on maternal
weight gain, the number of viable litters, the number of live pups born, birth
weight and tnrlght gain of the pups, or the survival of offspring. Although
the pups were not examined for abnormalities, the authors stated that
malformations observed In a conventional developmental toxicity study would b«
expressed in this system as reduced litter size, decreased birth weight, and
impaired survival. Therefore, 2,4-DNT was not considered to be teratogenlc m
this study. However, the authors recommended the use of this screening ttst
VI-47
-------
as a tool for prioritizing conventional testing rather than as a predictive
test for teratogenicity.
b. 2.6-DNT
No data on the developmental toxici.ty of 2,6-DNT were found in the
available literature. ,
c. to-DNT .
In a study by Price et al. (1985), the teratogenlc potential of technical
grade ONT was Investigated in Fischer 344 rats. The test material was a
mixture of 2,4-DNT (76%), 2,6-DNT (19%), 3,4-DNT (.2.4%K 2,3-DNT (1.5%),
2,5-ONT (<1%), and 3,5-DNT (<1%). This study was conducted in two phases to
evaluate the possible teratogenicity of ONT as well as ONT effects on
postnatal development. For the teratogenlc phase, groups of 5 to 20 time-
mated rats were administered, by gavage, ONT in com oil at 0, 14, 35, 37.5,
75, 100, or 150 mg/kg/day on gestation days 7-20. A group of 20 time-mated
females was administered 200 mg hydroxyurea/kg/day and served as positive
controls. In addition to collection of other maternal and fetal data at
terminal sacrifice on gestation day 20, blood was collected from dams and
fetuses (pooled by litter) in the vehicle and positive control groups and in
the 100-mg/kg/day group. Methemoglobin content and reticulocyte, red blood
cell (RBC), and platelet counts were the hematologic parameters measured.
Mean corpuscular volume (MCV) and red blood cell distribution width (ROW) wtrt
calculated.
Mortality was high at the 150-mg/kg/day level; 46% of the dams died prior
to gestation day 18. Mortality was similar among controls and other treatatnt
groups. Clinical signs of toxicity, including lethargy, rough hair coat, tnd
weakness of the hind legs, were observed in dams given 150 mg/kg/day beginning
on gestation day 11. Body weight and corrected body weight gain (minus gravid
uterine weight) were significantly decreased (p <0.01) In dams administered
150 mg/kg/day. Corrected body weight gain was also significantly reduced in
dams receiving 14 (p <0.05) or 100 (p <0.01) mg/kg/day. A significant
VI-48
-------
increase (p <0.01) in relative (to body weight) liver weight was observed in
dams administered 75 or 100 mg/kg/day. An increase in relative liver weight
was also observed at 150 mg/kg/day, but the difference was not significant.
Relative spleen weights were significantly increased in dams receiving 35,
37.5, 75, 100, and 150 mg/kg/day when compared with controls. Dams
administered 100 mg/kg/day exhibited significant increases in methemoglobln
(p <0.01), retlculocyte count (p <0.05), MCV (p <0.05), ROW (p <0.05), and
platelet count (p <0.05). In addition, significant decreases (p <0.05) in RBC
count and hematocrit were observed In the DNT-dosed dams when compared with
controls. No differences in the number of corpora lutea, implantations, or
live and dead fetuses were observed among the vehicle control and test groups.
An increase in the percent resorptions was observed at the high-dose level;
16.8, 2.3, 4.1, 14.6, 11.0, 12.7, and 46.OX resorptions were noted in dams
administered the vehicle or DNT at levels of 0, 14, 35, 37.5, 75, 100, and
150 mg/kg/day, respectively. Although the difference was not statistically
significant, the Increase was Indicative of a compound-related embryotoxic
effect. No differences in Utter size, sex ratio, fetal weight, crown-rump
length, or placental weight were observed among vehicle control and treatment
groups. The Incidences of malformations and variations were similar among
vehicle control and test groups. All abnormalities observed were spontaneous
in nature. Therefore, ONT was not teratogenlc in rats. However, some
developmental effects were noted. Relative liver weight was significantly
reduced (p <0.01) for fetuses from the 14-mg/kg/day group and significantly
increased for fetuses from the 35-mg/kg/day group. Significant Increases in
relative spleen weight were observed in fetuses from the 35- and 75-mg/kg/day
groups. Retlculocyte count (.p <0.05) and RBC count (p <0.01) were
significantly Increased (p <0.05) In the fetuses of dams administered
100 mg/kg/day when compared with the vehicle control group.
. ,~**-~- '
For thrpostnatal phase, groups of 5-7 time-mated rats were administered,
by gavage, ONT In corn oil (2 ml/kg) at 14, 35, 37.5, 75, or 100 mg/kg/day on
gestation days 7-20; in addition, groups of 11-14 time-mated females were
similarly administered corn oil (vehicle control) or 200 mg/kg hydroxyurea
(positive control). The dams were allowed to deliver their Utters and were
sacrificed on postnatal day 30. The following data were collected for each
VI-49
-------
pup on postnatal day 0: sex, body weight, crown-rump length, external
abnormalities, clinical signs of toxicity, and mortality. The litters were
then culled to .eight pups (four/sex), if possible. Liver and spleen weight
and blood samples were collected from randomly selected, culled pups on
postnatal day 0. The postnatal physical (e.g., righting reflex, pinna
\
unfolding, hair growth) and neurobehavtoral development (e.g., open field
testing) of surviving pups were evaluated during the remainder of the study.
Pups were randomly sacrificed on postnatal day 10, 25, or 50, and remaining
pups were sacrificed on postnatal day 60. Liver and spleen weights were
collected for one pup/Utter and for all dams sacrificed at the above-
specified Intervals during the study. In addition, testlcular weights from
pups sacrificed on pos.tnatal day 60 were recorded. Blood was collected from
dams and pups administered DNT at 75 or 100 mg/kg/day, hydroxyurea, or the
vehicle at sacrifice, and the above-sped fled hematological parameters were
analyzed.
Three females (one from each group) from the 14-, 35-, and 100-mg/kg/day
groups died during the study. No other deaths were reported. As seen during
the teratogenic phase, corrected body weight (minus gravid uterine weight) was
significantly reduced at 100 mg/kg/day during gestation. Reduced body weight
was still evident on postnatal day 15 in dams receiving 100 mg/kg/day, but the
reduction apparently was not evident at postnatal day 30 (final sacrifice),
indicating that some recovery had occurred. The only other maternal' effect
was a significant decrease (p <0.05) in reticulocyte count at 75 mg/kg/day on
postnatal day 30. Other hematologic parameters, Including methemoglobin
levels, were similar among dams from the vehicle control and from the 75- and
100-mg/kg/day groups. Relative liver weights were slightly, but
nonsignificantly, higher In dams receiving DNT at 35 mg/kg/day or at greater
levels than controls. Relative spleen weights were similar among the vehicle
control and test groups.
No dose-related pattern* 1n developmental effects were observed in pups
from dams administered DNT when compared with the vehicle controls. A
significant decrease (p <0.01) in female pup weight was observed at
75 mg/kg/day, but this was associated with a significant Increase (p <0.05) in
VI-50
-------
litter size at this dose level and was not compound related. A slight, but
nonsignificant, increase in postnatal death was observed in offspring at
100 nig/kg/day. However, this was apparently due to the loss of one entire
litter and did not appear to be related to ONT administration. Significant
increases (p <0.05-0.01) in relative liver weight were observed 1n pups from
all DNT-dosed groups on postnatal day 0 and in pups from the 37.5- and
75-mg/kg/day groups on postnatal day 50. However, relative spleen and
testicular weights were comparable among the vehicle control and ONT-dosed
groups. In addition, physical and neurobehavloral development were similar
among offspring from the vehicle control and test groups; no significant
differences were noted. Changes in hematologlc parameters were observed in
offspring, but they were not considered to be dose related. Reticulocyte
count was significantly Increased (p <0.01) in pups from the 75-mg/kg/day
group and significantly decreased (p <0.01) In pups from the 100-mg/kg/day
group. In addition, a significant increase (p <0.05) in MCV was observed in
pups from the 75-mg/kg/day group. Methemoglobin levels were similar among
vehicle control and ONT-treated pups.
The maternal NOAEL and LOAEL for the study were 14 and 35 mg/kg/day,
respectively, based on a significant increase in relative spleen weight in
dams receiving DNT at levels of 35 mg/kg/day or greater (Price et al., 1985).
Changes in hematologic parameters, including methemoglobin content, were
observed in dams administered 100 mg/kg/day. However, analysis of blood was
conducted only in dams receiving the vehicle or DNT at 100 mg/kg/day, and
therefore, a definitive assessment of hematologlc effects could not be
accomplished. The NOAEL and LOAEL for developmental toxicity were 14 and
35 mg/kg/day, respectively, based on significant Increases 1n relative liver
and spleen weight In the fetuses of dams administered ONT at levels of
35 mg/kg/day or greater. However, the numbers of animals per group for each
phase of this study were not adequate to evaluate the teratogenlc potential of
DNT.
VI-51
-------
5. Carcinoqenicitv
a. 2.4-DNT
When fed 1n the diet in 2-year bioassays, 2,4-DNT induced benign tumors
in Fischer 344 rats (males and females), benign and malignant tumors in CO
rats (males and females), and malignant tumors in CD-I mice (males only). No
treatment-related increase in tumors was observed in B6C3F, mice, in beagle
dogs, or in F344 rats fed 2,4-DNT for 1 year. These studies have been
reviewed in detail in Section VI.B.2.b, Lifetime Studies, and careinogenicity
findings are discussed below.
In an NCI (1978) 'bioassay; groups of 50 male and 50 female Fischer 344
rats were fed diets containing 0.008 or 0.02% 2,4-DNT (80 or 200 ppm; >95%
purity) for 78 weeks and observed for another 26 weeks. Assuming that rats
consume approximately 5% of their body weight in food each day, the diets were
equivalent to 4 and 10 mg/kg/day of DNT, respectively .(Lehman, 1959).
Corresponding control groups containing 25 (low-dose control) and 50
(high-dose control) rats of each sex were assigned because the low- and high-
dose groups were tested at different times. At termination, gross and
microscopic histopathological examinations were conducted on major tissues. w
The survival rate for the two treatment groups did not differ significantly
from their respective controls, with 52-64% of the males and 48-62% of the
females surviving until the end of the study. Reduced body weight gain in the
high-dose groups indicated that a Maximum Tolerated Dose (MTD) had been,
approached. As shown in Table VI-15, a significantly Increased incidence of
fibroma of the skin and subcutaneous tissue occurred in both high- and low-
' ' '
dose males/and a significantly increased incidence of fibroadenoma of the
mammary gland was observed in high-dose females. These benign lesions were
the only tumors reported to have incidences significantly greater than the
respective control groups; these lesions are within the range of .those
reported for strain-matched historical controls (Goodman et al., 1978).
A similar protocol was used for B6C3F, mice (NCI, 1978). Fifty mice of
each sex were exposed to 2,4-DNT at dietary concentrations of 0, 0.008, or
VI-52
-------
Table VI-15. Tumor Incidence in F344 Rats Fed 2,4-DNT for 78 Weeks1
Control Low dose High dose
Lesion Sex Low dose High dose (80 ppm) (200 ppm)
Subcutaneous
tissue or skin
fibroma M 0/46 0/25 7/49 13/49
(p - 0.008)" (p » 0.003)'
Mammary gland
fibroadenoma F 9/48 4/23 12/49 23/50
NSC (p = 0.016)°
'Incidence given as number of tumor-bearing animals/number of animals examined at
that site.
"Significantly different from respective control by Fisher's Exact test.
CNS - Not statistically significant.
SOURCE: Adapted from NCI (1978).
VI-53
-------
0.04% (80 or 400 ppm) for 78 weeks followed by 13 weeks without treatment.
Assuming that mice consume approximately 15% of their body weight in food each
day, the diets were equivalent to 12 and 60 mg/kg/day DNT, respectively
(Lehman, 1959). Because the low- and high-dose groups were tested at
different times, each was assigned its own control group. Survival rates were
similar for treated and control mice of both sexes, ranging between 74 and 90%
for males and between 70 and 84% for females. Mean body weight depression was
observed in all treated groups by week 30. No statistically significant
increase in tumors was observed in mice of either sex.
Ellis et al. (1979) evaluated the carcinogenldty of 2,4-DNT (*98.5%
2,4-DNT and *1.5% 2,6-ONT) in rats, mice, and dogs. The study in rats was
also reported by Lee et al. (1985); the study in mice was also reported by
Hong et al.. (1985); and the study in dogs was also reported by Ellis et al.
(1985).
In the study with rats (Ellis et al., 1979; Lee et al., 1985), diets
containing 0, 0.0015, 0.01, or 0.07% 2,4-DNT (0, 15, 100, or 700 ppm) were
administered to groups of 38 male and 38 female CD (Sprague-Dawley) rats,
yielding an average intake of 0.57, 3.9, or 34 mg/kg/day, respectively, for
males and 0.71, 5.1, or 45 mg/kg/day, respectively, for females.. Eight males
and eight females from each group were killed for necropsy after 12 months,
and the surviving rats were sacrificed after 24 months. Unscheduled deaths
for both sexes began after 12 months of treatment and increased until the end
of the study. Cumulative deaths reached 100% (high dose), 75% (mid dose), and
55% (low dose and controls) for male rats and 97% (high dose), 65% (mid dose),
and 60% (low dose and controls) for females. Histopathology data obtained
from rats that died after 12 months were used in calculating tumor incidences.
An increased incidence of hepatocellular carcinoma was seen in high-dose male
and female rats, although statistical significance (p <0.05) was reached only
in females (Table VI-16). Mammary tumors were prevalent in all female dose
groups; benign lesions (fibroadenoma, adenoma, and fibroma) predominated. The
incidence of these mammary gland lesions reached statistical significance only
for the high-dose .group. The frequency of malignant tumors, specifically
adenoma-carcinoma, was consistently low In all treatment groups.
VI-54
-------
Table VI-16. Tumor Incidence in CD Rats Fed 2,4-DNT for 24 Months'
Males (mg/kg/dav)
Lesion
Hepatocellular neoplastic
nodule
Hepatocellular carcinoM
Neoplastic nodule or
carcinooa •
NaMaary gland tusorsc
< Skin tutors'
•— «
en Pituitary adenoMa
en '
0
1/25 (4)
1/25 (4)
2/25 (8)
0/25
2/25 (8)
9/22 (41)
0.57
2/28 (7)
0/28
2/28 (7)
0/28
4/28 (14)
14/23 (61)
3.9
1/19 (5)
1/19 (5)
2/19 (10)
0/19
3/19 (16)
7/14 (50)
34
2/29 (7)
6/29 (21)
(p - 0.08)"
8/30 (27)
(p - 0.07)"
2/30 (7)
17/30 (57)
2/20 (10)
0
0/23
0/23
0/23
11/23 (48)
-1/22 (5)
18/23 (78)
Females (mg/kg/dav)
0.71
3/35 (9)
0/35
3/35 (9)
12/35 (34)
3/35 (9)
24/30 (80)
S.I
2/27 (7)
1/27 (4)
3/27 (11)
17/27 (63)
0/27
20/24 (83)
45
6/34 (18)
(p = 0.07)"
18/34 (i-3)
(p « 4.3 >: 10 V
23/35 (66)
(p - 9.5 > 10'V
33/35 (94)
(p i O.OOC1)"
6/35 (17)
7/23 (30)
'••••d on data fro* anioals fed 2,4-DNT for More than 12 Months. Incidence given as the number of tuaor-bearing rats/nunber of rats examined at that site
(percent incidence). Rats that died before week 52 are not included in the calculation of tunor incidence.
"fisher's Exact test perforated by Syracuse Research Corporation (SRC), as cited in U.S. EPA (1986).
'includes benign and Malignant tusors froM epithelial or Mesenchyaal cells.
"fisher's Exact test performed by reviewers.
'includes epidermal, epithelial, and subcutaneous Mesenchymal tuners.
SOURCE: Ellis et al. (1979).
-------
In the mouse,study (Ellis et al., 1979; Hong et al., 1985), diets
containing 0, 0.01, 0.07 or 0.5% 2,4-DNT (0, 100, 700, or 5,000 ppm,
respectively) were administered to groups of 38 male and 38 female CD-I mice
yielding an average intake of 0, 14, 95, or 898 mg/kg/day, respectively, using
procedures similar to those described for rats by Lee et al. (1985).
Unscheduled deaths occurred early in the high-dose mice; all males in this
group died by month 18 and all females died by month 21. Since most of the
high-dose mice died before 12 months (31/38 males and 23/38 females), this
group was not considered to be representative of a lifetime exposure and was
excluded from further analysis. Approximately 70% of the control and low-dose
and 85% of the mid-dose males and 80% of the control and low- and mid-dose
females died before scheduled sacrifice. Nice that died after 12 months were
used in calculating tumor incidences. There was no evidence of a treatment-
related increase in tumor frequency in any tissue in female mice. Among
males, a very high incidence of renal tumors, including cystic papillary
adenomas, solid renal cell carcinomas, and cystic papillary carcinomas, was
seen in the mid-dose (700 ppm) group (Table VI-17). Similar lesions were
observed in the low-dose males, but the Incidence was not significantly
different from controls. Liver tumors, described as most probably hepatomas,
were observed in male mice of all dose groups, but no significant difference
.from controls was seen.
In the study with dogs (Ellis et al., 1979; Ellis et al., 1985), groups
of six male and six female beagle dogs received daily doses of gelatin
capsules containing 0, 0.2, 1.5, or 10 mg 2,4-ONT/kg. The high-dose level ««s
toxic to all dogs and proved lethal to five. The mid-dose level was toxic to
some of the dogs, whereas the low-dose level had no apparent adverse effects.
A thorough clinical and Mstopathological examination was performed on each
dog following- sacrifice. No evidence of cardnogenicity was seen in any of
the dogs fed 2,4-DNT for 2 years. :
In a 12-month study, Leonard et al. (1987) observed significant
reductions in body weight gain (p <0.05) and increases in liver weight
(approximately 150% of control) following dietary administration of purlf'td
2,4-DNT (>99.4% pure) to 20 male CDF (F344)/CrlBR rats at concentrations
VI-56
-------
Table VI-17. Tumor Incidence in Male CD-I Mice Fed 2,4-DNT for 24 Months*
Dose fmaAa/dav)
Lesion
Liver tumorb
Kidney
Benign
Malignant
Benign or malignant
All tumors
(Kidney + liver)
0
7/33
0/33
0/33
0/33
7/33
14
9/33
3/33
5/33
8/33
17/33
95
8/28
3/28 (p - 0.014)c
16/28 (p - 1.45 x 10'J)C
19/28 (p - 1.32 x 10'V
(p - 0.059)c
24/28 (p - 2.17 x 10'5)c
"Incidence given as the number of tumor-bearing mice/number of mice examined at that
site. Animals that died before week 52 are not included in the calculation of tumor
incidence. Since most of the high-dose mice died before 1 year, the entire group
has been excluded. .
n"umor type not specified.
cFisher's Exact test performed by SRC, as cited in U.S. EPA (1986).
SOURCE: Ellis et al. (1979).
VI-57
-------
yielding doses of 27 mg/kg/day. One neoplastic nodule of the liver was found.
Due to some limitations (i.e., the short study duration and the low number of
animals), the study failed to demonstrate the carcinogenic activity of the
chemical.
b. 2.6-DNT .
In a 12-month study, Leonard et al. (1987) administered purified 2,6-ONT
(>99.4% pure) to 20 male COP (F344)/CrlBR rats at dietary concentrations
yielding doses of 0, 7, or 14 mg/kg/day. At both dose levels, 2,6-ONT
significantly reduced body weight gain and Increased liver weight.
Hepatocellular carcinomas were found in 17/20 (85%) low-dose and in 19/19
(100%) high-dose male rats, and pulmonary metastases were found In 3/20 (15%)
low-dose and in 11/19 (58%) high-dose rats. Cholangiocarclnoraas were reported
in 2/20 (10%) low-dose and 0/19 high-dose males. No neoplastic lesions were
found in the control group.,
c. to-DNT
In a 12-month study, Leonard et al. (1987) observed significant
'. . '
reductions in body weight gain and increases in liver weight following dietary
administration of a technical grade mixture of dinitrotoluenes to 20 male CDF
(F344)/CrlBR rats at concentrations providing a dose of 35 mg/kg/day. The
tg-ONT was prepared by mixing purified DNT isomers in a ratio representative
of a standard technical grade, with a final composition of 76.5% 2,4-DNT,
18.8% 2,6-ONT, 2.43% 3,4-DNT, 1.54% 2,3-DNT, 0.69% 2,5-DNT, and 0,04% 3,5-DNT.
Hepatocellular carcinomas were observed in 9/19 (47%) and cholangiocarcinomas
in 2/19 (11%) of the male rats fed tg-ONT for 1 year. No neoplastic lesions
were found In the control group of 20 males.
The carcinogenic effects of tg-DNT (composition not reported) were
evaluated in Charles River CDF (F344) rats (130 rats/sex/group) following
administration at dietary levels providing 0, 3.5, 14.0, and 35.0 mg/kg/day
(CUT, 1982). Sacrifice and necropsy of 10 rats/sex/group occurred at weeks
26 and 52; 20 rats/sex/group were sacrificed at week 78, all surviving high-
dose rats at week 55, and all surviving animals at termination after 104
VI-58
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weeks. A statistically significant dose-related decrease in body weight gain
was seen in all treated groups throughout the first year of the study. After
54 weeks, a dose-related decrease in mean body weight was observed at all dose
levels in both sexes.
Hepatocellular carcinomas were first evident in 2/10 high-dose males
sacrificed at 26 weeks. At 52 weeks, a high Incidence of hepatocellular
carcinomas and hepatic neoplastic nodules was evident in the high-dose group
and the mid-dose males.. Two cholangiocarcinomas observed in the high-dose
males were considered to be treatment-related because this neoplasm 1s unusual
in CDF rats. Biliary hyperplasia with atypia of bile duct epithelium that
occurred in two high-dose and one mid-dose male may have been precursor
lesions of cholangiocarcinoma. All high-dose rats were terminated at week 55
because of a significant decrease in survival. Histological examination
revealed hepatocellular carcinomas in all males (20/20) and most females
(11/20), with hepatic neoplastic nodules in 5/20 males and 12/20 females.
Cholangiocarcinomas were observed in three males, and biliary hyperplasia with
atypia of bile duct epithelium was observed in two males. At 78 weeks, a high
incidence of hepatocellular carcinomas and hepatic neoplastic nodules was
observed in the mid-dose animals and to a much lesser degree in the low-dose
animals. Increased Incidence of cholangiocarcinomas and probable precursor
lesions was seen in the mid-dose males along with some benign lesions
including mammary fibroadenomas and subcutaneous fibromas. The significance
of pituitary chromophobe adenomas observed in mid-dose females was equivocal.
At 104 weeks, a high incidence of hepatocellular carcinomas and hepatic
neoplastic nodules was seen In mid-dose animals and to a lesser degree In low-
dose animals (Table VI-18). The incidence of hepatocellular carcinomas shoved
a dose-related increase in males, and pulmonary metastatases were found in
seven mid-dose males and females. The incidence of hepatic neoplastic nodules
was dose-related in both males and females. Increased Incidences in the
relatively common benign neoplasms were also observed at 78 weeks. Thest
included mammary fibroadenomas In mid-dose rats and low-dose males;
subcutaneous fibromas (a dose-related Increase), particularly in male rats;
and testicular mesothelioma in mid-dose males (equivocal). An exacerbation of
chronic interstitial nephritis was observed, particularly in females, with «
VI-59
-------
Table VI-18. Incidence of Liver Tumors in CDF Rats Fed tg-DNT for up to 2 Years'
at
o
Duration
of treatment
(weeks) Lesion" 0
52 NN
HC •' -
NN or HC 0/10
55£ NN
HC
NN or HC
78 NN
HC
NN or HC 0/20
104 NN 9/61
HC 1/61
NN or HC 10/61
U0d NN
HC .
NN or HC 0/19
Males
3.5
0/10
,
1/20
11/70
9/70'
19/70
3/20
1/20
4/20
(mg/kg/day)
14 35
4/10' 3/10
. 3/10 10/10"
5/10* 10/10"
5/20
20/20"
20/20"
11/20"
19/20"
20/20"
16/23"
22/23"
23/23"
31/65"
57/65"
60/65"
Females (mg/kg/day)
0
0/10
0/20
5/57
0/57
5/57
1/23
0/23
1/23
3.5
0/10
2/20
0/20
2/20
12/61
0/61
12/61
1/29
1/29
2/29 ,
. 14 35
B/10"
4/10'
0/10 9/10"
12/20"
11/20"
19/20"
10/20"
0/20
10/20
53/68"
40/68.
66/68
"12/22"
l/22>t
13/22
'incidence given as the number of tumor-bearing animals/number of animals examined at that site. Statistical significance determined by SRC using
bFisher's Exact test.-as cited in U.S. EPA (1986). .
bNN, neoplastic nodule; HC. hepatocellular carcinoma.
CA1I high-dose animals were sacrificed at 55 weeks, and 20 rats of each sex were examined histologically. No other dose group was examined at this time.
Statistics for this group are based on comparison with controls at 52 weeks.' '
d(JO - Unscheduled deaths. Results include all animals found dead or sacrificed moribund throughout the study. •
"p <0.05; "p <0.001.
SOURCE: CUT "(1982).
-------
resultant increase in parathyroid hyperplasio and auenuma, and .in exacerbation
of degenerative changes in the adrenal glands was reported in mid-dose rats.
Histopathologic examination of the major tissues supported the above
observations and revealed, In addition, an Increased incidence of
neurofibromas and subcutaneous sarcomas In mid-dose males.
d. DNT Isomers
Hoi en et al. (1990) studied the possible relationship between
carcinogenicity of DNT Isomers and the response in the Syrian hamster embryo
cell (SHE) using two short-term in vitro assays. The 2,6- and 2,4-DNT Isomers
were 10 times less toxic than the 2,3- and 3,4-ONT Isomers on the relative
survival of SHE cells. Also, none of these four Isomers Increased
morphological cell transformation of SHE cells relative to the control cells.
None of the Isomers possessed promoter or Initiator effects 1n the
transformation assay. Because of lack of effect, four oxldatlve or reductive
metabolites of DNT were examined. All four metabolites were less toxic than
the parent DNT, and none (together with technical grade (tg) DNT) Induced art
increase In the morphological transformation of SHE cells. Intercellular
communication, based on a dye transfer, was Inhibited by 2,4- and 2,6-DNT
isomers and tg-DNT but only at toxic concentrations. It was suggested that
this effect was similar to data reported In vivo Indicating a similar promoter
effect of these compounds. The 2,3- and the 3,4-DNTs did not affect this
assay,
6. Genotoxlcitv
a. Mlcroblal systems
Both 2,4- and 2,6-DNT are weak mutagens in Salmonella test systems.
Because of this weak activity, some of the studies using plate Incorporation
assays gave equivocal and conflicting results. Hodgson et al. (1976) reported
that 2,4-DNT was mutagenlc 1o.£. tvohimurlum strain TA1535. Ch1u et al.
(1978) reported that 2,4-DNT was not mutagenlc in strain TA98 or TA100. Mori
et al. (1982) found .that mutation frequency was less than 3 times the control
rate in Salmonella strains TA98 and TA100 at 2,4-DNT concentrations between
100 and 300 M9/plate. Toklwa et al. (1981) found a modest response 1n strain
VI-,61
-------
TAiOO but negative responses in strain IA96 with 500 or 1,000 jig/plate 2,4-
DNT or 2,6-DNT. Spanggord et al. (1982) reported positive dose responses with
2,4- and 2,6-DNT in Salmonella strain TAIOO with or without S9 activation but
no mutageniclty in strain TA1535, TA1537, TA1538, or TA98; the 2,5- and 3,5-
isomers were more mutagenic in strain TAIOO with peak activities at 200 and
500 M9/plate, respectively. 2,4- and 2,6-DNT were tested at 100, 500, and
1,000 /ig/plate. Neither isomer showed mutagenic activity in a nitro
reductase-deficient strain (TAIOO NRS). The 1nterstra1n variability in these
studies may have been partly accounted for by exceeding the narrow
concentration range for cytotoxldty.
Couch et al. (1981) carried out a comprehensive analysis of the mutagenic
potential of technical.DNT and each of the six pure isomers of DNT in
Salmonella. A liquid suspension assay was used, with a 3-hour Incubation of
bacteria with test compound with or without S9 mix prior to plating.
Cytotoxicity was quantitated, and dose-response data for mutation frequency
i " ~ • . .
were developed. Technical grade DNT and all the pure Isomers of DNT were
mutagenic to one or more strains of S_. tvphimurium (TA1535, T.A1537, TA1538,
TA98, or TAIOO). Mutation frequency was not high compared with other
mutagens, but it was reproducible and concentration related. The most
mutagenic Isomer, 3,5-DNT, was almost an order of magnitude more potent than
any of the other Isomers and was mutagenic without S9 activation in strains
TA1538, TA98, and TAIOO. In dose-response experiments 1n strain TA98, 2,4-
and 2,6-DNT and tg-DNT all caused about a fivefold Increase in mutation
frequency at concentrations of 500 ng/mL when compared with the DMSO control.
When added, S9 did not appreciably affect the mutageniclty. In an
,8-azaguanine forward mutation assay in S. tvphimurium TM677, similar mutation
frequencies were noted for tg-DNT and 2,4- and 2,6-DNT.
/ ,- - • '
Because the weak mutagenic activity did not correlate with
carcinogeniclty of tg-DNT, studies have been performed to test the
mutagenicity of DNT metabolites and possibly to identify the proximate
carcinogen. Spanggord et al. (1982) initially showed that nitrpreductase
activity was required for mutagenic activity in S.. tvphimurium TAIOO. Couch
et al. (1987) found that 2,4-dinitrobenzyl alcohol, 2-am1no-4-n1trotoluene,
and 2-n1troso-4-n1trotoluene were more mutagenic that 2,4-DNT In
S. tvphimurium strain TA98 (Table VI-19). The first compound 1s a major
VI-62
-------
Table VI-19. Comparative Mutageriicity of 2,4-DNT and Its Metabolites
Revertants/viable cell/mmol (x 107)1
Compound
2,4-Dinitfotoluene
2,4-Diaminotoluene
2,4-Dinitrobenzoic acid
2-Amino-4-nitrotoluene
2-Amino-4-nitrobenzoic acid
2:Nitroso-4-nitrotoluene
2,4-Dinitrobenzyl alcohol
-S9
1.7
0.1
1.6
12.4
5.9
47.5
13.5
+S9
1.8
0.7
1.5
2.9
40.6
25.9
, 27.2
'Calculated from the slopes of the concentration versus mutant fraction by
linear regression analysis.
SOURCE: Adapted from Couch et al. (1987).
VI-63
-------
urinary metabolite in rats, and the,2-amino and 2-nitroso derivatives are
formed from 2,4-ONT by anaerobic microorganisms from rat cecum (see Chapter V,
Pharmacokinetlcs). Mori et al. (1982, 1985), in similar studies on the
mutagenicity of 2,4-DNT metabolites and derivatives, found that
2,4-dinitrophenyl alcohol was a strong mutagen in strains TA98 and TA100 but
that the corresponding aldehyde was 25 to 40 times more active than the
alcohol; they suggested that this correlated with the carcinogenicity of
2,4-DNT. Both oxidative and reductive metabolism may be involved in the
production of mutagenic metabolites.
Simmon et al. (1977) reported that 2,4- and 2,6-DNT did not produce
mitotic recombination in Saccharomvces cerevisiae 03; However, insufficient
data were provided to 'determine if the dose used was adequate.
b. Mammalian cells in vitro . ,
Abernethy and Couch (1982) found that 2,4- and 2,6-DNT were not mutagenic
in the HGPRT system in Chinese hamster ovary cells. The isomers were tested
at their limit of solubility in culture medium (3.0 mM 2,4-ONT or 2.5 mM
2,6-DNT) in the presence or absence of S9 from livers of Aroclor-treated rats.
Styles and Cross (1983) tested tg-ONT and 2,4- and 2,6-DNT in the P388
lymphoma assay. Both tg-DNT and 2,6-DNT gave a negative response to +/- S9
activation. 2,4-DNT gave a weak but positive result at 200 and 1,000 ng/mL
but only in the absence of S9 activation; the response was weaker than with
trinitrotoluene.
2,4-DNT was also reported to be negative in the C3H10P/4 transformation
assay system (Rickert et al.; 1984).
c. In vivo assays
v y \
Ellis et al. (1979) carried out cytogenetic examinations of primary
cultures of bone marrow and kidney cells from rats fed 2,4-DNT in the diet
VI-64 .
-------
(100 or 700 ppm) for 2 years. No clear effects on ploidy, chromosome number,
or chromosomal abnormalities were observed.
Scares and Lock (1980) found that 2,4-DNT and tg-DNT were negative in the
sperm morphology test performed 8 weeks after two dally intraperitoneal doses
of 250 mg/kg. Styles and Penman (1985) found that 2,4-DNT was negative 1n the
mouse spot assay. No mutant clones of pigment cells were found in embryos
after dams were intraperitoneally dosed with 100 mg/kg on gestation day 10.
Soares and Lock (1980) similarly found a negative response in the mouse spot
test with 100 mg/kg tg-DNT.
Negative results from tg-ONT and 2,4-DNT have been found In the dominant
lethal test in mice and rats. Soares and Lock (1980) dosed male DBA/Jmice
intraperitoneally or by oral gavage with a single dose or two dally doses of
250 mg/kg tg-ONT or pure 2,4-DNT and mated them after 48 hours. Lane et al.
(1985) dosed Sprague-Oawley rats by oral gavage for 5 days at 2,4-DNT levels
between 60 and 240 mg/kg/day. Ellis et al. (1979) evaluated rats fed 200 or
2,000 ppm 2,4-DNT for 10 to 13 weeks. In all of these studies, adverse
effects on fertility, but no dominant lethal mutations, were seen.
2,4-DNT was tested in the recessive lethal test and reciprocal
translocation test in Drosophila (Woodruff et al., 1985). Negative results
were found after feeding at 1,000 ppm or injection of 200 or 10,000 ppm.
d. Unscheduled DNA synthesis (UPS)
Bermudez et al. (1979) found that technical grade DNT (10~4 and 10"5M) and
2,4- or 2,6-ONT (10~3 or 10"4M) gave a negative response for UOS 1n primary
hepatocyte cultures from Fischer 344 male rats using an assay based on
autoradiography. Negative results for 2,4- and 2,6-ONT were also reported for
UDS in primary cultures of human hepatocytes (Butterworth et al., 1989).
Working and Butterworth (1984) reported negative results for UDS in cultured
rat spermatocyte at 10 or 100 urn 2,4- or 2,6-DNT with or without S9
activation.
VI-65
-------
Mirsalis et al. (1982) utilized an j_n vivo/in vitro assay for UDS in
which male Fischer 344 rats were dosed by oral gavage with 2,4- or 2,6-DNT
(99.9% pure). Hepatocytes were isolated by liver perfusion 12 hours after
dosing, and UDS was measured in primary culture by incubation with
3H-thymidine (with or without hydroxyurea) by determining nuclear grain counts
with autoradlography. A positive response was found 12 hours after dosing
with 200 mg 2,4-DNT/kg or 20 mg 2,6-DNT/kg. Mirsalis and Butterworth (1982)
assayed tg-DNT in this system. A sharp dose-response curve was seen with a
marked Increase in UOS between 50 and 100 mg/kg. UDS activity peaked 12 hours
after dosing, decreased substantially by 24 hours, and returned to background
levels by 48 hours. Hydroxyurea (10 or 20 mM) suppressed the number of cells
in S-phase at 48 hours but had no effect on UDS. This confirms that the
increase in grains 1s not due to replicative synthesis but is due to repair
synthesis. The potency of tg-DNA and of 2,4- and 2,6-DNT was compared. The
2,4-DNT gave a weaker response at 10 mg/kg than the tg-ONT (33 versus 73% of
cells in repair), and 2,6-DNT gave a strong response at 20 mg/kg (79% cells in
repair) but was toxic at 100 mg/kg. Female rats gave a much lower level of
UDS than males at 100 mg/kg. When 0.1% tg-DNT was administered in the diet
for 1, 2, or 4 weeks, a modest but significant Increase was seen in UOS in
cultured hepatocytes. Metabolism of DNT by gut flora is necessary for UOS
expression, since the effects could not be elicited in germ-free rats
(Mirsalis et al.,.1982). The activation of DNT probably requires
P-450-dependent hepatic metabolism to the benzyl alcohol, excretion into the
bile after glucurondation, metabolism in the gut, and reabsorption and further
hepatic action (Popp and Leonard, 1982).
e. Initiation, promotion, and DNA binding
Dor-man and Boreiko (1983) found that tg-DNT and 2,4-DNT did not inhibit
metabolic cooperation between 6-thioguanine resistant and sensitive V79
Chinese hamster lung cells. This test is positive with several tumor
promoters. The compounds were tested at levels of 10f3 to 10>9M; toxicitjr •«*
seen at levels between 5 x 10'4 and 10>3M.
VI-66
-------
Leonard et al. (1983, 1986) studied tg-ONT and purified 2,4-DNT and
2,6-DNT 1n initiation-promotion protocols in which the increase in liver foci
staining for gamma glutamyltranspeptidase (GGT) was the endpoint (also
discussed in Popp and Leonard, 1983). In the 1983 study, initiation potential
was studied in nonhepatectomized rats and at various periods after partial
hepatectomy. Technical grade DNT did not prove to be a reproducible Initiator
of GGT* foci except when administered as a single oral dose of 75 mg/kg
12 hours after partial hepatectomy. Under the same conditions, 2,6-DNT was
the only isomer that Induced GGT" foci. A dose-response Increase was seen
between 37.5 and 150 mg/kg. However, both tg-ONT and 2,6-DNT were weak
initiators in comparison with other initiators previously studied. In the
1986 study, male Fischer 344 rats were initiated with a single intraperltoneal
dose of dimethylnitrosamine (a necrogenic dose) and allowed to recover for
2 weeks. The rats were then fed diets containing tg-ONT, 2,4-DNT, 2,6-DNT, or
phenobarbital to test their promoting activity. The dietary levels were 14 or
35 mg/kg/day for tg-ONT, 2.8, 7 or 14 mg/kg/day for 2,6-DNT, 27 mg/kg/day for
2,4-DNT, and 0.05X of diet for phenobarbital. Treated animals and appropriate
controls were killed at 3 and 6 weeks (tg-ONT) or 6 and 12 weeks (2,4- and
2,6-DNT, and phenobarbital) and liver GGT* foci were quantified. Technical
grade ONT caused a dose-dependent increase in foci; 2,6-DNT produced a time-
and dose-related Increase as well as an increase in foci and noninitiated
controls, but 2,4rONT and phenobarbital produced only time-related Increases.
The 2,6-DNT Isomer was approximately 10 times more potent than the 2,4-DNT
isomer. Under the conditions of this study, 2,4-DNT appears to be a pure
promoter, whereas 2,6-DNT has Initiating as well as promoting activity
(complete hepatocarcinogen). Although the specificity of GGT* foci as an
early endpoint marker of hepatocarcinogenidty has not been adequately
established in animal and human studies, the results of these studies on
initiation-promotion using GGT* foci as a short-term endpoint correlate with
the results of cardnogenicity bloassays of tg-ONT and 2,4-DNT.
Kedderis et al. (1984) showed that when radloactlvely labeled 2,4-DNT or
2,6-DNT were administered orally to rats, radioactive residues were bound to
liver macromolecules. Binding of radioactivity from 2,6-DNT to liver DNA was
abolished by prior administration of the sulfotransferase inhibitors
VI-67
-------
pentachlorophenol or 2,6-dichloro-4-nitrophenol. A similar finding was
reported for 2-nitrotdluene but not the 3- or 4-nitrotoluene isomers (Rickert
et al., 1984). It was suggested that the action of sulfotransferase in the
metabolism of dlnltrophenols was required for formation of an electrophilic
compound capable of reacting with hepatic DMA. Metabolic pathways for 2,6-DNT
and 2,4-DNT are similar (biliary excretion of glucuronide of the nitrobenzole
acid and resorption and recirculation to the liver), and both compounds are
potent Inducers of DNA repair In the in vivo/1n vitro hepatic UDS assay (Popp
and Leonard, 1982; Mirsalis et al., 1982; Doolittle et al., 1983).
7. Neurotoxicitv
No neurotoxicity studies per se were found in the available literature.
However, subacute (4-week) and subchronic (13-week) studies in dogs
administered 2,4- or 2,6-DNT showed some evidence of neurotoxicity following
oral (capsule) administration (Lee et al., 1978; Ellis et al., 1985). These
studies are discussed in detail in Sections VI.B.I, Short-term Exposure, and
VLB.2, Long-term Exposure. Groups of four male dogs and four female dogs
were given 0, 1, 5, or 25 mg 2,4-ONT/kg/day for 4 or 13 weeks. After 4 or
13 weeks of exposure, two dogs/sex/dose were sacrificed, and treatment was
discontinued for the remaining dogs. These dogs were sacrificed following a
4-week recovery period. No neurotoxic signs were observed in dogs given 1 or
5 mg 2,4-ONT/kg/day for 4 or 13 weeks. Dogs exposed to 25 rag/kg/day showed
neurotoxic symptoms after 12 weeks of treatment. Neuromuscular system effects
(incoordination and paralysis) were characteristic. The first manifestation,
stiffness and incoordination of the hind legs, was followed by difficulty in
maintaining balance, which culminated with the animals lying on their sides;
the muscles appeared to be stiffened, and the legs were extended and arched.
The stiffness progressed upward from hind legs to trunk, forelegs, neck, and
head. Several dogs exhibited occasional tremors, and one dog had a grand mal-
type convulsion. Two dogs treated for 13 weeks developed transient blindness;
one dog was functionally blind with widely dilated pupils unresponsive to
light. Vertical or oval nystagmus was present when the dog's position was
changed or when she was stimulated by a loud noise. Three dogs (sacrificed at
weeks 6, 7, and 13) showed mild to moderate gliosis, edema, and demyelination
VI-68
-------
in the cerebellum, brain stem, or spinal cord. Oemyelination of the optic
nerve was observed in only one of the two dogs that suffered from transient
blindness. Allowed to recover for 4 weeks, one dog developed a minor balance
problem and some demyelination in the cerebrum and optic nerve. Two dogs kept
for 8 months completely recovered from all effects (Lee et al., 1978).
t
Ellis et al. (1985) dosed groups of six male and six female beagle dogs
with 0, 0.2, 1.5, or 10 mg 2,4-DNT/kg/day in capsules for up to 24 months.
This study 1s discussed in Section VLB.5, Cardnogeniclty. The first
neurotoxic symptoms were seen in dogs dosed at 1.5, 5, or 10 mg/kg/day after
465, >91, and 52 days of dosing, respectively. Individual variation in
susceptibility to these effects was observed; some animals were moribund while
others were barely affected. The minimal signs, of neurotoxicity were
incoordination and stiffness. The hind legs were affected to the greatest
extent; abnormal gait was pronounced when running. More severely affected
dogs experienced rigid paralysis of the hind legs that progressed upward
affecting the forelimbs and eventually the neck; during this stage, the dogs
assumed a rigid horizontal position. In some dogs (number not specified),
paralysis regressed after affecting the hind limbs; the dogs were without
toxic signs within days to weeks. Neurotoxic signs were also observed in tht
lips and tongue; the affected dogs were unable to pick up food.
Histopathology revealed central nervous system lesions that included
generalized yacuolat1on, hypertrophy and mitosis of the endothelium, focal
gliosis in the cerebellum, and some perl vascular hemorrhages in the cerebcllua
and brain stem. These lesions were most severe In dogs that developed toxic
signs late in the study. During the recovery period beginning 4 weeks afUr
treatment and continuing for 12 or 24 months, the less affected dogs recovtrtd
sooner than those severely affected.
/
Lee et al. (1978) gave groups of four male and four female dogs doses of
0, 4, 20, or 100 mg 2,6-DNT/kg/day in capsules for 4 or 13 weeks. The
experimental design and procedures were identical to the studies with 2,4-ONT
described above. No neurotoxic signs were seen in dogs dosed at 4 or
20 mg/kg/day. However, dogs dosed at 100 mg/kg/day showed listlessness,
incoordination, lack of balance, and general weakness, particularly of th«
VI-69
-------
hind limbs. " The clinical symptoms were not substantiated by any
histopathological lesions of the central nervous system.
VI-70
-------
VII. HEALTH ADVISORY DEVELOPMENT
A. SUMMARY OF HEALTH EFFECTS DATA
In humans, the toxic effects of DNT are on the central nervous system
and also may be on the on the heart and circulatory system. Chronic DNT
exposure, primarily via the inhalation route, is characterized in munitions
workers by nausea,.vertigo, methemoglobinemia, cyanosis, pain or paresthesia
in extremities, tremors, paralysis, chest pain, and unconsciousness (Etnier,
1987; U.S. EPA, 1980, 1986; Levine et al., 1985a; Ellis et al., 1979).
Following a latency period of 15 years, workers exposed to 2,4-DNT and tg-DNT
exhibited excessive mortality from ischemic heart disease and residual
diseases of the circulatory system (Levine et al., 1986a,b). Limited evidence
\
suggests that DNT,does not result in adverse effects on human reproductive
performance (Hamill et al., 1982; Ahrenholz and Meyer, 1982).
Acute oral toxicity studies indicate that rats are more susceptible to
2,4-DNT than mice. The LDM values ranged from 1,340 to 1,954 nig/kg in mice
and from 270 to 650 mg/kg in rats (Lee et al., 1975; Vernot et al., 1977).
Both species exhibited ataxia and cyanosis. When mice, rats, and dogs were
administered 2,4-DNT in the diet for 4 weeks, dogs were found to be most
sensitive, exhibiting incoordination and paralysis at doses of 25 mg/kg/day;
CMS effects were not observed in rodents (Lee et al., 1978; Ellis et al.,
1985). Similar CNS effects were seen when dogs were orally administered 20 mg
2,6-DNT/kg/day. Methemoglobinemia, reticulocytosis, and Heinz body formation
resulted from oral administration of 37.5 mg tg-DNT/kg/day to F344 rats for
4 weeks (CUT, 1977).
Subchronic toxicity was assessed in mice and rats administered 2,4-DNT
via the diet and in dogs administered 2,4-DNT via capsules for 13 weeks. In
mice of both sexes, anemia and reticulocytosis were observed at a dose level
of 413 (males) and 468 (females) mg/kg/day (Hong et al., 1985). In rats,
mortality occurred prior to study termination in 100% of females and 75% of
males fed 145 (females) to 266 (males) mg/kg/day (Lee et al., 1985). Anemia
and reticulocytosis were observed at dose levels as low as 93 (males) to 108
VII-1
-------
(females) mg/kg/day; splenic hemosiderosis and depressed spermatogenesis were
found in th.ese animals. In dogs, neuromuscular incoordination and paralysis,
methemoglobinemia, aspermatogenesis, hemosiderosis of the spleen and liver,
cloudy swelling of the kidneys, and lesions of the brain were observed in
males and females at a dose level of 25 mg 2,4-DNT/kg/day (Ellis et al.,
1985). Similar effects were seen when mice, rats, and dogs were orally
administered 2,6-DNT for 13 weeks (Lee et al., 1975). All species exhibited
methemoglobinemia, anemia, bile duct hyperplasia sometimes accompanied by
hepatic degeneration, and depressed spermatogenesis. Incoordination, rigid
paralysis, and renal degeneration occurred in dogs at a dose level of 20 mg
2,6-DNT/kg/day.
Lifetime feeding studies in rats and mice and a 2-year study in dogs
showed increased mortality, weight loss, anemia, neurotoxicity,
hepatotoxicity, renal toxtcity, and testicular atrophy (Ell 1s et al., 1979;
Ellis et al., 1985; Lee et al., 1985; Hong et al., 1985). In CD (Sprague-
Oawley) rats fed 2,4-ONT in the diet at a level giving a daily intake of 34
(males) and 45 (females) mg/kg/day, respectively, lifespan was shortened; the
incidence of hepatocellular carcinomas was increased (6/30 and 19/35 high-dose
. i
males and females, respectively, as compared with 1/25 and 0/23 concurrent
controls); seminiferous tubules atrophied resulting in almost complete
cessation of spermatogenesis; and excessive pigmentation accumulated in the
/ ;
spleen. An increased incidence of benign mammary gland tumors (33/35 as
compared with 10/23 in controls) Was exhibited in females. Anemia, partially
compensated, and testicular atrophy were evident at 3.9 mg/kg/day, and
hepatocellular alterations were observed at 0.57 mg/kg/day, the lowest dose
tested (Lee et al., 1985). In a study in CD-I mice, all the animals fed
898 mg/kg/day died by month 18 (males) or month 21 (females). Effects at
14 mg/kg/day, the lowest dose tested, included testicular atrophy, decreased
body weight in males, and hemosiderosis of many organs, primarily the liver
and spleen. The incidence of malignant renal tumors was elevated in males fed
95 mg/kg/day (15/1.7 as compared with 0/20 concurrent controls) (Hong et al.,
1985). In a study in dogs, 2,4-DNT administered daily in capsule form caused
neurotoxicity (incoordination and paralysis, often leading to death) in all
* - ' '
dogs at 10 mg/kg/day, and anemia (partially compensated) and bi.liary tract
VII-2
-------
hyperplasia at 1.5 mg/kg/day. No adverse effects were observed at
0.2 mg/kg/day (Ellis et al., 1985).
Male and female CDF (F344) rats fed tg-DNT in the diet at a level to
give a daily intake of 35 mg/kg/day were sacrificed in extremis after 55 weeks
(Leonard et al., 1987). At 14 mg/kg/day, 96% of the males and 59% of the
females developed hepatocellular carcinomas. After 78 weeks, decreased body
weight, increased liver weight, hepatotoxicity (increased incidence of
hepatocellular carcinomas, 9/19) renal toxicity, and parathyroid hyperplasia
were reported at 3.5 mg/kg/day, the lowest dose tested.
In a limited 1-year study, 47% of male CDF (F344) rats given tg-DNT at
dose levels providing 35 mg/kg/day (the only dose tested) developed
hepatocellular carcinomas (0/20 control, 9/19 treated) (CUT, 1982). Under
similar conditions, 2,6-DNT induced hepatocellular tumors in 100% of the
high-dose (14 mg/kg/day) and 85% of the low-dose (7 mg/kg/day) males. Rats
exposed to 2,4-ONT (35 mg/kg/day) did not develop tumors (Leonard et al.,
1987). Although limited in duration and in the number of animals tested,
these data may indicate that the 2,6-DNT isomer causes much of the
carcinogenic activity in the previously tested mixed-isomer DNT bioassays.
DNT is classified as Group 82: Probable Human Carcinogen.
Both 2,4- and 2,6-DNT are weak mutagens in Salmonella test systems.
However, metabolites of 2,4-DNT are mutagenic without metabolic activation,
particularly the 2,4-nitrobenzyl alcohol and the 2-amino- and 2,nitroso-4-
nitrotoluenes (Couch et al., 1987). The dinitrotoluenes are also negative
genotoxins in mammalian cells in vitro, in the dominant lethal test in mice
and rats, and in Drosoohila systems (Abernethy and Couch, 1982; Styles and
Cross, 1981; Scares and Lock, 1980; Lane et al., 1985; Ellis et al., 1979;
Woodruff et al., 1985). Technical grade DNT gave negative responses for
unscheduled DNA synthesis (UDS) except when an in vivo/in vitro testing system
was used (Bermudez et al., 1379; Mirsalis and Butterworth, 1982a). It was
concluded that biliary excretion, metabolism by gut flora, and resorption from
the intestine are prerequisites for genotoxic activity (Mirsalis et al.-, 1982;
Popp and Leonard, 1982). Metabolites of 2,4-DNT can bind to liver DNA, and
VII-3
-------
2,4-DNT appears to act as a promoter, inducing gamma glutamyl transferase
positive foci ..in the livers of rats initiated with dimethylnitrosamine
(Leonard et al., 1983, 1986): On the other hand, 2,6-ONT has both initiation
and promoting activity (Popp and Leonard, 1982; Mirsalis et al., 1982;
Doolittle et al., 1983).
The 2,4-DNT isomer causes severe reproductive effects in rats, mice, and
dogs (El11s et al., 1979; Lee et al., 1985; Hong et al., 1985; Ellis et al.,
1985). Oral exposure to 2,4-DNT results in testicular atrophy and
degeneration as well as reductions in spermatogenesis in males. In females,
oral exposure resulted in cessation of follicular function and reduction in
the number of corpora lutea. Consequently, fertility is reduced in both
sexes. Also, 2,4-DNT causes reduced viability as well as decreases in the
body weight of offspring at birth and weaning. No data on the reproductive
effects of 2,6-DNT or technical grade DNT were found in the available
literature. Limited avaialable data suggest that 2,4-DNT is not teratogenic'
in mice following ingestion (Hardin et al., 1987). Technical grade DNT was
not teratogenic to rats administered oral doses, although embryotoxicity was
observed at maternally toxic levels (Price et al., 1985). In addition, the
administration of technical grade DNT to pregnant rats also resulted in
changes in relative organ weights, and hematologic parameters in their fetuses.
No data on the developmental effects of 2,6-DNT were found in the available
literature.
B. QUANTIFICATION OF TOXICOLOGICAL EFFECTS
Health Advisories are generally determined for One-day, Ten-day, Longer-
term (approximately 7 years), and Lifetime exposures if adequate data are
available that identify a sensitive noncarcinogenic endpoint of toxicity. The
HAs for noncarcinogenic toxicants are derived using the following formula:
HA - fNOAEL or LOAEL) (bwl - mg/L (
(UF) ( L/day)
vir-4
-------
where:
NOAEL or LOAEL » No- or Lowest-Observed-Adverse-Effect Level
(in rag/kg bw/day).
bw - assumed body weight of a child (10 kg) or an adult
(70 kg).
UF - uncertainty factor'(10, 100, or 1,000) chosen in
accordance with NAS/OW guidelines.
L/day « assumed daily water consumption of a child (1 L/day) or
. an adult (2 L/day).
1. 2.4-DNT
a. Qne-dav Health Advisory
A 5-day oral reproduction study in male Sprague-Oawley rats resulted in
the deaths of 8/15 rats dosed at 240 mg/kg/day, the highest dose tested (Lane
et al., 1985). Rats dosed at this same level exhibited body weight loss and
cyanosis; a sharp decrease in the mating index and in the number of resulting
sperm-positive and pregnant females was observed at 240 mg/kg/day. Cyanosis
was also exhibited at 180 mg/kg/day. Since only one sex was tested and
precedence in the use of a reproduction study for setting an HA has not been
established, this'study was considered to be inappropriate for deriving the
One-day HA.
Since these data were not judged suitable for determining a'One-day HA
value for 2,4-ONT, it is recommended that the Ten-day HA for a 10-kg child
(0.5 mg/L) be used as a conservative estimate for the One-day HA value.
b. Ten-dav Health Advisory
The 14-day study with 2,4-ONT in Sprague-Dawley rats (McGown et al.,
1983) is acceptable for derivation of the Ten-day HA. Body weight gain and
food consumption were decreased in a dose-related manner in males and females.
A number of serum chemistry parameters (cholesterol, glucose, alanine
VII-5
-------
aminotransferase) were significantly elevated in dosed males and/or females.
Hyaline droplets were found histologically in the epithelium of the proximal
convoluted tubules of the kidneys of dosed r,ats of both sexes, with males
being more susceptible than females. Oligospermia was found in a dose-related
manner in males with accompanying degenerative changes of the testes. Based
on decreased body weight gain, decreased, food consumption, and changes in
serum chemistry levels in males and females and testicular lesions in males,
the LOAEL is 45 mg/kg/day, the lowest dose tested. -
The Ten-day HA for a 10-kg child is calculated as follows:
(45 mq/kq/dav (10 ka) * 0.45 mg/L (rounded to
(1,000). (1 L/day) 0.5 mg/L or 500
where:
45 mg/kg/day - LOAEL, based on decreased body weight gain, decreased
food consumption, and changes in serum chemistry levels
in males and females, and testicular lesions in males
following 14-day dietary dosing.
10 kg * assumed weight of a child.
1,000 » uncertainty factor, chosen in accordance with ODW/NAS
guidelines using a LOAEL from an animal study.
1 L/day - assumed water consumption of a 10-kg child.
c. Longer-term Health Advisory
The 13-week feeding study in CD rats by Lee et al. (1985) will be used
to derive the Longer-terra HA. The 2,4-ONT isomer was administered to malt
rats at 0, 34, 93, or 266 mg/kg/day and to female rats at 0, 38, 108, or
145 mg/kg/day. Mortality occurred in 100% of the high-dose females and 75% of
the high-dose males prior to study termination. Body weight gains and food
consumption decreased in a dose-related manner in males and females.
Hematologic indices (erythrocyte counts, hematocrit, hemoglobin) indicated
anemia with concurrent reticulocytosls in mid- and high-dose males and .
females. • Absolute liver and kidney weights were slightly decreased in
VII-6
-------
mid-dose males; relative liver and kidney weights of these animals were
significantly, increased. Histopathological examination revealed splenic
hemosiderosis in mid- and high-dose rats of both sexes, decreased
spermatogenesis in mid-dose males, and aspermatogenesis in high-dose males.
Oemyelination was found in the brain stem and cerebellum of one high-dose
male. Based on dose.-related decreases in body weight gain and food
consumption, the LOAEL was 34 mg/kg/day for male rats and 38 mg/kg/day for
i
female rats, the lowest doses tested. The LOAEL for males, 34 mg/kg/day, will
be used as the most conservative LOAEL for derivation of the Longer-term HA.
i < • , i
Results of the 13-week feeding study in dogs (Ellis et al., 1985)
indicated the primary target organs to be the neuromuscular system,
erythrocytes, and testes following dosing with 25 mg 2,4-DNT/kg/day. Dogs fed
this same dose exhibited body weight loss, decreased food consumption,
neuromuscular incoordination, and paralysis; dogs appeared to be in poor
nutritional condition. Hematologic indices indicated the presence of
methemoglobinemia, anemia, and Heinz bodies. Histopathology revealed
hemosiderosis of the liver and spleen, cloudy swelling of the kidneys, and
brain lesions including gliosis, edema, and demyelination of the cerebellum,
brain stem, and spinal cord in high-dose males and females. In addition,
aspermatogenesis was found in males of this dose group. The LOAEL is
25 mg/kg/day and .the NOAEL is 5 mg/kg/day. Even though the dog appears to be
the species most sensitive to DNT treatment, the study with rats will be used
for derivation of the Longer-term HA. Since the number of dogs/group tested
in this study (one dog/sex/group sacrificed at 13 weeks) was less than that
required by EPA for subchronic dog studies, this study is considered to be
unacceptable but supports the level of toxicity found in the rat study.
The Longer-term HA for the 10-kg child is calculated as follows:
(34 mo/kQ/dav) (10'kg) - 0.34 mg/L (rounded to
(1,000) „(! L/day) 0.3 mg/L or 300 jug/L)
VII-7
-------
where:
34 mg/kg/day » LOAEL, based on dose-related decreases in body weight
gain and food consumption in males and females following
13-week dietary dosing.
10 kg - assumed weight of a child.
1,000 - uncertainty factorv chosen.in accordance with ODW/NAS
guidelines using a LOAEL from an animal study.
1 L/day » assumed water consumption of a 10-kg child.
•-,.
The Longer-term HA for a 70-kg adult is calculated as follows:
(34 mq/kq/davl .f7Q kal » 1.19 mg/L (rounded to 1.0 mg/L
(1,000) (2 L/day) or 1,000 ug/L)
where: .
34 mg/kg/day * LOAEL, based on dose-related decreases in body weight
gain and food consumption in males and females following
13-week dietary dosing.
70 kg » assumed-weight of an adult.
1,000 - uncertainty factor, chosen in accordance with ODW/NAS
guidelines using a LOAEL from an animal study.
2 L/day - assumed water consumption of a.70-kg adult.
- d. Lifetime Health Advisory
The Lifetime HA represents that portion of an individual's total
exposure that is attributed to drinking water and is considered protective of
noncarcinogenic adverse health effects over a lifetime exposure. The Lifetime
HA is derived in a three-step process. Step 1 determines the Reference Dose
(RfD), formerly called the Acceptable Daily Intake (ADI). The RfD is an
estimate of a daily exposure to the human population that is likely to be
without appreciable risk of deleterious effects over a lifetime, and is
derived from the NOAEL (or LOAEL), identified from a chronic (or subchronic)
study, divided by an uncertainty factor(s). From the RfD, a Drinking Water
Equivalent Level (DUEL) can be determined (Step 2). A DWEL is a
VII-8
-------
medium-specific (i.e., drinking water) lifetime exposure .level, assuming 100%
exposure from that medium, at which adverse, noncarcinogenic health effects
would not be expected to occur. The DWEL is derived from the multiplication
of the RfD by the assumed body weight of an adult and divided by the assumed
daily water consumption of an adult. The Lifetime HA is determined in Step 3
by factoring in other sources of exposure, the relative source contribution
(RSC). The RSC from drinking water may be based on actual exposure data or,
if data are not available, a value of 20% is assumed. If the contaminant
is classified as a Group A or B carcinogen, according to the Agency's
classification scheme of carcinogenic potential (U.S. EPA, 1986), then caution
should be exercised in assessing the risks associated with lifetime exposure
to this chemical.
Three 24-month continuous feeding studies, one in CO rats, one in CD-I
mice, and one in beagle dogs, were considered for the Lifetime HA of 2,4-ONT.
The LOAELs and NOAELs established are presented in Table VII-I.
In a 2-year chronic toxicity study in which 2,4-DNT was fed daily to
beagle dogs in gelatin capsules, four of six males receiving 10 mg/kg died or
were sacrificed moribund by study week 19 after exhibiting progressive
paralysis. Neurotoxic effects, characterized by incoordination and paralysis,
were exhibited in all dogs at this dose level within 6 months of study
initiation and during month 16 in one dog receiving 1.5 mg/kg/day. CNS
lesions included vacuolization, endothelial proliferation, and gliosis of the
cerebellum. Methemoglobinemia including the presence of Heinz bodies and
reticulocytosis was evident within 3 months in dogs fed 1.5 and 10 mg/kg/day;
these hematologic effects were minimal during study year 2, presumably as a
result of an adaptive response by the dogs. Biliary tract hyperplasia was
exhibited 1n dogs fed 1.5 and 10 mg/kg/day; brown epithelial pigmentation »as
found in the gallbladder, kidneys, and spleen of two dogs receiving
10 mg/kg/day and in three dogs receiving 1.5 mg/kg/day. No adverse effects
were observed at 0.2 mg/kg/
-------
Table VI1-1. Summary of Candidate Studies for Derivation of the Drinking Water Equivalent Level (DUEL) for 2.4-DNT
Species/strain Route
Rat/CD Oral /diet
House/CD-I Oral /diet
Doo/beagle Oral/capsule
f
NOAEL
Duration Endpoint (mg/kg/day)
Lifetime Hepatocellular
alteratton
Anemia 0.57
Testicular 0.57
atrophy
Lifetime Generalized
abnormal
pigmentation
of organs
i
Hepatocellular
dysplasia
Decreased body
weight in
males
2 years I ncoordi nation 0.2
Anemia 0.2
Bile duct 0.2
hyperplasia .
LOAEL
(mg/kg/day) Reference
0.57 Ellis et al. (1979);
Lee et al . (1985)
3.9
3.9
14 Ellis et al. (1979);
Hong et al . (1985)
14
14 ' .
1.5 Ellis et al. (1979);
Ellis et al. (1985)
1.5
1.5
-------
In the study with rats by Ellis et al.' (1979) and Lee et al. (1985),
Sprague-Dawley rats were fed doses of 2,4-DNT at 0.57, 3.9, or 34 mg/kg/day or
0.71, 5.1, or 45 mg/kg/day for males and females, respectively, for 24 months.
Based on a dose-related increase in the incidence of testicular atrophy
(seminiferous tubules) in males and increased incidence of hyperplastic foci
in the liver of females, the LOAEL is 0,57 mg/kg/day, the lowest dose tested.
In a 2-year feeding study in CD-I mice, testicular and ovarian atrophy
were found in males and females receiving 898 mg/kg/day and in males receiving
95 mg/kg/day. An increased incidence of hepatocellular dysplasia and renal
tumors was seen in males receiving 14, 95, or 898 mg/kg/day and in females
receiving 898 mg/kg/day. A dose-dependent increase in the amount of'an
unidentified brown pigment was most prominent in the liver and spleen but was
also found in the lungs, kidneys, adrenals, bone marrow, heart, gonads, and
CNS of all dosed males and females.
The study by Ellis et al. (1985) has been selected for calculation of
the DWEL, since it is considered to be well conducted and has the lowest NOAEL
(0.2 mg/kg/day).
Using this study, the DWEL is derived as follows:
Step 1. Determination of the Reference Dose (RfD)
RfD - fO.2 mq/kQ/davl - 0.002 mg/kg/day
100
where:
0.2 mg/kg/day » NOAEL, based on neurotoxicity, Heinz bodies, biliary
tract hyperplasia, and organ pigmentation.
100 - uncertainty factor, chosen in accordance with OW/NAS
guidelines using a NOAEL from a chronic animal study
(10x for intraspecies variation and lOx for interspeeies
variation).
VII-11
-------
Step 2. Determination of the Drinking Water Equivalent Level (DWEL)
DUEL - (0.02 mo/ka/dav) (70 kal - 0.07 mg/L (rounded to 0.1 mg/L
(2 L/day) or 100 M9/L)
where:
0.002 mg/kg/day - RfD.
70 kg - assumed body weight of an adult.
2 L - assumed daily water consumption of an adult.
Step 3. Determination, of Lifetime HA
Dinitrotoluene is classified B2: Probable Human Carcinogen; thus, a
Lifetime HA is not recommended. The estimated excess cancer risk associated
with lifetime exposure to drinking water containing 2,4-ONT at the level of
the DWEL (100 M9/L) 1s 2 x 10"3. This estimate represents the upper 95%
confidence limit from extrapolations prepared by EPA's Carcinogen Assessment
Group using the linearized multistage model. The actual risk 1s unlikely to
exceed this value.
2. 2.6-DNT
a. One-dav Health Advisory
No studies were suitable for determining a One-day HA value for 2,6-DNT.
It is recommended that the Longer-term HA for a lO-^kg child (0.4 mg/L) be used
as a conservative estimate for the One-day HA value.
b. Ten-dav Health Advisory
No studies were suitable for determining a Ten-day HA value for 2,6-DNT.
It is recommended that the Longer-term HA for a 10-kg child (0.4 mg/L) be used
as a conservative estimate for the Ten-day HA value.
VII-12
-------
c. Longer-term Health Advisory
A 13-week study (Lee et al., 1976) with several species is used to
derive the Longer-term HA.
Dogs (4/sex/dose) were given 0, 4» 20, or 100 mg/kg/day 2,6-DNT in
capsules for 13 weeks. Dogs in the low-dose group showed no adverse effects.
All high-dose dogs (4/4 of each sex) died between weeks 2 and 8; two out of
four mid-dose females died during study week 9. Toxic effects included
decreased food consumption leading to weight loss, listlessness, and
incoordination leading to rigid paralysis with occasional tremors. Elevations
were seen in serum alkaline phosphatase, alanine aminotransferase (ALT),
and/or blood-urea-nitrogen (BUN). Hematological effects included
methemoglobinem.ia with Heinz body formation, anemia with reticulocytosis and
extramedullary hematopoiesis, and lymphoid depression leading to peripheral
lymphocytopenia. Both sexes developed bile duct hyperplasia and
degenerative/inflammatory changes in the liver and kidneys. Males exhibited
degeneration and atrophy of spermatogenic cells. Effects in high-dose animals
were more pronounced and appeared earlier than those in the mid-dose animals.
Effects were partially reversed in 4 weeks and completely reversed in 19 weeks
following cessation of treatment. The LOAEL is 20 mg/kg/day based on weight
loss, blood and neurological effects, and histopathology. The NOAEL is
4 mg/kg/day.
Groups of mice (16/sex/dose) were fed diets containing 2,6-DNT (>99%) at
0, 0.01, 0.05, or 0.25% for 13 weeks. The corresponding daily intakes were 0,
11, 51, or 289 mg/kg/day for male mice; and 0, 11, 55, or 299 mg/kg/day for
female mice. Mice in the low-dose group did not .exhibit any treatment-related
effects, and only a few in the mid-dose group showed weight loss. At the
high-dose level, 2,6-DNT produced decreased body weight gain and food
consumption, hematopoiesis in the liver and/or spleen, and bile duct
hyperplasia in both sexes. -Depression of spermatogenesis and atrophy of the
testes were seen in high-dose males. After 4 weeks, there was partial
recovery as demonstrated by weight gain and the disappearance of testicular
lesions; however, liver and spleen hematopoiesis continued. The LOAEL is
VII-13
-------
51 mg/kg/day based on weight, food consumption, histopathology, and testicular
effects in male mice. The NOAEL is 11 mg/kg/day.
Groups of rats (16/sex/dose) were fed diets containing 2,6-DNT (>99%) at
0, 0.01, 0.05, or 0.25% for 13 weeks. The corresponding daily intakes were 0,
7, 35, or 145 mg/kg/day for male rats; a,nd 0, 7, 37, or 155 mg/kg/day for
female rats. No adverse effects were seen in rats in the low-dose groups.
Mid-dose rats of both sexes had decreased body weight gain (approximately
22-26%) and food consumption, extramedul1ary hematopoietic activity in the
liver.and spleen, and bile'duct hyperplasia; males also exhibited depressed
spermatogenesis and testicular atrophy. High-dose rats of both sexes were
affected severely with effects that included decreased body weight and body
weight gain (approximately 41-46%), decreased food consumption,
methemoglobinemia, Heinz body formation, anemia, reticulocytosis, splenic and
liver extramedullary hematopoiesis, bile duct hyperplasia, and renal
degeneration. After 4 weeks, only partial recovery was observed. The LOAE1
is 35 mg/kg/day based on weight loss, blood effects, and histopathology in
male rats. The NOAEL is 7 mg/kg/day.
\
In this case, the dog was considered to be the most sensitive species
from which to derive a HA. The 20 mg/kg/day is a Frank-Effect Level (PEL)
based on neurotox.icity, Heinz bodies, bile duct hyperplasia, liver and kidney
histopathology, and death. A LOAEL is not identified for this study. The
NOAEL is 4 mg/kg/day.
.The Longer-term HA for the 10-kg child is calculated as follows:
(4 mo/kQ/dav) (10 kg) - 0.4 mg/L (400 jigA)
(100) (1 L/day)
VII-14
-------
where:
4 mg/kg/day - NOAEL, based on neurotoxicity, Heinz bodies, bile duct
hyperplasia, liver and kidney histopathology, and death.
10 kg = assumed weight of a child.
100 - uncertainty factor, chosen in accordance with OW/NAS
guidelines using a-NOAEL from an animal study.
1 L/day » assumed water consumption of a 10-kg child'.
The Longer-term HA for a 70-kg adult is calculated as follows:
(4 mq/kg/dav) (70 kg) - 1.4 mg/L (rounded to 1.0 mg/L or
(100) (2 L/day) 1,000 jug/L)
where:
4 mg/kg/day » NOAEL, based on neurotoxicity, Heinz bodies, bile duct
hyperplasia, liver and kidney histopathology, and death.
70 kg - assumed weight of an adult.
100 - uncertainty factor, chosen in accordance with OW/NAS
guidelines using a NOAEL from an animal study.
2 L/day * assumed water consumption of a 70-kg adult.
d. Lifetime Health Advisory
The Lifetime H'A represents that portion of an individual's total
exposure that is attributed to drinking water and is considered protective of
noncarcinogenic adverse health effects over a lifetime exposure. ' The Lifetime
HA is derived in a three-step process. Step 1 determines the Reference Dose
(RfD), formerly' called the Acceptable Daily Intake (ADI). The RfD is an
estimate of a daily exposure to the human population that is likely to be
without appreciable risk of deleterious effects over a lifetime, and is
derived from the NOAEL (or LOAEL), identified from a chronic (or subchronic)
study, divided by an uncertainty factor(s). , From the RfD, a Drinking Water
Equivalent Level (DWEL) can be determined (Step 2). A DWEL is. a medium-
specific (i.e., drinking water) lifetime exposure level, assuming 100%
exposure from that medium, at which adverse, noncarcinogenic health effects
VII-15
-------
would not be expected to occur. The DWEL is derived from the-multiplication
of the RfD by the assumed body weight of an adult and divided by the assumed
daily water consumption of an adult. The Lifetime HA is determined in Step 3
by factoring 1n other sources of exposure, the relative source contribution
(RSC). The RSC from drinking water may be based on actual exposure data or,
if data are not available, a value of 20% is assumed. If the contaminant is
classified as a Group A or B carcinogen, according to the Agency's
classification scheme of carcinogenic potential (U.S. EPA, 1986), then caution
should be exercised in assessing the risks associated with lifetime exposure
to this chemical.
Three 13-week continuous feeding studies, one in CD rats, one in CD-I
mice, and one in beagle dogs, were considered for the Lifetime HA of 2,6-DNT.
The studies are discussed in detail in section VII.B.2.c (2,6-DNT, Longer-term
Health Advisory). The LOAELs and NOAELs established are presented in
Table VII-2. '
The dog is considered the most sensitive species to derive a HA. The
20 mg/kg/day is a Frank-Effect Level (PEL) based on neurotoxicity, Heinz,
bodies, bile duct hyperplasia, liver and kidney histopathology, and death. A
LOAEL is not identified for this study. The NOAEL is 4 mg/kg/day.
Using this study, the DWEL is derived as follows:
/
Step 1. Determination of the Reference Dose (RfD)
RfD - U ma/ko/dav) - 0.001 mg/kg/day
3,000
VII-16
-------
Table VII-2. Summary of Candidate Studies for Derivation of the Drinking
Water Equivalent Level (DWEL) for 2,6-DNT
Species
Dog
Beagle
Rat
CO
Exposure
Duration/
Route
13 weeks/
diet
13 weeks/
diet
MOAEL LOAEL
Effects (ing/kg/day)
testicular atrophy; hepatic 4 20
degeneration; anemia;
paralysis; renal
inf lamnation
decreased body weight; 7 35
hepatic and splenic
Reference
Lee et al., 1976
Lee et al., 1976
hetnosiderosis; bile duct
hyperplasia; anemia; Heinz
bodies
House
Swiss-
Albino
13 weeks/
diet
decreased body weight;
hepatic and splenic
hematopoiesis; bile duct
hyperplasia; testicular
atrophy
11
51
Lee et al., 1976
VII-17
-------
where:
4 mg/kg/day - NOAEL, based on neurotoxicity, Heinz bodies, bile duct
hyperplasia, liver and kidney histopathology, and death.
3,000 =• uncertainty factor, chosen in accordance with OW/NAS
guidelines using a NOAEL from an animal study (lOx for
intraspecies variation, lOx for interspecies variation,
and lOx for use of a*less-than-lifetime study). An
additional factor of 3 1s used to account for the
limited database.
Step 2. Determination of the Drinking Water Equivalent Level (DWEL)
, DWEL - (0.001 mg/ko/dav) (70 kg) - 0.035 mg/L (rounded to
. (2 L/day) 0.04 mg/L or 40 M9/L)
where:
0.001 mg/kg/day - RfD.
70 kg » assumed body weight of an adult.
2 L « assumed dally water consumption of an adult.
Step 3. Determination of Lifetime HA
ONT is classified B2: Probable Human Carcinogen; thus, a Lifetime HA is
not recommended. The estimated excess cancer risk associated with lifetime
exposure to drinking water containing 2,6-DNT at the level of the DWEL
(40 M9/L) is 1 x 10"3. This estimate represents the upper 95% confidence
limit from extrapolations prepared by EPA's Carcinogen Assessment Group using
the linearized multistage model. The actual risk 1s unlikely to exceed this
value.
\
C. QUANTIFICATION OF CARCINOGENIC POTENTIAL
Leonard et al. (1987) treated four groups of 20 F344 male rats with
various isomers and Isomer mixtures of DNT for 1 year. Technical grade DNT
(76% 2,4-DNT, 19% 2,6-DNT) (35 mg/kg/day) induced hepatocellular tumors and
cholangiocarcinomas in 47 and 11%, respectively, of the treated males.
2,6-DNT (99.9% purity) induced hepatocellular carcinomas in 100% of the
VII-18
-------
high-dose group and 85% of the low-dose group (14 and 7 mg/kg/day,
respectively); pulmonary metastases were found in 58% of the high-dose and 15%
of the low-dose male rats. No tumors were found in rats exposed to 2,4-DNT
(99.9% purity) at 35 mg/kg/day in this study. Although these studies were
limited in duration (1 year) and in number of animals on test, the data may
indicate that the 2,6- isomer accounts fpr much of the carcinogenic activity
in the previously tested mixed-isomer ONT bioassays.
1. Dose-Response Data fCarcinoaenicitv. Oral Exposure)
Based on monitoring and production data, 2,6-ONT usually occurs in the
presence of 2,4-DNT, with 2,4-DNT the more significant component by volume.
For example, tg-DNT is composed of approximately 76.5% 2,4-DNT and 18.8%
2,6-DNT; 2,6-DNT is unlikely to occur alone. In addition, available data on
the long-term effects of 2,6-DNT and tg-DNT are limited. For these reasons,
the carcinogenic assessment for lifetime exposure of DNT was determined using
a chronic toxicity/oncogenicity study conducted with 2,4-DNT; the assessment
is representative of the 2,4-/2,6-DNT mixture.
,, i
<
Tumor type -- liver: hepatocellular carcinomas, neoplastic nodules; mammary
gland: adenomas, fibroadenomas, fibromas, adenocarcinomas/carcinomas
Test Animals -- ra.t/Sprague-Dawley, female
Route -- oral, diet
Reference -- Ellis et al., 1979 /
Admin-
istered
(ppm)
0
15
100
700
Human
Equivalent*
(mg/kg/day)
0
0.129
0.927
7.557
i umur
Incidence
11/23
12/35
17/27
34/35
*Administered dose + (70 kg/0.425 kg)
0 JJ
VII-19
-------
The human equivalent dose was determined using a standard surface area
correction factor. The administered study dose (ppm) was converted to
mg/kg/day (1 ppm - 0.05 mg/kg/day for the aging rat; Lehman, 1959); this dose
conversion was divided by the ratio of the human weight (70 kg) to the aging
rat weight raised to the 1/3 power. Transformed doses reflect the measured
weight of the rats for each treatment per.iod (0.425 kg control and low dose,
0.410 kg medium dose, 0.325 kg high dose). The tumor incidences were combined
for quantitative purposes because the report by Ellis et al. (1979) provided
pathology data for the individual animals. The unit risk should not be used
if the water concentration exceeds 500 M9/L, since above this concentration
the slope factor may differ from that stated.
2. Summary of Risk Estimates
Oral Slope Factor (mg/kg/day)" -- 6.8E-1
Drinking Water Unit (/ig/L) Risk -- 2.0E-5
Extrapolation Method -- Linearized multistage procedure
3. Drinking Water Concentrations at Specific Risk Levels
Risk Level Concentration
E-4 (1 in 10,000) 5.0
E-5 (1 in 100,000) 0.5
E-6 (1 in 1,000,000) 0.05
The multistage model was used for high- to low-dose extrapolation (Crump
and Watson, 1979; Howe and Crump, 1982). GLOBAL83 was used to fit the data in
the experimental dose range and to obtain upper 95% confidence limits on risk.
The multistage model conforms to a biological model of tumor initiation and
promotion (Crump et al., 1977) and provided an adequate fit to the dose-
response data for 2,4-DNT. The relationship of the concentration (/ig/L) of a
chemical in drinking water to cancer risk is expressed as follows:
VII-20
-------
35.000.x R - C
where:
35,000 * conversion factor for mg to M9 and exposure
. assumption that a 70-kg adult consumes 2 L of
water/day.
qt* - (mg/kg/day)"1, human slope factor.
R - 10'4, 10"5, 10'6, etc.
C • concentration of chemical in
For comparison purposes, drinking water concentrations associated with an
excess cancer risk of 10"6 are 8 x 10"8 ng/L, 5 x 10"2 M9/L. 2 x 101 Atg/L,
2 x 10"1 M9/L, and 2 x -10~* A*g/L for the one-hit, multihit, probit, logit, and
Wei bull models, respectively. The parameter estimates for these models were
computed with RISK81 (Kovar and Krewski, 1981) and MULTI80 (Rai and VanRyzin,
1980; Lavenhar, 1986).
\ ' •
4. Discussion of Confidence fCarcinoaenicitv. Oral Exposure)
Relatively few animals were observed for a period of time approximating
the lifespan of the animals. A slope factor of 3.9E-l/mg/kg/day based on
renal tumors in male CD-I mice (Ellis, 1979) is supportive of the risk
estimate.
The NOAEL for the 13-week oral toxicity study in CD rats conducted by Lee
et al. (1978) was 7 mg/kg/day. In a 12-month oral toxicity study in CDF
(F344)/CrlBR rats conducted by Leonard et al. (1987), 7 mg 2,6-DNT/kg/day was
found to produce hepatocellular carcinomas in 85% (17/20) of the rats tested;
hepatocellular carcinomas were found in 100% (19/19) rats fed 14 mg/kg/day.
Beagle dogs fed 20 mg 2,6-DNT for 13 weeks exhibited weight loss,
hematological effects, neuromuscular incoordination, and lesions of the liver,
spleen, kidneys, and testes; two of eight dogs died during study week 9. In
the absence of sufficient data and because 2,6-DNT usually occurs in the
VII-21
-------
presence of 2,4-DNT, with 2,4-DNT the more significant component by volume,
the cancer risk assessment for the 2,4-/2,6-DNT mixture is used as a
conservative approach to the determination of a cancer risk assessment for
either 2,4-DNT or 2,6-ONT, as well as the mixture (Table VII-3).
VII-22
-------
Table VII-3. Cancer Risk Data for Exposure to 2,4-DNT, 2,6-DNT, and tg-DNT
Potential Exposure
Risk Data
2,4-DNT
2,4-/2,6-m1xture (tg)
2,6-DNT
Health Advisory + cancer risk assessment
for 2,4-/2,6-DNT mixture.
Cancer risk assessment for 2,4-/2,6-DNT
mixture.
Default to cancer risk assessment for
2,4-/2,6-DNT mixture.
VII-23
-------
VIII. OTHER CRITERIA, GUIDANCE, AND STANDARDS
A number of regulations and guideline values have been established for
2,4- and 2,6-ONT by various U.S. and State agencies. These values are listed
in Table VIII-1.
VIII-1
-------
Table VIII-1. Regulations and Guidelines Applicable to 2.4- and 2.S-ONT*
Agency
Description
Value
Regulations
Occupational Safety and
Health Administration
(OSHA)
U.S. Environmental
Protection Agency, Office
of Water Regulations and
Standards (EPA/OWRS)
U.S. Environmental
Protection Agency. Office
of Environmental Rules and
Regulations (EPA/OERR)
Guidelines
American Conference of
Governmental and Industrial
Hygienists (ACGIH)"
National Institute of
Occupational Safety and
Health (NIOSH)
EPA/OWRS
EPA
State Environmental
Agencies
Permissible Exposure Limit (PEL)/
Time-Weighted Average (TVA) (DNTs) (skin)
Reportable Quantity (ONTs)
Reportable Quantity (2,4-OMT)
Reportable Quantity (proposed)
2.4-ONT
2,6-OHT
Threshold Limit Value-Time Weighted
Average (TLV-TWA)
Recommended Exposure Limit for
Occupational Exposure (DNTs)
[(mediately Dangerous to Life
or Health (ILOH) (ONTs)
Ambient Water Quality Criteria to
Protect Human Health (2.4-ONT)
Ingestion of Water and Organisms
Ingestion of Organisms Only
Carcinogenic Classification
2.4-ONT and 2.6-ONT as a mixture
Drinking Water Standards and
Guidelines <
2,4-ONT
Kansas
Minnesota
2.6-ONT
Kansas
1.5 mg/mj
1.000 Ib
1.000 Ib
10 Ib
100 Ib
1.5 mg/rn1
Potential
carcinogen.
Reduce exposure to
lowest feasible
level.
200 wgV
1.1 x 10"'
91 x 10'*
1,1 •*',.
1.1 »*-
0.4
• -ued)
VIII-2
-------
aril
Agency
Description
Value
State Environmental
Agencies (cont.)
Acceptable Ambient Air
Concentration Guidelines
or Standards
2.4-ONT
Connecticut
Nevada
ONTs
Virginia
15 /jg/m' (8 hr)
0.036pg/m' (8 hr)
25 fjq/m' (24 hr)
Agency for Toxic Substances
and Disease Registry, U.S.
Public Health Service
Minimum Risk Level (MRL)
2,4-DNT
Short-term exposure in food
Long-term exposure in food
Acute oral exposure
Intermediate oral exposure
Chronic oral exposure
2.6-OMT
Long-term exposure in food
Intermediate oral exposure
1 ppre
1 ppm
0.6 mg/kg/day
0.05 mg/kg/day
0.002 mg/kg/day
1 ppm
0.04 mg/kg/day
'SOURCE: ATSOR (1989).
'Listed In a notice of Intended changes for 1991-92 to lower the TLV-TWA to 0.15 mg/m* and to classify ONT as a
"Suspected Human Carcinogen" (ACGIH, 1991; Oinitrotoluene, 1992).
VIII-3
-------
IX. ANALYTICAL METHODS
Published analytical methods for 2,4- and 2,6-DNT for a variety of
situations refer predominantly to gas chromatography (GC) and high performance
liquid chromatography (HPLC); however, other methods include electron spin
resonance spectrometry, tandem mass spectrometry (MS), and cluster analysis.
A. GC ANALYSIS
Gas chromatography has been studied by a number of scientists utilizing
' ' ' i' •
different detection methods for various situations. Hartley et al. (1981)
studied the separation and identification of DNT isomers in water by GC using
an electron capture detector (ECO). Extraction was performed using hexane.
Retention times for 2,4- and 2,6-DNT were 25.43 and 16.55 minutes,
rf
respectively, while corresponding detection limits were 25.0 x 10'' and
62.5 x 10"* jig. This method was judged to be adequate to monitor DNT in
environmental samples and laboratory toxicity studies. .
t
Bel kin et al. (1985) also studied the quantitative analysis of water for
2,4- and 2,6-DNT using GC coupled with ECD. The study was designed to detect
ppb levels of the compounds., The DNT isomers were extracted from water
samples with toluqne, analyzed by capillary GC, and detected with an ECD. The
DNT isomers were found to be stable in water for 33'days at pH values below 3.
Recoveries for 2,4- and 2,6-DNT ranged from 98.4 to 106.0 and from 95.1 to
97.4%, respectively, at concentration levels of 1, 10, or 100 ppb.
Eichelberger et al. (1983) utilized both packed column GC (method 1) and
fused silica capillary GC (method 2) coupled with MS to determine the presence
of a number of compounds in water. The extraction procedure involved
methylene chloride at different pH levels for both methods. Mean recoveries
for methods 1 and 2 for 2,4- and 2,6-ONT, respectively, were 35 and 70%, *n
-------
Capillary GC or capillary GC/MS was used in conjunction with robotics to
analyze wastewater samples for a variety of compounds including 2,4- and
2,6-DNT. The sample was extracted with methylene chloride. Although the
robotics reduced the analysis time by half, the detection limits provided are
adequate only for routine monitoring of biological systems; they do not equal
those found in EPA procedures (Hornbrook.and Ode; 1987).
A procedure for the determination of munitions in water utilizes a
macroreticular resin for extraction, elution with ethyl acetate, concentration
of the eluate, separation by GC, and detection by ECO (Richard and Junk,
1986). Recovery values for 2,4- and 2,6-DNT ranged from 89 to 102% and from
33 to 91%, respectively. The results obtained were, similar to methylene
chloride extraction results, but with a savings in time and solvent,/
transportation costs. < ^
Gas chromatography has also been used to analyze sludge samples. A
method for the determination of 2,4- and 2,6-DNT In biosludges was described
by Phillips et al. (1983). A shakeout centrifugation procedure was used to
isolate DNT isomers from the sludge, and quantitation was performed using GC
and a thermal energy analyzer (TEA). The detection limit was 0.05 mg/L for
both isomers, and the precision was 9-14% at the detection limit. The authors
stated that the use of TEA eliminates the need for further sample cleanup.
Lichtenberg et al. (1987), in a study utilizing GC in conjunction with
either electron capture detection (ECO) or flame ionic detection (FID), stated
that parameters in providing successful GC are the column1 packing, temperature
conditions, and selection of a detector as specific to the analyte as
possible. The use of selective detectors in conjunction with GC/MS gives a
higher reliability in the identification and quantitation of toxic organic
substances In complex matrices such as wastewater and sludge samples (Giabbai
et al., 1983).
A method for the determination of 2,4- and 2,6-DNT in airborne
particulates using GC was developed by Matsushita and lida (1986). Samples
are collected on a quartz filter and-extracted with a benzene/ethanol mixture.
IX-2
-------
DNTs in the fraction are analyzed by GC in conjunction with a flame
thermoionic detector (FTD). The authors stated that this method has high
separation efficiency and reproducibility for both retention time and peak
height.
B. HPLC ANALYSIS
Krull et al. (1981) studied the use of HPLC with electron capture
detection (ECO) and HPLC with GC/ECO for the analysis of 2,4-DNT. The authors
state that ECO 1s perhaps the optimal detection system for use with HPLC owing
to its sensitivity and specificity for nltro derivatives. HPLC with ECO did
not produce optimum results. However, the detection limit for 2,4-DNT
(1.0 x 10"4 g) obtained with HPLC coupled with GC/ECD suggests that this
method may be more suitable for real-world application.
Lloyd (1983) described a technique that was developed primarily as a
basis for the screening of samples for trace amounts of explosives. 2,4-DNT
was analyzed using a pendant mercury drop electrode (PMOE) 1n conjunction with
HPLC. The detection limit with this method was 12.0 x 10"4 g and was similar
to that obtained by GC/electron capture detection. Characteristics of the
PMDE method are Its high reproducibility, renewal of the electrode at the
start of a chromatogram, and Us resistance to contamination problems.
Reverse phase HPLC has been used in the analysis of munitions wastewater
samples. Bauer et al. (1986) stated that this method was developed
specifically to provide Industrywide uniformity for quality control for
compliance monitoring but was deemed applicable for the determination of
munitions residues. DNT was recovered quantitatively, and the repeatability
value was 7 nq/l. Jenkins et al. (1986), using the same method,
conservatively estimated the detection limit to be 10 ng/l with a standard
deviation of 4.6 M9/L. This method was found to perform adequately for load,
pack, and manufacture wastewaters and contaminated groundwater.
The HPLC method (USATHAMA Method SM02) generally has been accepted as the
standardized method for measurement of TNT and,related metabolites in soils
IX-3
-------
and wastewaters. However, in certain instances ami nodinitrotoluenes are
present and may interfere with the quantitation of dinitrotoluene. Therefore,
a modification to method SM02 has been made (Preslan et al., 1991) which
involves an Intermediate derivatization step that ensures separation of
2-am1no-4,6-d1nitrotoluene, 4-amino-2,6-dinitrotoluene, and DNT Itself. Thus,
greater accuracy is assured in the measurement of these compounds.
Bongiovanni et al. (1984) used a combination of HPLC and ultraviolet (uv)
light to detect and analyze explosive-bearing soils for trace amounts of
2,4-and 2,6-DNT. DNT was extracted from the soil with acetonitrile, separated
by reverse phase HPLC, and detected by ultraviolet spectrometry at a
wavelength of 254-nm. Estimated detection limits for 2,4- and 2,6-DNT were
0.58 and 0.87 g, respectively. Corresponding average recovery values were
100.4 and 99.6%. This method provided good accuracy and low detection limits.
The authors stated that it is particularly suitable for routine short
turnaround analyses with minimum operator attention.
C. OTHER METHODS
Yinon (1989) analyzed and Identified a number of 2,4-DNT metabolites
using electron Impact and chemical ionization mass spectrometry. The
metabolites were differentiated by the electron-Impact Ions that are specific
to the position of the methyl group in relation to, the nitro group.
Hable et al. (1991) detected 2,4- and 2,6-DNT In drinking water at levels
below those measured by gas chroraatography. Using electron-capture detection
together with a DB-1301 widebore-fused silica capillary column, 2,6-DNT was
detected at 0.003 g/L; 2,4-DNT was detected at 0.04 g/L. This method may be
advantageous over other detection techniques.
Burns et al. (1987) studied the possibility of identification and
determination of 2,6-DNT by electron spin resonance spectrometry. DNT was
quantitatively converted to the corresponding anion radical form adsorbed on
magnesium oxide. Linear plots indicated that determination of 2,6-DNT by
electron spin resonance spectrometry Is possible.
IX-4
-------
McLuckey et al. (1985) studied the use of tandem MS for the analysis of
explosives. Tandem MS employs the use of two stages of MS in which the first
stage serves as a separator for the second. Three detection methods (electron
impact ionization, isobutane chemical ionization, and negative chemical
ionization) were used to determine the most suitable method in conjunction
with tandem MS. Isobutane and negative chemical ionization methods were
suitable; the latter was the most sensitive for nitroaromatic compounds such
as 2,4-DNT. Tandem MS facilitates rapid screening for particular types of
explosives.
Spanggord and Suta (1982) studied the use of cluster analysis to
characterize the distribution of waste components resulting from the
/
production and purification of TNT. This method selectively groups samples
together according to the observed concentration values of all components in
the sample. The relative concentrations of 2,4- and 2,6-DNT in average
cluster analysis were 49.3 and 21.7%, respectively. This approach can be used
to determine a representative ratio of components in complex industrial
wastewater.
IX-5
-------
X. TREATMENT TECHNOLOGIES
Treatment technologies found in the available literature include
adsorption, chlorination, ozonation, ultraviolet radiation, and several lesser
used techniques.
The use of activated carbon for the adsorptive displacement of 2,4- and
2,6-DNT has been investigated. Activated carbon adsorption is the technique
most frequently used to clean nitroorganic-contaminated wastewater in military
munitions plants. When the carbon becomes exhausted, it must be disposed of
at an approved hazardous waste disposal site. Ho and Daw (1988) investigated
the possibilities of regenerating spent carbons. Solvents tested for
extracting the adsorbed ONT were water, acetone, methanol, and mixtures of the
solvents. Both acetone and methanol were effective for the removal of ONT
from activated carbon.
Thakkar and Manes (1987) also studied the adsorptive displacement of 2,4-
and 2,6-DNT. After being preloaded onto activated carbon, the compounds were
equilibrated with benzo[a]anthracene-7,12-dione in methylene chloride/
methanol. The 2,6-DNT isomer showed essentially complete displacement, while
2,4-DNT exhibited nonlinear displacement.
The use of resin for the adsorption of munitions components in aqueous
solutions followed by desorption in acetone was studied by Maskarinec et al.
(1984) and Richard and Junk (1986). Resin adsorption techniques appear to
offer several advantages: specific sorptivity toward nitro groups, increased
stability,^and field sorption to ensure sample integrity.
Lloyd (1985) determined the distribution coefficients of DNT for the
adsorption of 10 representative adsorbents used in cleanup procedures. The
adsorbents were placed in solutions of methanol, and the ONT was loaded into
the solution. Two of the adsorbents gave nonsignificant results, three showed
negative selectivity, and the remaining five gave distribution coefficients of
0.129 to 0.356.
X-l
-------
The effects of chlorination and ozonation on 2,4- and 2,6-DNT were
studied by Lee and Hunter (1985). Concentrations of 21.3 (ozone) and
45.5 mg/L (chlorine) were added to compound concentrations of 100 mg/L and
observed. Reduction recoveries of 2,4-DNT by chlorine and ozone at 1 hour
were 35 and 60%, respectively. Corresponding values for 2,6-DNT were 17 and
13%. .
•i
Ho (1986) studied the synergistic effect of hydrogen peroxide and
ultraviolet (uv) radiation on the decomposition of 2,4-DNT in water and found
that at molar ratios of H202/DNT between 26 and 52, DNT disappeared very
rapidly. The degradation rate of DNT in aqueous solution was also found to be
affected by the energy of the incident light.
Other treatment methods investigated include solvent and sediment .
extraction and partial reduction. Hwang (1981) conducted a literature review
to determine the feasibility of solvent extraction as a treatment method for
separating organic materials in wastewater effluent. He found that solvent
extraction can remove up to 99.9% of targeted materials. Lopez-Avila et al.
(1983) used an extraction technique involving the homogenization of a sedinent
sample with dichloromethane at dual pH and phase separation by centrifugation
to determine priority pollutants in a standard reference sediment sample.
Total recoveries for 2,4- and 2,6-DNT were 95 and 93%, respectively. Ono and
Kitazawa (1983) obtained a successful partial reduction of 2,4-DNT by mild
reduction under controlled conditions with a metal and organic acid systea.
The reduction products obtained were 4-methyl-3-nitroan1line and
2,4-diaminotoluene. This method is useful for the "recycling" of 2,4-DNT.
In addition, biodegradation and photolysis techniques may be considered as
\
treatment technology alternatives because of the rapid degradation of DNT by
these two processes (Liu et al., 1984; Davis et al., 1981; Hall as and
Alexander, 1983).
X-2
-------
XI. CONCLUSIONS AND RECOMMENDATIONS
Advisory values for 2,4- and 2,6-DNT are summarized in Table XI-1. Based
on decreased body weight gain, decreased food consumption, and changes in
serum chemistry levels in male and female Sprague-Dawley rats and testicular
lesions in male rats administered 2,4-DNT in the diet'for 14 days, the One-day
and Ten-day HAs for exposure in a 10-kg child are 0.5 mg/L (500 M9/L). Based
on dose-related decreases in body weight gain and food consumption in rats
administered 2,4-DNT in the diet for 13 weeks, the Longer-term HA for exposure
in a 10-kg child is 0.30 mg/L (300 M9/1); the Longer-term HA for exposure in
a 70-kg adult is 1.0 mg/L (1,000 ng/L). The Drinking Water Equivalent Level
(DWEL) for 2,4-DNT is 0.1 mg/L (100 M9/L), derived from a Reference Dose
(RfD) of 0.002 mg/kg/day, based on neurotoxicity, Heinz bodies, and biliary
tract hyperplasia in dogs dosed orally with 2,4-DNT for 2 years.
The critical effects for 2,6-DNT were neurotoxicity, Heinz bodies,
biliary tract hyperplasia, liver and kidney histopathology, and death. The
One- and Ten-day HAs and the Longer-term HA for exposure in a 10-kg child are
0.4 mg/L (400 fig/I). The Longer-term HA for exposure in an adult is 1.0 mg/L
(1,000 M9/L). The Drinking Water Equivalent Level (DWEL) for 2,6-DNT is
0.04 mg/L (40 Mg/L), derived from a Reference Dose (RfD) of 0.001 mg/kg/day.
i
Dinitrotoluene is classified B2: Probable Human Carcinogen; thus a
Lifetime HA is not recommended. The estimated excess cancer risk associated
with lifetime exposure to drinking water containing 2,4-DNT at the level of
the DWEL (100 Mg/L) is 2 x 10"*. The estimated excess cancer risk associated
with lifetime exposure to drinking water containing 2,6-ONT at the level of
the DWEL (40 ng/L) is 1 x 10"*. The cancer risk assessment is for the
2,4-/2,6-DNT mixture and, therefore, is used for the cancer risk assessment
for both 2,4- and 2,6-DNT.
A companion report titled "Data Deficiencies/Problem Areas and
Recommendations for Additional Data Base Development for 2,4- and 2,6-DNT"
(Appendix) summarizes the scope of existing data reviewed for this HA. This
companion report delineates the areas where additional data and/or a
clarification of existing data would be appropriate for a second HA.
XI-1
-------
Table XI-1. Drinking Water Advisory Values for 2,4- and 2,6-Dinitrotoluene
Classification 2,4-DNT 2,6-DNT
One-day HAa (mg/L)
Ten-day HA (mg/L)
Longer-term HA:
Child (mg/L)
Adult (mg/L)
Lifetime HA (mg/L)
DWELC (mg/L)
Cancer Unit Risk Factord
0.5
0.5
0.3
1.0
NAb
0.1
0.68
0.4
0.4
0.4
1.0
NA
0.04
0.68
(mg/kg/dayp
Excess Cancer Risk at the 2xlO'3 IxlO'3
OWEL
aHealth Advisory.
bOinitrotoluene is classified as a Group B2: Probable Human Carcinogen; therefore
a Lifetime HA is not recommended.
cDrinking Water Equivalent Level.
dAlso referred to as "Human Potency Factor" and "q".
XI-2
-------
VXII. REFERENCES
Abernethy DJ, Couch OB. 1982. Cytotoxicity and mutagenidty of
dinitrotoluenes in Chinese hamster ovary cells. Mutat. Res. 103: 53-59.
ACGIH. 1991. American Conference of Governmental Industrial Hygienists.
1991-1992 Threshold limit values for chemical substances and physical agents
and biological exposure indices. Cincinnati, OH: American Conference of
Governmental Industrial Hygienlsts.
Ahrenholz SH. 1980. Health hazard evaluation determination. Report No. HE
79-113-728. 01 in Chemical Company, Brandenburg, KY. Cincinnati, OH: National
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i • " •
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/
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APPENDIX
Data Deficiencies/Problem Areas and Recommendations for
Additional Data Base Development for 2,4- and 2,6-DNT
-------
DATA' BASE DEVELOPMENT
A. OBJECTIVES
The objective of this document is to provide an evaluation of data
deficiencies and/or problem areas encountered in the review process for
2,4-/2,6-DNT and to provide recommendations, as appropriate, for additional
data base development. This document is presented as an independent analysis
of the current status of 2,4-/2,6-DNT toxicology, as related to its possible
presence in drinking water, and includes a summary of the background
information used in development of the Health Advisory (HA). For greater
detail on the toxicology of 2,4-/2,6-DNT, the Health Advisory on 2,4-/2,6-DNT
should be consulted.
B. BACKGROUND '
. . •
Dinitrotoluene, commonly known as ONT, is a white- to buff-colored solid
at room temperature and exists as a mixture of two or more of its six isomers;
the 2,4- and 2,6-DNT isomers are the most significant and have been used in
military munitions, dye manufacture, and the synthesis of toluenediamine (the
organic intermediate in. the production of polyurethane). Both isomers are
soluble in water and are found in industrial wastewater. DNT is removed from
water by rapid photochemical transformation or biodegradation. Biodegradation
occurs via cometabolic transformation in local eutrophic waters; wastewater
and municipal sludge utilize microbial and/or fungal populations under
aerobic/anaerobic conditions (Bausum et al.', 1991; Davis et.al.-, 1981; Hallas
and Alexander, 1983; Hashimoto et al., 1982; Liu et al., 1984; Small and
Rosenblatt, 1974; Spanggord et al., 1981; Tabak et al., 1981). DNT is best
detected at low concentrations in water media by high-performance liquid
chrbmatography (HPLC) or gas chromatography (GC) in combination with electron-
capture detection (ECD) or pendant mercury drop electrode (PMDE) detection.
Detection limits range from 12.0 x 10" to 6.25 x 10'* jig (Belkin et al., 1985;
Hartley et al., 1981; Lloydr-1983).
A-l
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The pharmacokinetics of primarily the 2,4-DNT isomer has been studied
extensively in laboratory animals. Data indicate that approximately 70 to 90%
of orally administered 2,4- or 2,6-DNT is eliminated in the urine of CD and
Fischer rats, New Zealand white rabbits, beagle dogs, rhesus monkeys, and A/J
mice (Rickert et al., 1984; Schut et al., 1983; Ellis et al., 1985, 1979; Lee
et al., 1975, 1978). Fecal excretion generally accounts for less than 10%.
Results of one study indicated that absorption of 2,6-DNT by rats is somewhat
slower than that of 2,4-DNT (Lee et al., 1975); a series of other studies
demonstrated that urinary excretion of 2,4-DNT by two common strains of mice
(CD-I and B6C3F,) is only 8 to 12% following oral exposure (Shoji et al.,
1985; Lee et al., 1978; Mori et al., 1977). Percent absorption of either
isomer is difficult to estimate since biliary excretion 1s significant in most
animals. Male rats usually excrete less of the administered dose of 2,4-DNT
in the urine and more in the feces than females, a difference due most likely
to higher biliary excretion in males (Medinsky and Dent, 1983; Bond and
Rickert, 1981; Ellis et al., 1980; Lee et al., 1978). No sex-related
differences in the absorption or elimination of 2,6-DNT have been observed in
animals or humans (Levine et al., 1985b; Turner et al., 1985; Schut et al.,
1983; Long and Rickert, 1982; Ellis et al., 1980; Lee et al., 1978). Fecal
metabolites have not been identified in either animals or humans exposed to
DNT. Elimination of either compound via the lungs is minimal. Workers
exposed to technical grade DNT absorb the munitions chemicals via the
inhalation and dermal routes, but the extent of absorption cannot be
determined (Levine et al., 1985b; Turner et al., 1985; Woollen et al., 1985).
The data on distribution of 2,4- and 2,6-DNT suggest that accumulation of
these compounds in the body following a single exposure is, at most, minimal
(Schut et al., 1981, 1982, 1983; Ellis et al., 1980; Rickert et al., 1980;
Lee et al., 1975, 1978; Mori et al., 1977, 1980). Repeated oral exposure in
CD rats indicates preferential uptake of 2,4-DNT and/or its metabolites by the
i
liver, kidneys, brain, lungs, and skeletal muscle, although total retention in
the body is low (Mori et al... 1980; Ellis et al., 1979; Lee et al., 1978).
Limited data suggest that placental transfer of 2,4-DNT occurs in rats
(Rickert et al., 1980).
A-2
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The metabolism of DNT isomers has been studied in a variety of
experimental systems and animals (Etnier, 1987; Guest et al., 1982; Bond et
al., 1981; Rlckert et al., 1981; Rickert and Long, 1981; Ellis et al., 1980;
Lee et al., 1978). The first step in the metabolism of DNT appears to involve
hepatic oxidation of the methyl group to form 2,4- or 2,6-dinitrobenzyl
alcohol (2,4- or 2,6-DNBalc). Sulfate or glucuronate conjugation occurs next.
Metabolites that are eliminated in the bile and into the gut are hydrolyzed
and reduced by intestinal microflora to aminonitrobenzyl alcohols. Many of
these compounds, in turn, are reabsorbed from the gut into the systemic
circulation and then oxidized in the liver. Biliary excretion of these
metabolites Into the gut results in additional reduction by Intestinal
bacteria. Hepatic metabolism probably plays only a minor role in the
reduction of either isomer. The only remarkable sex-related difference In the
metabolism of DNT is the high rate of biliary excretion of 2,4-dinitrobenzyl
alcohol glucuronide (2,4-DNBalcG) in male rats.
For most animals (A/J mice, CD and Fischer 344 rats, New Zealand rabbits,
beagle dogs, and rhesus monkeys) and humans, significant portions of orally or
intraperitoneally administered 2,4-DNT are converted to 2,4-DNBalc and/or its
glucuronide (i.e., 2,4-DNBalcG) (Turner, 1986; Levine et al., 1985b; Schut
et al., 1985; Woollen et al., 1985; Rickert et al., 1981). Other primary
metabolites excreted by these animals include 2-am1no-4-nitrobenzyl alcohol
(2A4NBalc) and 4-amino-2-nitrobenzyl alcohol (4A2NBa1c) (Schut et al., 1985;
Lee et al., 1978); humans excrete only the former in large quantities. For
Fischer rats and humans, another major product of 2,4-DNT metabolism is
2,4-din1trobenzoic add (2,4-DNBac1d) (Levine et al., 1985b; Rickert et al.,
1981; Rickert and Long, 1981). A significant portion (4 to 9%) of a single
oral dose of 2,4-DNT is reduced in vivo to 2,4-diaminotoluene (2,4-DAT) by
rats, rabbits, dogs, and monkeys, but none is found in the urine of
occupational^ exposed humans (Levine et al., 1985b; Woollen et al., 1985;
Lee et al., 1978). In contrast, metabolism of 2,4-DNT by CD-I mice and Wistar
rats is not extensive, and .only small amounts of an oral dose of 2,4-DNT given
to these animals are oxidized to one of the benzyl alcohol metabolites;
reduction of 2,4-ONT to 2,4-DAT 1s negligible in mice (Schut et al., 1985;
A-3
-------
Siioj i el di., 1935; Mori et a"\ ., i96i; tee et ai., 1978). Sex-related
differences in the metabolism of. 2,4-DNT have been reported in only Fischer
rats and humans. Exposure in rats was by the oral route, whereas human
exposure was via the inhalation and dermal routes. For both species, females
produce up to three times more 2,4-DNBalc and/or 2,4-DNBalcG than males, and
in humans only, men excrete nearly twice as much 2,4-DNBacid as females
(Levine et a1.,'1985b; Turner et al., 1985; Rickert et al., 1981; Rickert and
Long, 1981).
The metabolism of 2,6-DNT has not been studied extensively. As with
2,4-DNT, Fischer rats convert the 2,6-DNT isomer to the corresponding
dinitrobenzyl alcohol glucuronide and dinitrobenzoic acid; however, only one
,'
metabolite (2-amino-6:nitrobenzoic acid, 2A6NBacid) results from the in vivo
reduction of 2,6-DNT (Long and Rickert, 1902). Similarly, only three major
metabolites of 2,6-DNT (2,6-DNBalc, 2,6-DNBalcG, and 2,6-DNBacid) have, been
recovered from the urine of men and women; women appear to excrete more
2,6-DNBalcG than men,(Levine et al., 1985b; Turner et al., 1985). No other
sex-related differences in the metabolism of 2,6-DNT have been reported.
In humans, the toxic effects of DNT are on the heart, the circulatory
system, and the central nervous system. Chronic DNT exposure, primarily via
the inhalation route (although percutaneous absorption and ingestion are
considered to be secondary and tertiary routes of exposure) has been
characterized in munitions workers by nausea, vertigo, methemoglobinemia,
cyanosis, pain or paresthesia in extremities, tremors, paralysis, chest pain,
and unconsciousness (Etnier, 1987; U.S. EPA, 1980, 1986; Levine et al., 1985a;
Ellis et al., 1979). Following a latency period of 15 years, workers exposed
to 2,4-DNT and tg-DNT exhibited excessive mortality from ischemic heart
disease and residual diseases of the circulatory system (Levine et al.,
1986a,b). A more recent study failed to demonstrate excess mortality from
ischemic heart di.sease and cardiovascular diseases in DNT-exposed workers
(Stayner et al., 1989); however, it did suggest a possible increase in
mortality from liver and biliary tract cancer (Stayner, 1989). Limited
evidence suggests that DNT does not result in adverse effects on human
reproductive performance (Hamill et al., 1982; Ahrenholz and Meyer, 1982.).
, A-4
-------
Acute oral toxici.ty studies indicate that rats are more susceptible to
2,4-DNT than mice. The LD50 values ranged from 1,340 to 1,954 mg/kg in mice
and from 270 to 650 mg/kg in rats (Lee et al., 1975; Vernot et al., 1977).
Both species exhibited ataxia and cyanosis. When mice, rats, and dogs were
administered 2,4-DNT in the diet for 4 weeks, dogs were found to be most
sensitive, exhibiting incoordination and paralysis at doses of 25 mg/kg/day;
CNS effects were not observed in rodents (Lee et al., 1978; Ellis et al.,
1985). Similar CNS effects were seen when dogs were orally administered 20 mg
2,6-DNT/kg/day. Methemoglobinemia, reticulocytosis, and Heinz body formation
resulted from oral administration of 37.5 mg tg-DNT/kg/day to F344 rats for
,4 weeks (CUT, 1977).
Subchronic toxicity of mice and rats administered 2,4-DNT via the diet,
and in dogs administered 2,4-DNT via capsules for 13 weeks, was assessed. In
mice of both sexes, anemia and reticulocytosis were observed at a dose level
of 413 (males) to 468 (females) mg/kg/day (Hong et al., 1985). In rats,
mortality occurred prior to study termination in 100% of females and 75% of
./ ,
males fed 145 (females) to 266 (males) mg/kg/day (Lee et al., 1985). Anemia
and reticulocytosis were observed at dose levels as low as 93 (males) to 108
(females) mg/kg/day; splenic hemosiderosis and depressed spermatogenesis were
found in these animals. In dogs, neuromuscular incoordination and paralysis,
methemoglobinemia, aspermatogenesis, hemosiderosis of the spleen and liver,
cloudy swelling of the kidneys, and lesions of the brain were observed in
males and females at a dose level of 25 mg 2,4-DNT/kg/day (Ellis et al.,
1985). Similar effects were seen when mice, rats, and dogs were orally
administered 2,6-DNT for 13 weeks (Lee et al., 1975). All species exhibited
methemoglobinemia, anemia, bile duct hyperplasia sometimes accompanied by
hepatic degeneration, and depressed spermatogenesis. Incoordination, rigid
paralysis, and renal degeneration occurred in dogs at a dose level of 20 mg
2,6-DNT/kg/day.
Lifetime feeding studies in rats and mice and a 2-year study in dogs
showed increased mortality, weight loss, anemia, neurotoxicity,
hepatotoxicity, renal toxicity, and testicular atrophy (Ellis et al., 1979,
A-5
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1985; Lee et al., 1985; Hong et al., 1985). In CD (Sprague-Dawley) rats fed
2,4-DNT in the diet at a level giving a daily intake of 34 (males) and 45
(females) mg/kg/day, respectively, lifespan was shortened, the incidence of
hepatocellular carcinomas was increased (6/30 and 19/35 high-dose males and
females, respectively, as compared with 1/25 and 0/23 concurrent controls),
seminiferous tubules atrophied resulting, in almost complete cessation of
spermatogenesis, and excessive pigmentation accumulated in the spleen. An
increased incidence of benign mammary gland tumors (33/35 as compared with
10/23 in controls) was exhibited in females. Anemia, partially compensated,
and testicular atrophy were evident at 3.9 mg/kg/day, and hepatocellular
alterations were observed at 0.57 mg/kg/day, the lowest dose tested (Lee
et al., 1985). In a study in CD-I mice, all the animals fed 898 mg/kg/day
died by month 18 (males) or month 21 (females). Effects at 14 mg/kg/day, the
lowest dose tested, included testicular atrophy, decreased body weight in
males, and nemosiderosis of many organs, primarily the liver and spleen. The
incidence of malignant renal tumors was elevated in males fed 95 mg/kg/day
(15/17 as compared with 0/20 concurrent controls) (Hong et al., 1985). In a
study in dogs, 2,4-DNT administered daily in capsule form caused neurotoxiclty
(incoordination and paralysis, often leading to death) in all dogs at
10 mg/kg/day, and anemia (partially compensated) and biliary tract hyperplasia
at 1.5 mg/kg/day. No adverse effects were observed at 0.2 mg/kg/day
(Ellis et al., 198,5).
Male and female CDF (F344) rats fed tg-DNT in the diet at a level to g1v«
a daily intake of 35 mg/kg/day were sacrificed in extremis after 55 weeks
(Leonard et al., 1987). At 14 mg/kg/day, 96% of the males and 59% of the
females developed hepatocellular carcinomas. After 78 weeks, decreased body
weight, increased liver weight, hepatotoxicity (increased Incidence of
hepatocellular carcinomas; 9/19), renal toxicity, and parathyroid hyperpUsU
were reported at 3.5 mg/kg/day, the lowest dose tested. In a limited l-yt«r
study, 47% of male CDF (F344) rats given tg-DNT at dose.levels providing
35 mg/kg/day (the only dose tested) developed hepatocellular carcinomas (0/20
control, 9/19 treated) (CUT, 1982). Under similar conditions, 2,6-DNT
induced hepatocellular tumors in 100% of the high-dose (14 mg/kg/day) and 85%
A-6
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of the low-dose (7 nig/kg/day) males. Rats exposed to 2,4-ONT (35 mg/kg/day)
did not develop tumors (Leonard et a!., 1987). Although limited in duration
and in the number of animals tested, these data may indicate that the
2,6-DNT isomer causes much of the carcinogenic activity in the previously
tested mixed-1somer ONT bioassays. DNT is classified as Group 82: Probable
Human Carcinogen.
2,4- and 2,6-DNT are weak mutagens in Salmonella test systems. However,
metabolites of 2,4-ONT, particularly the 2,4-nitrobenzyl alcohol and the
2-amino- and 2-nitroso-4-nitrotoluenes, are mutagenic without metabolic
activation (Couch et al., 1987). The dinitrotoluenes are also negative
genotoxins in mammalian .cells in vitro, in the dominant lethal test in mice
and rats, and in Drosobhila systems (Abernethy and Couch, 1982; Styles and
Cross, 1985; Soares and Lock, 1980; Lane et al., 1985; Ellis et al., 1979;
Woodruff et al., 1983). Technical grade ONT gave negative responses for
unscheduled DNA synthesis (LIDS) except when an in vivo/1n vitro testing system
was used (Bermudez et al., 1979; Mirsalis and Butterworth, 1982a). It has
been concluded that biliary excretion, metabolism by gut flora, and resorption
from the Intestine are prerequisites for genotoxic activity (Mirsalis et al.,
1982b; Popp and Leonard, 1982). Metabolites of 2,4-ONT can bind to liver ONA,
and 2,4-ONT appears to act as a promoter, inducing gamma glutamyl transferase-
positive foci in the livers of rats initiated with dimethylnitrosamine
(Leonard et al., 1983, 1986). On the other hand, 2,6-DNT has both Initiation
and promoting activity (Popp and Leonard, 1982; Mirsalis et al., 1982;
Doolittle et al., 1983).
2,4-Dinitrotoluene causes severe reproductive effects In rats, mice, and
dogs (Ellis et al., 1979; Lee et al., 1985; Hong et al., 1985; Ellis et al.,
1985). Oral exposure to 2,4-DNT results in testicular atrophy and
degeneration, as well as reductions in spermatogenesis in males. In females,
oral exposure resulted in cessation of follicular function and reduction in
the number of corpora lutea. Consequently, fertility is reduced in both
sexes. Also, 2,4-DNT causes reduced viability as well as decreased body
weight of offspring at birth and weaning. No data on the reproductive effects
of 2,6-DNT or tg-ONT were found in the available literature. Limited
A-7
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available data suggest that 2,4-ONT is not teratogenic in mice following
ingestion (Hardin et al., 1987). Technical grade DNT was not teratogenic to
rats administered oral doses, although embryotoxicity was observed at
maternally toxic levels (Price et al., 1985). In addition, the administration
of tg-DNT to pregnant rats also resulted in changes in relative organ weights
and hematologic parameters in their fetuses. No data on the developmental
effects of 2,6-DNT were found in the available literature.
The One-day Health Advisory (HA) and Ten-day HA for 2,4-DNT for a 10-kg
child are 0.5 mg/L {500 M9/L), and the Longer-term HA for exposure in a 10-kg
child 1s 0.30 mg/L (300 M9/L). The Longer-term HA for exposure in an adult
is 1.0 mg/L (1,000 M9/L). The Drinking Water Equivalent Level (DWEL) for
2,4-DNT is 0.1 mg/L (100 M9/L), derived from a Reference Dose (RfD) of
0.002 mg/kg/day. The One- and Ten-day, and the Longer-term HAs for 2,6-DNT
exposure in a 10-kg child 1s 0.4 mg/L (400 M9/L), and the Longer-term HA for
an adult is 1.0 mg/L (1,000 M9/L). The Drinking Water Equivalent Level
(DWEL) for 2,6-DNT Is 0.04 mg/L (40 M9/L), derived from a Reference Dose
(RfD) of 0.001 mg/kg/day. DNT is classified B2: Probable Human Carcinogen;
thus a Lifetime HA 1s not recommended. The estimated excess cancer risk
associated with lifetime exposure to drinking water containing 2,4-DNT at the
level of the DWEL (100 M9/L) 1s 2 x 10~3. The estimated excess cancer risk
associated with lifetime exposure to drinking water containing 2,6-DNT at the
level of the DWEL (40 M9/L) Is 1 x 10'3.
C. DISCUSSION
Available data on the pharmacokinetics, health effects, analysis, and
treatment of ONT have been reviewed.
Pharmacok1net1c studies in rodents, dogs, and monkeys Indicate that DNT
is effectively absorbed via the oral route. Despite the significant role of
gut tnicroflora and biliary excretion in the metabolism of DNT, fecal
metabolites have not been identified. Limited data indicate that 2,4-DNT is
absorbed by fetal rats; further studies on placental transfer and fetal
A-8
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accumulation are needed. Humans absorb tg-DNT primarily via the inhalation
and dermal routes; however, studies on the pharmacokinetics of DNT in animals
using these routes of exposure are not available. Data on the
pharmacokinetics of 2,6-DNT are limited;, additional single- and multiple-dose
studies should be conducted, particularly in light of the carcinogenic and
mutagenic properties of the isomer.
Available acute toxicity studies include oral L0,0s in mice and rats.
Subacute (5-, 8-, 14-, 21-, and 30-day) studies were available for Sprague-
Dawley rats, CD-I mice, Swiss mice, and beagle dogs administered 2,4-DNT.
Mouse and rat studies appeared to be adequate; dog studies were not conducted
with sufficient numbers of animals (one dog/sex/dose sacrificed at dosing
termination, one dog/sex/dose sacrificed following 4 weeks of recovery).
Four-week subacute studies were available for Sprague-Dawley rats, Swiss mice,
and beagle1 dogs administered 2,6-DNT, and a 4-week study was available, for
F344 rats administered tg-DNT; studies appeared to be adequate.
Subchronic (13-week) studies were available for Sprague-Dawley rats, CD-I
mice, and beagle dogs administered 2,4-DNT or 2,6-DNT. Mouse and rat studies
appeared to be adequate; dog studies conducted with 2,4-DNT were not conducted
with sufficient numbers of animals. Subchronic studies were not conducted
using tg-DNT.
i
Chronic toxicity/oncogenicity studies were conducted in Sprague-Dawley
rats, CD-I mice, and beagle dogs administered 2,4-DNT. Further chronic
studies are unlikely to yield additional data pertinent to the development of
HA values for 2,4-DNT. However, chronic/oncogenicity rodent studies on
2,6-DNT and tg-DNT were limited to 1 year in duration as a result of high
tumor incidence; additional lifetime rodent studies with 2,6-DNT or tg-DNT
should be conducted at low-dose levels.
The data currently available are adequate to determine the reproductive
effects of 2,4-DNT in animals. However, since there is sufficient evidence in
animal studies to conclude that 2,4-ONT adversely affects reproductive
performance and fertility and causes testicular degeneration, more information
A-9
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on health effects associated with human exposure is needed to evaluate the
reproductive effects on humans. No data are currently available on the
reproductive toxicity potential of 2,6-DNT or tg-DNT in animals or humans.
Therefore, a multigeneration reproductive toxicity study in animals,
preferably rats, should be conducted. In addition, since 2,4-DNT has been
shown to cause reductions in reproductive performance and fertility in
animals, as well as testicular degeneration, information on the reproductive
effects associated with human exposure is needed.
The data currently available are not adequate to assess the developmental
toxicity of 2,4-DNT or tg-DNT in animals or humans. The only available animal
study found for 2,4-DNT was a screening test using mice, and this was
insufficient to adequately assess developmental effects. The only available
animal study for tg-ONT tested insufficient numbers of animals and was
considered to be inadequate. Therefore, two developmental toxicity studies
using 2,4-DNT in two different species, preferably in rats and rabbits, are
needed. Since no data are currently available on the developmental toxicity
potential of 2,6-DNT in animals or humans, two developmental toxicity studies
in two different species of animals, preferably rats and rabbits, should be
conducted. In addition, information on the developmental effects associated
with human exposure to 2,6-DNT are needed.
Further investigation of initiation/promotion activity for 2,4-DNT and
2,6-DNT should be conducted.
D. CONCLUSIONS/RECOMMENDATIONS
The following conclusions/recommendations are based upon the above
discussion:
1. The available studies on 2,4-DNT toxicity are generally considered to be
adequate for development.of Health Advisories useful in dealing with the
potential contamination of drinking water; available studies on 2,6-DNT
and tg-DNT are limited.
'A-10
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2. It is recommended that single- and multiple-dose delayed neuropathy
studies be performed on 2,4-DNT, 2,6-DNT, and tg-ONT in hens (sensitive
animal model).
3. It is recommended that.additional pharmacokinetics studies be conducted
on 2,4-DNT, Including identification of fecal metabolites, placental
transfer, fetal accumulation, and metabolism of animals exposed to tg-ONT
by the inhalation and dermal routes. In addition, single- and multiple-
dose pharmacokinetics studies should be conducted on 2,6-DNT.
4. It is recommended that the 13-week study in which beagle dogs were
administered 2,4-DNT be repeated using at least four animals/sex/group,
since this species was the most sensitive species treated.
5. It is recommended that adequate lifetime chronic toxicity/oncogenicity
studies be performed in rats and mice using 2,6-DNT at dose levels up to
7 mg/kg/day.
i
6. It 1s recommended that adequate reproduction studies be performed using
2,6-DNT in at least one rodent species.
7. " It is recommended that adequate developmental toxicity studies be
performed using 2,4-DNT and 2,6-DNT in at least one rodent species.
8. It is recommended that further investigation of initiation/promotion
activity for 2,4-DNT and 2,6-DNT be conducted.
The recommended order of completion for these studies is as follows:
pharmacokinetics, initiation/promotion activity, subchronic dog study, chronic
toxicity/oncogenicity, developmental toxicity, reproduction, and delayed
neuropathy.
A-ll
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