November 1988
HEALTH ADVISORY FOR
HEXAHYDRO-1,3 ,5-TRINITRO-1,3,5-TRIAZINE
(ROX)
CRITERIA AND STANDARDS DIVISION
OFFICE OF DRINKING WATER
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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Novenber 1988
HEALTH ADVISORY FOR
HEXAHY0RO-1,3,5-TRINITRO-1,3,5-TRIAZINE
(RDX)
AUTHORS:
William L. McLellan, Ph.D.
William R. Hartley, Sc.D.
Margaret E. Brower, Ph.D.
PROJECT OFFICER:
Krishan Khanna, Ph.D.
Criteria and Standards Division
Office of Drinking Water
U.S. Environmental Protection Agency
Washington, DC 20460
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PREFACE
This report was prepared in accordance with the Memorandum of Understanding
between tne Department of the Army, Deputy for Environment Safety and
Occupational Health (0ASA(I&L)), and the U.S. Environmental Protection Agency
(EPA), Office of Drinking Water (ODW), Criteria and Standards Division, for
tne 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 whicn adverse health effects would not be anticipated and
which include a margin of safety so as to protect the most sensitive memDers
of tne 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, 10-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).
The report is the product of the foregoing process. Available toxicological
data, as provided by the Army, on the munitions chemical hexahydro-1,3,5-trinitro-
l,3,b-tnazine (RDX) 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
to critical internal and external review by the EPA.
A companion document, "Data Deficiencies/Problem Areas and Recommendations for
Additional Data Base Development for RDX" is included in this report.
I would like to thank the authors (Dr. William L. McLellan, Dr. William R.
Hartley and Dr. Margaret E. Brower) who provided the extensive technical skills
required for the preparation of this report. I am grateful to the members of
the EPA Tox-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,
Health Effects Branch, and Dr. Joseph Cotruvo, Director, Criteria and Standards
Division, for providing me with the opportunity and encouragement to be a part
of this project.
The preparation of this Advisory was funded in part by Interagency Agreement
(IAG) between the U.S. EPA and the U.S. Army Medical Research and Development
Command (USAfflDC). This IAG was conducted with the technical support of the
U.S. Anmy Biomedical Research and Development Laboratory (USABRDL).
Krishan Khanna, Ph.D.
Project Officer
Office of Drinking Water
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TABLE OF CONTENTS
Page
LIST OF TABLES v
EXECUTIVE SUMMARY vi
I. INTRODUCTION 1-1
II. GENERAL INFORMATION ll-l
III. SOURCES OF EXPOSURE 111 -1
IV. ENVIRONMENTAL FATE IV-1
V. PHARMACOKINETICS V-l
A. Absorption V-l
B. Distribution V-2
C. Metabolism and Excretion V-6
VI. HEALTH EFFECTS VI-I
A. Humans VI -1
B. Animal Experiments VI-5
1. Short-tenm Exposure VI-5
a. Acute VI-5
b. Primary Irritation and Sensitization VI-10
2. Longer-term Exposure VI-13
a. Ten- to 13-week Studies VI-13
b. Lifetime Studies VI-21
3. Reproductive Effects VI-29
4. Developmental Toxicity VI-30
5. Carcinogenicity VI-33
6. Genotoxicity VI-39
7. Neurotoxicity VI-40
VII. HEALTH ADVISORY DEVELOPfCNT VII-1
A. Summary of Health Effects Data VII-1
B. Quantification of Toxicological Effects VII-3
1. One-day Health Advisory VII-3
2. Ten-day Health Advisory VII-4
3. Longer-term Health Advisory VI1-4
4. Lifetime Healtn Advisory V11 - 5
C. Quantification of Carcinogenic Potential VI1-9
iii
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TABLE OF CONTENTS (continued)
page
VIII. OTHER CRITERIA, GUIDANCE, AND STANDARDS VIII-1
IX. ANALYTICAL METHODS IX-1
X. TREATMENT TECHNOLOGIES X-l
XI. CONCLUSIONS AND RECOMMENDATIONS XI-1
XII. REFERENCES XII-L
Appendix A: Data Deficiencies/Problem Areas and Recommendations for
Additional Data Base Development for RJX
1 v
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LIST OF TABLES
Table No. page
11 -1 General Chemical and Physical Properties of RDX .... 11 - 2
V-l Mean (i SE) RDX Concentration in Plasma, Urine,
and Tissues of Rats at Various Times After Oral
Administration of 100 mg RUX/kg V-3
V-2 Mean (± SE) RDX Concentration in Plasma, Urine,
Feces, and Tissues of Miniature Swine 24 Hours
After Oral Administration of 100 ng ROX/kg V-5
V-3 ROX Concentration of Tissues From Rats Provided With
Drinking Water Spiked With [^4C] RDX (bO to 70 ug
RDX/mL) at 30, 60, and 90 Days V-7
V-4 Daily Recovery of Radioactivity at Specific Intervals
During Administration of Drinking Water Spiked With
c 14C]RDX to Rats V-3
VI-1 Acute LD50 Values for RDX in Laboratory Animals .... VI-6
VI-2 Representative Results of Mortality and Percent
Survival in Rats Fed RDX in the Diet for 104 Weeks. . . V1-22
VI-3 Histologic Lesions in Male F-344 Rats Fed RDX for
2 Years VI - 2 5
V1-4 Representative Results of Mortality and Percent
Survival in Mice Fed RDX in the Diet for 104
Weeks V1-27
V1-5 Fetal Weight and Length (£ SD) in Progeny of
Rats Administered RDX VI - 34
VI-6 Incidence of Neoplastic Lesions in Male B6C3Fi Mice
Fed RDX 1n the Diet for 24 Months VI-3b
VI-7 Incidence of Neoplastic Lesions in Female B6C3Fi
Mice Fed RDX in the Diet for 24 Months V1-36
V1-8 Incidence of Primary Liver Tumors in Historical
Control 86C3Fi Mice V1-38
V11-1 Summary of Candidate Studies for Derivation of
the Drinking Water Equivalent Level for RDX VI1-7
v
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EXECUTIVE SUMMARY
Hexahydro-1,3,5-tri ni tro-1,3,5-triazine, commonly known as RDX (British
code name for Research Department Explosive or Royal Demolition Explosive), is
a white crystalline solid that has been extensively used in military munitions
formulations.
The pharmacokinetic properties of RDX have been extensively studied in
rats. RDX was found to be completely absorbed via the oral route; the rate
of absorption was reported to be a direct function of the particle size of
the powder in the slurry administered. The rate of gastrointestinal absorption
was found to be faster in rats than in humans or miniature swine; in rats, peak
plasma levels were reached in 2 to 3 hours, whereas in swine and probably in
humans, plasma levels peaked approximately 12 hours after dosing. Absorbed RDX
is rapidly cleared from the plasma and distributed to tissues. The half-lives
of clearance of RDX from plasma are of a similar order of magnitude in rats and
humans: the t^/2 was found to be 10.1 hours in rats and 15.1 hours in the one
available human study. The highest RDX levels are found in the kidneys, followed
by the levels in the liver, brain, and heart. RDX is metabolized by the liver,
and its metabolites are excreted primarily in the urine. The metabolites have
not been identified or characterized.
In humans, the toxic effects of RDX have been on the central nervous system
(CNS). Exposure of workers in a munitions plant via inhalation of dust
containing RDX has resulted in nausea, irritability, convulsions, unconsciousness,
and amnesia. Military personnel have been exposed to RDX while burning
composition C-4 explosives in the field to heat food; inhalation of the smoke
resulted tn clonic/tonic convulsions. Ingestion of RDX has caused similar CNS
effects.
vi
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Acute toxicity studies indicated oral LD50 values of about 80 mg RDX/kg in
mice and lid my RDX/kg in rats. Intravenous administration of single doses of
rfDX to oeagle dogs caused convulsions and death at a dose of 40 mg/kg, central
nervous system hyperactivity and nonlethal convulsions at a dose of 20 mg/kg,
and decreased blood pressure and erratic electroencephalographic patterns at
doses of 3.37 and 6.78 mg/kg.
Subchronic 90-day feeding studies in mice and rats indicate effects on tne
blood and liver. In mice of both sexes, increased liver weights were noted in
groups receiving 320 mg RDX/kg/day, and anemia was seen in males receiving
160 mg RDX/kg/day. In rats, anemia was observed at a dose level of 28 mg
RDX/kg/day in males, and increased liver weight was noted at a dose level of
100 mg RDX/ky/day in females. In a 10-day oral gavage study in monkeys, vomiting
and convulsions were seen in five of six animals dosed with RDX at 10 mg/kg/day,
but no central nervous system effects were observed at 1 mg/kg/day.
Lifetime feeding studies in rats and mice produced CNS effects, increased
mortality, weight loss, anemia, hepatotoxicity, renal toxicity, testicular
degeneration, and inflammation of the prostate. In male and female rats fed
RDX in the diet at a level to give a daily intake of 40 mg/kg, tremors and
convulsions, increased mortality, and enlargement of the liver were observed.
Anemia and enlargement of the kidneys accompanied by histologic changes were
also found in males receiving 40 mg RDX/kg/day. Inflammation of the prostate
was found when RDX was administered at 1.5, 8, and 40 mg/kg/day; no effects
were noted at a dose of 0.3 mg/kg/day. When mice were administered 175 mg
RDX/kg/day, increased mortality was seen within 10 weeks. The high dose was
reduced to 100 mg RDX/kg/day. Decreased weight gain was seen in females
receiving 100 mg RDX/kg/day between 10 weeks postadministration and study
vi i
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termination. Increased liver weights were found in males and females
receiving RDX at 100 my/ky/day, and testicular degeneration was found in
males receiving 35 or 101) mg/kg/day; no important toxic effects were observed
at 7 mg/kg/day.
RDX was not found to be mutagenic in bacteria and gave negative results in
the domi nant-1 etnal test and in an unscheduled ONA synthesis assay. RDX was
not carcinogenic in rats. In B6C3F^ mice, a significant increase was observed
in the combined incidence of hepatocellular carcinomas and adenomas in females
receiving RDX at 7, 35, or 100 mg/kg/day for 2 years. Mortality in mice
receiving the highest dose was excessive, and the dose was lowered from 175 to
100 mg/kg at week 11. RDX is classified as Group C: Possible Human Carcinogen.
In a two-generation reproduction study in rats, decreased fertility was
observed at 50 mg ROX/kg/day. Developmental effects (decreased pup weights)
were seen at 16 and 50 mg ROX/kg/day; there were no effects at 5 mg/kg/day. ROX
was found to be embryotoxic in rats at 20 mg/kg/day but was not found to be
teratogenic. In a study in rabbits, RDX caused maternal toxicity at 20 mg/kg/day,
and there was suggestive evidence for a teratogenic effect at 2 and 20 mg/kg/day.
Based on these findings and on the results of a 90-day oral toxicity study
in monkeys where convulsions occurred in five of six animals administered 10
mg RDX/kg/dty but no CNS effects were seen in monkeys administered 1 mg/kg/day,
the Longer-term Health Advisory (HA) for a 10-kg child has been determined to
be 0.1 mg/L (100 ug/L). In the absence of adequate animal data to determine
a One-day or Ten-day Health Advisory, the Longer-term HA for a 10-kg child, 0.1
mg/L (100 ug/L), is used as a conservative estimate of the One-day or Ten-day
HA. The Longer-term HA for an adult was established at 0.35 mg/L (400 ug/L).
A Lifetime HA of 0.002 mg/L (2 ug/L) for an adult was determined based on a
vi i ^
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Drinking Water Equivalent Level (DUEL) of 0.100 mg/L (100 >jg/L). The DWEL is
based on a 'deference Dose (RfD) of 0.U03 mg/kg/day where the effect was
suppurative inflammation of the prostate of male rats fed R0X for 2 years.
1 x
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I. INTRODUCTION
T'ie Health Advsory (HA) Program, sponsored by the Office of PrTik-ng wafer
(ODW), provaes information on the health effects, analytical methodology, and
treatment technology that would be useful 'n deal'ng wth the contam^nat'on
of drinking water. Health Advisories describe nonregulatory concentrations
of dr'nk'-ng water contaminants at which adverse health effects would not
anfcpated to occur over specf'c exposure durafons. Health Adv-sor^es
contain a margin of safety to protect sens't've 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 nor to he
construed as leyally enforceable Federal standards. HAs are subject to change
as new information becomes available.
HAs are developed for One-day, Ten-day, Longer-term (approximately 7
years, or 1U% of an individual 's lifetime), and Lifetime exposures hased on
data describing noncarcinogenic endpo^nts of toxicity. For those substances
that are known or probable human carcinogens, according to the Agency
classification scheme (Group A or B), Lifetime HAs are not recommended. The
chemical concentration values for Group A or B carcinogens are correlated w->th
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 linear multistage
model with 95% upper confidence limits. 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 One-hit, Weibull, Logit, and Probit models. There is no
1-1
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current understand1 ng of the b,ologi'cal mechanisms Tivolved ""n cancer to sugges
that any one of these models is ablp to pred^t r->sk morp accurately than
another. Because each model is based upon d^ffer^ng assumpt'ons, the esfmates
that are delved can differ by several orders of magnitude.
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II. GENERAL INFORMATION
Hexahydro-1,3,b-trirr,tro-l ,3,5-tr1 az1 ne (CAS No. 121-32-4), an explosive
polyn1 tram1ne , ""S commonly known as RDX (Rffsh code name for Resparc^
Department Explosive or Royal Demol1t',on Explosive).
RUX, a wivte crystalline solid, is a completely N-n-'trated, s^x-menher
heterocyclic r--ng compound. It has been extensively used as a h->gh-Tipact
explosive in military munitions formulations during and since World War II.
RUX is also used as a rat poison (ACGIH, 1986; Windholz, 1983).
RDX is generally manufactured by the nitration of hexamethylene tetramine
(C6H12N4). In the United States, RDX is mainly manufactured at the Holston
Army Ammunition Plant using the continuous Bachmann process (Pal and Ryon,
1986). This method involves the nitration of hexamine with ammonium nnratp
and nitric acid in an acetic acid-acetic anhydride solvent (Sullivan et al.,
1979).
General chemical and physical properties of RDX are presented in Table
11-1. RDX (molecular weight 222.26) has approximately 130% the explosive power
of trinitrotoluene (TNT) (Sullivan et al., 1979). It is considered a stable,
relatively insensitive explosive, and can be stored up to 10 months at 85°C
without perceptible deterioration (Meyer, 1977; Pal and Ryon, 1986). The low
solubility of RDX in water (7.6 mg/L at 25°C and 1.3 g/L at 83°C) indicates
that much of the compound detected in wastewater consists of undissolved
particulates (Pal and Ryon, 1986). RDX preparations contain approximately 9%
octahydro-1,3,5,7-tetranitro 1,3,5,7-tetrazocine (HMX).
II-l
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Table 11-1. General Chemical and Physical Properties of RDX
CAS No.
Synonyms
Molecular weight
Empirical formula
Chemical structure
121-82-4
Cyclonite/Hexogen
Cyclotrimetnylenetrinitrami ne
Hexahydro-1,3,5-trim tro-1,3,5-triazine
RDX
sym-Trimethylenetrim tramine
T4
1,3,5-Tn ni trohexahydro-s-tri azi ne
222.26
C3H5N5O6
3JG
Physical state
Specific gravity
Melting point
Heat of combustion
Solubility characteristics:
Water
Cycl ohexanone
Cyclopentone
Acetone
Nitrobenzene
Methylisobutenyl ketone
Methylacetate
Acetic anhydride
Conversion factors (air)
White crystalline solid-orthorhombic crystal
1.816 0 20°C
204.1°C
2,259.4 cal/g
0.00076% w/v 0 25°C (7.6 mg/l) to
42.3 mg/L (20°C) reported
12.7% w/w 0 25°C
9.9% w/w 0 25°C
8.3% w/w 0 25°C
1.5% w/w 0 25°C
3.0% w/w @ 25°C
1.9% w/w 0 20°C
4.9% w/v 0 30°C
1 ppm = 9.09 mg/m3
SOURCE: Adapted from Hawley (1977); Small and Rosenblatt (1974); Windholz (1983);
Sullivan et al. (1979); Etnier (1986).
11-2
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III. SOURCES OF EXPOSURE
Occupafonal exposure may occur dumng manufacture and mun-'fons
1 ncorporat1 on of RDX. Human -"llness results from repeated exposure va the
respTatory and yastro1 ntesf nal tracts and by sk-'n absorption (Kaplan er al.,
196b; ACGIH, 19d6.) Reports of tox-'Cty to humans were anecdotal and occurred
¦"n the early 194Us *n Italy and Germany. All 'ncdents were probably attr-1 huta^l e
to poor ',ndustrial hyyene procedures and -"nadequate vent11 at1 on, wh-'ch resuir^i
'n contam1 nat1 on of workroom a'r wth RDX dust. In one situation, no further
cases of tox-'city were observed when remedial protectee measures were enforced
(Kaplan et al., 1965). No air mom tor-rig sampling was performed 'n the early
studies. Hathaway and Buck (1977) surveyed U.S. munitions plants between
1972 and 1974 and found no adverse effects *n workers w»th average ^-*our
time-weighted exposures to RDX of 0.28 mg/m3 and maximum levels up to 1.57
mg/m3. Exposure to RDX in an occupational setting with standard hygiene
and working procedures (e.y., protective clothing) is principally v'a airborne
dust. In limited geographical areas, humans may be potentially exposed to RDX
as an envronmental contaminant.
RDX manufacturing operations such as recrystal 1 i zation, dewatpMng, and
incorporation are primary sources of RDX in wastewater. Load, assemble, and
pack (LAP) facilities also discharye RDX-contaminated wastewater from such
operations as washdown, explosive melting, and reject warhead steam cleaning.
These wastewaters could contaminate groundwater and public drinking water
supplies (Sullivan et al., 1979).
Wastewaters resulting from manufacture and loading of ROX may be discharged
into the environment and may present a potential for aquatic pollution. Sediment
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deposits in Army ammunition plants may also pose an environmental problem
because seepage into the groundwater may occur (Etnier, 1986).
111-2
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IV. ENVIRONMENTAL FATE
The environmental fate of RDX has been thoroughly -eviewed by Etnie- (1936).
In experimental studies on migration of [14c]RDX in soils of various pH, textj-e,
and organic matter typically found in the United States, RDX was associated
with downward movement and a very low leachate level (less than 0.5 ppm, wivch
was the level of detection). Biological degradation to near the surface of
the soil was also observed. RDX is resistant to aerobic bacterial degradation
in soil. However, in activated sludge systems, 97% of a 20-mg/L solution of RDX
was degraded in 5 days. Bacterial degradation results in reduction to mono-,
di-, and fi-nitroso derivatives, in reduction to a triazine, and in hydrolytic
cleavage of the triazine. Formaldehyde, methanol, and COg are end products.
Photolysis rapidly degrades RDX. Volatilization is not significant ;n the
environmental fate of RDX. Sediment absorption will not lead to a significant
loss in the aquatic environment.
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V. PHARMACOKINETICS
A. A6SURPTION
A dose, by yavage, of a slurry of coarse yranular RDX did not cause
convulsions in 10 Sprague-Dawley rats administered 100 mg/ky, whereas a gavage
dose of a slurry of finely ground RDX caused convulsions in all 10 rats 2 hours
after dosing at a level of bO mg/ky. Measured particle sizes were not reported.
The plasma concentration, 24 hours after dosing with the coarser RDX, was
3.04 ^y/mL, whereas it was 4.7 ug/mL 24 hours after the smaller dose of
finely ground RDX was administered. The IO5Q value for the slurry of finely
ground RDX was equivalent to that of a dimethyl sulfoxide (DMSO) solution of ROX
(Schneider et al., 1977). In preliminary studies with individual rats orally
given a single 50-mg/kg dose of [^CjKDX in DMSO, 83% of the administered
radioactivity was found in the gut after 4 nours, 39.3% was found after 24 nours,
and only 2.1% was found after 48 hours. Only 1 or 2% of the dose was found in
the feces at 24 or 48 nours (Schneider et al., 1976).
Cholakis et al. (1980) dosed mice, by oral gavage, with much higher
concentrations of RDX. Death occurred within 5 to 10 minutes at doses of 180,
225, and 350 mg/kg and within 30 minutes following a 140-mg/kg dose, indicating
rapid absorption. From data of Schneider et al. (1977), it can be estimated
that deatn would occur at plasma levels of about 14 ug/mL.
Absorption was slower in miniature swine than in rats. The peak level
(4.7 ug/mL) was found 1n the plasma of swine 24 hours after a 100-mg/kg dose
of finely ground RDX. Schneider et al. (1977) suggested that the rate of
absorption via the gut in humans may be more comparable to the rate in swine,
because the latent period preceding RDX convulsions In humans (Stone et al.,
V-l
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1969) is of the same order as cue period observed in swine and much longer than
the period observed in rats.
3. 0 IS Fr4 IBUT ION
Schneider et al. (1977 , 1978) studied the tissue distribution, metabolism,
and excretion of RDX in rats after single doses administered via parenteral and
oral routes and after multiple oral doses. Ten Sprayue-Dawl ey rats (botn sexes)
received a single intraperitoneal (ip) dose of 500 mg RDX/kg. Eight of the 10
rats died within 6 hours after experiencing severe clonic-tomc convulsions.
The two rats that survived were sacrificed at 3.5 and 6.5 hours. The mean RDX
plasma level (± SE) for all rats was 13.8^2.6 ug/g. The ratio of tissue
concentration to plasma concentration was approximately 4.8 for kidney, 3.3 for
liver, and 2.3 for heart or brain tissue. Two hours after an ip dose of SO mg
RDX/kg, the plasma level was 1.1 ug/g, and the tissue-to-plasma ratios for
kidney, liver, heart, and brain tissue were 8.8, 5.7, 2.6, and 3.4, respectively.
No convulsions were observed at the lower dose (Schneider et al., 1977).
After intravenous injection of 5 to 6 mg RDX/kg in 10 rats, there was a
biphasic disappearance from the plasma. The half-life of the distribution phase
was 6.32±0.18 minutes, the elimination phase had a half-life of 10.1*0.32
hours, and the apparent volume of distribution was 2.18 L/kg (Scnneider et al.,
1977). This high apparent volume of distribution may indicate significant binding
to tissue.
The tissue distribution and RDX levels in the urine' and plasma of rats at
various times after an oral dose of 100 mg coarse granular RDX/kg are shown in
Table V-l. Tissues were homogenized and extracted with benzene, and RDX was
quantitated following gas chromatography with electron capture detection. The
V-2
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Table V-l. Mean (i SE) KDX Concentration in Plasma, Urine, and
Tissues of Rats at Various Times After Oral Administration
of 100 mg R0X/kga
Time
(hr)
PI asm a
(ug/g)b
Uri ne
(ug/mL)
Br ai n
(uy/g)
Heart
(ug/g)
Li ver
(ug/g)
K i d n ey
2
1.5010.26
2.45±0.30
10.36il.24
7.97rl.ll
4.34r0.90
12.36-1.-
4
2.0910.09
5.46±0.96
7.71-0.98
6.49±0.96
2.1610.56
12.JO-1.~
6
1.78-0.15
5.02i0.81
7.5U0.57
7.13±0.71
0.51i0.34
13.5d-1.1
8
2.36±0.22
7.3U0.85
b.57±0.67
3.82t0.52
0.15=0.Id
10.90-0.:
12
2.26±0.16
5.49U.03
11.28H.60
11.08H.82
8.51i2.24
22.02-2.D
18
2.03-0.10
5.58-0.28
6.3U-0.20
b.5812.24
0.4810.20
12.12i0.-
24
3.0410.48
6.87±0.84
8.91U.07
7.8910.83
2.56H.15
16.35iO.e
aRats were fasted 24 hours prior to dosing.
^Conversion factor (micrograms per milliliter to micrograms per gram wet
weight) = 0.9737.
S0U>
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levels in kidney tissue were highest, followed by those in brain, heart, and
liver tissue.
Tissue levels after oral dosing of rats with 50 my/kg finely powered
C14C3RDX were also studied (Schneider et al., 1977). A time course of tissue
concentrations was not presented. Concentrations of tissue radioactivity 24
hours after a gavage dose of 50 mg [^C]RDX (finely powered material) were
compared with a study using 100 mg of coarse granular RDX. It was assumed that
all radioactivity was associated with RDX, and quantitation was accomplished Dy
dividing the amount of radioactivity of tissue by the specific radioactivity of
tne administered RDX. No correspondence was observed between the 24-hour
tissue levels in this experiment and those determined after gas-chromatographic
quantitation of the 100-mg/kg nonradioactive dose. Since it has been shown
that [^C]RDX is metabolized to ^C02» it is conceivable that labeled activated
one-carbon fragments are biosynthetically converted to tissue components
(Schneider et al., 1977), thereby causing the apparent discrepancy.
Ten female miniature swine (21 to 60 kg) were fasted for a day and then
administered 10U mg RUX/kg by oral gavage; four convulsed and two died at 22
hours. Plasma RDX concentrations reached about 1.8 ng/mL 1 hour after dosing;
the level (measured at 30-minute intervals) remained fairly constant over
6 hours, then increased to 4.7 ug/mL prior to sacrifice at 24 hours postdosing.
Tissue concentrations at 24 hours are shown in Table V-2. No marked accumulation
of RDX was found in any tissue, except stomach and colon, when compared to
plasma levels. This result is in contrast to that found for rats (Schneider et
al., 1977). A time course of tissue radioactivity was not available for swine.
V-4
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TaDle V-2. Mean (i SE) RDX Concentration in Plasma, Urine, Feces, and
Tissues of Miniature Swine 24 Hours After Oral Administration
of 100 mg RDX/kg
RDX
Tissue
concentration3
Brai n
7.0il.9 (10)b
Heart
5.3il.4 (10)
Li ver
4.4±1.7 (10)
Renal cortex
9.U4.3 (10)
Renal medulla
b.6rl.8 (10)
Spinal colon
18.7sll.O (6)
Stomacn
42.b±14.4 (6)
Fat
8.7±2.b (9)
Feces
2.5±0.6 (6)
PI asm a
4.7±2.1 (a)
Uri ne
3.6il.l (9)
aTissue RUX concentrations are expressed in micrograms per gram of wet weight.
Urine and plasma ROX are expressed in micrograms per milliliter.
bThe number of animals sampled is in parentheses.
SOURCE: Adapted from Schneider et al. (1977).
V-5
-------
In a 90-day study conducted by Schneider et al. (1978), groups of 18
Sprague-Oawl ey rats (botn sexes) were provided with drinking water spiked witn
[14C] RDX (50 to 70 uy/mL containing 7 to 18 nCi/mL). Based on measured
water consumption, the daily intake of RDX was between 5 and 8 iny/ky. Groups
of six rats were sacrificed at 30, 60, and 90 days to determine tissue distriDution
and body burden of RDX. Data for tissue levels are summarized in Table V-3.
No accumulation of RDX was found in any of the tissues examined after 90 days,
the levels in fat and brain tended to be slightly higher than those in other
tissues. No overt toxic signs were reported.
In another 90-day study, 30 rats were administered, by gavage, 20 my
ROX/kg/day. Tissues were collected from groups of 10 animals sacrificed at
30, 60, and 90 days postdosing. In the brain, heart, liver, and Kidney tissues,
the RDX levels increased only slightly from 30 to 90 days. The mean tissue
levels at 90 days (estimated from a bar graph) were approximately 10 ug/y for
brain, heart, liver, and kidneys. The RDX level in fat was 7 to 8 ug/g dry
weight at 30 and 60 days, and increased to approximately 20 ny/g dry weiyht at
90 days (Schneider et al., 1978).
C. METABOLISM AND EXCRETION
In a 90-day study, groups of six Sprague-Dawley rats (both sexes) were
provided with drinking water spiked with RDX (SO to 70 ug/mL containing 7
to 8 uCi/mL [14C]RDX). At weeks 1, 4, 8, and 13, radioactivity in exhaled
^COg, as well as in urine and feces, was determined. The results expressed as
percent of administered daily dose are presented in Table V-4. From 27 to 50%
of a daily dose was metabolized to ^COg and exhaled, 22 to 35% was excreted in
the urine, and 4 to 5% was excreted in the feces. The nature of the urinary and
fecal radioactivity was not investigated. Some variability in recovery, which
ranged from b4 to 89% (Schneider et al., 1978), was observed.
V-6
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Table V-3. RDX Concentration of Tissues From Rats
Provided With Drinking water Spiked with
[14C] KOX (60 to 70 uy RDX/mL) at 30,
60, and 90 Days
RDX
concentrations
(ug/g)
30 days
60 days
90 days
Brain
0.59=0.31a
0.40=0.23
0.65=0.15
Heart
0.84=0.67
0.20=0.09
0.45=0.07
Li ver
0.80=0.64
0.09=0.04
0.20=0.05
Kidney
0.30±0.14
0.12=0.04
0.47=0.11
Stomach
0.13=0.06
0.11=0.05
0.35=0.08
Colon
0.27=0.14
0.11=0.04
0.40=0.10
Fat
NDb
ND
0.57=0.04
aThe values are mean = SE for six rats for each
determination.
bND = Not determined.
SOURCE: Adapted from Schneider et al. (1978).
V-7
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TaDle V-4. Daily Recovery of Radioactivity at Specific
Intervals During Administration of Drinkiny
Water Spiked With [^C]RQX to Rats
Week of
study
Percent of
admi ni stered
dai ly dose3
Tot al
recovery
U)
Exhal ed
CO2
Uri ne
Fee es
1
35.4±2.7
35.3±2.5
4.0±0.8
74.7
4
34.8t2.U
24.611.6
4.0t0.3
63.4
8
50.5t3.9
33.7H.3
5.3£0.8
89.4
13
27.4r0.8
22.2±1.5
4.3±0.8
53.9
a Mean ± SE.
SOURCE: Adapted from Schneider et al. (1978).
v-a
-------
A 7-day study was carried out in five rats administered, by gavage, 20 ng/nj
[14C]RDX dissolved in DMSO. The percent of radioactivity recovered in exnaled
CO2, urine, and feces was 38.9^3.9, 33.6i2.3, and 4.8^0.8, respectively;
total recovery was 77.3% of the daily administered radioactivity (Schneider et
al., 1978).
The urinary metabolites of ROX have not been identified. However, less
tnan 3% of a single dose of 50 mg/kg is excreted in the urine unchanged (Scnneider
et al., 1977). The residual RDX remaining in the carcass 4 days after a single
dose was 0.30±0.05 ny/g, which corresponds to 0.6% of the original dose.
However, when radioactivity was used to monitor residues in the carcass after 4
days, 9.b±0.03% of the radioactivity was found. This would suggest that
metabolites such as bicaroonate and formaldehyde or formate (one-carbon fragments)
may be biosynthetically incorporated into the tissues of the rats.
One study was found in which rates of excretion were determined in numans
(Woody et al., iy86). A 3-year-old cnild weighing 14.5 kg ingested pellets of
composition C-4 (91% RDX). The peak RDX concentration in serum was 10.74 my/L
at 24 hours and dropped to 3.56 and 0.66 mg/L at 48 and 96 hours, respectively.
Disappearance of RDX from serum followed a monophasic exponential pattern, with
a half-life of 15.06 hours. The concentration in the cerebrospinal fluid (CSF)
was 8.94 mg/L at 24 hours, giving a ratio of 0.832 for CSFrserum. The rate of
fecal excretion was slower than the rate of urinary excretion; peak fecal
concentrations (4.49 mg/g) were reached at 96 hours compared with 48 hours for
\
peak urine concentrations (38.4 mg/L). Using an assumed value of 2.2 L/kg for
volume of distribution, it was estimated that a total of 1.23 g had been ingested
(84 mg/kg).
V-9
-------
\lI. HEALTH EFFECTS
A. HUMANS
Chronic RDX exposure v^a the inhalation route has been character1'zed 'n
munitions workers by epi 1 ept1 form seizures (convulsions) and unconscousness
(Ryon et al., 1984). These convulsions may be preceded hy insomnia,
restlessness, and irritability, and they may be followed by temporary annes-a
and d1 sor^'entafon. Convulsions have been reported primarily from exposure
the powdered form of RDX; complete recovery has been found to occur upon removal
from exposure.
Kaplan et al. (1965) reported five case histories involving convuls'ons
or unconsciousness among 26 employees in an explosives plant that was processing
RDX. These cases occurred within 3 months of startup of RDX-processing
operations. There were few prewarning symptoms; some workers had short per-ods
of headache, dizziness, nausea, or vomiting. Two convulsed and became unconscious
while working. One to several hours after leaving work, "three others became
unconscious without experiencing convulsions. These individuals were hospitalized
and treated, and they generally recovered within 1 day. No abnormalities were
observed in blood counts or urinalyses, and no symptoms other than CNS effects
were noted. Workers were exposed to finely powdered RDX dust, and absorption
was probably primarily via inhalation. When hygienic measures were taken in
the plant, no further adverse effects were seen. Kaplan et al. (1965) cited
previously reported cases involving industrial exposure to RDX in Russia,
Italy, and Germany that resulted in epileptiform symptoms and unconsciousness.
Ketel and Hughes (1972) reported 40 cases of RDX intoxication in U.S.
soldiers in Vietnam. In these cases, exposure was primarily via inhalation since
VI-1
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the soldiers frequently burned composition C-4 explosives to heat food in the
field. Composition C-4 contained 91% RDX, 2.1% polyisobutylene, 2.1% motor
oil, and 5.3% sebacate (a piasticizer). The patients showed symptoms of CrtS
toxicity ranging from confusion to multiple seizures followed by amnesia.
Most patients (88%) experienced frequent nausea and vomiting. Three patients
had one generalized clonic/tonic convulsion, and 44% had intermittent generalized
convulsions that occurred from 24 to 36 hours after hospitalization. Phenytoin
did not control the convulsions, but phenobarbital controlled myoclonic activity.
Electroencephalograms (EEGs) were performed on 18 patients. EEGs performed at
the time of convulsions showed bilateral synchronous spike and wave complexes (2
to 3/second) on the frontal areas; there was also diffuse slow wave activity.
Eight days later, spike and wave complexes were absent, but the slow frequency
background rhythm persisted. EEGs usually normalized within 1 to 3 months.
Hollander and Col bach (1969) also reported five cases of convulsions in
which soldiers burning composition C-4 to heat food in the field were exposed
to KDX or its combustion products.
Systemic intoxication was not found at a World War II munitions plant that
employed a closed system of production. However, primary irritation and
sensitization dermatitis of the face and eyelids occurred in workers exposed to
production-related fumes. Patch testing with solid ROX failed to produce local
skin reactions; the irritation and dermatitis experienced were determined to be
related to an unidentified component in the fumes (Sunderman, 1944, as cited in
Ryon et al., 1984).
Hathaway and Buck (1977) conducted a study of 93 munitions plant workers
exposed to RDX and HMX; 69 workers were exposed to primarily ROX. The workers
were evaluated for abnormalities of the hematologic, hepatic, and renal systems,
V1-2
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and for the presence of autoimmune disease. Atmosphere samplmy of RDX was
conducted intermittently for several hours over a 6-week period; sampl-rg of
HMX was not perfonmed. Exposures ranged f'-om undetectable to 1.57 my RDX/in3,
with a mean detectable exposure of 0.28 my RDX/m3 . Duration of occupational
exposures was not "eported. Results of the fluorescent antinuclear ant'body
test, which had previously been used to demonstrate evidence of autoimmune
disease (systemic lupus erythematosus or SLE), were found to be higher m tne
nonexposed controls (3.5%) than in the RDX-exposed employees (2.2%). Results
of other blood chemistry and hematological tests were found to be similar
between controls and RDX-exposed workers. The authors concluded that medical
testing of RDX-exposed workers failed to reveal evidence of adverse health
effects for exposures up to 1.57 mg RDX/m3. Three cases of SLE previously
found at one ammunition plant were reported to be unrelated to RDX exposure.
Stone et al. (1969) presented case reports on four military personnel wno
ingested from 25 to 180 g of C-4 plastic explosive (91% RDX). All were admitted
to the hospital because of generalized convulsions. Between convulsions, the
state of consciousness of the patients varied from coma to lethargy. Symptoms
included muscular twitching, hyperactive reflexes, headaches, nausea, vomitiny,
hematuria, low-grade fever, and loss of memory. Although serum glutamic-
oxaloacetic transaminase was elevated, there were no abnormal findings in liver
function tests, and liver biopsies appeared nonnal . Kidney biopsies did not
reveal any permanent histologic change. Recovery of mental capacity was complete
after i to 2 months. No fatalities resulted.
Knepshield and Stone (1972) reported six cases of RDX intoxication in U.S.
soldiers in Vietnam who Ingested unreported concentrations of composition C-4.
All patients exhibited symptoms of CNS toxicity including convulsions (100%),
VI-3
-------
coma (67%), disorientation and confusion. Mental capacfes were reported to
normal'ze w'th'n one to two months. NeuromuSCul ar 1rr1tab11 1ty (muscular
tw tch1 ng) , gastrcntesfnal symptoms, hematuria and fever abated w't^n
forty-eight hours of ingesf'on. Bifrontal headaches, occurring 50% of the
pafents, persisted for one to three weeks. The ingestion of larger concentrar1 ons
of RDX were reported to produce extended periods of mental confusion and anem-'a.
Similarly, Merrill (1968) reported case studies on two military personnel who
¦jested unreported concentrations of composition C-4. Symptoms of CNS tox^cty
(hyper1 rri tabi 1 1 ty, muscle twitching, convulsions, mental confusion and amnes'a^,
renal damage (oliguria, hematuria, elevated BUN) and hepatic involvement normal1 zen
wth^n 10 days of ingestion.
Woody et al . (1985, 1986) reported a case of a 3-year-old, 14.7-kg ch->ld who
was hospitalized in status epilepticus. It was determined that the child had
ingested pellets of plasticized RDX explosive. The child's mother worked in a
munitions plant, and clumps of the explosive had adhered to her boots and
clothing. Blood, ur^ne, and stool samples were taken serially, and samplps of
cerebrospinal fluid (CSF) were analyzed. CAT scans of the head were normal.
An EEG revealed no evidence of epileptiform activity; however, it showed defuse
wave slowing, predominantly in the occipital regions. Hematology parameters
and urinary function were normal. Serum glutamic-oxaloacetic transaminase
increased from an initial value of 46 IU to 60 IU at 24 hours. Other serum
chemistry parameters were normal. The CSF showed slight increases in protein
and glucose compared with normal values. The level of RDX
-------
B. ANIMAL EXPERIMENTS
1. Short-term Exposure
a. Acute
The acute oral LD5Q values for RDX in mice and rats range from 59 to 97 mg/'
-------
qui c • i - * • r\^ u uc 'aiucb r\u>A »u LdUOPdCOfy MM I ind 1 S
Species
Strai n
Sex
Route Vehicle
ld5 0
(mg/kg)
Reference
Mouse
Mouse
Mouse
Rat
Rat
Rat
Rat
Swiss-Webster M
F
86C3Fi M
F
Combined
..b
Sprague-Dawl ey M
Sprague-Uawl ey M/F
Fischer 344
rt
Combined
Rat
Guinea pig
Oral
Oral
Intra-
venous
Oral
Sprague-Dawl ey M/Fc Oral
Oral
Oral
Oral
Corn oil
Methyl -
eel 1 ul ose
Tween-80
DMSO
Corn oil
DMSO or
saline
slurry
Coarse
powder
Methyl -
eel 1ulose
Gum acacia
Intra- DMSO
venous
<7b
86 a
(8-124)
97.2±8.7
(81.6-115.8)
58.9i26.8
(24.8-139.5)
80.319.6
(55.3-99.2)
18.71
(15.66-22.24)
71
(56-85)
71
(50-75)
100
300
119.0+4.6
(110.4-128.3)a
8.7i4.5
(108.0-128.9)
U8.U2.8
(111.8-124.1)
200*
25.1
(20.0-31.6)
Oi 11 ey st
al . (19 7a)
Choiaki s er
al. (19d0)
McNamara e*
al. (1974)
Oi 11 ey et
al. (1973)
Schneider «
al. ( 1977 )
Cholakis e1
al. (198J)
Von
Oettingen ¦
al. (1949)
McNamara er
al. (1974)
a95% confidence limits.
^Oata not reported.
cEstimated LDgQ for male or female.
'Approximate minimum lethal dose.
VI-6
-------
system s1 §ns that incluoed gasp'ng/labored breathing, Straub ta^-l^ke synnrones,
and clon1c/ton1c convulsions prior to death. In rats, all the animals rpcp'vng
the 150-, ltfi)-, and 250-mg/kg doses, anrl 95% of those rece'vng the 20n-mg/'
-------
hepatocyte alterations and proli feration of the SER, indicate a possible -nducfo
of the mixed function oxidase (MFU) system. However, no data on MFO levels
we^e presented.
In a study with doys (breed and sex not reported), oral administration by
stomach tube of 5 or 15 mg of RDX/kg caused little, if any, changes m the
animals' blood pressure, nespiratory rate, minute volume, and spinal fluid
pressup-e (Von Oettingen et al., 1949).
McNamara et al . ( 1974) investigated the toxicity of RDX afte" mfavenous
(iv) dosing in mice, guinea pigs (st-ain and sex not specified), and beagle
dogs. LD50 values are presented in Table VI-1.
Beagles were injected intravenously with solutions containing 33% RDX in
DMSO, 7.5% in cyclohexanone, and 5.4% in acetone (w/v) in single doses of 0.125
mL/kg. Two dogs dosed at 40 mg RDX/kg in DMSO exhibited subconvulsive jerking,
twitching, and convulsions 15 to 30 minutes after dosing; both dogs died withm
90 minutes. The convulsions were cyclic, and dogs exhibited inadequate respi"ato
movements, deceased blood pressure (BP), and a flat line on the EEG. When
administered a dose of 20 mg/kg, one dog exhibited CNS hyperactivity within
15 seconds after dosing and hyperreflexia for 1 hour; the other dog convulsed
within 90 seconds and did not recover until 16 hours later. Dogs given 4.7 or
9.4 my RDX/ky 1n cyclohexanone showed marked decrease in BP, cardiac arrest,
respiratory inhibition, and low-frequency EEG discharge. Dogs were semicomatose
to comatose, eyes were dilated, and the pain threshhold was elevated. Dogs
given 3.37 or 6.78 my RDX/kg in acetone showed decreased BP and erratic EEG
disturbances (McNamara et al., 1974). The Lowest Observed Adverse Effect Level
(LOAEL) for this study was 3.37 mg/kg; a No Observed Adverse Effect Level
(NOAEL) was not established.
VI-8
-------
Qi 11 ey et al. (1982) investigated tne snort-term oral toxicity of a syntnetic
LAP (load, assemble, pack) munition plant wastewater mixture of u-TNT and X
at a ratio of 1:0.62. A pnotolyzed mixture containing 10% rtOX and 0.32% a-T^7
by weignt, was designated LAP(l). The dose levels ranged from 234 to 119U
mg/kg and 178 to 1780 mg/kg for Sprague-Dawl ey rats and Swiss-WeDSter mice
administered LAP, respectively, and 2b0 to 1UUU mg/kg for Swiss-Webster mice
administered LAP(l); these LAP mixtures were dispersed uniformly in corn oil.
Tne single-dose oral LD^gs for LAP were 947 (95% C.I. 707-1090) and 1130 (9b?* 2.1.
946-1340) mg/kg in male and female mice, respectively, and 574 ( 952, C.I. 482-bod)
and 594 (9b% C.I. 502-678) mg/kg in male and female rats, respectively. All
deaths occurred within 24 hours of dosing; many animals exhibited aggressive
behavior, depressed activity and discolored urine and many of those dosed at _>
790 mg/kg exhibited convulsions prior to death. Longer dispersion periods
during preparation of the mixture lowered the LD^s presumably because of increased
dissolution of RDX, considered to be the more toxic component. The LO^ys for
LAP(l) were 585 (9b% C.I. 472-680) and 684 (95% C.I. 568-841) mg/kg for male
and female mice, respectively. Deaths occurred between 0.5 and 1 hour of
dosing; animals exhibited convulsions, squealing, depressed activity, and
discolored urine.
Sprague-Dawley rats, Swiss-Webster mice and beagle dogs were treated in a
repeated dose exposure study. Groups of 5 male and 5 female beagle dogs were
dosed with gelatin capsules containing 0, 0.5, 5.0 or 50 mg LAP/kg/day. Several
high-dose males exhibited convulsions following the second dose; one death
resulted. Depressed body weight, depressed food intake, anemia and alterations
in spleen (hemosiderosis), liver (hepatomegaly), and testes (atrophy) were
exhibited at the high dose. The L0EL was 5.0 mg/kg/day based on signs of
anemia; the NOEL was 0.5 mg/kg/day. Groups of 20 male and 20 female rats were
V1-9
-------
treated tor 13 weeks with 0, 0.005, 0.05 or 0.5% LAP in the diet; mice were
treated in the same manner with an additional group dosed at 0.2b% LAP. The
compound-related effects exhioited in rats and mice were similar to those found
in dogs with the addition of uterine hypoplasia exmbited in female rats.
Discolored urine, depressed activity and several biochemical changes were
exhibited in all species. The NOEL for rats was 3.6 mg LAP/kg/day and that of
mice was 8.3 mg LAP/kg/day, the lowest doses tested. A 4-week study in which
irradiated LAP(l) was fed to rats at dosages of 0.003, 0.03 or 0.3% in the diet
indicated that the irradiated mixture was less toxic than the unirradiated
mi xture.
b. Primary irritation and sensitization
In an acute dermal toxicity study, groups of five male and five female New
Zealand albino rabbits received a single dermal application of 2 g ROX/kg in 1%
carboxymethylcel lulose solution; five additional males were retested. Animals
were observed for 14 days; the toxicological endpomts included body weight
measurements (on days 1, 3, 8, and 15) and observations of clinical signs and
mortality. Except for a slight, transient loss in body weight, no other
signs of toxicity were observed. Two males died on days 3 and 9; however, the
cause of death (Tyzzer's disease) was unrelated to treatment. In the group of
five retested males, there were no mortalities (Furedi-Machacek et al., 1984).
McNamara et al. (1974) studied the acute dermal toxicity of RDX (suspended
in three solvents) in rabbits, guinea pigs (strain and sex not specified), and
beagles. RDX was dissolved in UMS0, cyclohexanone, and acetone at concentrations
of 33, 7.5, and 5.4% (w/v), respectively.
In a single-dose study, 1 mL of each mixture was applied to the shaved
backs of six rabbits; controls received 1 mL of the solvent alone. All animals
VI-10
-------
were oDserved for skin irritation and systemic toxicity for 30 days. Blood
samples drawn from each raDbit (time not specified) were analyzed for the
following parameters: red blood cell count, wmte blood cell count, nematocn:,
nemoglobin, alkaline phosphatase, serum glutamic-oxaloacetic transaminase iSGOT),
blood urea nitrogen, creatinine, sodium, potassium, chloride, and carbon dioxide.
Two treated rabbits and one control raDbit were sacrificed at 1 hour, 3 days,
and 3U days after dosing for patnological evaluation. Topical applications of
ROX in the three solvents did not cause skin irritation, deaths, systemic
toxicity, or changes in any of tne hematologic and blood chemistry parameters
in treated raDbits. However, dermatitis was observed histologically in raboits
receiving RDX irrespective of the solvent. Dermatitis persisted for up to 30
days in rabbits treated with 33% RDX in 0MS0.
In a repeated dose study, RDX (in the three solvents, as described aDove)
was applied as a 0.1- or 1-mL dose to six rabbits/mixture/volume, 5 days/week
for 4 weeks; controls received solvent alone. All animals were observed daily
for 30 days, and blood samples were analyzed as described in the single-dose
study. Pathological examination was conducted in one control and two treated
rabbits sacrificed on days 7, 14, and 28. No skin irritation or changes in
blood parameters were observed. One rabbit died after the fifth 0.1-mL dose,
and one died after the tenth application of a 1.0-mL dose of t>.4% RDX in acetone.
Another rabbit died after the eighth 1.0-mL application of 7.b% RDX in
cyclohexanone. Histopathology revealed skin lesions at the site of RDX application
that were characterized by a thickening or reddening and inflammation. Minimal
dermatitis was observed in animals receiving the RDX mixtures as well as in
some of the solvent controls. Rabbits treated with either 1, 10, or 20 1-mL
doses of RDX in DMSO had dermatitis at necropsy, while the DMSO controls
receiving the same doses did not develop dermatitis. Two animals that received
VI-11
-------
one 1.0-mL dose of RDX in acetone and two that received 20 1-mL doses of RUX in
cyclohexanone developed dermatitis, and the solvent controls did not (McNamara
et al ., 1974).
Groups of four guinea pigs received a single topical application of a 3%
(w/v) KDX/DMSO solution at doses of 316, 510, 1,000, or 2,000 mg/kg, or three
applications at a dose of 1,000 mg/kg. Single doses of 316 or 510 mg/kg had no
effect, while slight erytnema was observed at the 1,000- or 2,000-mg/kg dose.
When applied three times at 1,000 mg/kg, slight erythema was observed after
the first application; later applications showed no further erythema. Applications
of 2 mL OMSO alone produced no effects (McNamara et al., 1974).
Beagle dogs (sex not specified) received a single topical application of
1 mL of 33% RDX in DMSO (289.0 mg/kg), 7.5% KOX in cyclohexanone (65.7 mg/kg),
or 5.4% ROX in acetone (47.3 my/kg); each mixture was applied to two dogs. Two
additional dogs received 480 mg/kg of 33% ROX in DMSO daily for 3 consecutive
days. Treatment produced no consistent increase or decrease in any of the
physiological parameters. One of the dogs at the 480-mg/kg dose was slightly
more irritable and hyperactive for 20 to 30 minutes after the first and second
applications; dogs appeared normal during the 2-week observation period (McNamara
et al., 1974).
Groups of two to four beagles (sex not specified) received daily topical
applications (on the dorsal area) of 1 mL of ROX in the three solvents (as
described earlier), 5 days/week, for 4 weeks; control dogs received 1 mL of the
solvent alone on the same schedule. Slight erythema and desquamation of the
back was observed in some dogs (number not specified) during the second or
third week of application of either OMSO alone or 289.0 mg/kg ROX in DMSO; no
V1-12
-------
effects were seen at a dose of 65.7 my/kg ROX in cyclohexanone or 47.3
RDX in acetone (McNamara et al., 1974).
McNamara et al. (1974) tested ROX for evidence of sensitization in male
and female guinea pigs using topical or intradermal injections of ROX in tne
three solvents. During the sensitization phase, topical (0.5 mL) or intradermal
(0.1)5 mL) doses were applied to the clipped dorsal thorax, 3 days/wee* for 3
weeks. Following a 2-week rest period, animals were challenged with a single
topical or intradermal dose of RDX prepared in 1:1 (v/v) solvent-saline mixture
(intradermal) and polyethylene glycol (topical). Although one route per animal
was used for sensitization, both routes were used for challenge, one on eacn
thigh at different times. Topical or intradermal injections of RDX in the
three solvents produced no evidence of sensitization.
2. Longer-term Exposure
a. Ten- to 13-week studies
Schneider et al. (1978) dosed 30 Sprague-Oawley rats (sex not specified)
orally with RDX in an isotonic saline slurry at 20 mg/kg/day for 90 days. A
control group of 10 rats received isotonic saline on the same schedule. RDX
did not induce convulsions or overt neurologic signs. Rats became lethargic,
lost weight, developed rough hair coat, and had a blood-tinged exudate around
the external nares immediately prior to death. During the study, eight dosed
rats died (between days 42 and 77), apparently from exacerbation of chronic
respiratory disease.
Cholakis et al. (1980) conducted 90-day subchronic dietary toxicity studies
in male and female B6C3Fi mice and Fischer 344 rats. RDX mixed in rodent
laboratory chow was fed to the animals for 13 weeks; the RDX concentrations
V1—13
-------
were adjusted weekly based on the animals' body weight and food consumption.
The RDX used in the study was contaminated with 9% HMX.
In the study with mice, groups of 10 male and 10 female mice, 5 weeks of
age, received RDX doses of 10, 14, 20, 28, or 40 mg/kg/day for 13 weeks. A
control group of 10 males and 10 females was given a normal diet. No meaningful
changes were observed in clinical signs, mean body weight and food consumption,
nematoloyy, and clinical chemistry values or terminal organ weivjnts in treated
mice. Histopathology was not performed.
In a followup study (Cholakis et al., 1980), groups of 10 male and \i
female mice were fed 40, 60, or 8U mg/kg/day RDX in the diet for 2 weeks,
followed by 80, 160, or 320 mg/kg/day RDX, respectively, for 11 weeks. An
equal nunber of mice served as controls and received a normal diet. Animals
were observed daily, and body weiyht and food consumption were measured weekly.
Hematology and clinical cnemistry tests were conducted at termination. Tissues
from the control and high-dose groups were examined histopatnologically.
Mortality occurred in 4 of 10 males (40%) and 2 of 12 females (16.7%) at
the highest dose (320 mg/kg/dty). One female died during week 6 of the study;
the other mortalities occurred during week 11. No toxic signs were noted prior
to death. Hyperactivity and/or nervousness were seen in 50% of the males at
the highest dose during weeks 7 and 8; these signs were not observed in the
females. Hales fed 320 mg/kg/dty RDX exhibited a significant (p <0.05)
increase in mean body weight gain during weeks 10, 12, and 13; females at this
dose showed a 5.8% increase during week 13 when compared to controls. No
changes were observed in mean food consumption during the 13-week period.
Statistically significant (p <0.05) changes were observed in some of the
hematology parameters. Males fed 160 mg/kg/day RDX showed a 12% decrease in
VI-14
-------
erythrocyte count, a 7* decrease in hemoglobin concentration, and an 3% increase
in mean corpuscular volume (MCV); however, there were no effects on these
parameters at the higher dose (320 ppm). The mean corpuscular hemoglobin
(MCHB) was increased 6 and 5i in females fed ROX at 80 or 160 my/kg/day,
respectively. This is not considered of toxicologic importance. In addition,
intermittent cnanges in platelets and neutrophils (increased) and in serum
glutamic-pyruvic transaminase (SGPT) (decreased) values were found in both sexes
of treated mice. The effects of SGPT are not considered to be of toxicologic
importance because the values were within the normal range and only increased
values indicate an adverse effect.
Dose-related increases in mean absolute liver weight and mean liver weight
relative to body weight and brain weight were seen in both sexes of mice; the
increases were significant (p <0.05) at 320 mg/kg/day. Histopathology revealed
minimal focal myocardial degeneration (five males and two females), mild tubular
nepnrosis (four males and one female), and periportal hepatocellular vacuol ization
(five males) in the high-dose mice. Other tissue changes observed in treated
mice were mild fatty infiltration and mild focal subscapular fibroplasia of the
adrenals, mild microgranuloma, increased (minimal) karyomeyaly of hepatocytes
in the liver, and a dilated lumen of the uterus. The L0AEL for tne study based
on anemia in males was 160 my/kg/dty, and the NOAEL was 80 mg/kg/day.
In the 13-week study with Fischer 344 rats (Cholakis et al., 1980), 60
male and 60 female rats were divided into six groups, each consisting of 10
males and 10 females. Five groups were fed diets that provided an ROX intake of
10, 14, 20, 28, or 40 mg/kg/day. The sixtn group, serving as controls, was
given a normal diet. Rats were observed daily, and body weight and food
consumption were measured weekly. Standard hematology tests were conducted at
V1—15
-------
30 days, 60 days, and at termination, and serum chemistry tests were conducted
at termination. Tissues from controls and the two highest doses (28 and 40
mg/kg/day) were evaluated histopathological ly.
No mortality occurred in the treated rats. The mean body weignt of males
fed RDX at 4J mg/kg/day was significantly (p <0.05) lower than that of controls
during weeks 2 to 13; at termination, the weight gam was 8.4% lower when
compared to controls. Females exhibited dose-related decreases in body weight,
the weight gain at termination was 5.5% lower in high-dose females than in
controls. Mean food consumption was significantly (p <0.05) reduced in males
fed the high dose during weeks 1, 7, 10, 11, and 13; a slight, but nonsignificant,
reduction in food consumption was observed in the females at this level.
In males fed 40 mg ROX/kg/day, significant (p <0.05) decreases in
hematocrit and hemoglobin (at 30 and 60 days) and increases in reticulocytes
(at termination) were observed. In addition, a dose-related increase in platelet
counts was observed at termination. After 3U days, females fed RDX at 28
mg/kg/day showed significant (p <0.05) increases in reticulocytes and decreases
in liCV and MCHB. The high-dose (40 my/kg/day) females showed a significant
(p <0.0b) increase in leukocytes. After 2 months, the reticulocytes in females
fed 28 mg/kg/day returned to normal level, but the reticulocyte count decreased
(p <0.05) in females fed 40 mg RDX/kg/day. No significant changes were observed
in any of the parameters for females at termination. Significant (p <0.05)
decreases were observed in glucose, StiPT, and serum potassium (males), and in
SGOT and serum sodium (females) at termination; however, these values were
reported to be within the normal historical ranges.
There was an apparent dose-related reduction in absolute and relative (to
body weight and Drain weight) terminal heart weight in both sexes fed 4U
VI-16
-------
mg/kg/day ROX. The organ weight changes correlated with an increase in the
incidence of myocardial degeneration (minimal) in females, 6/10 at the high
dose compared witn 2/10 in controls. In addition, there was an increased
incidence (minimum degree) of liver portal inflammation in females receiving 40
mg/kg/day (7/10) as compared to controls (1/10). Based on anemia, the LOAEL
was 28 mg/kg/day, and the NOAEL was 20 mg/kg/day.
Levine et al. (1981) fed ROX in the diet to groups of 10 male and 10
female Fischer 344 rats for 13 weeks at doses of 10, 30, 100, 300, or 600
mg/kg/day; a control group of 30 males and 30 females was given a normal diet.
At termination, hematology and clinical chemistry tests were conducted, and all
major oryans (not specified) were weighed; 25 tissues from controls, from 30-
and 100-mg/kg/day-dosed animals, and all major organs from the remaining groups
were evaluated histologically. Male and female rats fed ROX at 300 or 600
mg/kg/day died within 1.5 weeks of dosing. The mean survival times were inversely
correlated with the dose level, and there was no apparent sex difference. A
dose-related and significant (p £0.05) reduction in body weight gain was observed
in males but not in females. Food consumption was significantly (p £0.05)
reduced in both sexes fed 100 mg/kg/day or above during study week 1, and in
males (100 mg/kg/day) during study weeks 5 and 9. Dose-related and significant
(p £0.05) decreases in serum triglyceride levels were seen in both sexes; the
levels were significantly (p <0.05) lower than those of controls in the groups
receiving 30 and 100 mg/kg/day. A slight but significant (p £0.05) increase in
leukocyte count was observed in females at all doses; however, this is of
doubtful toxicologic importance because there were no effects on differential
white blood cell ratios. Males showed slight increases in leukocyte counts,
but the effect was not significant. No other hematology or serum chemistry
changes were observed. The absolute and relative liver weights were significantly
V I-17
-------
(p <0.0b) increased in females fed 100 mg/kg/day; however, histopathology
indicated no treatment-related changes in the liver. Based on effects on liver
weights, the LOAEL was 100 mg/kg/day and the NOAEL 30 mg/kg/day.
Von Oettmgen et al. (1949) conducted longer term toxicity studies with KdX
in rats (strain and sex not specified) and dogs (breed not specified), broups
of lb rats received ROX in the feed at doses of lb, 50, or 100 mg/kg/day for 10
weeks. KOX concentrations were based on the weekly body weight and food
consumption of the rats. No deaths occurred at the 15-mg/kg/day dose; however,
the mortality rate was 60 and 86.6% in the bO- and- 100-mg/kg/day dose groups,
respectively. Most of the rats died during the first month. Necropsy revealed
congestion of the lungs and gastrointestinal tract. Mean body weight of rats
fed ROX at bO or 100 mg/kg/day was lower than that of controls, while rats at tne
lb-mg/kg/day level showed normal weight gain. Hyperirntabi 1 ity and clonic/tonic
convulsions were seen in rats fed bO or 100 mg/kg/day ROX. No abnormal behavioral
activity was seen at the lower dose. Based on CNS effects, the LOAEL was 50
mg/kg/day and the NOAEL was 15 mg/kg/day.
In a followup study, groups of 20 rats were administered ROX in the diet
at doses to provide an intake of 15, 2b, or bO my RDX/kg/day for 12 weeks; a
control group of 20 rats received a normal diet. Mortality occurred in 1, 8,
and 8 rats fed 15, 25, or 50 my/ky/daty, respectively. Necropsy revealed
conyestion of the lungs and gastrointestinal tract in most animals that died.
The death in the low-dose group was not compound related. Body weight loss,
hyperirritability, and clonic/tonic convulsions were observed in rats fed the
two highest doses; these effects were not seen in rats fed 15 my RDX/kg/day.
Erythrocytes and hemoglobin content were reduced in all rats, including controls,
during study weeks 3 and 4. However, normal values were observed in all rats
by study week 8, except in rats fed 25 my ROX/kg/day; recovery in these rats
VI-18
-------
was not complete until termination. No treatment-related cnanges were observed
in rats sacrificed at termination (Von Oettingen et al., 1949). The LUAEL
based on mortality and body weight loss was 2b mg/kg/day, and the NUAEL was lo
my/kg/day.
In the study with dogs, seven healthy females were force-fed RDX (molded
in a moistened pellet) at 50 my/kg/day, 6 days/week for 6 weeks. One treated
dog that died at the end of week 5 had many congested areas on the walls of the
small intestines. Treatment caused hyperi rri tabi 1 ity, convulsions, and weight
loss. The gross and microscopic changes in the organs and tissues were negligiole
(Von Oettingen et al., 1949).
Hart (1974) observed no signs of toxicity, except for temporary episodes
of vomiting in beagles following dietary administration of RUX for 90 days.
The vomiting only occurred sporadically and during the first 2 weeks. Groups
of three male and three female dogs were fed RDX at 0.1, 1, or 10 mg/kg/day
for 90 days; an equal number of control animals received a normal diet. All
animals were observed daily, and body weights were recorded prior to initiation
and at weekly intervals. Ophthalmoscopic examinations were conducted prior to
initiation and at termination. Hematology, clinical chemistry, and urinalysis
tests were performed on all dogs twice prior to initiation and during study
weeks 4, 8, and 13. At necropsy, the heart, kidneys, liver, thyroids, adrenals,
spleen, and testes (with epididymis) were weighed. Histopathological examinations
of 12 selected tissues from the control and high-dose animals were conducted.
No signs of toxicity, except for temporary episodes of emesis, were seen.
Laboratory tests, ophthalmoscopic examination, and organ weights, as well as
gross and microscopic examination, revealed no important differences from
controls.
V1-19
-------
Martin and Hart (1974) conducted a 90-day subchronic oral toxicity study
in cynomolgus monkeys. The animals ranged in age from 36 to 54 months; tne
males weighed 2.6 to 4.6 kg, and females weighed 2 to 4.2 kg. Groups of tnree
male and three female monkeys were administered oral gavage doses of rtDX in a
1% aqueous suspension of methyIcellulose at U.l, 1, or 10 mg/kg/day for 90 days.
An equal number of monkeys, serving as controls, received a 1% aqueous suspension
of methylcellulose alone on the same schedule. Animals were observed daily,
and body weights were recorded prior to initiation and at weekly intervals.
Ophthalmoscopic examinations were conducted prior to initiation and at termination.
Standard hematology, clinical chemistry, and urinalysis tests were conducted
before study initiation, at study weeks s and 9, and at termination. The
bromosulphalein (BSP) liver function test was conducted twice before initiation
and during study weeks 4, and 13. Additionally, plasma levels of RDX were
determined for each monkey at study weeks 5 and 9, and at termination. At
necropsy, the thyroid, heart, liver, kidneys, and adrenals were weighed.
Selected tissues and lesions from the control group and from the animals
administered 10/mg/kg/day, and liver, kidneys, spleen, and gross lesions from
the animals administered U.l and 1 mg/kg/day, were examined histopathological ly.
Vomiting and central nervous system disturbances were the major toxic signs
observed in dosed monkeys. Vomiting occurred in one low-dose, three mid-dose,
and five high-dose animals. Five of the six monkeys receiving 10 mg/kg/day
showed 12 instances of CNS disturbances, usually involving tonic-type convulsions.
One male animal at the high-dose level was sacrificed during study week b due
to severe CNS disturbances. Eye examination, hematology and clinical chemistry
parameters, urinalysis, BSP test, and organ weights showed no toxicologically
significant changes. Body weights of treated and control monkeys were comparable.
Histologic examination revealed necrotic and degenerative megakarocytes in bone
marrow sections, and increased amounts of iron-positive material in liver cord
VI-20
-------
cytoplasm in monkeys given 10 my RDX/kg/day. However, the authors were uncertain
reyarding the toxicological significance of these findings. Based on effects on
the CNS, tne LOAEL was 10 my/kg/day and the NOAEL was 1 my/ky/day.
b. Lifetime studies
Levine et al. (1983) evaluated tne chronic effects of RDX in yroups of db
male and female Fisher 344 rats fed doses of 0.3, l.b, 8.0, or 40.0 my/kg/day
for 24 months. RDX purity ranged from 89.2 to 98.7%, and particle size ranged
from <22 nm (51.7%) to 440 um. Mortality was increased in high-dose males and
females throughout the study (e.y., 88 and 41%, respectively, at week 88 as
compared to 32 and 33% for respective controls) (Table V1-2). Tremors and
convulsions were frequently observed prior to deaths of high-dose males and
females beginning at week 25. Behavioral hypersensitivity to stimuli resulted
in fighting among cohabited high-dose males. Histologic evaluation failed to
detect lesions of the central nervous system. The incidence of cataracts was
significantly increased (p <0.05) in high-dose females at weeks 78 and 104; the
eyes of high-dose males appeared normal. These observations were considered to
be treatment related.
Significant dose-related reductions (p <0.05) in body weight gain were
seen throughout the study for males receiving ROX at 8 mg/kg (5 to 6%) and 40
my/kg (20 to 30%) when compared to controls. Females were less affected than
males. Sporadic reductions in body weight gain were found in females receiviny
8 mg/kg (5 to 6%) and 40 mg/kg (10 to 15%). Food consumption was slightly but
significantly reduced for high-dose males at various intervals throughout the
study; sporadic increases and decreases were reported for other dosed males and
femal es.
V1-21
-------
Table VI - 2. Representat1ve Results of Mortality and Percent Survval
'n Rats Fed RDX -"n the Diet for 104 Weeks
Dose group Mortality (percent survival) at week
(mg/kg/day) 26 52 66 88 104
Mai es
0
3
(96)
16
(79)
21 (72)
24
(68)
39
(48)
0.3
3
(96)
16
(79)
21 (72)
27
(64)
39
(48)
1.5
4
(95)
19
(75)
24 (68)
31
(59)
48
(36)
8.0
3
(96)
17
(77)
2(1 (73)
24
(68)
46
(39)
40.U
7
(91)
39
(48)
52 (31)
66
(12)
72
(4)
Females
0
3
(96)
18
(76)
22 (71)
25
(67)
36
(52)
0.3
3
(96)
17
(77)
20 (73)
23
(69)
33
(56)
1.5
3
(96)
16
(79)
22 (71)
25
(67)
35
(53)
8.0
4
(95)
17
(77)
21 (72)
23
(69)
36
(52)
40.0
3
(96)
22
(71)
28 (63)
31
(59)
49
(35)
a
Percent survival was based on 75 rats/sex/group.
SOURCE: Adapted from Levine et al. (1983).
VI—22
-------
Hemoglobin and nematocrit values, and erythrocyte counts, were decreased
in high-dose males and females tnrougnout tne study; males were affected to a
greater extent than females. The study autnors reported tne anemia to be mild,
since compensatory mechanisms (e.g., reticulocytosis) were not ooserved.
Reticulocyte counts were not reported. Bone marrow was reported as appearing
to be within normal limits. Spleens were reported to appear enlarged, with
secondary splenic lesions in hi gn-dose mal es and females (e.g., extramedu 11 ary
hematopoiesis and sinusoidal congestion); however, spleen weights were not
significantly increased. Individual pathology data were not available for
revi ew.
Hepatotoxicity, primarily at 40 mg/kg/day, was evidenced by hepatomegaly
(although histological changes were not reported to be apparent), hypocnol esteremi a,
hypotnglyceridemi a, reduced serum albumin/total protein levels, and increased
lactic dehydrogenase (LDH) levels. More specifically, cholesterol and triglyceride
levels were significantly decreased in high-dose males and females throughout
the study. Total protein and albumin were decreased in females receiving 6 and
40 mg/kg at weeks s2, 78, and 104; total protein was decreased in nign-dose
males at s2 weeks. LDH levels were increased in high-dose males at weeks 13 and
26 and decreased in mid- and high-dose females at week 26; LDH levels were not
reported for weeks 52, 78, and 104. Absolute liver weights were increased for
high-dose females, and relative liver weights were increased for high-dose
males throughout the study.
Compound-induced renal toxicity was found primarily in high-dose males.
Absolute kidney weights were significantly increased in high-dose females, and
relative kidney weights were significantly increased in high-dose males throughout
the study. Absolute and relative kidney weights were sporadically increased in
males and females receiving 8 my/ky. Blood urea nitrogen (BUN) levels were
V1-23
-------
significantly increased in high-dose females at week 104. Histologic examination
of the kidneys showed renal medullary papillary necrosis in 15/19 males that
received 40 my/kg for more than 6 months and died by 12 months, but not in the
same group sacrificed at 12 months or in any controls.
In hign-dose males that died after 12 months or were sacrificed at
termination, there was a significant (p <0.05) increase in kidney lesions as
medullary papillary necrosis, pyelitis, and uremic mineralization. Distension
and cystitis of the bladder were also observed in high-dose males (Table VI - 3).
No corresponding changes were found in the urogenital system of females. Males
receiving 1.5, 8.0, and 40.0 mg/kg/day exhibited increased pigment in the
spleen (possibly a hematopoietic response and not adverse) and suppurative
inflammation of the prostate (Table VI - 3). The only significant (p <0.05)
histologic change in females was an increase in lenticular cataracts (32/48 in
the high-dose group compared with 15/53 in controls).
Additional toxic effects found in high-dose males and females included
hypoglycemia and thrombocytosis. Glucose levels were significantly decreased
at this dose level throughout the study. White blood cell counts and platelet
counts were increased in high-dose males and females throughout the study.
Adrenals were found to be enlarged in males receiving 40 mg/kg/day, although
histologic changes were not reported; adrenal weights were increased in high-
dose males at 27 weeks and in high-dose females at 52 and 104 weeks. Absolute
brain weights of high-dose females and relative brain weights of high-dose
males were significantly increased tnroughout the study.
In summary, when KDX was fed at levels of 0.3, 1.5, 8.0, or 40.0 mg/kg/day
to Fischer 344 rats for 24 months, major toxic effects included increased
mortality, weight loss, hepatotoxicity, hypoglycemia, anemia with secondary
splenic lesions, renal toxicity, urogenital lesions, testicular degeneration,
V1—24
-------
Table VI - 3. Histologic Lesions i-n Male F-344 Rats Fed RDX for 2 Yearsa
Dose level fmq/kq/day)
Oryan/lesion 0 0.3 1.5 3.0 40
Kidney
(55)b
(54)
(52)
(55)
(31)
Medullary pap^lary necrosis
0
1
0
U
13*
Pyel1 cs
0
1
0
1
5*
Uremic mi neral1 zaf on
1
1
2
0
13*
Bladder
(54)
(55)
(52)
(51)
(3i r
Distension
0
2
1
3
25*
Cysti tis
0
2
1
1
13*
Prostate
(54)
(55)
(52)
(55)
(31)
SuDpurative inflammation
2
4
9*
12*
19*
Spleen
(55)
(52)
(52)
(55)
(31)
Increased pigment
3
1
11*
15*
13*
alncludes rats that died or were sacrificed moribund after 12 months and those
sacrificed at termination.
bThe numbers in parentheses are the numbers of animals for wh-'ch a specie
tissue was examined microscopically.
~Significantly different from control incidence, p £0.05.
SOURCE: Levine et al. (1983).
VI -25
-------
CNS effects, and cataracts. Based on suppurative inflammation of the prostate
of males receiving 1.5 mg/kg/day and above, the LOAEL was 1.5 mg/kg/day and
the NOAEL was 0.3 mg/kg/day.
Hart ( 1977 ) fed ROX to male and female Sprague-Dawl ey rats at doses of
l.U, 3.1, or lu mg/kg/day for 24 months. Survival was comparable to controls
in high-dose males and females. Body weights in females receiving 3.1 and
10 mg/kg/day were significantly decreased (p <0.0b) from week b6 to week dd,
nonsignificant decreases in body weights were seen in males receiving 10 mg/kg/day
from week 60 to termination. The study authors, however, considered these
findings to be toxicological ly insignificant. Histopathologic evaluations
were comparable between control and dosed animals. The LOAEL was determined
to be 3.1 mg/kg/day and the NOAEL to be 1 mg/kg/day based on decreased body weijnts
in females.
Lish et al. (1984) evaluated the chronic effects of ROX in groups of 35
male and female B6C3Fi mice fed doses of 1.5, 7.0, 35.0, or 175 mg/kg/day for
24 months. RDX purity ranged from 89.2 to 98.7%; particle size ranged from
<22 um (51.7%) to 440 um. The high dose was reduced from 17b mg/kg/day to
100 my/kg/day dunny week 11 due to high mortality; mortality rates were 35 and
42% for high-dose males and females, respectively, at week 12 (Table V1-4).
Survival was similar between dosed and control groups from week 12 to study
termination. A nigh incidence of skin lesions (associated with fighting) was
found in high-dose males during the first year of the study; study authors
attributed this finding to treatment-related behavioral changes. During the
second year, all dosed male and control groups exhibited this behavior; histology
revealed chronic dermatitis and skin ulcers in all dosed males. This was
reported to be a secondary treatment-related effect. Convulsions were exhibited
VI-26
-------
Table VI-4. Representative Results of Mortality and Percent S'irvval
m M^ce Fed RDX 4n the Diet for 104 Weeks
Dose kjroup
(my/kg/day)
Mortal
¦•ty (percent survival)
a
at week
6
12
35
89
104
Mai es
0
0
(100)
1
(99)
11 (87)
32
(62)
39 (54)
l.b
0
(100)
0
(100)
13 (85)
34
(60)
43 (49)
7.0
0
(100)
0
(100)
13 (85)
31
(64)
44 (48)
J5.0
0
(100)
0
(100)
13 (85)
33
(61)
48 (44)
100.0
18
(79)
30
(65)
46 (46)
60
(29)
63 (26)
Females
0
0
(100)
0
(100)
10 (88)
29
(66)
36 (58)
1.5
0
(100)
0
(100)
11 (87)
25
(71)
40 (53)
7.0
0
(100)
0
(100)
11 (87)
24
(73)
33 (61)
35.0
0
(100)
0
(100)
10 (88)
29
(66)
39 (54)
100.0
25
(71)
36
(58)
44 (48)
56
(34)
60 (29)
a
Percent survival was based on 85 mice/sex/group.
SOURCE: Lish et al. (1984).
VI —27
-------
during month 24 in one male receiving 35 mg/kg and in one female receiving 100
mg/kg.
The body weight gains of nigh-dose females were significantly decreased
(p <0.05) from week 10 to study termination; the oody weight gains of high-dose
males were significantly decreased (p
-------
B1ood-urea-mtrogen (BUN) levels were slightly but significantly increased
(p
-------
males and 21 females, was fed RDX in the diet at nominal doses of 0, b, 16, or
50 mg/kg/day for 13 weeks. After treatment, the animals were mated and the
dams were allowed to litter. After weaning, groups of 26 males and 26 females
(FjJ were fed the same dietary concentrations of RDX as their parents for 13
weeks and were allowed to mate following completion of treatment. The dams
were allowed to litter, and after weaning, male and female rats (F2) were
necropsied for histopathological examination. Parental toxicity was demonstrated
at bU my/kg by increased Fy mortality (18 versus U% in the other yroups) and
significantly reduced body weights and food consumption. Transient reductions
in oody weight and food consumption were also noted at 5 and 16 mg/kg.
Reproductive and developmental effects noted at 50 mg/kg were reduced mating
and fertility rates and decreased litter size and pup survival. The numbers of
live F]_ litters were 17, 21, 19, and 10 in the 0-, 5-, 6-, and bO-mg/kg groups,
respectively; the respective mean litter sizes were 11.7, 13.8, 12.8, and 7.6.
Only one high-dose litter survived to produce a second generation. Significantly
reduced pup weights reflected developmental toxicity at both the 16- and bO-mg/kg
dose levels; mean F^ values at day 25 were S3, 47, 43, and 41 g in the 0-, 5-,
16-, and 50-mg/kg groups, respectively. The LOAEL for reproductive effects was
50 mg/kg/day, and the NOAEL was 16 mg/kg/day. The LOAEL for developmental
toxicity was 16 mg/kg/day, and the NOAEL was 5 mg/kg/day.
4. Developmental Toxicity
Cholakls et al. (1980) conducted developmental toxicity studies in Fischer
344 rats and New Zealand rabbits. RDX did not induce a teratogenic effect. RDX
was suggested to be teratogenic in the rabbits, but only at maternally toxic doses.
In the study with rats, groups of 24 or 25 rats received RDX, by gavage,
at 0, 0.2, 2, or 20 mg/kg/day on days 6 through 19 of gestation. Maternal
VI-30
-------
toxicity was demonstrated at 20 mg/kg/day by mortality, hyperacr1 vty, convulsions,
reduced body weight gaTis (adjusted for graved uterine we^yhts), and decreased
food consumpf on. Although one dam receivng 2.0 mg/kg/day had convulsions,
Cholakis et al. (1980) reported no compound-related effects at 2.0 or 0.2
mg/ky/day. Embryotox^cty was demonstrated at 20 mg/kg/day by increased resorption
rates. The means of posfmpl antat^on loss per letter were 6.8, 2.A, 5.1, and
18.6» at the 0-, U.2-, 2.0-, and 20-my/kg/day groups, respectively. Teratogen-,cry
was not demonstrated at any of the dose levels tested. Rased on emhryotoxicty
and maternal toxicity, the L0AEL was 20 mg/kg/day and the N0AEL was 2 mg/kg/day.
This study *s not suitable for calculation of a Ten-day Health Advsory due to
the severity of the effects (convulsions) at 20 mg/kg/day.
In the study with rabbits, dams (number not reported) receded R0X, by
gavage, at levels of 0, 0.2, 2, or 20 mg/kg/day on days 7 through 29 of qestat'on,
and 11 to 12 litters/group were delivered by cesarean section on day 30.
Maternal toxicity was evidenced at 20 mg/kg/day by nonsignificantly reduced
weight gains. Uterine implantation data and fetal weights were comparable for
all groups; however, a nonsignificantly increased incidence of fetal abnormalities
(e.g., spina bifida, meningocele, misshapen/enlarged eye bulges, gastrnschis^s,
limb reductional defects, tail abnormalities) suggested developmental toxicity
at 2U my/kg. Cleft palate was not found on gross examination of 94 to 110
fetuses in 11 to 12 litters at doses of 0.2, 2.0, or 2U mg/kg/day, but it was
found on soft tissue examination in 1/46 fetuses at 0.2 mg/kg/day and in 2/44
and 2/52 fetuses irv the 2.U- and 20.0-mg/kg/day groups, respectively. No cleft
palates were found in controls; the normal control incidence in rabbits is
about 0.1%. Spina bifida was found in 3/110 fetuses (two litters) from dams
dosed at 20.U mg/kg/day; this anomaly was not found in controls or at the two
lower doses. No maternal or developmental effects were noted at 0.2 or 2.0
VI —31
-------
mg/kg/day (Cholakis et al., 1980). Thus, based on maternal tox1c,ty and
developmental effects, the LOAEL was 20 mg/kg/day and the NOAEL was 2.0 ma/kg/nay.
Angerhofer et al. (1986) ""nvesfgated the developmental tox'-c effects of
RDX ¦'n rats. The authors conducted a range-f""nd'og study usi-ng groups of s'x
pregnant rats treated with RDX, by gavage, at dose levels of 0, 10, 20, 40, 80,
or l^u my/ky/day on gestational days 6 through 15. Treatments w'th 40, 30, and
120 mg/kg/day were lethal in all females. Animals in the 20-mg/kg/day gro'jp
exhibited urogenital and nasal discharge throughout the dosing period; no s^gns
of compound-related toxicity were noted at 10 mg/kg/day. Fetuses from the
yroups dosed with RDX had significantly reduced body weights when compared wth
controls. The teratogenic1ty study consisted of 39, 40, 40, and 51 mated
females dosed with RDX on gestational days 6 through 15 at 0, 2, 6, and 20
mg/kg/day, respectively. A total of 0, 1, 1, and 16 animals, respect1vely,
died in the above groups. No clinical s^ns were noted for the animals dosed
at 2 and 6 mg/kg/day; however, antemortem signs for animals in the 20-mg/kg/day
group included urogenital discharge, nasal and oral exudate, convulsions, and
prostration. Necropsy findings did not provide evidence of specie causes for
the deaths of these animals.
Among the surviving animals, maternal body weights for the high-dose group
were significantly (p £0.05) reduced when compared with controls at gestational
days 10, 13, and 16; however, body weights in this group were comparable with
those of controls on gestation day 20.
The incidence of fetal resorptions was slightly increased for all groups
dosed with RDX, but these fetal effects were not significantly different from
those of controls and the effects were not dose related. Fetal weights and
lengths were reported to be significantly (p <0.05) reduced at all dose levels
VI—32
-------
when compared with controls. However, when statistical analyses was repeated
on the data, using the litter as the bas^s, 't was found that fetal we'ght and
length were significantly decreased compared to controls only nn fefuses from
dams dosed at 2U mg/kg/day (Table VI-5). The male-to-female rat'os were 0.33,
0.88, U.94, and U.99 for the 0-, 2-, 6-, and 20-mg/kg/day groups, respect1vely.
No teratogenic effects were noted 'n this study. Based on maternal deaths and
toxicity and a decrease nn fetal weight and fetal length, the LOAEL ns ?0
my/kg/day and the NOAEL ^s 6 mg/kg/day.
5. Care noqenicity
RDX was not found to be carcinogenic when fed to rats (two strains); however,
it was found to produce hepatocellular and alveolar/bronchiolar carc'nomas and
adenomas when fed to mice.
Levine et al. (1983) evaluated the incidence of tumor formation male
and female Fischer 344 rats fed RDX in the diet at doses of 0.3, 1.5, 8.0, or
40.0 mg/kg/day for 24 months. Following treatment, animals were examined for
occurrence of tumors in various organs/systems. RDX was not found to be
carcinogenic in rats at doses up to 40.0 mg/kg/day. This study is reviewed in
detail in Section VI.B.2.b.t Lifetime Studies.
RDX was not found to be oncogenic in male and female Sprague-Dawley rats
fed RDX in the diet at doses of 1.0, 3.1, or 10 mg/kg/day for 24 months (Hart,
1977). The incidence of neoplasms was found to be comparable between dosed and
control animals.
Lish et al. (1984) evaluated the incidence of tumors in groups of 85 male
and female B6C3Fj mice fed RDX at doses of 1.5, 7.0, 35.0, or 100 mg/kg/day for
24 months (Table VI-6 and Table VI-7, respectively). The incidence of
VI-33
-------
Table VI-5. Fetal Weight and Length (± SD) in Progeny
of Rats Administered RDX
Dose
1 evel
Weight (g)
Length (cm)
0
3.81*0.47
3.7U0.16
2
3.70±U.39
3.68^0.17
6
3.72±0.34
3.67iO.15
20
3.45±0.43*
3.52r0.21*
*Significantly different from control value using analysis of
variance and Duncan's test for multiple comparisons, p <0.05; the
analysis is on a litter basis.
SOURCE: Adapted from Angerhofer et al. (1986).
V1-34
-------
Table VI-6. Incidence of Neoplasfc Lesions Male B6C3F} M^ce
Fed RDX ^n the D'et for 24 Months
Dose
level (mq/kg/day)
Oryan/f1nd1nga
0
1.5
7.0
35.0
100.0
L1 ver
(6 3) b
(60)
(62)
(59)
(27)
Malignant lymphoma
0
(0*)c
1
(1.7%)
A
(6.4%)
5
(3.5*)
0
Hepatocellular carcnoma
13
(20.6%)
20
(33.3%)
16
(25.8%)
13
(30.5*)
6
(22.2*)
Hepatocellular adenoma
8
(12.7%)
6
(10.0%)
1*
(1.6%)
7
(11.9%)
7
(25.9*-)
Hepatocellular adenoma and
carcinoma combined
21
(33.3%)
26
(43.3%)
17
(27.4%)
25
(42.4%)
13
(48.1--)
Lung
(63)
(60)
(62)
(59)
(27)
Alveolar/bronchiolar carcinoma
3
(4.8%)
6
(10.0%)
3
(4.8%)
7
(ll.Q%)
5
(18.5%)
Alveolar/bronchiolar adenoma
6
(9.5%)
5
(8.3%)
5
(8.1%)
7
(11.9%)
1
(3.7%)
Alveolar/broncniolar adenoma
and care noma combined
9
(14.3%)
11
(18.3%)
8
(12.9%)
14
(23.7 f.)
6
(22.2%)
Kidney
(63)
(60)
(62)
(59)
(27)
Malignant lymphoma
1
(1.6%)
2
(3.3%)
4
(6.4%)
4
(6.8%)
1
(3.7%)
alncludes animals sacrificed at study ternrnafon and those that died or were
sacrificed moribund in the course of the study.
bNumber of animals with specific tissue examined histologically.
cPercentage of incidence.
*Significantly different from control value (p <0.05).
SOURCE: Adapted from Lish et al. (1984).
V1—35
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Table VI-7. Incidence of Neoplastic Lesions in Female B6C3Fj M->ce
Fed RDX ti the D'et for 24 Months
Dose level (mq/kq/day)
Oryan/f1nd,nga
1.5
7.0
35.0 100.0
L1 ver
Hepatocellular carcinoma
Hepatocellular adenoma
Hepatocellular adenoma and
carcinoma combined
Lung
A1veolar/bronchiolar carcinoma
A1 veolar/broncmolar adenoma
A1 veolar/bronchiolar adenoma
and carcinoma combined
(65)b (62) (64) (64)
nn
0 4
(0%)c (6.4%)
1 1
(1.5%) (1.6%)
1** 5
(1.5%) (8.1%)
3 6 3
(4.7%) (9.4%) (9.7?J
6 6 3
(9.4%) (9.4%) (9.75M
9* 12* 6*
(14.1%) (18.8%) (19.4%)
(65) (62) (64) (64) (31)
3 1
(4.6%) (1.6%)
4 2
(9.2%) (3.2%)
7 3
(10.8%) (4.8%)
3 3 4
(4.7%) (4.7%) (12.9%)
5 9 3
(7.8%) (14.1%) (9.7-.)
8 12 7
(12.5%) (18.8%) (?2.5%)
alncludes animals sacrificed at study termination and those that died or were
sacrificed moribund in the course of the study.
bNumber of animals with specific tissue examined histologically.
cPercentage of incidence.
~Significantly different from control value (p <0.05), Fisher's Exact test.
"Significant trend (p£0.05), Cochran-Armitage test; analysis by reviewers.
SOURCE: Adapted from Lish et al. (1984).
VI-36
-------
hepatocellular carcinomas and adenomas combined was reported to oe significantly
increased (p <0.0b) in females receiving RDX at 7.0, 35.U, and 1UU.0 mg/fcg
(14.1, 18.8, and 19.4%, respectively) when compared to concurrent (1.5%) or
historical (7.9%) controls (Tables VI-7 and V1-8). These findinys were
considered to be compound related in females. In addition, it was reported
that the incidence of hepatocellular adenomas in females receiving 7.0 and
but not 100 my/kg/day was significantly (p <0.0s) increased when conpared to
historical controls. Historically, the incidence of combined hepatocellular
adenomas and carcinomas in untreated male 86C3F^ mice is 31.1% as compared to
7.9% for untreated females of this strain (Table V1-8) (Haseman et al., 1984).
The incidence of combined hepatocellular adenomas and carcinomas in niyh-dose
males was 48.1% as compared to 33.3% for the concurrent control group; the
increase was not significant (Table VI-6). A nonsignificant increase in alveolar
and bronchiolar carcinomas was found in high-dose males and females; histiocytes
were reported to be increased in the lungs of high-dose females. Malignant
lymphoma of the kidney was reported to be slightly increased (nonsignificantly)
in males receiving RDX at levels of 1.5, 7.0, and 35.0 mg/kg (3.3, 6.4, and
6.8%, respectively) when compared with concurrent controls (1.6%). This finding
was not seen in female mice. In summary, the incidence of hepatocellular
carcinomas and adenomas (combined) was significantly Increased in ROX-treated
female mice. Based on EPA Guidelines for Carcinogen Risk Assessment, ROX is
classified 1n Group C: Possible Human Carcinogen.
This study had some technical flaws. The high dose was lowered from 17t>
mg/kg/day to 10U mg/kg/day during week 11 due to high mortality in both sexes.
This mortality substantially reduced the number of high-dose animals at risk
(see Table V1—4). Since there were four doses in the study, the high dose
could be removed for quantitation purposes and still provide an acceptable
V1—37
-------
Table VI-8. Incidence of Primary Liver Tumors
in Historical Control B6C3F], Mice
Tumor
incidence
Finding
Mai es
Femal es
Number of tissues examined
(2,334)
(2,469)
Hepatocellular carcinoma
498
101
(21.3%)
(4.1%)
Hepatocellular adenoma
240
98
(10.3%)
(4.0%)
Hepatocellular adenoma
725
196
and carcinoma comDined
(31.1%)
(7.9%)
SOURCE: Adapted from Haseman et al. ( 1984).
V1-38
-------
potency estimate. The RDX used contained 3 to 10% HMX. The increases in
hepatocellular adenomas or carcinomas were not significantly (p >0.05) increased
in dosed females wnen analyzed separately Out were significant when comomed.
6. Genotoxicity
ROX gave neyative results in all genotoxicity studies conducted.
The mutagenic potential of RDX was evaluated in two studies using tne
Salmonel 1 a/microsomal prei ncubation assay. Salmonel 1 a strains TA98, TA100,
TA1535, TA1537, and TAlb38 were exposed to RDX at concentrations of 1, 10, 100,
300, or 1,000 uy/plate in one study (Cholakis et al., 1980), and at 0.625 or
1.25 my/plate in the second study (Whong et al., 1980). The assays were
conducted with and without metabolic activation (S-9 fraction from livers of
Aroclor-induced rats). In both studies, ROX was nonmutagenic.
Simmon et al. (1977) tested the mutagenicity of several munitions
wastewater chemicals before and after chlorination or ozone treatment. ROX was
evaluated in Salmonel 1 a strains TA98, TA100, TA1535, TA1537, and TA1538 at
several concentrations ranging from 0.24 to 14 uy/plate, and in Saccharomyces
cerevisi ae strain 03 at concentrations ranging from 0.U0004 to 0.0023%. The
assays were conducted with or without metabolic activation (S-9 fraction from
livers of Aroclor-induced rats). ROX was nonmutayenic before and after
chlorination in these assays.
In a dominant lethal assay, male Fischer 344 rats were fed ROX-contaimng
diets providing 0, 5, 16, or 50 mg/kg/day for 13 weeks and then mated with
virgin females. ROX did not Induce a dominant lethal effect. There was no
evidence of a preimplantation loss of blastocysts or postimplantation of embryos
(Cholakis et al., 1980).
V1-39
-------
KDX gave negative results in an unscheduled UNA synthesis assay usiny
W1-38 (human fibroblasts) when tested at a maximum concentration of
4,DUO uy/mL, with or without metabolic activation (Oilley et al . , 1973).
7. Neurotoxicity
MacPhail et al. (1985) studied neurobehavioral effects after acute or
subchromc oral administration of rtDX to Sprague-Dawl ey rats. In acute studies,
ROX in corn oil was administered, by gavage, in doses of 12.5, 25, or 50 my/kg.
Testing for behavioral effects was conducted 2 and 24 hours after administration.
In subchromc studies, doses of 1, 3, or 10 mg/kg/day were yiven for 30 days,
and testing was performed prior to dosing and after 16 and 30 days of dosing.
Between 24 and 64 rats of each sex were studied in a series of neurobenavioral
tests. After acute dosing, a wide range of sensory, motor, and cognitive
effects were found in the rats, but no overt neurotoxic effects were found.
Little evidence of behavioral toxicity was observed after subchronic dosing.
Motor activity was tested in a figure-8 maze with blind alleys that
converged on a central open area; photoelectric cells monitored total activity
as well as temporal and spatial distribution of motor activity. In the acute
study, there was a significant dose-related decrease in activity (all doses)
with some indication of a residual effect at 24 hours. Landing footspread,
measured in the same groups of rats, was consistently decreased at 2 hours, but
no dose-related effect was observed. In an acoustical startle response test,
a 13-kHz startle stimulus was presented 10 times in each testing session in the
presence of three different background noise intensities. A dose-related increase
in response latency was observed after single doses of ROX (MacPhail et al., 1985).
VI-40
-------
In a schedule-controlled behavior test, male rats were trained to press a
key to deliver sweetened condensed milk in response to a light cue over the
milk dipper. KOX administration decreased the overall operant response rate
and, at the hignest dose, recovery to baseline performance did not occur until
3 days after dosing. There was a dose-response effect on recovery. In a
flavor aversion conditioning test, KDX-treated rats preferred water to a
saccnarin-f1avored solution.
In the subchronic studies, no clear behavioral effects were observed in any
of the above tests after daily dosing at levels of up to 10 mg/kg/day.
Burdette et al. (1988) studied spontaneous seizure activity, audiogenic
seizure activity, and tne phenomenon of kindling in Long-Evans male rats
after oral administration of RUX. Oral gavage doses of KDX (class u particle
size) dispersed in carboxymethylcellulose by ultrasonic treatment were
administered to groups of 10 to 12 rats, and the animals were observed for
8 hours. Spontaneous convulsions occurred in about 20% of the rats dosed at
12.5 mg/kg ROX within 2 hours and subsided by 4 hours. At 2b- and 50-mg/kg
doses, spontaneous convulsions occurred in 80% of the animals and seizure
activity persisted for 2 and 6 hours, respectively. The tnreshold plasma level
of RDX at seizure was 3.1 pg/mL. Susceptibility to audiogenic seizures,
elicited by exposure to an ultrasonic source, was significantly increased
with dose and persisted for up to 300 seconds after a 60-mg/ky dose of ROX;
this dose resulted in death of 6 of 16 rats.
To study the kindling phenomenon, electrodes were implanted in the
amydalold nucleus of the brain. Kindling Is characterized by development of
seizure activity by repeated daily electric stimulation of the amydaloid at
a current that is a fraction of the normal threshold current for seizures.
VI-41
-------
RDX given at nonconvulsive daily doses of 6 my/kg/day acted as a chemical
kindlmy agent. Convulsions could be induced by low currents a week after
cessation of ROX dosmy, indicating that tne effect was not due to accumulation
of rtUX in tne body. The results indicated to Burdette et al. (1988) that tne
primary target for RDX toxicity may be tne limbic system of the brain.
VI-42
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VII. HEALTH ADVISOKY DEVELOPMENT
A. SUMMARY OF HEALTH EFFECTS DATA
In humans, the major toxic effects of KOX have been on the central nervous
system (CNS). Exposure of workers in a munitions plant via inhalation of oust
containing KDX has resulted in nausea, irritability, convulsions, unconsciousness,
and amnesia (Kaplan et al., 1965). Military personnel have been exposed to RJX
and its decomposition products while burning composition C-4 explosives in tne
field to heat food; inhalation of the smoke resulted in clonic/tomc convulsions
(Ketel and Hughes, 1972; Hollander and Colbach, 1969). Ingestion of HDX has
caused similar CNS effects (Stone et al., 1969, Woody et al., 1986).
Acute toxicity studies by Cholakis et al. (1980) indicated oral LO5J values
of about 80 mg/kg in mice and 118 mg/kg in rats. It has been estimated that
convulsions occur in rats when plasma levels reach 13.8 ug/mL (Schneider et
al., 1977).
Subchronic 90-day feeding studies in mice and rats indicate effects on the
blood and liver. In both sexes of mice, increased liver weights were seen in
groups receiving ROX at 320 mg/kg/day, and anemi a was seen in males receiving
160 my/kg/day (Cholakis et al., 1980). In rats, anemia was seen at a dose
level of 28 mg/kg/day in males (Cholakis et al., 1980), and increased liver
weight was seen at a dose level of 100 mg/kg/day in females (Levine et al.,
1981). In a 10-day oral gavage study in monkeys, vomiting and convulsions were
seen in five of six animals receiving RDX at 10 mg/kg/day, but no central
nervous system effects were seen at 1 mg/kg/day (Martin and Hart, 1974).
VII-1
-------
Lifetime feeding studies in rats and mice produced CNS effects, increased
mortality, weight loss, anemia, hepatotoxicity, renal toxicity, testicular
defeneration, and inflammation of tne prostate. In male and female rats fed
RJX in tne diet at a level to give a daily intake of 40 my/ky, tremors and
convulsions, increased mortality, and enlargement of the liver were observed.
Anemia and enlargement of the kidneys accompanied by histologic changes were
also found in males receiviny 40 mg/kg/day. Inflammation of the prostate was
found at l.b, 8, and 40 mg/kg/day, no effects were noted at a dose of 0.3
mg/kg/day (Levine et al., 1983). In a study in mice by Lish et al. (1984),
increased mortality was seen in the first 10 weeks of the study when mice
received 17b mg/kg/day. The high dose was reduced to 100 mg/kg/day. Decreased
weight gain was observed in females receiving 100 mg ROX/kg/day between 10
weeks and study termination. Increased liver weights were found in males and
females receiving 100 mg/kg/day. The males receiving 3b or 100 mg/kg/day
exhibited testicular degeneration. No important toxic effects were found at
7 mg/kg/day.
RDX was not found to be mutagenic in bacteria (Whong et al., 1980, Simmon
et al., 1977). It gave negative results in the dominant-lethal test (Cholakis
et al., 1980) and in an unscheduled ONA synthesis assay (Oilley et al., 1978).
RDX was not carcinogenic in rats (Levine et al., 1983; Hart, 1977). In
86C3Fi mice, there was a significant increase in the incidence of hepatocellular
carcinomas and adenomas (combined) in females receiving 7, 35, or 100 mg/kg/day
for 2 years. Mortality 1n mice receiving the highest dose was excessive and
the dose was lowered from 175 to 100 my/kg at week 11. RDX is classified
as Group C: Possible Human Carcinogen.
V11—2
-------
In a rwo-generation reproduction study in rats, decreased fertility was
observed at bO mg/kg/day. Developmental effects (decreased pup weights) were
seen at 16 and bU my/kg/day; no effects were observed at b mg/kg/day (Cnolakis
al., 1980). RDX was found to be embryotoxic in rats at 2D my/kg/day (Cholakis
et al., 1980; Angernofer et al., 1986) but was not found to be teratogenic.
B. QUANTIFICATION OF TOXICOLOGICAL EFFECTS
Healtn 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 noncarcinogemc endpoint of toxicity. The
HAs for noncarci nogemc toxicants are derived using the following formula:
HA = (NOAEL or LOAEL) (BU) = my/L ( y/L)
(UF) ( L/day)
where:
NOAEL or LOAEL = No- or Lowest-Observed-Adverse-Effect Level
(in mg/ky 8W/day).
BW = assumed body weight of a child (10 kg) or an adult (7U
kg).
UF = uncertainty factor (10, 100 or 1,000), in accordance with
NAS/ODW guidelines.
L/day = assumed daily water consumption of a child (1 L/day) or
an adult (2 L/day).
1. One-day Health Advisory
In humans, ROX intoxication results in central nervous system effects such
as convulsions and unconsciousness. Good quantitative data on the minimum dose
that causes the effects are not available. One study on acute intoxication of
3-year-old child who consumed ROX measured blood and spinal fluid levels of ROX
V11—3
-------
and estimated the dose ingested (Woody et a 1., 1986). However, since
assumptions were made to estimate the dose, and a single individual was exposed,
tnis study was judged to have inadequate data for HA development. In a stJdy
in dogs, a single intravenous dose of 3.37 mg kDX/kg was reported to cause an
erratic el ectroencepnalogram (EEG) pattern and decreased blood pressure.
However, graphic data for this dose (the lowest dose tested) were not presented,
only one dog was tested, a NOAEL was not established, and the route of exposure
may not nave been appropriate for setting a One-day HA value for RDX.
Since these data were not judged suitable for determining a One-day HA
value for KDX, it is recommended that the Longer-term HA for a 10-kg child (0.1
mg/L) be used as a conservative estimate for the One-day HA value.
2. Ten-day Health Advisory
No data found in the available literature were suitable for determination
of the Ten-day HA value for KDX. It is therefore recommended that the Longer-
term HA for a 10-kg child (0.1 mg/L) be used as a conservative estimate for the
Ten-day HA value.
3. Longer-term Health Advisory
In a 90-day study in cynomolgus monkeys, convulsions occurred in five of
six animals receiving RDX, by gavage, at 10 mg/kg/day. No CNS effects were
seen in three males and three females receiving 1 mg/kg/day (Martin and Hart,
1974). Based on this study, the LOAEL is 10 mg/kg/day and the NOAEL is 1
mg/kg/day. In rats, tne LOAEL for CNS effects was 25 my/kg/day and the NOAEL
was lb mg/kg/day in a 90-day feeding study (Von Oettingen et al., 1949).
LOAELs and NOAELs were available for effects on liver weight and liver pathology
in mice (Cholakis et al., 1980) and in rats (Levine et al., 1981), but they
V11-4
-------
were not considered because the values were much less sensitive than those for
CNS effects in monkeys. The Cholakis et al. (1980) developmental toxicity
study was considered but rejected due to the seventy of the effects (convulsions)
at the LOAEL (21) my/kg/day).
The Longer-term HA for the 10-kg child is calculated as follows:
(1 mg/kg/day) (10 kg) = U.l mg/L (100 ug/L)
(1 L/day) (100)
where:
1 my/kg/day = NOAEL, based on CNS effects in monkeys following 9U-day oral
dosing at 10 my RDX/kg/day.
10 kg = assumed weight of a child.
1 L/day = assumed water consumption of a 10-kg child.
1UU = uncertainty factor, chosen in accordance with ODW/NAS
guidelines using a'NOAEL from an animal study.
The Longer-term HA for a 70-kg adult is calculated as follows:
(1 mg/kg/day) (70 kg) = 0.35 mg/L (rounded to 400 uy/L)
(2 L/day) (100)
where:
1 mg/ky/day = NOAEL, based on CNS effects in monkeys followiny 90-day oral
dosing at 10 mg ROX/kg/day.
70 kg = assumed weiyht of an adult.
2 L/day = assumed water consumption of a 70-kg adult.
4. Lifetime Health Advisory
The Lifetime HA represents that portion of an individual's total exposure
that is attributed to drinking water and is considered protective of noncarcinoyenic
VI1-5
-------
adverse health effects over a lifetime exposure. The Lifetime HA ¦•s de^v<"i
'n a three step process. Step 1 determines the Reference Dose (RfD), formerly
called the Acceptable DaMy Intake (ADI). The RfD ""s an estimate of a da^ly
exposure to the human population that is 1 -¦ ke 1 y to be without appreciable r^sk
of deleterious effects over a lifeline, and 's derived from the NOAEL (nr
LOAEL), identified from a chrome (or subchron^c) study, d^ded by •in uncerta1nry
factor(s). From the RfD, a Drinking Water Equivalent Level (DWEL) can
determined (Step 2). A DWEL IS A MEDIUM-SPECIFIC (I.E., DRINKING WATER 1 LIF^timf
EXPOSURE LEVEL, ASSUMING 100% EXPOSURE FROM THAT MEDIUM, AT WHICH AnvERSF,
NONCARCINOGENIC HEALTH EFFECTS WOULD NOT BE EXPECTED TO OCCUR. The RWEL 'S
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 IS BASED
ON ACTUAL EXPOSURE DATA OR, IF DATA ARE NOT AVAILA8LE, A VALUE OF 20% IS ASSUMED.
If the contaminant i's classified as a Group A or 8 carcinogen, according to the
Agency's classification scheme of carcinogenic potential (U.S. EPA, 1986), thpn
caution should be exercised in accessing the risks associated with lifetime
exposure to this chemical.
Three 24-month continuous feeding studies, one in B6C3Fj mice and two sn
rats (F-344 and Sprague-Dawley), were considered. The LOAELs and NOAELs
established are presented in Table VII-1.
In the study with rats by Levine et al. (1983), F-344 rats were fed doses
of RDX at 0.3, 1.5, 8.0, or 40 mg/kg/day for 24 months. Based on a dose-related
increase in the incidence of suppurative inflammation of the prostate of males
receiving 1.5 mg/kg/day and above, the LOAEL is 1.5 mg/kg/day and the NOAEL is
0.3 mg/kg/day. This study is the most sensitive for deriving a DWEL.
VI1-6
-------
Table VII-1. Summary of Candidate Studies for Derivation of the
Drinking Water Equivalent Level (DUEL) for RDX
Speci es/
strain
Route
Duration
Endpoint
NOAEL LOAEL
(mg/kg/day)
Reference
Rat/F-344
Oral/
di et
Li fetime
Inflammation
of prostate
0.3
1.5
Levine et al . ( 1933)
Rat/S-D
Oral/
di et
Lifetime
Decreased body
weight in
females
1.0
3.1
Hart et al. ( 1977 )
Mouse/
B6C3Fi
Oral/
diet
Lifetime
Testicular
degenerati on
7.0*
35
Lish et al . ( 1984)
*The NUAEL is 7.0 mg/kg/day for systemic effects based on the absence of
systemic effects at this dose and the separation by EPA of tumongemc and
systemic changes; it is recognized that hepatocellular adenomas and carcinomas
were exhibited in female mice at this dose level.
VI1-7
-------
In c 24-month feeding study in Sprague-Dawley rats (Hart et al., 1977), no
histologic changes were seen at the doses tested (1, 3.1, and 10 mg/kg/day).
However, significant decreases were seen in body weight gain in females at 3.1
and 10 mg/kg/day. Thus, a NOAEL of 1.0 mg/kg/day was determined.
In a 2-year feeding study in B6C3Fi mice, an increase in testicular
degeneration was found in males receiving 35 mg/kg/day but not in those receiving
a dose of 1.5 or 7.0 mg/kg/day (Lish et al ., 1984). There was an increase in
absolute and relative liver weights in males and females administered 100
mg/kg/day and an increase in combined hepatocellular tumors in females receiviny
7.0, 35, or 100 mg/kg/day.
Oata on pharmacokinetics in humans are inadequate, and no data are available
for mice. Therefore, it cannot be determined which species most closely
resembles man in absorption, metabolism, and excretion of RDX.
The study by Levine et al. (1983) has been selected for calculation of the
DUEL, since it has the lowest NOAEL (0.3 mg/kg/day) among all the studies.
Using this study, the DUEL is derived as follows:
Step 1. Determination of the Reference Dose (RfD)
RfD = (°*3 ^/kg/day) = 0f003 mg/kg/day
where:
0.3 mg/kg/day = NOAEL, based on increased incidence of suppurative
inflammation in the prostate of males receiving 1.5
mg/kg/day.
100 = the uncertainty factor, chosen in accordance with 0DW/NAS
uidelines using a NOAEL from a chronic animal study
10X for intraspecies variation and 10X for interspecies
variationTT
VI1-8
-------
Step 2. Determination of the Drinking Water Equivalent Level (DUEL)
DWEL = (0.003 mq/kq/day) (70 kg) = 0.105 mg/L
2 L/day (rounded to 100 ug/L)
where:
0.003 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 Health Advisory
Lifetime HA = (0.105 mq/L) (0.2) = 0.002 mg/L (2 ng/L)
10
where:
0.105 mg/L = Drinking Water Equivalent Level (DWEL).
0.2 = Assumed Relative Source Contribution (RSC) if actual
data are not available.
10 = 0DW policy, additional factor of 10 to account for equivocal
evidence of carcinogenicity for Group C chemicals.
C. QUANTIFICATION OF CARCINOGENIC POTENTIAL
The carcinogenic potential of RDX has been evaluated in Fischer 344 rats
(Levine et al., 1983), Sprague-Dawl ey rats (Hart, 1976) and B6C3F1 mice (Lish
et al., 1984). RDX was not found to be carcinogenic when fed to the rats (both
strains). However, it was found to produce significant combined hepatocellular
adenomas/carcinomas in B6C3F1 female mice. RDX is classified Group C:
Possible Human Carcinogen.
RDX was not found to be oncogenic in male and female Sprague-Dawley rats
fed RDX in the diet at doses of 1.0, 3.1, or 10 mg/kg/day for 24 months (Hart,
1976).
VII —9
-------
levne et al . (1983) evaluated the carcinogenicity of ROX ti male and
female Fischer 344 rats fed doses of 0.3, 1.5, 8.0, or 40.0 mg/kg/day °DX for
24 montns. The major tox^c effects observed included anemia wth secondary
splenic lesions, hepatotox-1 c'ty and urogenital lesions. Based on adverse
systemic effects at 1.5 mg/kg/day, the MTD was achieved.
L^sh et al. (1984) evaluated the incidence of tumors 'n groups of ^5 nale
and female B6C3F1 nrce fed doses of 1.5, 7.0, 35.0, or 100 mg/kg/day RDX for 2^
months. The ,nc,dence of hepatocellular carcnomas and adenomas combined was
significantly increased (p <0.U5) in females receding 7.0, 35.0, and 100.0 mg
RUX/kg (14.1, 18.8, and 19.4%, respectively) when compared to concurrent (1.5*)
or historical (7.9%) controls. These findings were considered to be compound-
related in females. Historically, the incidence of combined hepatocellular
adenomas and carcinomas in untreated male B6C3F1 mice is 31.6% as compared to
7.y% for untreated females of this strain (Haseman et al., 1984). In add:t'on,
the incidence of hepatocellular adenoma in females receding 7.0 and 35.0 but
not 100 mg/ky/day was significantly (p <0.05) increased when compared to
historical controls.
The incidence of combined hepatocellular ademomas and carcinomas sn h^'gh-
dose males was 48.1% as compared to 33.3% for the concurrent control group; the
increase was not significant. A nonsignificant increase in alveolar and
0
bronchiolar carcinomas was found in high-dose males and females; histocytes
were reported to be increased in the lungs of high-dose females. Malignant
lymphoma of the kidney was reported to be slightly increased (nonsignificant)
in males receiving 1.5, 7.0, and 35.0 mg/kg when compared with concurrent
controls. This finding was not seen in female mice.
VII-10
-------
Ths study had some techn'cal flaws wfrch may have compromised '•ts
sens1 tivity to evaluate oncogerccty. The frvgh-dose was lowered from 175
mg/kg/day to 100 mg/kg/day during week 11 due to Ivgh mortality "•n both sexes.
Th^s mortality substantially reduced the number of Ivgh-dose an'mals. The RQX
used contained 3 to 10% HMX. The increase in hepatocellular adenomas or
carcnomas were not s^n^ficantly (p >0.05) increased (when compared to
concurrent controls) 'n dosed females when analyzed separately but only when
comb1ned.
DOSE-RESPONSE DATA (CARCINOGENICITY, ORAL EXPOSURE)
Tumor Type -- l^ver
hepatocellular carcinoma
and adenomas (combined)
Test An'mals -- mice/B6C3Fl/females
Route -- d^et/oral
Reference — Lish et al. (1984)
Dose
Human*
Administered Equivalent Tumor
(mg/kg/day) (mg/kg/day) Incidence
0.0 0.0 1/65
1.5 0.13 5/62
7.0 0.58 9/64
35.0 2.90 12/64
100.0 8.30 6/31
* Administered dose * (70 kg/.040 kg).33
The human equivalent dose was determined using a standard surface area
correction factor. The animal study dose is divided by the ratio of the human
weight (70 kg) to the mouse weight (.040 kg) raised to the 1/3 power. The high-
dose data was not used in the slope factor calculation since it was lowered
from 175 mg/kg/day to 100 mg/kg/day during week 11. The unit risk should not
VII-11
-------
be used if tne water concentration exceeds 3,000 ug/L. Above this concentration
the slope factor may differ from that stated.
SUMMARY OF RISK ESTIMATES
Without High-dose
Oral Slope Factor (mg/kg/day)"* 1.1E-1
Drinking Water Unit (ug/L) Risk 3.1E-6
Extrapolation Method Linearized Multistage
Procedure, extra ris<
Drinking Water Concentrations at Specific Risk Levels:
Risk Level Concentrations ug/L)
E-4 (1 in 10,000) 30.0
£-5 (1 in 100,000) 3.0
E-6 (1 in 1,000,000) .3
The multistage model was used for high-to-low dose extrapolation (Crump
and Watson, 1979; Howe and Crump, 1982). G1obal83 was used to fit the data in
the experimental dose range and to obtain upper 951 confidence limits on risk.
Tne 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 ROX. The relationship of the concentration (.ug/L) of a chemical in
drinking water to cancer risk is expressed as follows:
35000 x R 3 C
Where:
qj* » (mg/kg/day)"^, human slope factor
R = (10-4, 10"5, 10"6, etc.)
VI1-12
-------
C = concentration of chemical in ug/L
35000 = conversion factor for mg to ^g and exposure
assumption that a 70 kg adult drinks 2L of water/day
DISCUSSION OF CONFIDENCE (CARCINOGENICITY, ORAL EXPOSURE)
There are three animal bioassay studies to evaluate the carcinogenic
potential of ROX. Both well conducted rat studies (Fischer 344 rat and Sprayue-
Dawley rat) were clearly negative. The ROX was found to be carcinogenic in
the female B6C3F1 mice. This study had some previously discussed technical
problems but is clearly positive for liver carcinomas and adenomas combined.
OTHER CANCER RISK MODELS
For comparison purposes the drinking water concentration associated with
an excess cancer risk of 10"® was 0.3 ug/L for the One-hit model. The
drinking water concentrations associated with an excess cancer risk of 10-6
for the other models (Multihit, Logit, Probit, and Weibull) were all
<0.002 ug/L. The estimates for these models were calculated with RISK81
(Kovar and Krewski, 1981).
V11-13
-------
VIII. OTHER CRITERIA, GUIDANCE, AND STANDARDS
Trie thresnold limit value (TLV) for ROX recommended by the American
Conference of Government Industrial Hygienists is 1.5 mg/m^ (notation skin)
(ACGIH, 1986), and tne snort-term exposure limit (STEl) is 3 mg/m^. OSHA nas
also recommended a TLV of 1.5 mg/m^ (National Researcn Council, 1982).
Schneider et al. (1978) suggested an ingestion limit of 0.1 my/kg/day or 2
to 3 ppm in potable water.
In 198U, the United States Army Medical Sioengineering Research and
Development Laboratory recommended an RDX limit of 0.03 my/L in drinking water
as an interim standard (National Research Council, 1982).
VIII-1
-------
IX. ANALYTICAL METHODS
Published methods for analyzing RDX fall into two broad categories:
analysis of bulk material and trace analysis. Volumetric methods involving
reduction, hydrolysis, or acid-base titrations are used for bulk analysis.
Methods for analyzing trace quantities (below 10 ppm) of RDX include the
following: thin-layer chromatography (TLC), hi gh-pressure liquid chromatography
(HPLC), gas-liquid chromatography (GLC), and single-sweep polarography.
Polarography has a detection limit of 0.05 mg/L for RDX, an analysis time
of approximately 5 minutes, and reproducibi1ity of t 10%. However, metal ions
and some anions can interfere with the analysis. Since these interferences
have not been completely characterized, polarography is not routinely used for
ROX analysis (Whitnach, 1976, as cited in Sullivan et al., 1979).
Since the detection limit for ROX using TLC is 25 my/L for direct
determination (Leach and Hash, 1972, as cited in Sullivan et al., 1979), TLC is
generally not used for quantitative determination of ROX (Sullivan et al.,
1979). TLC may be useful for qualitative identification of RDX when it is
present in mixtures with other munitions components (Sullivan et al., 1979).
GLC is one of the two preferred analytical techniques for determination of
RDX at low concentrations (below 10 ppm). Each of the GLC methods involves
extraction of RDX into a solvent prior to determination. Hoffsomer et al.
(1977, as cited in Sullivan et al., 1979) extracted RDX with benzene/acetone
followed by GLC determination usiny an electron capture (EC) detector; the
detection limit for the method is 0.002 ppm. Jain (1976, as cited in Sullivan
et al., 1979) used benzene as the extraction solvent and either an EC or a
flame ionization detector (FID); the detection limit was 0.03 ppm. Sullivan
IX-1
-------
et al. ( 1977 , as cited in Sullivan et al., 19 7y) used ethyl acetate as the
extraction solvent and either EC or an alkaline earth FID; the detection limits
were 0.U05 ppm for water samples and 0.2 ppm for sediment samples. One
disadvantage of the GLC methods is that other, less volatile munition components
(e.g., HMX) that may be present in the same sample cannot be codetermined
(Sul1lvan et al., 1979).
HPLC methods would appear to be the methods of choice for detection of rfLK
at low concentrations. Even though the detection limits are not quite as low
for HPLC (U.Ob to 0.4 ppm) as for GLC (0.002 to 0.2 ppm), advantages include
low operating temperatures and determination of RDX with less volatile components.
The HPLC methods have the following features in common: solvent extraction, an
aqueous methanol mobile phase, and use of an ultraviolet (UV) detector. Hale
et al. (1978, as cited in Sullivan et al., 1979) obtained a detection limit of
0.05 ppm for RDX in both soil and water samples using ether extraction, a mobile
phase of 30% methanol in water, Zorbax-ODS stationary phase, and detection at
230 nm. Spanggord et al. (1978, as cited in Sullivan et al., 1979) determined
RDX levels in "load, assemble, and pack" (LAP) wastewater using a mobile phase
of 60% methanol :40% water and a stationary phase of M-Bondapak L^j; detection
limits were 0.4 ppm at 254 nm and 0.2 ppm at 210 nm. Stilwell et al. (1977)
determined RDX in wastewater from the Holston Army Ammunition Plant followiny
extraction with ethyl acetate. A mobile phase of 40% methanol :60% water was
used with a stationary phase of Partisil 10-0DS; the detection limit was 0.05
ppm at 230 nm. HMX was determined concurrently with a detection limit of 0.05
ppm.
IX—2
-------
X. TREATMENT TECHNOLOGIES
Noss and Chyrek (1984) stud1ed the deyradafon of RDX usTig ultraviolet
rad^t'on, hydrogen peroxide add-on, and ultrasoijnd cavitation. Hydrogen
peroxide alone had no effect on munitions degradation. Similarly, ultrasound
cavitational processes yielded no benefit when used alone or when combined with
other treatments. Hydrogen peroxide applied at initial concentrations less
than 0.01% enhanced RDX decomposition by ultraviolet photolysis. During treat-
ment wnth ultraviolet radiation (n combination with 0.01% hydrogen peroxide,
RDX (18.9 mg/L) was degraded rapidly (half-life = 8.0 minutes).
The use of an ultraviolet light-hydrogen peroxide system for treatment of
RDX i" wastewater was studied by the Naval Weapons Support Center (ESR, 198S).
RDX and its organic reaction products were completely destroyed ussng a system
with 0.05 to 0.15% hydrogen peroxide and a minimum of 10 megawatt-minutes of
ultraviolet light at 254 nm/mole of explosive. Kubose and Hoffsommer (1977,
as cited in Sullivan et al., 1979) have shown that concentrations of 20 to
40 my RDX/L in aqueous solution could be reduced 98% by photolysis using the
full spectral output from a medium-pressure mercury vapor lamp (220 nm to
1367 nm) with irradiation periods of 15 seconds.
Kubose and Hoffsommer (1977, as cited in Sullivan et al., 1979) have
indicated that strongly basic ion exchange resins may be a potential single-
step waste treatment method for the degradation of RDX. This process involves
RDX adsorption onto the resin and reaction with quaternary ammonium hydroxide.
Sullivan et al. (1979) commented that this procedure is only feasible for
aqueous solutions that have fairly low acid and/or anion concentrations.
X-l
-------
Chemical oxidation using potass^m d^chromate, potass^m permanganate, or
calcum hypochlorate w'll destroy n1 tram1 nes. Jackson et a 1 . ( 1976 , as c'ted
'n Sull'van et a 1 ., 1979) found that concentrations of Ca(OC1 )2» K2i-r2l^7» nr
KMn04 at 1,000 my/L completely oxid1zed 50 mg ROX/L wuhm 72, <18, or 24 hours,
respect!vely.
Chemical coagulation us^'ng lime was also stud^-ed by Jackson et ai. (1Q75,
as cted ti Sullivan et al., 1979). Ninety percent RDX removal was achieved
using a 500-mg/L lime dose. Castonna et al . ( 1977, as cited Sullivan et al.,
1979) have caufoned against the use of polymer flocculants because of
incompatibilities with RDX.
Jackson et al. (1976, as cited 4n Sullivan et al., 1979) reported that
complete adsorptive removal of RDX (1.5 to 12 mg/L) was achieved us^ng activated
charcoal and Rohm and Haas XAD-2 resins. Activated carbon was found to
be superior to res^n at a hydraulic loading of 10 gpm/ft3. Vlahak^s (1974, as
cited in Sullivan et al., 1979) found that activated carbon had a capacity of
125 mg RDX/g carbon for RDX alone and 76 mg RDX/g carbon when ^n the presence
of TNT.
nata regarding aerobic microbiological degradation of RDX are m^xed.
Pilot plant studies by Green (1972, as cited in Sullivan et al., 1979) suggested
that RDX could be at least partially degraded by aquatic microflora. Green was
able to remove 42% of the RDX from manufacturing wastes using an activated
sludge reactor dominated by Pseudomonas and Alcaligenes. Conversely, Soli
(1973) concluded that aerobic heterotrophs could not degrade RDX.
McCormick et al. (1981) reported that RDX is biodegraded under anaerobic
conditions, yielding the following products: hexahydro-l-nitroso-3.S-din*tro-
X-2
-------
1,3,5-triazine; hexahydro-1,3-dimtroso-5-nitro-l,3,5-triazine; hexanydro-1,3,5-
trinitroso-l,3,5-tn azine; hydrazine; 1,1-dimethylhydrazine; 1,2-dmetnylhydra-
zine; formaldehyde, and methanol. Jackson et al . ( 1976, as cited in Sullivan et
al., 1979) achieved 100% removal of RDX by anaerobic fermentation from waters
containing 50 my ROX/L in conjunction with supplemental carbon sources: sucrose,
methanol, and hydroxyethyl cellulose. Times required for complete removal of
ROX ranged from 1 day for hydroxyethyl cellulose-supplemented wastewater to ^
days for sucrose-supplemented water.
X-3
-------
XI. CONCLUSIONS AND RECOMMENDATIONS
Based on the available animal data and on adverse effects on the central
nervous system of monkeys administered RDX by oral gavage for 90 days, the
Longer-term HA for a 10-kg child has been determined to De 0.1 mg/L (100 uy/L).
In the absence of adequate animal data to determine a One-day or Ten-day Healtn
Advisory, the Longer-term HA for a 10-kg child, U.l mg/L (100 ug/L), is used
as a conservative estimate of the One-day or Ten-day HA. The Longer-term HA
for a 70-kg adult was determined to be 0.35 mg/L (400 ug/L). A Lifetime HA
of 0.002 mg/L (2 ug/L) for a 70-kg adult has been determined, based on a
Drinking Water Equivalent Level (DWEL) of 0.100 mg/L (100 ug/L). The QUEL is
based on a Reference Dose of 0.003 mg/kg/day where the effect was suppurative
inflammation of the prostate of male rats fed RDX for 2 years. Based on the
study by Lish et al. (1984), RDX is classified as Group C: Possible Human
Carcinogen. The classification of RDX in EPA Group C is based upon limited
animal data. A quantitative cancer risk assessment, based on the limited
animal data, is provided to support selection of the uncertainty factors for
the recommended lifetime HA.
A comparison report "Data Deficiencies/Problem Areas and Recommendations
for Additional Data Base Development for RDX" (Appendix A) summarizes the scope
of existing data reviewed for this HA. This comparison report delineates the
areas where additional data and/or a clarification of existing data would be
appropri ate.
XI-1
-------
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following ingestion of C-4 plastic explosive. Arch. Intern. Med. 124:726-730.
Sullivan JH Jr., Putnam HD, Keirn MA, Pruitt BC Jr., Nichols JC, McClave JT.
1979. A summary and evaluation of aquatic environmental data in relation to
establishing water quality criteria for munitions-unique compounds. AD A087683
Part 4: RDX and HMX. Water and Air Research, Inc., Gainesville, FL. U.S. Army
Medical Research and Development Command Contract No. DAMD17-77-C-7U27.
U.S. EPA. 1986. U .S.-Environmental Protection Agency. Guidelines for
carcinogen risk assessment. Federal Regi ster 51(185):33992-34003.
Vogel W. 1951. Hexogen poisoning in human beings. Zent. F. Arbeitsmed. U.
Arbeitsschutz (German) 1:51-54.
Von Oettmgen WF, Donahue DO, Yagoda H, Monaco AR, Harris Mr. 1949. Toxicity
and potential dangers of cyclotrimethylenetnnitramine (RDX). J. Ind. Hyg. Tox.
31:21-31.
Whong WZ, Speciner ND, Edwards GS. 1980. Mutagenic activity of tetryl, a
ni troaromatic explosive, in three microbi al test systems. Toxicol. Lett.
5:11-17.
Windholz M, ed. 1983. The Merck Index, 10th Edition. Rahway, NJ: Merck and
Co. pp. 392-393.
Woody RC, Kearns GL, Brewster MA, Turley CP, Lake R, Sharp G, Lake RS. 1985.
Neurotoxicity of cyclotrimethylenetrinitramine (RDX) in a child: A clinical
and pharmacokinetic evaluation. Vet. Hum. Toxicol. 28(4):303-304 [abstract
No. 64].
X11-4
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Woody RC, Kearns GL, Brewster MA, Turley CP, Lake R, Sharp G, Lake RS. 1986.
Neurotoxic! ty of cycl otrimethylenetrini tramine (RDX) in a child: A clinical
and pharmacokinetic evaluation. Clin. Toxicol. 24(4):305-319.
XI1-5
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APPENDIX A
Data Deficiencies/Problem Areas and Recommendafons
for Additional Data Base Development for RDX
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DATA BASE DEVELOPMENT
A. OBJECTIVES
The objecfve of trvs document to prov'de an evaluafon of data def1-
cencies and/or problem areas encountered ""n the revew process for RDX and to
make r
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dosing. Absorbed RDX is rapidly cleared from the plasma and distributed to
issues. When drinking water saturated wth RDX (7.5 mg/L) was offered to rafs
for 90 days, there was no accumulation of RDX 'n the fssues. The half-l^vps
of clearance of RDX from plasma are of a s^lar order of magnitude 'n rats a"d
humans. The l\/2 was 10.1 hours 4n rats (Schneider et al., 197 7 ) and was 15.1
hours 'n the one available human study (Woody et al. , 1986). RDX ^s metahnl-zed
by the l^ver, and its metabolites are excreted primarily ¦•rt the ur'np. The
metabolites have not been identified and characterized.
In humans, the toxic effects of RDX have been on the central nervous system
(CNS). Exposure of workers "in a munitions plant via inhalation of dust con-
taining RDX has resulted in nausea, irritability, convulsions, unconciousness,
and amnesia (Kaplan et al., 1965). Military personnel have been exposed to RDX
while burning composition C-4 explosive in the field to heat food; inhala^-on
of the smoke resulted in clomc/tonic convulsions (Ketel and Hughes, 1972;
Hollander and Colbach, 1969). Ingestion of RDX has caused similar CNS effects
(Stone et al., 1969; Woody et al., 1985, 1986).
Acute toxicity studies by Cholakis et al. (1980) indicated oral LD50 values
of about 80 mg/kg in mice and 118 mg/kg in rats. Intravenous administration of
single doses of RDX to beagle dogs caused convulsions and death at a dose of an
mg/kg, central nervous system hyperactivity and nonlethal convulsions at a dose
of 20 mg/kg, and decreased blood pressure and erratic electroencephalograph^
patterns at doses of 3.37 and 6.78 mg/kg (McNamara et al., 1974).
Subchronic 90-day feeding studies in mice and rats indicate effects on the
blood and liver. In both sexes of mice, increased liver weights were observed
in groups receiving 320 mg/kg/day, and anemia was seen in males receiving 160
mg/kg/day (Cholakis et al., 1980). In rats, anemia was observed at a dose level
A-2
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of 28 mg/kg/day 'n males (Cholakis et al., 1980), and Ticreased l'ver wp^ht
was seen at a dose level of 100 mg/kg/day in females (Levne et al., 1981). In
a 10-day oral gavage study in monkeys, vonr't',ng and convulsions occurred 1 n
f1ve of s1x animals at 10 mg/kg/day, but no central nervous system effects were
seen at 1 mg/kg/day (Martin and Hart, 1974).
L^fefme feeding stud^s '•n rats and rmce produced CMS effects, increaserl
morta'Mty, weight loss, anemia, hepatotoxic5ty, renal tox^cty, testicular
degenerat1on, and inflammation of the prostate. In male and female rats fed
RDX in the d^et at a level to give a daily intake of 40 mg/kg, tremors and
convulsions, increased mortality, and enlargement of the liver were observed.
Anemia and enlargement of the kidneys accompanied by histologic changes were
also found in males receiving 40 mg/kg/day. Inflammation of the prostata was
found at 1.5, 8, and 40 mg/kg/day; no effects were noted at a dose of 0.3
mg/kg/day (Levine et al ., 1983). In a study in mice by L-»sh et al. (1984),
increas.ed mortality was seen in the first 10 weeks of the study when nrce
received 17b my/ky/day. The high dose was reduced to 100 mg/kg/day. Decreased
weight gain was seen in females receiving RDX at 100 mg/kg/day between 10 wepks
postadministrafon and study termination. Increased liver weights were found
in males and females receiving 100 mg/kg/day. The males receiving 35 or 100
mg/kg/day exhibited testicular degeneration. No important toxic effects were
found at 7 mg/kg/day.
RDX was not found to be mutagenic in bacteria (Whong et al., 1980; Simmon
et al., 1977). It yave negative results in the dominant-lethal test (Cholakis
et al., 1980) and in an unscheduled DNA synthesis assay (Dilley et al., 1978).
RDX was not carcinogenic in rats (Levine et al., 1983; Hart, 1977). In
B6C3Fj mice, a significant increase was observed in the incidence of
A-3
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hepatocellular carcinomas and adenomas (combined) in females receiving 7, 3b,
or 1U0 mg/kg/day for 2 years. However, mortality in mice receiving the highest
dose was excessive, and the dose was lowered from 175 to 100 mg/kg at week 11.
RDX is classified as Group C: Possible Human Carcinogen.
In a two-generation reproduction study in rats, decreased fertility was
oDserved at 50 mg/kg/day. Developmental effects (decreased pup weights) were
seen at 16 and 5U mg/kg/day; no effects were observed at 5 mg/kg/day (Cnolaicis et
al., 1980). RDX was found to be etnbryotoxic in rats at 20 mg/kg/day (Cholakis
et al., 1980; Angerhofer et al., 1986) but was not found to be teratogenic. In
a study in rabbits, RDX caused maternal toxicity at 20 mg/kg/day, and there was
suggestive evidence for a teratogenic effect at 2 and 20 mg/kg/day (Cholakis
et _ .980).
Based on the findings in these studies, One-day and Ten-day HA values
were established at 0.1 mg/L, and the Longer-term HA value was established
for an adult at 0.3b mg/L (400 pg/L). The Lifetime HA for an adult is 0.002
mg RUX/L (2 ug/L).
Methods of chemical analysis utilizing extraction and gas-liquid
chromatography reviewed by Sullivan et al. (1979) are adequate for detection of
RDX at low concentration in water. RDX in wastewater can be degraded by
ultraviolet irradiation in the presence of hydrogen peroxide (ESB, 1985).
Other methods using ion exchange resins or charcoal have been reviewed by
Sullivan et al. (1979).
C. DISCUSSION
Available data on the pharmacokinetics, health effects, analysis, and
treatment of RDX have been reviewed.
A-4
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Pharmacokinefc studies in rats ind'cate that RDX is effect'vely absorbed
va tne oral route, and ¦•t 'S rapidly metabol1 zed and excreted. Available dar.a
indicate tnat the rate of clearance 'n humans 's approximately of the same
order of magn^ude as that *n rats. Further studies 'n animals are unlikely fo
yeld add^fonal data perfnent to the development of HA values.
Ava^aDle acute toxicity stud^s of RDX ¦'nclude oral LD5QS 'n m-'ce and
rats and a study fn dogs us^ng the intravenous route, ""n wh^ch a LOAEl for
nonlethal CNS effects was established but a NOAEL was not achieved. S'x longer
term studies (90 days) were available *n mice, rats, dogs, and monkeys. In
five of these studies, LOAELs and NOAELs were established for various endpo^nts:
CNS effects, anemia, liver weight changes, and histologic effects on the l^ver.
Three lifetime studies were available, two in rats and one in nrce; LDAELs and
NUAELs were established. Additional short-term, longer term, and lifetime
studies are not likely to be pertinent in development of HA values. A
carcinogenicity study in mice was flawed because the high dose exceeded the
maximum tolerated dose (MTO) and was lowered from 175 to 100 mg/kg/dfiy at 11
weeks. Althouyh mortality was high at this dose, sufficient animals were
available to assess carcinogenicity (Lish et al., 1984). There was a carcinogenic
response, but time-to-tumor could not be analyzed from the available data. The
animals with hepatocellular tumors could be identified from individual animal
histopathology tables, but the identification numbers for histopathology d-"d
not correspond with the animal numbers on the disposition tabulation.
A two-generation reproduction study and several mutagenicity studies were
available. Not all categories of genotoxicity were investigated. A mutagenicity
test in a eukaryotic microorganism with and without activation and a test for
chromosome aberrations and sister chromatid exchange would fulfill this data
A-5
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gap. Two studies on developmental tox^cty in rats were available t^at
indicated embryotox^c, but not teratogenic, effects. A study *n rahb'ts
(Cholatc^s et al ., 19BU) gave results that suggested a possible teratogenic
response. Cleft palate was seen on soft tsssue analyses of fetuses from dosed
females. However, it was not recorded on gross examination or found 'n the
fetuses examined for skeletal anomalies. It is recommended that a teratology
study be repeated in rabbits using 20 pregnant dams/dose group to reeval'jat?
the teratogenic potential of RDX.
The methods for analysis of RDX in wastewater appear to he adequate to
detect levels that may be considered hazardous to health. The methods of
treating RDX-contaminated water appear to be adequate.
E. CONCLUSIONS/RECOMMENDATIONS
Based on the above discussion, the following conclusions/recommendations
can be made:
1. The available studies on RDX toxicity are generally considered
adequate for development of Health Advisories useful in dealing with
the potential contamination of drinking water.
2. It is recorrenended that a teratology study in rabbits be repeated.
Additional genotoxicity studies in eukaryotes and a test for chromosome
aberrations are recommended to fill data gaps in genotoxic'ty.
3. Additional card nogen"1 city bioassays should be considered to clarify
the carcinogenic potential of RDX.
A-6
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Ground, MD. Study No. 75-51-0573-86.
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11 ey JV, Tyson CA , Newell GW. 1978. Mamma^an toxicological evaluation of
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Hollander AI, Colbach EM. 1969. Composition C-4 induced seizures: A report
of five cases. U.S. Army Vietnam Med. Bull. 14(31):1529-1530.
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Environ. Health 10:877-883.
Ketel WB, Hughes JR. 1972. Toxic encephalopathy with seizures secondary to
ingestion of composition C-4: A clinical and ejectroencephalographic study.
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study of hexahydro-1,3,5-trinitro-l ,3,5-triazine (RDX) in Fischer 344 rats.
Toxicol. Lett. 8:241-245.
Levine BS, Furedi EM, Rac VS, Gordon DE, Lish PM. 1983. Determination of the
chronic mammalian toxicological effects of RDX: Twenty-four month chron'c
toxicity/carcinogenicity study of hexahydro-1,3,5-trinitro-l,3,5-triazine (RDX)
in the Fischer 344 rat. Phase V. Vol.1. AD A160774. Chicago, IL: 11T Resparch
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Lish PM, Levine BS, Furedi EM, Sagartz EM, Rac VS. 1984. Determination of the
chronic mammalian toxicological effects of RDX: Twenty-four month chronic
toxicity/carcinogenicity study of hexahydro-1,3,5-trinitro-l ,3,5-triazine (RDX)
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Martin OP, Hart ER. 1974. Subacute toxicity of RDX and TNT in monkeys.
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No. NU0014-7 j-C-0162; NR108-985. AD A044650.
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Renystorff RH, B^kup RK. 1974. The toxicology of cyclotr-'methylenetr1 n•> tne
(RDX) and eye 1 otetramethyl enetetram trarrr ne (HMX) solufons 'n d^methylsul fox-dp
(OMSO), eye 1ohexanone, and acetone. AD 780010. Edyewood Arsenal, Aberdeen
Proviny Ground, MD. Report No. EB-TR-73040.
Schneider NR, Bradley SL, Anderson ME. 1977. Toxicology of cyclotrTnet^yle^e-
tr1 ntram-1 ne: D'str^ut'on and metabolism 1 n the rat and the m-,n,aturp swnp.
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Schneider NR, Bradley SL, Anderson ME. 1978. The d'str^tifon and meta^ol'sm
of cyclotr-,methylenetrimtram",ne (RDX) in the rat after subchron1 c adm1 n-< sfav .
Toxicol. Appl. Pharmacol. 46:163-171.
Summon VF, Spanygord RJ , Eckford S, McClury V. 1977. Mutagenicity nf some
munition wastewater chemicals and chlorine test reagents. SRI Internafonal ,
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No. DAMD17-76-C-6013.
Stone WJ, Paletta TL, Herman EM, Bruce JI, Knepsh^eld JH. 1969. Tox^'c effects
followng ingestion of C-4 plastic explosive. Arch. Intern. Med. 124:726-730.
Sullivan JH Jr., Putnam HD, Keirn MA, Pruitt BC Jr., Nichols JC, McClav® JT.
1979. A summary and evaluation of aquatic environmental data *n relation to
establishing water quality criteria for munitions-unique compounds. Part 4:
RDX and HMX. AD A087683. Water and Air Research, Inc., Gainesville, FL. U.S.
Army Metfical Kesearch and Development Command Contract No. DAMD17-77-C-7027.
Whony WZ-, Speciner ND, Edwards GS. 1980. Mutagenic activity of tetryl , a
n1troaromatic explosive, in three microbial test systems. Toxicol. Lett. 5:11-17.
Woody RC, Kearns GL, Brewster MA, Turley CP, Lake R, Sharp G, Lake RS. 1985.
Neurotoxicity of cyclotrimethylenetrinitramine (RDX) in a child: A clinical
and pharmacokinetic evaluation. Vet. Hum. Toxicol. 24(4) :303-304 (^abstract
No. 64].
Woody RC, Kearns GL, Brewster MA, Turley CP, Lake R, Sharp G, Lake RS. 1986.
Neurotoxicity of cyclotrimethylenetrinitramine (RDX) in a child: A clinical
and pharmacokinetic evaluation. Clin. Toxicol. 24(4):305-319.
A-8
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