Screening-Level Hazard Characterization 4-Nitro-N-methylphthalimide (CASRN 41663-84-7)

U.S. Environmental Protection Agency
Hazard Characterization Document
September, 2014
SCREENING-LEVEL HAZARD CHARACTERIZATION
4-Nitro-N-methylphthalimide
(CASRN 41663-84-7)
The High Production Volume (HPV) Challenge Program1 was conceived as a voluntary initiative
aimed at developing and making publicly available screening-level health and environmental
effects information on chemicals manufactured in or imported into the United States in quantities
greater than one million pounds per year. In the Challenge Program, producers and importers of
HPV chemicals voluntarily sponsored chemicals; sponsorship entailed the identification and
initial assessment of the adequacy of existing toxicity data/information, conducting new testing if
adequate data did not exist, and making both new and existing data and information available to
the public. Each complete data submission contains data on 18 internationally agreed to "SIDS"
(Screening Information Data Setl1'2) endpoints that are screening-level indicators of potential
hazards (toxicity) for humans or the environment.
The Environmental Protection Agency's Office of Pollution Prevention and Toxics (OPPT) is
evaluating the data submitted in the HPV Challenge Program on approximately 1400 sponsored
chemicals by developing hazard characterizations (HCs). These HCs consist of an evaluation of
the quality and completeness of the data set provided in the Challenge Program submissions.
They are not intended to be definitive statements regarding the possibility of unreasonable risk of
injury to health or the environment.
The evaluation is performed according to established EPA guidance2'3 and is based primarily on
hazard data provided by sponsors; however, in preparing the hazard characterization, EPA
considered its own comments and public comments on the original submission as well as the
sponsor's responses to comments and revisions made to the submission. In order to determine
whether any new hazard information was developed since the time of the HPV submission, a
search of the following databases was made from one year prior to the date of the HPV
Challenge submission to the present: (ChemID to locate available data sources including
Medline/PubMed, Toxline, HSDB, IRIS, NTP, AT SDR, IARC, EXTOXNET, EPA SRS, etc.),
STN/CAS online databases (Registry file for locators, ChemAbs for toxicology data, RTECS,
Merck, etc.), Science Direct and ECHA4. OPPT's focus on these specific sources is based on
their being of high quality, highly relevant to hazard characterization, and publicly available.
OPPT does not develop HCs for those HPV chemicals which have already been assessed
internationally through the HPV program of the Organization for Economic Cooperation and
Development (OECD) and for which Screening Initial Data Set (SIDS) Initial Assessment
Reports (SIAR) and SIDS Initial Assessment Profiles (SIAP) are available. These documents are
presented in an international forum that involves review and endorsement by governmental
authorities around the world. OPPT is an active participant in these meetings and accepts these
documents as reliable screening-level hazard assessments.
1 U.S. EPA. High Production Volume (HPV) Challenge Program; http://www.epa.gov/chemrtk/index.htm.
2 U.S. EPA. HPV Challenge Program - Information Sources; http://www.epa.gov/chemrtk/pubs/general/guidocs.htm.
3 U.S. EPA. Risk Assessment Guidelines; http://cfpub.epa.gov/ncea/raf/rafguid.cfm.
4 European Chemicals Agency, http://echa.europa.eu.

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These hazard characterizations are technical documents intended to inform subsequent decisions
and actions by OPPT. Accordingly, the documents are not written with the goal of informing the
general public. However, they do provide a vehicle for public access to a concise assessment of
the raw technical data on HPV chemicals and provide information previously not readily
available to the public.
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Chemical Abstract
Service Registry Number
	(CASRN)	
41663-84-7
Chemical Abstract Index
Name
lH-Isoindole-l,3(2H)-dione, 2-methyl-5-nitro-
Structural Formula
o
o
N	
O
O
0=ClN(C(=0)c2ccc([N+](=0)[0-])ccl2)C
Summary
4-Nitro-N-methylphthalimide is a solid substance with moderate water solubility and low
vapor pressure that hydrolyzes in water. 4-Nitro-N-methylphthalimide and its hydrolysis
product N-methyl-4-nitrophthalamic acid are expected to possess high mobility in soil. 4-
Nitro-N-methylphthalimide was not readily biodegradable using a standard OECD method;
however, it hydrolyzes after several hours at neutral pH and, therefore, is not persistent in the
environment. Volatilization is considered minimal. The rate of atmospheric photooxidation is
considered moderate. 4-Nitro-N-methylphthalimide is expected to have low persistence (PI)
and low bioaccumulation potential (Bl).
Acute oral and inhalation toxicity of 4-nitro-N-methylphthalimide to rats and acute dermal
toxicity of 4-nitro-N-methylphthalimide to rabbits is low. Following repeated oral
administration of 4-nitro-N-methylphthalimide to rats ,via gavage, for 13 weeks, increased
absolute liver and spleen weights and differences in hematology were observed at 10 mg/kg-
day; the NOAEL is 1 mg/kg-day. In an oral (gavage) modified combined
reproductive/developmental toxicity screening test in rats, decreased body weights were noted
in F0 males and increased pigment in the spleen was noted in F0 and adult F1 females with
decreased hemoglobin and hematocrit and increased spleen weight in adult F1 females at 25
mg/kg-day; the NOAEL for systemic toxicity is 2.5 mg/kg-day. No effects were observed on
any reproductive parameters; the NOAEL for reproductive toxicity is 25 mg/kg-day (highest
dose tested). In an oral (gavage) prenatal developmental toxicity study in rabbits, decreased
maternal body weight gain and body weight were observed in does at 50 mg/kg-day, with a
NOAEL for maternal toxicity of 10 mg/kg-day. Increased numbers of fetuses with vertebral
malformations were observed at > 50 mg/kg-day and additional effects (other malformations,
increased late resorptions) seen at 200 mg/kg-day; the NOAEL for developmental toxicity is
10 mg/kg-day. In an oral (gavage) prenatal developmental toxicity study in rats, dams
exhibited decreased body weight at > 50 mg/kg-day; the NOAEL for maternal toxicity is 10
mg/kg-day. Skeletal variations (i.e., incomplete ossification of the pubis, vertebrae or
sternebrae) were seen in fetuses at > 50 mg/kg-day with additional effects (soft tissue
variations, increased resorptions and decreased fetal weights) seen at the highest dose of 200
mg/kg-day; the NOAEL for developmental toxicity is 10 mg/kg-day. 4-Nitro-N-	
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methylphthalimide was mutagenic to both bacteria and mammalian cells in vitro, but did not
induce chromosomal aberrations in mammalian cells in vitro. 4-Nitro-N-methylphthalimide is
irritating to rabbit skin and eyes.
No adequate data are available to evaluate the potential acute and chronic toxicity of 4-nitro-
N-methylphthalimide to aquatic organisms.
Data gaps for the acute toxicity to fish and aquatic invertebrates and toxicity to aquatic plants
endpoints were identified under the HPV Challenge Program.	
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The sponsor, General Electric Company-Plastics (GE Plastics), submitted a Test Plan and Robust
Summaries to EPA for 4-Nitro-N-Methylphthalimide (CASRN 41663-84-7; CAName: 1H-
isoindole-l,3(2H)-dione, 2-methyl-5-nitro-]) on December 31, 2002. EPA posted the submission
on the ChemRTK HPV Challenge website on January 30, 2003
(http://www.epa.gov/chemrtk/pubs/summaries/4nitronm/cl4219tc.htm). EPA comments on the
original submission were posted to the website on June 11, 2003. Public comments were also
received and posted to the website. The sponsor submitted updated/revised documents on
August 5, 2003 and March 31, 2005, which were posted to the ChemRTK website on September
5, 2003 and May 31, 2005, respectively.
1. Chemical Identity
1.1 Identification and Purity
4-nitro-N-methylphthalimide is a solid substance that is manufactured as either a 0.3% slurry or
a 30-60% wet cake.
1.2 Physical-Chemical Properties
The physical-chemical properties of this compound are summarized in Table 1. 4-nitro-N-
methylphthalimide has low vapor pressure and hydrolyzes in water.
Table 1. Physical-Chemical Properties of 4-Nitro-N-methylphthalimide1
Property
Value
CASRN
41663-84-7
Molecular Weight
206.16
Physical State
Solid
Melting Point
181-182°C (measured)
Boiling Point
Decomposes at 300°C (measured)
Vapor Pressure
1.7xl06mm Hg at 25°C (estimated)2
Dissociation Constant
(pKa)
Not applicable
Henry's Law Constant
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2. General Information on Exposure
2.1 Production Volume and Use Pattern
4-Nitro-N-methylphthalimide had an aggregated production and/or import volume in the United
States between 10 to 50 million pounds during calendar year 2005.
Non-confidential information in the IUR indicated that the industrial processing and uses of the
chemical include other basic organic chemical manufacturing as intermediates and some
information was indicated as not readily obtainable (NRO). Also, non-confidential commercial
and consumer information for the chemical was reported as NRO.
2.2 Environmental Exposure and Fate
4-Nitro-N-methylphthalimide is moderately soluble in water and is expected to have high
mobility in soil. It achieved 8.7% of its theoretical biochemical oxygen demand (BOD) in 28
days using the closed bottle (OECD 301D) test; however, these results likely pertain to the
hydrolysis product N-methyl-5-nitrophthalamic acid. The hydrolysis half-life of 4-nitro-N-
methylphthalimide in buffered solutions containing 1.8% acetonitrile at 21°C was 54, 6.4, and
0.5 hours at pH 5, 7, and 9, respectively. Volatilization is considered minimal based on Henry's
Law constant and the fact that this substance hydrolyzes quickly. The hydrolysis product is a
phthalamic acid derivative that will exist as an anion under environmental conditions. The rate
of atmospheric photooxidation is considered moderate. Due to the rapid rate of hydrolysis, 4-
nitro-N-methylphthalimide is expected to have low persistence (PI) and low bioaccumulation
potential (Bl).
The environmental fate properties of this compound are provided in Table 2.
Conclusion: 4-Nitro-N-methylphthalimide is a solid substance with moderate solubility and low
vapor pressure that hydrolyzes in water. 4-Nitro-N-methylphthalimide and its hydrolysis product
N-methyl-4-nitrophthalamic acid are expected to possess high mobility in soil. 4-Nitro-N-
methylphthalimide was not readily biodegradable using a standard OECD method; however, it
hydrolyzes after several hours at neutral pH and, therefore, is not persistent in the environment.
Volatilization is considered minimal. The rate of atmospheric photooxidation is considered
moderate. 4-Nitro-N-methylphthalimide is expected to have low persistence (PI) and low
bioaccumulation potential (Bl).
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Table 2. Environmental Fate Properties of 4-Nitro-N-methylphthalimide 1
Property
Value
CASRN
41663-84-7
Photodegradation Half-life
8.2 hours (estimated)2
Hydrolysis Half-life
54 hours at pH 5 and 21°C (measured);
6.4 hours at pH 7 and 21°C (measured);
0.5 hours at pH 9 and 21°C (measured)
Biodegradation
8.7% after 28 days (not readily biodegradable, OECD 301D)1'3
Bioaccumulation Factor
BAF = 2.9 (estimated)2
Log Koc
1.4 (estimated)2
Fugacity
(Level III Model)2'4
Air (%)
Water (%)
Soil (%)
Sediment (%)
0.1
36.7
63.2
<0.1
Persistence5
PI (low)
Bi oaccumul ati on5
Bl (low)
General Electric Corporation. 2005. Revised Test Plan and Robust Summary for 4-Nitro-N-methylphthalimide.
Available online at http://www.epa.gov/chemrtk/pubs/summaries/4nitronm/cl4219tc.htm as of September 24,
2012.
2U.S. EPA. 2012. Estimation Programs Interface Suite™ for Microsoft® Windows, v4.10. U.S. Environmental
Protection Agency, Washington, DC, USA. Available online at
http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm as of September 24, 2012.
3The test substance hydrolyzes rapidly; therefore, these data primarily pertain to the hydrolysis product.
4Half-lives of 6.4 hours (hydrolysis half-life at pH 7) were used for the water, soil, and sediment compartment.
^Federal Register. 1999. Category for Persistent, Bioaccumulative, and Toxic New Chemical Substances. Federal
Register 64, Number 213 (November 4, 1999) pp. 60194-60204.
3. Human Health Hazard
A summary of the human health toxicity data for SIDS and other endpoints is provided in
Table 3.
Acute Oral Toxicity
Sprague-Dawley rats (5/sex/dose) were administered 4-nitro-N-methylphthalimide (in 0.5%
carboxymethylcellulose) via gavage at 2000, 3500 or 5000 mg/kg and observed for 14 days.
Mortality occurred at all doses (1, 8, and 10 rats at 2000, 3500, 5000 mg/kg, respectively).
LDso = 2800 mg/kg
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Acute Inhalation Toxicity
Sprague-Dawley rats (5/sex) were exposed to 4-nitro-N-methylphthalimide at concentration of
36 mg/L for 4 hours and monitored for 14 days following exposure. No mortalities occurred.
LCso > 36 mg/L
Acute Dermal Toxicity
New Zealand White rabbits (5/sex) were administered 4-nitro-N-methylphthalimide (moistened
with 0.5% carboxymethylcellulose) at 2000 mg/kg to clipped, abraded skin under occluded
conditions for 24 hours. Following exposure, application sites were rinsed with luke-warm water
and animals were monitored for 14 days. No mortality occurred.
LD50 > 2000 mg/kg
Repeated-Dose Toxicity
(1) In a 13-week repeated-dose toxicity study, Sprague-Dawley rats (10/sex/dose) were
administered 4-nitro-N-methylphthalimide (>95% purity) via gavage at 0, 0.1, 1.0 or 10 mg/kg-
day. Clinical signs, body weights and food consumption were evaluated. In addition,
ophthalmological examination, hematology, clinical biochemistry, gross necropsy and
histopathological examination of unspecified tissues were performed at study termination.
Weights of the spleen, liver, kidneys, testes, epididymides and adrenals were measured. All rats
survived the study period and there were no effects on food consumption or body weight. Males
had decreased eosinophils and females had increased reticulocytes at the highest dose. Increased
absolute liver and spleen weights were observed in high-dose females. Not treatment-related
histopathological changes were seen.
LOAEL = 10 mg/kg-day (based on increased absolute liver and spleen weights and changes in
hematology)
NOAEL = 1.0 mg/kg-day
(2) Sprague-Dawley rats (5/sex/dose) were administered 4-nitro-N-methylphthalimide (in 0.5%
carboxymethyl cellulose) via gavage at 0, 10, 100, 500 or 1000 mg/kg-day for 29 days. Clinical
signs and body weight were evaluated as well as organ weights and food consumption. In
addition, hematology, clinical biochemistry, gross necropsy and histopathology of selected
tissues were examined at study termination. This study was conducted as a dose range-finding
study for the 13 week study. At 1000 mg/kg-day, 1 male and 3 females died. Decreases in body
weight and body weight gain were seen at 500 and 1000 mg/kg-day and food consumption was
decreased at the highest dose (p < 0.05). Changes in clinical chemistry included decreased red
blood cell mass, bone marrow myeloid/erythroid ratios, increased reticulocyte fractions and
increased reticulocytes in peripheral blood were seen at > 100 mg/kg-day (p<0.05). The
increased reticulocytes in peripheral blood were originally misidentified as elevated white blood
cell counts. Decreased potassium and chloride was observed at > 500 mg/kg-day (p < 0.05).
Increased total bilirubin was seen (significant in males at > 100 mg/kg-day; p < 0.05). Males
also had increased calcium at 500 and 1000 mg/kg-day (p < 0.05). Gamma glutamyl
transpeptidase was increased at the highest dose (p < 0.05). Enlarged and darkly discolored
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spleens were seen at > 100 mg/kg-day and darkened kidneys and liver were seen at > 500 mg/kg-
day. Absolute and relative spleen weights were increased at > 100 mg/kg-day (p < 0.05).
Increased relative liver weights were seen at > 10 mg/kg-day (males) and >100 mg/kg-day
(females) (p < 0.05). Increase relative kidney weights were seen at > 100 mg/kg-day (p < 0.05).
Hemosiderin was seen in macrophages in the spleen, in Kupffer cells and in renal proximal
tubules at > 100 mg/kg-day. These effects were characterized by the report authors as a
significant level of hemolysis with deposition of hemosiderin in the spleen, liver and kidney
(TSCATS OTS0540088).
LOAEL = 100 mg/kg-day (based on hemolysis and deposition of hemosiderin in spleen, liver
and kidneys)
NOAEL = 10 mg/kg-day
(3) In a 4-week study, Sprague-Dawley rats (10/sex/concentration) were administered 4-nitro-N-
methylphthalimide at dietary concentrations of 0, 0.125%, 0.25% and 0.5%. For males,
equivalent doses were ~ 90-107 mg/kg-bw/day for the low dose (weeks 1-4), -206 mg/kg-day
for the middle dose (week 2 only) and -555 mg/kg-bw/day for high dose (week 2 only). In
females, the estimated doses in mg/kg-bw/day were - 102-130 for the low dose (weeks 1-4),
-192 for the middle dose (week 2 only) and was not estimated at the high dose. Clinical signs,
body weight, liver and kidney weights and food consumption were evaluated. In addition, gross
necropsy and histopathology of selected tissues were conducted at study termination. No clinical
chemistry or hematological examinations were conducted. High dose females had higher
mortality. Increased food spillage at both the 0.25% and 0.5% concentrations precluded
measuring food consumption for several weeks; the report authors suggested that the diet might
have been impalatible, resulting in inanition and hunching/thinness at these concentrations.
Decreased body weights (both sexes) were seen at all doses (p<0.05). Other effects included
decreased absolute liver and kidney weights at all concentrations (males) or at the high dose
(females). Also, females had decreased absolute kidney weights at the middle dose. Increases in
relative liver and kidney weights, however, were observed at either the highest or the two highest
doses in males and/or females (p <0.05). Spleen enlargement, increased incidences of stomach
lesions and small thymuses were observed (both sexes) and males showed a dose-related
decrease in testes and seminal vesicle size (TSCATS OTS0540088). Due to limited information
on equivalent doses, the LOAEL and NOAEL were not determined.
Reproductive Toxicity
In a modified combined reproductive/developmental toxicity screening test, Sprague-Dawley rats
(10/sex/dose) were administered 4-nitro-N-methylphthalimide (97.4% purity) (in 0.5%
carboxymethylcellulose) via gavage at 0, 0.25, 2.5, or 25 mg/kg-day for 4 weeks (males;
premating and mating) or approximately 10 weeks (females; premating, mating, gestation and
lactation). Five additional F0 males per group for the controls and 25 mg/kg-day rats were
observed without dosing for two additional weeks after the dosing period to evaluate recovery.
F1 offspring were dosed from weaning until sacrifice on postnatal day 85. In F0 animals,
decreased food consumption was observed in high-dose animals of both sexes and decreased
body weights were observed in high-dose males. Increased pigment was seen in the spleen of 4
high-dose females. No effects were observed following clinical chemistry, hematology, organ
weights, gross necropsy and histopathological examinations. In F1 adults, there were no
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differences between male animals among groups, while in females, decreased hemoglobin and
hematocrit, increased pigment in the spleen (mild to minimal severity) and increased relative
spleen weight were noted at the high-dose. No treatment-related effects were noted on any
reproductive parameter evaluated, including precoital interval, mating, fertility and gestational
indices, number of implantation sites, postimplantation loss, number of pups per litter, number of
live and dead pups per litter, number of pregnant females, live birth index, survival index, sex
ratio, anogenital distance, pup weights, age of vaginal opening, age of preputial separation,
estrous cycle length and sperm parameters. In the recovery group males, there were no effects
on body weight, organ weights, or necropsy compared with controls.
LOAEL (systemic toxicity) = 25 mg/kg-day (based on decreased body weight in F0 males and
increased pigment in the spleen of F0 and F1 adult females, decreased hemoglobin and
hematocrit and increased spleen weight in F1 females)
NOAEL (systemic toxicity) = 2.5 mg/kg-day
NOAEL (reproductive toxicity) = 25 mg/kg-day (highest dose tested)
Developmental Toxicity
(1) In a prenatal developmental toxicity study, pregnant female New Zealand White rabbits
(16/dose) were administered 4-nitro-N-methylphthalimide (>95% purity), in 0.5%
carboxymethyl cellulose, via gavage at 0, 10, 50 or 200 mg/kg-day on days 6 through 19 of
gestation. On gestation day 29, the animals were sacrificed and the uterus was excised, weighed
and examined. Viable fetuses were sacrificed, weighed, sexed and examined for external
abnormalities. Fetuses were also prepared for the evaluation of visceral and skeletal
malformations and variations. Anorexia was noted in all treated animals. A marked decrease in
mean maternal body weight was observed at 200 mg/kg-day compared to controls. A slight
weight loss was observed in mid-dose females on gestation days 6-11 and decreased body
weight gain was observed on gestation days 11 - 15. There were no treatment-related gross
pathological findings in dams at necropsy. At the high dose, there were increases in the number
of late resorptions, incidence of implants affected and total number of litters with affected
implantations as indicated in the robust summary. Information in the TSCATS submission
shows a positive significant trend test for skeletal malformations. Specific malformations
included vertebral anomaly with/without rib anomaly (2 and 4 fetuses affected at 50 and 200
mg/kg-day, respectively). These numbers translate to 0.14 and 0.29 fetuses/litter, respectively
versus none in controls. Other malformations seen at the highest dose included small forelimb,
agenesis of digit, hyperflexed forepaw, malformed scapula, bent clavicle and humerus
malaligned with scapula. A positive trend test for malformations was seen for both skeletal and
total malformations. In addition, at the highest dose, the number of litters with skeletal
malformations was higher (p<0.05) compared with controls (robust summary and TSCATS
OTS0000170-3).
LOAEL (maternal toxicity) = 50 mg/kg-day (based on decreased maternal body weight and
body weight gain)
NOAEL (maternal toxicity) = 10 mg/kg-day
LOAEL (developmental toxicity) = 50 mg/kg-day (based on increased vertebral
malformations)
NOAEL (developmental toxicity) = 10 mg/kg-day
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(2) In a prenatal developmental toxicity study pregnant female Sprague-Dawley rats (24/dose)
were administered 4-nitro-N-methylphthalimide (>95% purity) ,in 0.5% carboxymethyl
cellulose, via gavage at 0, 10, 50 or 200 mg/kg-bw/day on days 6 through 15 of gestation. On
gestation day 20, the animals were sacrificed and the uterus was excised, weighed and examined.
The ovaries of each female were examined for the number of corpora lutea. Viable fetuses were
sacrificed, weighed, sexed and examined for external abnormalities. Fetuses were also prepared
for the evaluation of visceral and skeletal malformations and variations. Clinical signs of
toxicity were seen in high-dose dams (i.e., thinness/anorexia, hunched posture, rough coat, urine
stains, sensitivity to touch, squinted eyes, rhinorrhea and vaginal discharge). Decreased body
weight gain and food consumption were observed at 50 and 200 mg/kg-day and reduced body
weight was observed at the highest dose. One dam at 200 mg/kg-day had tan and green material
in the uterine horns; one dam at 50 mg/kg-day had dilated renal pelvises. The mean gravid
uterine weight was decreased in the high-dose group compared to controls. At the high dose, an
increase in the percent of total resorptions per litter and decreases in mean male, female, and
combined fetal body weights were observed (p<0.05). In the high-dose group, four fetuses had
small or absent pinnae and one had a short tail. No soft tissue or skeletal malformations were
observed. Statistically significantly increased soft tissue variations consisting of small renal
papilla, no renal papilla and moderately or severely dilated ureters were observed at 200 mg/kg-
day (p level not stated). Statistically significant increases in skeletal variations consisting of
incomplete ossification of the pubis, vertebrae or sternebrae were observed at 50 and 200 mg/kg-
day (p level not stated).
LOAEL (maternal toxicity) = 50 mg/kg-day (based on decreased body weight gain, multiple
clinical signs)
NOAEL (maternal toxicity) = 10 mg/kg-day
LOAEL (developmental toxicity) = 50 mg/kg-day (based on incomplete ossification of the
pubis, vertebrae or sternebrae)
NOAEL (developmental toxicity) = 10 mg/kg-day
Genetic Toxicity — Gene Mutations
In vitro
In a reverse mutation assay, Salmonella typhimurium strains TA98, TA100, TA1535, TA1537
and TA1538 were exposed to 4-nitro-N-methylphthalimide (>95% purity) at concentrations of 0,
1, 10, 100, 500, 1000, 2500, 5000 and 10,000 |ig/plate in the presence and absence of metabolic
activation. The test was run in duplicate. Positive and solvent controls were tested concurrently
and responded appropriately. Cytotoxicity was observed at > 5000 |ig/plate and precipitation at
10,000 |ig/plate. The mutagenic effects were seen at >2500 |ig/plate with TA98 and TA 100 and
at 10,000 |ig/plate with TA1538 in the presence and absence of metabolic activation.
4-Nitro-N-methylphthalimide was mutagenic in this assay.
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Genetic Toxicity - Chromosomal Aberrations
In vitro
In an in vitro cytogenetic assay, Chinese hamster ovary (CHO) cells were exposed to 4-nitro-N-
methylphthalimide (>95% purity) in the presence (two trials) and absence (three trials) of
metabolic activation. The concentrations of 4-nitro-N-methylphthalimide used in the absence of
metabolic activation were 0, 300, 350, 400, 450, or 500 |ig/mL for trial 1, 0, 340, 388 or 485
|ig/mL for trial 2 and 600 - 1000 |ag/m L for trial 3. In the first trial without activation, there was
a slight increase in the number of aberrations, which was not reproducible in the second or third
trial. A white precipitate was seen in all cultures from the third trial. The first trial conducted in
the presence of metabolic activation was abandoned because of microbial contamination. In the
second trial, CHO cells were exposed to concentrations of 0, 600, 700, 800, 900 or 1000 |ig/mL.
No treatment-related increase in chromosome aberrations was found. Positive and negative
controls were tested concurrently and responded appropriately. The cytotoxic concentration of
4-nitro-N-methylphthalimide was 500 |ig/mL.
4-Nitro-N-methylphthalimide did not induce chromosomal aberrations in this assay.
Genetic Toxicity — Other
Mouse lymphoma cells heterozygous at the L5178 TK +/- locus were exposed to 4-nitro-N-
methylphthalimide at concentrations of 0, 62.5, 125, 250, 500 or 1000 |j,g/mL in the absence of
metabolic activation and 0, 0.977, 1.95, 3.91, 7.81 or 15.6D ng/mL (trial 1) or 0, 1.25, 2.5, 5, 10,
15 or 20 |j,g/mL (trial 2) in the presence of metabolic activation. Positive controls were tested
concurrently and responded appropriately. Cytotoxicity was observed at concentrations > 20
Hg/mL in the presence of metabolic activation and > 1000 |j,g/mL in the absence of metabolic
activation. Precipitation occurred at > 1000 |j,g/mL. 4-Nitro-N-methylphthalimide was not
mutagenic without metabolic activation, but was positive for mutagenicity in the presence of
metabolic activation. Information on colony sizes was not available and therefore, it is not
known whether the effects are related to gene mutations or chromosomal aberrations.
4-Nitro-N-methylphthalimide was mutagenic in this assay.
Additional Information
Skin Irritation
In six female Albino rabbits, 4-Nitro-N-methylphthalimide was applied to intact and abraded
skin (0.5 g/site) under occluded conditions for 24 hours. Slight erythema was observed at both
abraded and intact sites in one of six rabbits at 24 but not 72 hours after application (TSCATS
OTS0540086).
4-Nitro-N-methylphthalimide was slightly irritating to rabbit skin.
Eye Irritation
In each of 9 female Albino rabbits, 0.1 g of 4-Nitro-N-methylphthalimide was placed in the left
eyes with right eyes serving as controls. In three rabbits, eyes were rinsed 30-60 seconds after
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instillation. Slight conjunctival reddening at 24 hours was seen in one rabbit with rinsed eyes.
Conjunctival redness and discharge were also seen in rabbits whose eyes were not washed for up
to seven days and chemosis was seen in one of these rabbits at 24 hours (TSCATS
OTS0540086).
4-Nitro-N-methylphthalimide was slightly irritating to rabbits' eyes.
Conclusion: Acute oral and inhalation toxicity of 4-nitro-N-methylphthalimide to rats and acute
dermal toxicity of 4-nitro-N-methylphthalimide to rabbits is low. Following repeated oral
administration of 4-nitro-N-methylphthalimide to rats ,via gavage, for 13 weeks, increased
absolute liver and spleen weights and differences in hematology were observed at 10 mg/kg-day;
the NOAEL is 1 mg/kg-day. In an oral (gavage) modified combined reproductive/developmental
toxicity screening test in rats, decreased body weights were noted in F0 males and increased
pigment in the spleen was noted in F0 and adult F1 females with decreased hemoglobin and
hematocrit and increased spleen weight in adult F1 females at 25 mg/kg-day; the NOAEL for
systemic toxicity is 2.5 mg/kg-day. No effects were observed on any reproductive parameters;
the NOAEL for reproductive toxicity is 25 mg/kg-day (highest dose tested). In an oral (gavage)
prenatal developmental toxicity study in rabbits, decreased maternal body weight gain and body
weight were observed in does at 50 mg/kg-day, with a NOAEL for maternal toxicity of 10
mg/kg-day. Increased numbers of fetuses with vertebral malformations were observed at > 50
mg/kg-day and additional effects (other malformations, increased late resorptions) seen at 200
mg/kg-day; the NOAEL for developmental toxicity is 10 mg/kg-day. In an oral (gavage)
prenatal developmental toxicity study in rats, dams exhibited decreased body weight at > 50
mg/kg-day; the NOAEL for maternal toxicity is 10 mg/kg-day. Skeletal variations (i.e.,
incomplete ossification of the pubis, vertebrae or sternebrae) were seen in fetuses at > 50 mg/kg-
day with additional effects (soft tissue variations, increased resorptions and decreased fetal
weights) seen at the highest dose of 200 mg/kg-day; the NOAEL for developmental toxicity is 10
mg/kg-day. 4-Nitro-N-methylphthalimide was mutagenic to both bacteria and mammalian cells
in vitro, but did not induce chromosomal aberrations in mammalian cells in vitro. 4-Nitro-N-
methylphthalimide is irritating to rabbit skin and eyes.
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U.S. Environmental Protection Agency
Hazard Characterization Document
September, 2014
Table 3. Summary Table of the Screening Information Data Set under the
U.S. HPV Challenge Program - Human Health Data
Endpoint
4-Nitro-N-methylphthalimide
(41663-84-7)
Acute Oral Toxicity
LDso (mg/kg)
2800
Acute Inhalation Toxicity
LC50 (mg/L)
>36
Acute Dermal Toxicity
LD50 (mg/kg-bw)
>2000
Repeated-Dose Toxicity
NOAEL/LOAEL
Oral (mg/kg-day)
(rat; 13-wk)
NOAEL = 1.0
LOAEL = 10
(rat: 28-d)
NOAEL = 10
LOAEL = 100
Reproductive Toxicity
NOAEL/LOAEL
Oral (mg/kg-day)
Reproductive Toxicity
NOAEL = 25
(highest dose tested)
Developmental Toxicity
NOAEL/LOAEL
Oral (mg/kg-day)
Maternal and Developmental
Toxicity
(rabbit)
NOAEL = 10
LOAEL = 50
Maternal and Developmental
Toxicity
(rat)
NOAEL = 10
LOAEL = 50
Genetic Toxicity - Gene
Mutations
In vitro
Positive
Genetic Toxicity - Chromosomal
Aberrations
In vitro
Negative
Additional Information
Skin irritation
Eye irritation
Irritating
Irritating
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U.S. Environmental Protection Agency
Hazard Characterization Document
September, 2014
4. Hazard to the Environment
Acute Toxicity to Fish
No Adequate Data
Acute Toxicity to Aquatic Invertebrates
No Adequate Data
Toxicity to Aquatic Plants
No Adequate Data
Conclusion: No adequate data are available to evaluate the potential acute and chronic toxicity
of 4-nitro-N-methylphthalimide to aquatic organisms.
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