United States
Environmental Protection
1=1 m m Agency
EPA/690/R-09/040F
Final
1-08-2009
Provisional Peer Reviewed Toxicity Values for
Nitroguanidine
(CASRN 556-88-7)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

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ACRONYMS AND ABBREVIATIONS
bw
body weight
cc
cubic centimeters
CD
Caesarean Delivered
CERCLA
Comprehensive Environmental Response, Compensation and Liability Act

of 1980
CNS
central nervous system
cu.m
cubic meter
DWEL
Drinking Water Equivalent Level
FEL
frank-effect level
FIFRA
Federal Insecticide, Fungicide, and Rodenticide Act
g
grams
GI
gastrointestinal
HEC
human equivalent concentration
Hgb
hemoglobin
i.m.
intramuscular
i.p.
intraperitoneal
IRIS
Integrated Risk Information System
IUR
inhalation unit risk
i.v.
intravenous
kg
kilogram
L
liter
LEL
lowest-effect level
LOAEL
lowest-observed-adverse-effect level
LOAEL(ADJ)
LOAEL adjusted to continuous exposure duration
LOAEL(HEC)
LOAEL adjusted for dosimetric differences across species to a human
m
meter
MCL
maximum contaminant level
MCLG
maximum contaminant level goal
MF
modifying factor
mg
milligram
mg/kg
milligrams per kilogram
mg/L
milligrams per liter
MRL
minimal risk level
MTD
maximum tolerated dose
MTL
median threshold limit
NAAQS
National Ambient Air Quality Standards
NOAEL
no-ob served-adverse-effect level
NOAEL(ADJ)
NOAEL adjusted to continuous exposure duration
NOAEL(HEC)
NOAEL adjusted for dosimetric differences across species to a human
NOEL
no-ob served-effect level
OSF
oral slope factor
p-IUR
provisional inhalation unit risk
p-OSF
provisional oral slope factor
p-RfC
provisional inhalation reference concentration
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p-RfD
provisional oral reference dose
PBPK
physiologically based pharmacokinetic
ppb
parts per billion
ppm
parts per million
PPRTV
Provisional Peer Reviewed Toxicity Value
RBC
red blood cell(s)
RCRA
Resource Conservation and Recovery Act
RDDR
Regional deposited dose ratio (for the indicated lung region)
REL
relative exposure level
RfC
inhalation reference concentration
RfD
oral reference dose
RGDR
Regional gas dose ratio (for the indicated lung region)
s.c.
subcutaneous
SCE
sister chromatid exchange
SDWA
Safe Drinking Water Act
sq.cm.
square centimeters
TSCA
Toxic Substances Control Act
UF
uncertainty factor
l^g
microgram
[j,mol
micromoles
voc
volatile organic compound
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PROVISIONAL PEER REVIEWED TOXICITY VALUES FOR
NITROGUANIDINE (CASRN 556-88-7)
Background
On December 5, 2003, the U.S. Environmental Protection Agency's (EPA's) Office of
Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human
health toxicity values for Superfund risk assessments, establishing the following three tiers as the
new hierarchy:
1.	EPA's Integrated Risk Information System (IRIS).
2.	Provisional Peer-Reviewed Toxicity Values (PPRTV) used in EPA's Superfund
Program.
3.	Other (peer-reviewed) toxicity values, including:
~	Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR),
~	California Environmental Protection Agency (CalEPA) values and
~	EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in EPA's Integrated Risk Information System (IRIS). PPRTVs are
developed according to a Standard Operating Procedure (SOP) and are derived after a review of
the relevant scientific literature using the same methods, sources of data, and Agency guidance
for value derivation generally used by the EPA IRIS Program. All provisional toxicity values
receive internal review by two EPA scientists and external peer review by three independently
selected scientific experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the
multi-program consensus review provided for IRIS values. This is because IRIS values are
generally intended to be used in all EPA programs, while PPRTVs are developed specifically for
the Superfund Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a five-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV manuscripts conclude
that a PPRTV cannot be derived based on inadequate data.
Disclaimers
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and RCRA program offices are advised to carefully review the information provided
in this document to ensure that the PPRTVs used are appropriate for the types of exposures and
circumstances at the Superfund site or RCRA facility in question. PPRTVs are periodically
updated; therefore, users should ensure that the values contained in the PPRTV are current at the
time of use.
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It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
users are strongly encouraged to read the entire PPRTV manuscript and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center for OSRTI. Other EPA programs or external parties who may
choose of their own initiative to use these PPRTVs are advised that Superfund resources will not
generally be used to respond to challenges of PPRTVs used in a context outside of the Superfund
Program.
Questions Regarding PPRTVs
Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on
chemicals not covered, or whether chemicals have pending IRIS toxicity values) may be directed
to the EPA Office of Research and Development's National Center for Environmental
Assessment, Superfund Health Risk Technical Support Center (513-569-7300), or OSRTI.
INTRODUCTION
Nitroguanidine (CASRN 556-88-7) is a crystalline solid with explosive properties that
have led to military use in munitions and propellants. It is also used as an intermediate in the
synthesis of some pharmaceuticals (HSDB, 2007). The empirical formula for nitroguanidine is
CH4N4O2.
The U.S. Environmental Protection Agency (U.S. EPA)'s IRIS database (U.S. EPA,
1989) lists a chronic RfD of 0.1 mg/kg-day for nitroguanidine that references a Drinking Water
Health Advisory document (U.S. EPA, 1990a). This RfD is also included in the Drinking Water
Standards and Health Advisories list (U.S. EPA, 2006). Both IRIS (U.S. EPA, 1990b) and the
DWHA list (U.S. EPA, 2006) also include a Group D (not classifiable as to human
carcinogenicity) cancer classification for nitroguanidine. IRIS does not include an RfC
assessment for this chemical. Subchronic or chronic RfDs or RfCs, or cancer assessments for
nitroguanidine are not listed in the HEAST (HEAST; U.S. EPA, 1997). The CARA list
(U.S. EPA, 1991a, 1994) does not include nitroguanidine. No standards for occupational
exposure to nitroguanidine have been established by the American Conference of Governmental
Industrial Hygienists (ACGIH, 2007), the National Institute of Occupational Safety and Health
(NIOSH, 2007) or the Occupational Safety and Health Administration (OSHA, 2006). The
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ATSDR (ATSDR, 2007), International Agency for Research on Cancer (IARC, 2007), and the
World Health Organization (WHO, 2007) have not published toxicological reviews on
nitroguanidine.
Literature searches for studies relevant to the derivation of provisional toxicity values for
nitroguanidine were conducted in MEDLINE, TOXLINE special, and DART/ETIC
(1960s-July 2007); BIOSIS (August 2000-July 2007); TSCATS/TSCATS 2, CCRIS,
GENETOX, HSDB, and RTECS (not date limited); and Current Contents (6 months prior to
July 2007). An updated literature search (June 2007-November 2008) was conducted in
PubMed.
REVIEW OF PERTINENT DATA
Human Studies
No studies investigating the effects of subchronic or chronic oral or inhalation exposure
to nitroguanidine in humans were identified.
Animal Studies
Oral Exposure
Relevant information regarding the toxicity of nitroguanidine comes from a 90-day
dietary study in rats (Morgan et al., 1988a), a 90-day dietary study in mice (Frost et al., 1988), a
developmental toxicity study in rats (Coppes et al., 1988a), a developmental toxicity study in
rabbits (Coppes et al., 1988b), and a 2-generation reproductive toxicity study in rats
(Coppes et al., 1990). Information from these studies and from the Drinking Water Health
Advisory Document for nitroguanidine (U.S. EPA, 1990a) is presented below.
Subchronic Studies—Groups of Sprague-Dawley rats (15/sex/dose level) were
administered 0 (control), 100, 316, or 1000 mg/kg-day nitroguanidine (99.2-99.7% pure) in their
diets for 90 days (Morgan et al., 1988a, unpublished report). Food and water consumption were
measured on a weekly basis and the animals were observed twice daily for clinical signs. Body
weights were recorded weekly and on the days of sacrifice. On day 45, 10 rats (5 of each sex)
were sacrificed and submitted for necropsy. At the interim and terminal sacrifices, blood was
collected for comprehensive hematology and clinical chemistry testing. At the terminal sacrifice,
all major organs and tissues were weighted and processed for microscopic examination. There
were no deaths or clinical signs attributable to dosing with nitroguanidine during the study. Food
consumption was significantly reduced in the high-dose males on week 1 and in the high-dose
females on weeks 5 and 6 relative to the controls (p < 0.05), and it is not clear whether this is due
to food palatability issue. In general, water consumption was elevated in treated rats throughout
the study, and the difference from the controls (25-35% at the high dose) achieved statistical
significance for the mid- and high-dose rats. No significant differences in body weight were
exhibited in the male dose groups. Body weights of high-dose females were significantly lower
than controls at various times during the study and at termination, and the differences from
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control values at the terminal sacrifice were about 8.8%. Results from clinical chemistry tests
showed that there were no significant differences in serum electrolyte levels between the treated
groups and the control groups for either sex. There were some significant differences with the
controls in some serum biochemical parameters, e.g., increased cholesterol levels in high-dose
males at interim and terminal sacrifice, decreased lactate dehydrogenase (LDH) activity and total
protein levels in the mid-dose males at terminal sacrifice, and decreased triglyceride levels in
low- and high-dose females at terminal sacrifice. However, all these values were within normal
limits. Hematology tests were unremarkable. The mid-dose male rats showed a significant
decrease in absolute adrenal gland weight as compared to controls at interim sacrifice, but not at
the terminal sacrifice. There were no consistent, treatment-related changes in any organ weights
(including the adrenal gland) or organ-to-body weight or organ-to-brain weight ratios in male
rats. The females showed significantly decreased ovarian weights for all dose groups at interim
sacrifice, but no significant changes in the ovarian-to-body weight or ovarian-to-brain weight
ratios. The differences in ovarian weight do not appear to be dose related, and the same changes
were not observed at the terminal sacrifice. A statistically significant decrease in brain weight at
interim sacrifice (6%), and a significant increase in relative brain weight (9%) at terminal
sacrifice were noted in high-dose females; although the latter effect could have been related, at
least in part, to the reduced growth rate in high-dose females. Gross and microscopic
examination of the tissues and organs did not reveal any treatment-related lesions, including the
adrenal gland, ovaries, brain, and kidneys. Although there was a dose-related increase in water
consumption in both sexes, it is considered a compensatory effect that may result in an
accelerated excretion of the less soluble test compound compared to related urea which has been
used as an osmotic diuretic. In addition, there were no kidney pathological changes and no
evidence of disturbance of electrolyte levels in the serum. The difference in body weight
between high-dose female rats and controls was not considered adverse because this change was
less than 10% (a change commonly considered as an indication of reaching MTD), and the
decreased weight gain could be attributed to reduced food consumption during the study. The
significant decreases in ovarian weight were not considered adverse because several
considerations: (1) they were not dose related; (2) they were only observed at the interim
sacrifice (not at the terminal sacrifice); (3) there were no significant histo-pathological changes
in the same organ in any of the treated dose groups. Based on these considerations, the high dose
of 1000 g/kg-day can be defined as a freestanding NOAEL in this study.
In a similar study, groups of ICR mice (15/sex/dose-level) were administered 0 (control),
100, 316, or 1000 mg/kg-day nitroguanidine (the purity information was not available) in the diet
for 90 days (Frost et al., 1988, unpublished report). The study protocol and endpoints evaluated
were the same as in the rat study (Morgan et al., 1988a) summarized above. Treatment with
nitroguanidine produced no mortality, and no clinical signs attributable to dosing with
nitroguanidine were observed. Neither body weight gain nor food consumption was significantly
affected by dosing with nitroguanidine. Water consumption was significantly increased in
high-dose females during the second and third week of the study and in high-dose males
throughout most of the study. Hematology values were not significantly affected. In male rats,
the only significant changes in clinical chemistry values were increased aspartate
amino-transferase (AST) activity in the high-dose group at interim sacrifice and increased uric
acid in the mid-dose group at the terminal sacrifice. In female rats, the only significant clinical
chemistry changes were increased albumin levels and albumin-globulin ratio in mid- and
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high-dose groups at the interim sacrifice. However, all these changes remained within the
respective normal ranges and did not exhibit consistent dose-response relationships.
Organ-to-body-weight ratios and organ-to-brain-weight ratios were not significantly different
from the controls, with the exception of an increase in relative brain weight in the high-dose
males at interim sacrifice. Gross and microscopic examination of tissues and organs (including
the brain and kidneys) did not reveal any treatment-related alterations. Thus, the only
treatment-related change was increased water consumption. Similar to the consideration
mentioned in the rat study, this change is not considered an adverse effect due to a lack of
corresponding effects in kidney pathology and serum electrolyte levels. Therefore, the high dose
of 1000 mg/kg-day can be defined as a freestanding NOAEL in this study.
The U.S. EPA (1990a) summarized a lifetime study from the Russian literature
(Korolev et al., 1980), but it also indicated that the animal species, the method of administration,
and the duration of the study were not reported. Based on this information, this study is not
considered in this assessment.
Reproduction/Developmental Studies—Reproductive organs and sex glands have been
examined in both rat and mouse studies (Morgan et al., 1988a; Frost et al., 1988). No significant
alterations in organ weight or in the gross or microscopic appearance of the reproductive organs
from male and female Sprague-Dawley rats and ICR mice treated with up to 1000 mg/kg-day
nitroguanidine were reported in the 90-day dietary studies summarized above.
In a developmental study, groups of presumed-pregnant Sprague-Dawley rats
(23-27/dose level) were administered 0 (vehicle), 100, 316, or 1000 mg/kg-day nitroguanidine
(99.2% pure) in 1% carboxymethylcellulose sodium salt solution by oral gavage on gestation
days 6-15 (Coppes et al., 1988a, unpublished report). The dose for each female was based on
the body weight at gestation day 6, and that dose was used throughout the treatment period.
Maternal gravid body weight and food consumption were monitored on gestation days 0, 6, 10,
15, and 20. Dams were monitored daily for clinical signs of toxicity, abortion, or premature
delivery. On gestation day 20, the dams were sacrificed and the gravid uteri were examined for
number of implantation sites, resorptions, and live and dead fetuses. The fetuses, uterus, and
ovaries were removed; the corpora lutea were counted; and the dams were examined for gross
visceral signs of toxicity, and the remaining maternal body was weighed. Each fetus was sexed,
weighed, measured crown-to-rump, and examined externally. Half of the fetuses were assigned
to skeletal examination and the other half to visceral examination.
A total of 7 rats died during the study; five of these deaths were caused by difficulties
related to the administration of the test material, and the other two deaths occurred in the
high-dose group on gestation days 14 or 16. In addition, 1 dam from the high-dose group was
sacrificed due to a moribund condition. Relative to the controls, high-dose dams lost weight
(8.5%) during treatment days and showed decreased weight gain (49%) during gestation
day 0-20. Food consumption was also significantly reduced in high-dose dams during only the
gestation days 6-15 (46%). Clinical signs occurred in 100% of the high-dose group versus 39%
of the control group during the treatment period. These clinical signs included red urine,
dehydration, red material on the nose/whiskers, red material on the forelimbs, and hunched
posture. During the posttreatment period, 29% of the high-dose group showed clinical signs
versus 9% of the control group. Treatment with nitroguanidine had no significant effect on the
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number of corpora lutea, implantations, resorptions, or live and dead fetuses. The high-dose
fetuses were significantly lighter and shorter in length than the controls. Significant
treatment-related morphological alterations were limited to increased incidences of skeletal
variations in the high-dose fetuses, including fewer ossified sternebrae and caudal vertebrae, and
reduced ossification of the pubis. Treatment with nitroguanidine did not significantly increase
the incidence of external, visceral, or skeletal malformations in treated groups relative to
controls. Because the rats treated with the same 90-day dietary dose (1000 mg/kg-day)
(Morgan et al., 1988a) did not show increases in mortality rate, the two deaths and the one
moribund rat in the high-dose group suggested that the high concentrations of nitroguanidine
necessary to administer the 1000 mg/kg-day dose by oral gavage interfered with the digestive
processes of the animals in this group. This was supported by other signs of maternal toxicity
(e.g., decreased food consumption, decreased body weight gain, and increased incidence of
clinical signs) that were present in the same dose group. Based on increased maternal mortality,
the alterations in maternal body weight and food consumption, and the clinical signs observed in
high-dose dams, the 1000 mg/kg-day dose level can be considered a maternal LOAEL, and the
next lower dose of 316 mg/kg-day can be considered a maternal NOAEL. Based on decreased
fetal weights and increased incidence of skeletal variations, these dose levels also represent
developmental LOAEL and NOAEL values, respectively.
A similar study was conducted with New Zealand White rabbits (Coppes et al., 1988b,
unpublished report). Bred females (16-22/dose level) were dosed daily with 0 (vehicle), 100,
316, or 1000 mg/kg-day nitroguanidine (99.2% pure) in 1% carboxymethylcellulose sodium salt
solution by oral gavage on day 6 through day 18 of gestation. The dose for each female was
based on the gestation day 6 body weight and that dose was used throughout the treatment
period. Females were weighed on days 0, 6, 16, 23, and 29; they were observed daily for clinical
signs of toxicity, abortion, or premature delivery. Food consumption was calculated daily.
Sacrifices were conducted on day 29 of gestation. Non-pregnant rabbits were removed from the
study. Gravid uteri were examined for number of implantation sites, resorptions, and live and
dead fetuses. The fetuses, uterus, and ovaries were removed; the corpora lutea were counted;
each dam was examined for gross visceral signs of toxicity; and the remaining maternal body
was weighed. Each fetus was weighed, measured crown-to-rump, and examined externally. The
viscera from the fetuses were examined for anomalies and the sex was determined. The fetal
skeleton was prepared and examined for malformations, alignment, and degree of ossification.
A total of 6 high-dose rabbits died during the study between days 11 and 19, and
4 additional moribund rabbits were euthanized. All these animals had lost significant weight
between the first dosing day and the day of death. Food consumption in these animals was
significantly reduced. Clinical signs observed in these animals included thick, foamy, granular
orange-rust-colored urine, convulsions, hypertonia, and hunched posture. Gross necropsy
revealed congestion of the internal organs in most of these rabbits. In the high-dose animals that
survived, food consumption during the treatment period was significantly lower than in the
controls and weight loss was recorded. Thick, foamy, orange-rust-colored urine and decreased
amount of feces occurred frequently in the high-dose groups. Clinical signs occurring only in the
high-dose group included hunched posture, hypertonia, increased startle reflex, and death or
moribund condition. Nitroguanidine had no significant effect on the number of corpora lutea,
implantations, live and dead fetuses, or the sex ratio. Fetal weight was significantly reduced in
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the high-dose group compared to the control group. Evaluation of visceral and skeletal
development showed that the number of fetuses with skeletal variations in the high-dose group
was significantly higher than in the control group. The variants most frequently observed were
reduced ossification of the sternebrae, olecranon, patella, and phalanges. No treatment-related
malformations were observed.
The most significant finding of the study was an increase in the number of gravid females
with resorptions at examination on day 29 (3/13 [23%], 13/15 [87%], 7/15 [47%], and
5/11 [45%] in the control, low-, mid-, and high-dose groups, respectively) (Coppes et al., 1988b).
According to the investigators, the differences from the controls were statistically significant in
the low- and high-dose groups using Marascuilo's method of multiple comparison of
proportions. Fisher's Exact Test performed for this review found statistical significance in both
low- and mid-dose groups, but it did not show significant response in the high-dose group. In
addition, a Cochran-Armitage trend test for these data (gravid females with resorptions) did not
demonstrate statistical significance. The investigators also reported that the percent resorptions
per litter [(resorptions/implantations) x 100] was significantly higher in treated groups than in
controls (2.2%, 9.3%, 6.6%), and 17,9%' in the control, low-, mid-, and high-dose groups,
respectively). A further review of the number of resorptions by the study authors prompted a
letter from the investigators to the U.S. EPA providing additional information [Appendix B in
U.S. EPA (1990a)]. In the letter, the investigators stated that the percent resorptions per litter for
control rabbits (2.2%) was lower than found in historical control data for New Zealand White
rabbits (3.3—16%) from the recent literature (historical data were not available at the study
laboratory). The study investigators further stated, "While there is the possibility that the data
may be an accurate assessment of the effect of nitroguanidine on embryonic development in the
rabbit, it is more likely a statistical aberration (page B-l)." However, the preferential use of
literature derived historical control data from other laboratories over the concurrent control data
in the interpretation of study findings is in conflict with the principles and practices described in
the Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA, 1991b). Therefore, it is
not appropriate to use such background data as a criterion to discount the significance of the
observed resorptions in this study. In addition, it is not uncommon to see more sensitive
developmental responses in rabbits than in the rats, and there is no evidence suggesting the
contrary. The high percent resorptions per litter in the high-dose group were also consistent with
the apparent maternal toxicity evidenced by increased mortality and moribund condition and
clinical signs. Based on these considerations, it would appear that the low dose of
100 mg/kg-day could be considered a developmental, freestanding LOAEL on the grounds that it
caused a statistically significant increase in the percentage of resorptions compared with current
control group. This conclusion is in disagreement with the interpretation of this study in
U.S. EPA (1990a) and on IRIS (U.S. EPA, 1989). The highest dose level is a maternal frank
effect level (FEL) as it caused deaths in the dams; the maternal NOAEL is 316 mg/kg-day.
The reproductive and fertility toxicity of nitroguanidine was assessed in a two-generation
study (Coppes et al., 1990, unpublished report). Nitroguanidine (99.7% pure) was fed to
Sprague-Dawley rats (parental [F0] generation) in diet concentrations of 0, 1.3, 4.0, or 12.7 parts
1 Percent resorption in the high-dose group was reported as 9.7% in the original report (Coppes et al., 1988b). The
corrected value of 17.9% was reported by the researchers in a letter to U.S. EPA (see Appendix B of U.S. EPA,
1990a), and it was also confirmed with original data reported in the Appendix I of Coppes et al. (1988b).
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per thousand (ppt) starting at 56-58 days of age and continuing throughout mating
(24-25 pairs/dose mated after 10 weeks of treatment), gestation, and lactation. Pairs of Fi
offspring (19-26/dose) received the same treated feed as the F0 animals and were mated when
approximately 18-weeks old to produce an F2 generation, which was evaluated through weaning.
In young-adult rats, the consumption of nitroguanidine in the treated feed reportedly
approximated the 100, 316, and 1000 mg/kg-day dosages in the developmental studies in rats and
rabbits summarized above. Endpoints that were evaluated include clinical observations, food
consumption (adult and pup), body weight (adult, pup, and litter), male and female mating and
fertility indices, gestation length, numbers of pups delivered, litter size and number, and pup and
litter survival indices. Pathological examinations included gross pathology (all dose groups) and
histopathology including the reproductive system (control and high-dose groups) in F0 and Fi
adult rats at end of breeding (males) or end of lactation (females). Gross pathology was
evaluated in Fi and F2 rats at age 21 days (weaning).
Nitroguanidine had some effect on adult body weight, predominantly in the Fi generation
at 1000 mg/kg-day. Body weights in the 1000 mg/kg-day Fi males and females were
significantly lower than controls (7.6% and 4.9%, respectively) from postweaning week 10 until
week 18 or 19 (mating). Maternal gestation body weights were significantly lower than controls
in the Fo high-dose females on gestation days 7, 14, and 21 (7.7, 7.4, and 8.1%, respectively), in
the low-dose Fi females on gestation day 21 (6.6%) and in the high-dose Fi females on gestation
days 14 and 21 (7.8 and 10.5%, respectively). Maternal postpartum body weights were
significantly lower than controls in the F0 high-dose females on lactation days 0 {12%) and
14 (8.3%>) and in the Fi high-dose females on lactation days 0 (8.9%), 7 (11.4%), and 14 (8.1%).
Food consumption was reduced in both sexes at 1000 mg/kg-day for a few sporadic weeks
during the study. The only observed dose-related effect was reduced body weight in the F0 and
FI females during gestation and lactation; however, this effect is not considered adverse because
most of these changes were less than 10% compared to the control values. No dose-related toxic
effects on reproduction, fertility, or other outcomes were observed. Thus, a NOAEL of
1000 mg/kg-day for reproductive effects and systemic toxicity in adults can be defined in this
study.
Inhalation Exposure
No subchronic, chronic, developmental, or reproduction studies on inhaled nitroguanidine
in animals are identified.
Other Studies
Acute Studies
The information below has been extracted from U.S. EPA (1990a). In rats, the oral LD50
for nitroguanidine in water was 4640 mg/kg and in sunflower-seed oil it was 10,200 mg/kg. In
mice and guinea pigs, the oral LD50 in sunflower-seed oil was 3850 and 3120 mg/kg,
respectively. Other studies reported median lethal doses of >5620 mg/kg and 4340 mg/kg in
male and female ICR mice, respectively, administered nitroguanidine in methylcellulose/Tween
80 and a median lethal dose of >5620 mg/kg for male and female Sprague-Dawley rats also
treated with nitroguanidine in methylcellulose/Tween 80. Signs of CNS stimulation, such as
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seizures, were associated with high doses of nitroguanidine. In general, necropsies did not reveal
significant histopathology other than signs of gastric and intestinal irritation. In a 14-day dietary
study, doses of 1000 mg/kg-day nitroguanidine increased water consumption and decreased
serum calcium and potassium levels in Sprague-Dawley rats (Morgan et al., 1988b). In another
study, no significant effects on survival, food intake, weight gain, or gross pathology were noted
in male albino rats that received up to 930 mg/kg-day nitroguanidine for 30 days (American
Cyanamid, 1955).
Dermal application of a nitroguanidine paste in doses up to 10,000 mg/kg for 24 hours to
albino rabbits produced no signs of toxicity or primary skin irritation, and no significant gross
pathology was reported. Nitroguanidine was classified as a "nonirritating chemical" in New
Zealand White rabbits using the modified Draize method for skin irritation. In another study,
application of 500 mg of nitroguanidine to the shaved intact dorsal surface of New Zealand
White rabbits for 4 hours did not induce dermal irritation, erythema, or edema during a 14-day
observation period. Nitroguanidine was not a dermal sensitizer in a test conducted in Hartley
guinea pigs. Direct application of powdered nitroguanidine to the eye on New Zealand White
rabbits induced slight conjunctival inflammation 1 and 4 hours post-treatment, but nitroguanidine
was not found to be an eye irritant.
Genotoxicity Studies
Assays for the genotoxicity of nitroguanidine (U.S. EPA, 1990a) have generally yielded
negative responses, including assays for mutagenicity in Salmonella typhimurium and L5178Y
mouse lymphoma cells, a mitotic recombination assay in Saccharomyces cerevisiae, dominant
lethal assays in rats and mice, a sister chromatid exchange assay in Chinese hamster ovary cells
(Harbell et al., 1988), a sex-linked recessive lethal mutation assay with Drosophila melanogaster
(Gupta et al., 1993), and a DNA damage (unscheduled DNA synthesis) assay in human
embryonic lung WI-38 cells. However, there was limited evidence of chromosome aberrations
in Chinese hamster fibroblast (lung) cells at high doses that may have been cytotoxic (U.S. EPA,
1990a). Overall, the available data suggest that nitroguanidine is not a genotoxic hazard.
DERIVATION OF A PROVISIONAL SUBCHRONIC ORAL p-RfD
FOR NITROGUANIDINE
There is an RfD of 0.1 mg/kg-day for nitroguanidine on IRIS (U.S. EPA, 1989). The
RfD is based on a LOAEL of 1000 mg/kg-day and a NOAEL of 316 mg/kg-day for reduced
weight gain in female rats in a 90-day dietary study (Morgan et al., 1988a), maternal and fetal
toxicity in rats exposed during gestation (Coppes et al., 1988a), and evidence of maternal
mortality and developmental toxicity in rabbits (Coppes et al., 1988b). However, reanalysis of
the rabbit developmental data (Coppes et al., 1988b) in this assessment led to the conclusion that
the 100 mg/kg-day dose level was a freestanding LOAEL for developmental effects. Thus, the
subchronic oral p-RfD for nitroguanidine was derived using the LOAEL of 100 mg/kg-day as the
POD.
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Both 90-day studies evaluated clinical signs, hematology, clinical chemistry parameters,
and gross and microscopic appearance of tissues and organs. Nitroguanidine exhibited little
toxicity in these studies. High-dose female rats gained less weight than controls beginning on
week 5 of the study, but the difference between the two groups was less than 10%. Final body
weight in the high-dose females was 8.8% lower than controls. This effect may have been
related to a significant decrease in food consumption that occurred on weeks 5 and 6. Both
studies also showed treatment-related increases in water consumption; however, there was no
evidence of kidney pathology or disturbed serum electrolytes in the treated animals. In mice
(Frost et al., 1988), the only significant effect from nitroguanidine exposure was an increase in
the relative brain weight of male mice at interim sacrifice on day 45 of the study. In contrast to
the response in male mice, a similar effect was only observed in female rats (Morgan et al.,
1988a), and this effect could have been related to the reduced growth rate in treated rats. Neither
study reported positive pathological findings in the brain tissue. Based on the lack of any
biologically significant consistent adverse effect, the highest dose level of 1000 mg/kg-day
represents a NOAEL in these 90-day studies.
In the rat developmental study, nitroguanidine at 1000 mg/kg-day significantly reduced
food consumption and rats lost weight during treatment days (Coppes et al., 1988a). This could
have caused, at least in part, the fetuses in this treated group to be significantly lighter in weight
and shorter in length than those in the control group. Skeletal variations were also increased at
this dose level, and significantly fewer ossified sternebrae and caudal vertebrae were reported.
No developmental effects were observed in the 316 mg/kg-day dose group. In pregnant rabbits,
exposure to nitroguanidine in dose amounts of 1000 mg/kg-day produced clinical signs and death
in the does, reduced food consumption during the treatment period (gestation days 6-18), and
weight loss (Coppes et al., 1988b). This dose level significantly reduced fetal weight, but not
body length, and increased the incidence of skeletal variations. A significant finding in this
study is that treatment with nitroguanidine significantly increased the percentage of resorptions
per litter in all treated groups (as low as 100 mg/kg-day), although a dose-response trend was not
apparent as shown in a Cochran-Armitage trend test. As mentioned above, a further review of
the data revealed that the percent resorptions in the treatment dose as low as 100 mg/kg-day
should be considered significantly different from the controls, and as a freestanding LOAEL in
this study. The 2-generation reproductive study only identified a high NOAEL of
1000 mg/kg-day.
The lowest LOAEL in the overall database is 100 mg/kg-day for developmental effects
(increased resorptions) in rabbits. Although all the treated groups in the rabbit study showed
significantly increased resorptions, there is significant noise in the dose response relationship in
the data. For the litter-based response (percentage of gravid females with resorptions), the
lowest dose group presented a far greater response (87%) than those in the mid- and high-dose
groups (45-47%)). Similarly, the pup-based resorption rate in the low-dose group (9.3%>) was
higher than that in the mid-dose group (6.6%>). Thus, these data are not amenable to BMD
modeling. Therefore, the LOAEL of 100 mg/kg-day is used as the POD for the derivation of the
subchronic p-RfD.
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The subchronic p-RfD of 0.1 mg/kg-day is calculated by applying a composite
uncertainty factor of 1000 to the developmental POD of 100 mg/kg-day, as follows:
Subchronic p-RfD = LOAEL UF
= 100 mg/kg-day 1000
= 0.1 mg/kg-day or 1 x 10"1 mg/kg-day
The composite UF of 1000 was composed of the following:
•	An UF of 10 is applied for interspecies extrapolation to account for potential
pharmacokinetic and pharmacodynamic differences between laboratory animals
and humans.
•	An UF of 10 for intraspecies differences is used to account for potentially
susceptible individuals in the absence of information on the variability of
response in humans.
•	An UF of 10 is applied because a LOAEL, instead of NOAEL, was used as the
POD.
•	An UF is not applied to account for uncertainty in the database. The database
includes comprehensive subchronic studies in 2 animal species, 2 developmental
toxicity studies in 2-animal species and a 2-generation reproduction study in rats.
Confidence in the critical study is medium. It was a well conducted GLP study that had
an adequate number of animals and evaluated comprehensive toxicological endpoints. However,
the study did not identify a NOAEL, and the dose-response data for the critical effect (i.e.,
increased resorptions rate) may be confounded by high mortality and moribund rate in the
high-dose group. Confidence in the database is high. The available database is comprehensive,
including 2 subchronic studies in 2 animal species, 2 developmental studies in 2 species, and a
2-generation reproductive study. In considering the confidences in both the key study and the
database, the overall confidence in the resulted subchronic p-RfD is medium.
The chronic RfD of 0.1 mg/kg-day is currently listed on IRIS (U.S. EPA, 1989);
therefore, no chronic p-RfD is derived in this document. Note that the IRIS RfD is based on
systemic toxicity from several studies (reduced weight gain in female rats, developmental and
maternal toxicity at 1000 mg/kg-day body weight). This subchronic p-RfD is based on
reproductive effects at 100 mg/kg-day in one of the same studies.
FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION p-RfC VALUES FOR NITROGUANIDINE
No studies investigating the effects of subchronic or chronic inhalation exposures to
nitroguanidine in humans or animals are identified. The lack of suitable data precludes
derivation of subchronic and chronic p-RfCs for nitroguanidine.
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PROVISIONAL CARCINOGENICITY ASSESSMENT FOR NITROGUANIDINE
Weight-of-Evidence Descriptor
Studies evaluating the carcinogenic potential of oral or inhalation exposure to
nitroguanidine in humans or animals were not identified in the available literature. The available
genotoxicity data suggest that nitroguanidine is not a genotoxic hazard. Nitroguanidine is
classified on IRIS (U.S. EPA, 1990b) in Group D, not classifiable as a human carcinogen on the
basis that pertinent data regarding carcinogenicity were not located in the available literature.
Under the 2005 Guidelines for Carcinogenic Risk Assessment (U.S. EPA, 2005), nitroguanidine
is characterized as "Inadequate Information to Assess the Carcinogenic Potential."
Quantitative Estimates of Carcinogenic Risk
The lack of suitable data precludes the derivation of quantitative cancer risk estimates for
nitroguanidine.
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