SCREENING-LEVEL HAZARD CHARACTERIZATION
OF HIGH PRODUCTION VOLUME CHEMICALS
CHEMICAL CATEGORY NAME
Mononitroaniline Category
SPONSORED CHEMICALS
2-Nitrobenzeneamine (CAS No. 88-74-4)
[9th CI Name: Benzenamine, 2-nitro-]
4-Nitrobenzeneamine (CAS No. 100-01-6)
[9th CI Name: Benzenamine, 4-nitro-]
October 2007
INTERIM
Prepared by
High Production Volume Chemicals Branch
Risk Assessment Division
Office of Pollution Prevention and Toxics
Environmental Protection Agency
1200 Pennsylvania Avenue, NW
Washington, DC 20460-0001

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SCREENING-LEVEL HAZARD CHARACTERIZATION
OF HIGH PRODUCTION VOLUME CHEMICALS
The High Production Volume (HPV) Challenge Program1 is 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 sponsor chemicals; sponsorship entails the identification and
initial assessment of the adequacy of existing toxicity data/information, conducting new testing if adequate data do
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 Set1'2) 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. OPPT is using a hazard-
based screening process to prioritize review of the submissions. The hazard-based screening process consists of two
tiers described below briefly and in more detail on the Hazard Characterization website3.
Tier 1 is a computerized sorting process whereby key elements of a submitted data set are compared to established
criteria to "bin" chemicals/categories for OPPT review. This is an automated process performed on the data as
submitted by the sponsor. It does not include evaluation of the quality or completeness of the data.
In Tier 2, a screening-level hazard characterization is developed by EPA that consists of an objective evaluation of
the quality and completeness of the data set provided in the Challenge Program submissions. The evaluation is
performed according to established EPA guidance2'4 and is based primarily on hazard data provided by sponsors.
EPA may also include additional or updated hazard information of which EPA, sponsors or other parties have
become aware. The hazard characterization may also identify data gaps that will become the basis for a subsequent
data needs assessment where deemed necessary. Under the HPV Challenge Program, chemicals that have similar
chemical structures, properties and biological activities may be grouped together and their data shared across the
resulting category. This approach often significantly reduces the need for conducting tests for all endpoints for all
category members. As part of Tier 2, evaluation of chemical category rationale and composition and data
extrapolation(s) among category members is performed in accord with established EPA2 and OECD5 guidance.
The screening-level hazard characterizations that emerge from Tier 2 are important contributors to OPPT's existing
chemicals review process. These hazard characterizations are technical documents intended to support 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. The public, including
sponsors, may offer comments on the hazard characterization documents.
The screening-level hazard characterizations, as the name indicates, do not evaluate the potential risks of a chemical
or a chemical category, but will serve as a starting point for such reviews. In 2007, EPA received data on uses of
and exposures to high-volume TSCA existing chemicals, submitted in accordance with the requirements of the
Inventory Update Reporting (IUR) rule. For the chemicals in the HPV Challenge Program, EPA will review the
IUR data to evaluate exposure potential. The resulting exposure information will then be combined with the
screening-level hazard characterizations to develop screening-level risk characterizations4'6. The screening-level
risk characterizations will inform EPA on the need for further work on individual chemicals or categories. Efforts
are currently underway to consider how best to utilize these screening-level risk characterizations as part of a risk-
based decision-making process on HPV chemicals which applies the results of the successful U.S. High Production
Volume Challenge Program and the IUR to support judgments concerning the need, if any, for further action.
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. HPV Chemicals Hazard Characterization website (http://www.epa.gov/hpvis/abouthc.html).
4	U.S. EPA. Risk Assessment Guidelines; http://cfpub.epa.gov/ncea/raf/rafguid.cfm.
5	OECD. Guidance on the Development and Use of Chemical Categories; http://www.oecd.org/dataoecd/60/47/1947509.pdf.
6	U.S. EPA. Risk Characterization Program; http://www.epa.gov/osa/spc/2riskchr.htm.
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SCREENING-LEVEL HAZARD CHARACTERIZATION
Mononitroanilines Category
Introduction
The sponsor, Solutia, Inc., submitted a Test Plan and Robust Summaries to EPA for the Mononitroanilines Category
on November 15, 2002. EPA posted the submission on the ChemRTK HPV Challenge website on April 21, 2003
(http ://www.epa. eov/chemrtk/pubs/summaries/mntranlc/c 143 91 to.html. EPA comments on the original submission
were posted to the website on August 26, 2003. Public comments were also received and posted to the website. The
sponsor submitted updated/revised documents on April 14, 2006, which were posted to the ChemRTK website on
August 24, 2004. The mononitroanilines category consists of two chemicals:
2-nitrobenzeneamine (2-nitroaniline)	CAS No. 88-74-4
[9th CI name: benzenamine, 2-nitro-]
[4-nitrobenzeneamine (4-nitroaniline)	CAS No. 100-01-6
[ 9th CI name: benzenamine, 4-nitro-]
Category Justification
The mononitroanilines category is based on the structural similarities of the members and the demonstrated
similarities in their physicochemical and environmental fate properties, and toxicological and ecotoxicological
effects.
This screening-level hazard characterization is based primarily on the review of the Test Plan and Robust
Summaries of studies submitted by the sponsors) under the HPV Challenge Program. 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. The structures of the sponsored chemical(s)
are included in Appendix. Summary tables of SIDS endpoint data are included in the document. The screening-
level hazard characterization for environmental and human health toxicity is based largely on SIDS endpoints and is
described according to established EPA or OECD effect level definitions and hazard assessment practices.
Summary-Conclusion
The log Kow of 2-nilrobcnzcncaminc and 4-nilrobcn/cncamine indicate that their potential to bioaccumulale is
expected to be low. 2-Nitrobenzcncamine and 4-nilrobcn/cncamine are not readily biodegradable, indicating that
they have the potential to persist in the environment.
The evaluation of the available toxicity data for fish, aquatic invertebrates and aquatic plants indicates that the
potential hazard of 2-nilrobcnzcncaminc and 4-nitrobenz.cncaminc to aquatic organisms is low.
The acute oral, inhalation and dermal toxicity of the members of the mononitroanilines category chemicals is low.
Repeated-dose exposures of these chemicals to rats via the oral route resulted in pale eyes, elevated melhcmoglobin
values, increased spleen weights and iron deposition and extra medullary hemalopoicsis in the spleen. 4-
Nilrobcnzcncaminc resulted in reduced pregnancy rate and male fertility index. In developmental toxicity studies,
maternal effects (reduced body weight gains and reduced food consumption) were seen at oral doses > 300 mg/kg-
bw/day. Developmental effects included reduced fetal body weights, delayed ossification and skeletal and soft
tissue malformation at the highest tested dose (250 mg/kg-bw/day). Positive test results were seen in one tested
strain of Salmonella typhi murium (TA98) with 4-nilrobcnzcncaminc in the absence of metabolic activation. Neither
chemical increased micronuclci or induced chromosomal aberrations when tested in vivo.
The potential health hazard of mononitroanilines category members is high based on rcpcalcd-dosc. reproductive
and developmental toxicity. Available data suggest the members of the mononitroanilines category have the
potential to be genotoxic.
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No data gaps have been identified under the HPV Challenge Program.
1. Physical-Chemical Properties and Environmental Fate
A summary of physical-chemical properties and environmental fate data submitted is provided in Table 1. For the
purpose of the screening-level hazard characterization, the review and summary of these data was limited to the
octanol-water partition coefficient and biodegradation endpoints as indictors of bioaccumulation and persistence,
respectively.
Octanol-Water Partition Coefficient
2-Nitrobenzeneamine (CAS No. 88-74-4)
LogKow: 1.85 (estimated)
4-Nitrobenzeneamine (CAS No. 100-01-6)
LogKow: 1.39 (estimated)
Biodegradation
2-Nitrobenzeneamine (CAS No. 88-74-4)
A Semi-Continuous Activated Sludge (SCAS) Biodegradability study was conducted to determine the
biodegradation potential of 2-nitrobenzeneamine. Although this is not a ready biodegradability test, EPA found the
data presented adequate for the purposes of the HPV Challenge Program. 2-nitrobenzeneamine degradation was
poor throughout the 12-30 week testing period.
2-Nitrobenzeneamine is not readily biodegradable.
4-Nitrobenzeneamine (CAS No. 100-01-6)
A Semi-Continuous Activated Sludge (SCAS) Biodegradability study was conducted to determine the
biodegradation potential of 4-nitrobenzeneamine. Although this is not a ready biodegradability test, EPA found the
data presented adequate for the purposes of the HPV Challenge Program. 4-nitrobenzeneamine degradation was
highly variable throughout the 40 week testing period. Several other studies of the biodegradability of 4-
nitrobenzeneamine have been reported in the literature and generally indicate poor biodegradability of 4-
nitrobenzeneamine.
4-Nitrobenzeneamine is not readily biodegradable.
Conclusion: The log Kow of 2-nitrobenzeneamine and 4-nitrobenzeneamine indicate that their potential to
bioaccumulate is expected to be low. 2-Nitrobenzeneamine and 4-nitrobenzeneamine are not readily biodegradable,
indicating that they have the potential to persist in the environment.
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Table 1. Summary of Physical-Chemical Properties and Environmental Fate Data
Endpoints
2-Nitrobenzeneamine
(CAS No. 88-74-4)
4-Nitrobenzeneamine
(CAS No. l(KM) 1-6)
Melting Point (C)
71.5 (m)
146 (m)
Boiling Point (C)
284 (m)
332 (m)
Vapor Pressure
(hPa at 25C)
0.00115(e)
0.0000042 (e)
Log Kw
1.85 (e)
1.39(e)
Water Solubility
(mg/L at 25C)
1470(m)
724 (m)
Indirect (OH ) Photodegradation
Half-life (t1/2)
9.5 hours (e)
9.5 hours (e)
Stability in Water (Hydrolysis) (ti/2)
Resistant to hydrolysis
Resistant to hydrolysis
Fugacity
(Level in Model)
Air (%)
Water (%)
Soil (%)
Sediment (%)
<0.01
>99
<0.01
< 1
<0.01
>99
<0.01
< 1
Biodegradation at 28 days (%)
Not readily biodegradable
Not readily biodegradable
(m) = measured data (i.e., derived from experiment); (e) = estimated data (i.e., derived from modeling)
2. Environmental Effects - Aquatic Toxicity
A summary of aquatic toxicity data submitted for SIDS endpoints is provided in Table 2. The table also indicates
where data for tested category members are read-across (RA) to untested members of the category.
Acute Toxicity to Fish
2-Nitrobenzeneamine (CAS No. 88-74-4)
Zebrafish (Brachydanio rerio) were exposed to measured concentrations of 2-nitrobenzeneamine (test concentration
values were not provided) of under semi-static conditions for 96 hours.
96-h LC50 = 19.5 mg/L
4-Nitrobenzeneamine (CAS No. 100-01-6)
Rainbow trout (Salmo gairdneri) were exposed to nominal concentrationsof 4-nitrobenzeneamine ranging from 5.6
to 100 mg/L under static conditions for 96 hours. No deaths were seen at concentrations up to 32 mg/L. At 56
mg/L, mortality was 80% after 24 hours and 90% after 48 and 96 hours. Mortality was 100% at 100 mg/L at all
three time points.
96-h LCS0 = 45 mg/L
Acute Toxicity to Aquatic Invertebrates
2-Nitrobenzeneamine (CAS No. 88-74-4)
Daphnia magna were exposed to 2-nitrobenzeneamine at concentrations of 6.25, 12.5, 25, 50 and lOOmg/L under
static conditions for 48 hours.
48-h ECS0 =14.5 mg/L
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4-Nitrobenzeneamine (CAS No. 100-01-6)
Daphnia magna were exposed to 4-nitrobenzeneamine for 48 hours under static conditions using nominal
concentrations. The test concentrations ranged between 3.2 and 32 mg/L, spaced logarithmically.
48-h LCS0 = 20 mg/L
Toxicity to Aquatic Plants
2-Nitrobenzeneamine (CAS No. 88-74-4)
Green algae (Scenedesmus sp.) was exposed to 2-nitrobenzeneamine at five nominal concentrations under static
conditions for 48 hours. The 48-hour EC50 for growth rate inhibition was 64.5 mg/L. The submitted study duration
deviates from the standard 72- or 96-hour algal toxicity study duration. The sponsor supported the 48-h toxicity
value with a literature-reported 96-h algal growth inhibition (biomass) threshold of 20 mg/L for 4-
nitrobenzeneamine, which is consistent with the 48-h value in that it this threshold value is lower than the EC50
value. The 96-hour EC50 value for this endpoint estimated using ECOSAR (14 mg/L) also supports the 48-hour
EC50 value. All available information indicate low toxicity to algae.
48-h EC50 (growth) = 64.5 mg/L
96-h EC50 (growth) = 14 mg/L (estimated)
4-Nitrobenzeneamine (CAS No. 100-01-6)
Green algae (Scenedesmus sp.) was exposed to 4-nitrobenzeneamine at five nominal concentrations under static
conditions for 48 hours. The 48-hour EC50 for growth rate inhibition was 54.9 mg/L. The submitted study duration
deviates from the standard 72- or 96-hour algal toxicity study duration. T he sponsor supported the 48-h toxicity
value with a literature-reported 96-h algal growth inhibition (biomass) threshold of 20 mg/L for 4-
nitrobenzeneamine, which is consistent with the 48-h value in that it this threshold value is lower than the EC50
value. The 96-hour EC50 value for this endpoint estimated using ECOSAR (29 mg/L) also supports the 48-hour
EC50 value. All available information indicate low toxicity to algae.
48-h EC50 (growth) = 54.9 mg/L
96-h EC50 (growth) = 29 mg/L (estimated)
Conclusion: The evaluation of the available toxicity data for fish, aquatic invertebrates and aquatic plants indicates
that the potential hazard of 2-nitrobenzeneamine and 4-nitrobenzeneamine to aquatic organisms is low.
Table 2. Summary of Environmental Effects - Aquatic Toxicity Data
Endpoints
2-Nitrobenzeneamine
(88-74-4)
4-Nitrobenzeneamine
(100-01-6)
Fish
96-h LCS0 (mg/L)
19.5 (m)
45 (m)
Aquatic Invertebrates
48-h ECS0 (mg/L)
14.5 (m)
20.0 (m)
Aquatic Plants
96-h ECS0 (mg/L)
(growth)
64.5 (48-h) (m)
14 (96-h) (e)
54.9 (48-h) (m)
29 (96-h) (e)
(m) = measured data (i.e., derived from testing); (e) = estimated data (i.e., derived from modeling)
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3. Human Health Effects
A summary of health effects data submitted for SIDS endpoints is provided in Table 3. The table also indicates
where data for tested category members are read-across (RA) to untested members of the category.
Acute Oral Toxicity
2-Nitrobenzeneamine (CAS No. 88-74-4)
Sprague-Dawley rats (20/mixed sex/group) were administered 2-nitrobenzeneamine as a 10% corn oil solution via
gavage at doses of 1260, 1580, 2000 and 2510 mg/kg-bw and observed for 14 days. Signs of toxicity included
yellow colored urine and general weakness. Observations at autopsy included hemorrhagic lungs, liver hyperemia,
yellow stained abdominal cavity and gastrointestinal irritation; for survivors, viscera appeared normal.
LDS0 = 2050 mg/kg-bw
4-Nitrobenzeneamine (CAS No. 100-01-6)
Sprague-Dawley rats (25/mixed sex/group) administered 4-nitrobenzeneamine as a 20% solution-suspension in corn
oil at doses of 794, 1000, 1260, 1580 and 2000 mg/kg-bw and observed for 14 days. Signs of toxicity included
ocular discharge, tremors and convulsions. At necropsy, hemorrhagic lungs, liver discoloration and gastrointestinal
inflammation were observed; all survivors had normal viscera.
LDS0 = 1400 mg/kg-bw
Acute Inhalation Toxicity
2-Nitrobenzeneamine (CAS No. 88-74-4)
Ten Wistar rats (10/male and female/group) were exposed (nose-only) to a single aerosol concentration of
2-nitrobenzeneamine solution in polyethylene glycol at the maximum achievable concentration of 2529 mg/m3 (2.53
mg/L). A vehicle control group of rats was exposed to polyethylene glycol/acetone. The observation period was 14
days. No mortality was observed. Decreased body weight gain, hypothermia, distinct discoloration of urine and
bradypnea were observed during the study.
LC50 > 2.53 mg/L
Acute Dermal Toxicity
2-Nitrobenzeneamine (CAS No. 88-74-4)
Three New Zealand White rabbits were exposed dermally (intact skin) to 5010 and 7940 mg/kg-bw of
2-nitrobenzeneamine as 40% solution-suspension in corn oil under occluded conditions for 24 hours and observed
for 14 days. Yellow staining and reduced appetite and activity were seen during the first 3 days of the study. There
were no deaths.
LDS0 > 7940 mg/kg-bw
4-Nitrobenzeneamine (CAS No. 100-01-6)
Three New Zealand White rabbits were exposed dermally (intact skin) to 5010 and 7940 mg/kg-bw of
4-nitrobenzeneamine as 40% solution-suspension in corn oil under occluded conditions for 24 hours and observed
for 14 days. No toxic signs were observed during the study; all viscera were normal at necropsy. There were no
deaths.
LDS0 > 7940 mg/kg-bw
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Repeated-Dose Toxicity
2-Nitrobenzeneamine (CAS No. 88-74-4)
(1)	Sprague-Dawley rats (10/males/dose) were exposed to 2-nitrobenzeneamine via inhalation (whole-body
exposure) at 9.8 and 93 mg/m3 (0.0098 and 0.093 mg/L) for 6 hours/day, 5 days/week for 4 weeks. Minimal
changes in some hematological parameters (increases in methemoglobin and decreases in total leukocytes and
segmented neutrophils) were seen at 93 mg/m3.
LOAEL = 0.093 mg/L (based on effect on some hematological parameters)
NOAEL = 0.0098 mg/L
(2)	Sprague-Dawley rats (10/males/dose) were exposed to 2-nitrobenzeneamine via inhalation (whole-body
exposure) at 10, 27.5 and 73 mg/m3 (0.01, 0.028 and 0.073 mg/) for 6 hours/day, 5 days/week for 4 weeks. At the
highest dose, decreased leukocytes in males, reduced hemoglobin and erythrocytes in females, increased relative
liver weights in females and increased polychromia, anisocytosis and poikilocytosis in males and females were seen.
LOAEL = 0.073 mg/L (based on effect in some hematological parameters)
NOAEL = 0.030 mg/L
4-Nitrobenzeneamine (CAS No. 100-01-6)
(1)	In a 90-day study, Sprague-Dawley rats (20/sex/group) were administered 4-nitrobenzeneamine in corn oil via
gavage at 0, 3, 10 or 30 mg/kg-bw/day. At 30 mg/kg-bw/day, pale appearance around ears and marked increases in
urinary urobilinogen were seen. Increases in methemoglobin levels, red blood cell counts and hemoglobin1 levels
were also reported. All animals from this group had discolored spleens at necropsy, increased spleen weights,
splenomegaly and excessive splenic hemosiderin deposition. At 10 mg/kg-bw/day, increased methemoglobin,
decreased red blood cell counts and hemoglobin concentrations were noted. All animals had splenomegaly, elevated
splenic weights, discolored spleens and excessive hemosiderin deposits. At 3 mg/kg-bw/day, methemoglobin levels
were increased and excessive splenic hemosiderin deposits were seen.
LOAEL = 3 mg/kg-bw/day (based on effect on spleen and changes in some hematological parameters)
NOAEL = Not established
(2)	Male and female Sprague-Dawley rats were exposed to 4-nitrobenzeneamine via inhalation (whole-body) at 0,
10, 32 and 80 mg/m3/day (0, 0.01, 0.032 and 0.08 mg/L/day) for 6 hours/day, 5 day/week for 4 weeks. At 80
mg/m3/day, decreases in hemoglobin and hematocrit, increases in methemoglobin and higher incidence of
polychromasia and anisocytosis (females only) were seen. Increased absolute and relative spleen weights, iron
deposits in the spleen and extramedullary hematopoiesis in spleen and liver (females only) were seen. At 32
mg/m3/day, effects included decreased hemoglobin, increased levels of methemoglobin, higher incidence of
polychromasia and anisocytosis, increased spleen weight and increased iron deposits and extramedullary
hematopoiesis in the spleen. At 10 mg/m3/day, elevated methemoglobin, increased spleen weights and iron deposits
and extramedullary hematopoiesis in the spleen were seen.
LOAEL = 0.01 mg/L/day (based on effects on spleen and increased levels of methemoglobin)
NOAEL = Not established
1 EPA notes that this is the only increase in hemoglobin noted. At all other dosed in all the repeated dose studies
with both 2- and 4-nitrobenzeneamine, decreased hemoglobin was observed.
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Reproductive Toxicity
4-Nitrobenzeneamine (CAS No. 100-01-6)
In a two-generation study, Sprague-Dawley FO and F1 rats were administered 4-nitrobenzeneamine via gavage at 0,
0.25, 1.5 and 9 mg/kg-bw/day during a pre-mating (14 weeks for FO and 18 weeks for Fl), growth period, mating,
gestation and lactation intervals. Fl rats were continued on treatment during a post-weaning period of 30 days. No
mortality or adverse effects on body weights or food consumption or in-life evaluations were observed in either FO
or F1 adults. Mating indices were comparable to controls for both FO and F1 animals. A reduction in pregnancy
rate was observed in the 9 mg/kg-bw/day FO group. The male fertility index was slightly lower at 9 mg/kg-bw/day
in FO animals. No adverse effects were observed in mean length of gestation, number of live and dead pups, pup
weights during lactation or pup and litter survival. In the longest exposure to the test substance in Fl animals, there
was evidence of "brown pigment" in the spleen at the highest dose.
LOAEL (systemic toxicity) = 9 mg/kg-bw/day (based on effect on spleen)
NOAEL (systemic toxicity) = 1.5 mg/kg-bw/day
LOAEL (reproductive toxicity) = 9 mg/kg-bw/day (based on reduced male fertility index and pregnancy rate)
NOAEL (reproductive toxicity) = 1.5 mg/kg-bw/day
Developmental Toxicity
2-Nitrobenzeneamine (CAS No. 88-74-4)
Pregnant Sprague-Dawley rats (25 females/group) were administered 2-nitrobenzeneamine in corn oil via gavage at
doses of 0, 100, 300 and 600 mg/kg-bw/day from gestation days 6 to 15. Daily clinical signs were observed on
gestation days 6-21. Maternal toxicity was evidenced by reduced body weight gains, reduced food consumption
and piloerection in 300 and 600 mg/kg-bw/day groups. No effects were noted on pregnancy rates, mean number of
live and dead pups, resorptions, nidations or corpora lutea. The mean fetal weights were slightly lower than control
in the 600 mg/kg-bw/day group. No effects were seen on number of litters, fetuses or malformations.
LOAEL (maternal toxicity) = 300 mg/kg-bw/day (based on reduced body weight gain and food consumption)
NOAEL (maternal toxicity) = 100 mg/kg-bw/day
LOAEL (developmental toxicity) = 600 mg/kg-bw/day (based on reduced fetal weights)
NOAEL (developmental toxicity) = 300 mg/kg-bw/day
4-Nitrobenzeneamine (CAS No. 100-01-6)
(1)	Pregnant Sprague-Dawley rats (25/female/group) were administered 4-nitrobenzeneamine in corn oil via gavage
at doses of 25, 85 and 250 mg/kg-bw/day during gestation days 6 - 19. Maternal toxicity was evidenced by reduced
body weight gains, pale eye coloration and occasional convulsions after dosing, increased mean number of
resorptions and percent resorptions and increased mean spleen weights (absolute and relative). Reduced mean fetal
weights (both sexes), increased number of fetuses with delayed or no ossification and increased number of fetuses
with external, soft tissue or skeletal malformations (predominantly kinked or shortened tail, absence of kidneys or
ureter and fused ribs) were seen at 250 mg/kg-bw/day. At 85 mg/kg-bw/day, increased mean maternal spleen
weights and reduced mean fetal weights (both sexes) were observed. There were no observed or treatment-related
effects of maternal toxicity, embryotoxicity or fetotoxicity at 25 mg/kg-bw/day.
LOAEL (maternal toxicity) = 85 mg/kg-bw/day (based on increased spleen weights)
NOAEL (maternal toxicity) = 25 mg/kg-bw/day
LOAEL (developmental toxicity) = 85 mg/kg-bw/day (based on fetal effects of reduced body weights)
NOAEL (developmental toxicity) = 25 mg/kg-bw/day
(2)	Pregnant New Zealand White rabbits (18/female/group) were administered 4-nitrobenzeneamine in corn oil via
gavage at 0, 15, 75 and 125 mg/kg-bw/day during gestation days 7 - 19. At 125 mg/kg-bw/day, 7/18 females died.
Maternal toxicity was evidenced by grayish appearing eyes and body weight loss. No differences were observed in
mean number of implantations, resorptions or variable fetuses or mean fetal weights. At 75 mg/kg-bw/day, the only
observed effect was grayish eyes; there were no other effects on measured maternal, embryo or fetal parameters.
LOAEL (maternal toxicity) = 125 mg/kg-bw/day (based on mortality and body weight loss among survivors)
NOAEL (maternal toxicity) = 75 mg/kg-bw/day
LOAEL (developmental toxicity) > 125 mg/kg-bw/day
NOAEL (developmental toxicity) = 125 mg/kg-bw/day (based on no effects at the highest dose tested)
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Genetic Toxicity - Gene Mutation
In vitro
2-Nitrobenzeneamine (CAS No. 88-74-4)
In an Ames assay, Salmonella typhimurium strains (TA98, TA100, TA1535 and 1537) were exposed to 2-
nitrobenzeneamine up to 1500 |ig/plate in the presence and absence of metabolic activation. 2-Nitrobenzeneamine
did not show any marked increases in mutagenic activity for any of the four tested strains.
2-Nitrobenzeneamine was not mutagenic in this assay.
4-Nitrobenzeneamine (CAS No. 100-01-6)
In an Ames assay, S. typhimurium strains (TA98, TA100, TA1535, TA97 and/or 1537) were exposed to 4-
nitrobenzeneamine up to up to 10 mg/plate in the presence and absence of metabolic activation. There were no
marked increases in mutagenic activity for any of the four tested strains up to maximum concentration of 25 mg/spot
in the spot test. However, mutagenic activity was markedly elevated with TA98 strain with and without metabolic
activation in the plate incorporation assay. 4-Nitrobenzeneamine gave equivocal results in this assay.
4-Nitrobenzeneamine gave equivocal results in this assay.
Genetic Toxicity - Chromosomal Aberrations
In vivo
2-Nitrobenzeneamine (CAS No. 88-74-4)
In a micronucleus assay male/female mice were administered two single doses of 2-nitrobenzeneamine via
intraperitoneal injection at 0, 50, 250 and 500 mg/kg-bw, 24 hours apart. Bone marrow cells were evaluated for the
presence of micronuclei in erythrocytes. No increases in micronuclei were observed at any dose level. Positive
controls responded appropriately. Animals exhibited signs of listlessness and unresponsive behavior at all doses.
Females at 500 mg/kg-bw/day showed marked body weight loss.
2-Nitrobenzeneamine did not induce chromosomal aberrations in this assay.
4-Nitrobenzeneamine (CAS No. 100-01-6)
In a micronucleus assay male/female mice were administered two single doses of 4-nitrobenzeneamine via
intraperitoneal injection at 80, 400 and 800 mg/kg-bw, 24 hours apart. Bone marrow cells were evaluated for the
presence of micronuclei in erythrocytes. No increases in micronuclei were observed at any dose level. Positive
controls responded appropriately. One death and clear signs of toxicity (unresponsiveness and tremors up to 4 hours
after dosing) were noted at 800 mg/kg-bw; at 400 mg/kg-bw, listlessness and some tremors were seen occasionally
after dosing and at 80 mg/kg-bw, listlessness immediately after dosing was noted. No effects on body weight were
observed at any test level.
4-Nitrobenzeneamine did not induce chromosomal aberrations in this assay.
Conclusion: The acute oral, inhalation and dermal toxicity of the members of the mononitroanilines category
chemicals is low. Repeated-dose exposures of these chemicals to rats via the oral route resulted in pale eyes,
elevated methemoglobin values, increased spleen weights and iron deposition and extramedullary hematopoiesis in
the spleen. 4-Nitrobenzeneamine resulted in reduced pregnancy rate and male fertility index. In developmental
toxicity studies, maternal effects (reduced body weight gains and reduced food consumption) were seen at oral doses
> 300 mg/kg-bw/day. Developmental effects included reduced fetal body weights, delayed ossification and skeletal
and soft tissue malformation at the highest tested dose (250 mg/kg-bw/day). Positive test results were seen in one
tested strain of Salmonella typhimurium (TA98) with 4-nitrobenzeneamine in the absence of metabolic activation.
Neither chemical increased micronuclei or induced chromosomal aberrations when tested in vivo.
The potential health hazard of mononitroanilines category members is high based on repeated-dose, reproductive
and developmental toxicity. Available data suggest the members of the mononitroanilines category have the
potential to be genotoxic.
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Table 3.
Summary of Human Health Data

Endpoints
2-N itrobcn/cncaminc
(CAS No. 88-74-4)
4-Nitrobcnzcncaminc
(CAS No. 100-01-6)
Acute Oral Toxicity
LDS0 (mg/kg-bw)
2050
1400
Acute Inhalation Toxicity
LC50 (mg/L)
> 2.53 (4-h)
No Data
>2.53 (4-h)
(RA)
Acute Dermal Toxicity
LDS0 (mg/kg-bw)
>7940
>7940
Repeated-Dose Toxicity
NOAEL/LOAEL
Oral (mg/kg-bw/day)
No Data
NOAEL = Not established
LOAEL = 3
(RA)
NOAEL = Not established
LOAEL=3
Repeated-Dose Toxicity
NOAEL/LOAEL
Inhalation (mg/L)
NOAEL = 0.01
LOAEL = 0.093
NOAEL = 0.030
LOAEL = 0.073
NOAEL = Not established
LOAEL = 0.01
Reproductive Toxicity
NOAEL/LOAEL
Oral (mg/kg-bw/day)
Systemic Toxicity
No Data
NOAEL = 1.5
LOAEL = 9
NOAEL = 1.5
LOAEL = 9
Reproductive Toxicity
NOAEL = 1.5
LOAEL = 9
(RA)
NOAEL = 1.5
LOAEL = 9
Developmental Toxicity
NOAEL/LOAEL
Oral (mg/kg-bw/day)
Maternal Toxicity
NOAEL =100
LOAEL = 300
(Rat)
NOAEL = 25
LOAEL = 85
(Rabbit)
NOAEL = 75
LOAEL = 125
Developmental Toxicity
NOAEL =300
LOAEL = 600
(Rat)
NOAEL = 25
LOAEL = 85
(Rabbit)
NOAEL =125
LOAEL > 125
Genetic Toxicity - Gene Mutation
In vitro
Negative
Positive
Genetic Toxicity - Chromosomal Aberrations
In vivo
Negative
Negative
(m) = measured data (i.e. derived from testing); (RA) = Read Across
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4. Hazard Characterization
The log Kow of 2-nitrobenzeneamine and 4-nitrobenzeneamine indicate that their potential to bioaccumulate is
expected to be low. 2-Nitrobenzeneamine and 4-nitrobenzeneamine are not readily biodegradable, indicating that
they have the potential to persist in the environment.
The evaluation of the available toxicity data for fish, aquatic invertebrates and aquatic plants indicates that the
potential hazard of 2-nitrobenzeneamine and 4-nitrobenzeneamine to aquatic organisms is low.
The acute oral, inhalation and dermal toxicity of the members of the mononitroanilines category chemicals is low.
Repeated-dose exposures of these chemicals to rats via the oral route resulted in pale eyes, elevated methemoglobin
values, increased spleen weights and iron deposition and extramedullary hematopoiesis in the spleen. 4-
Nitrobenzeneamine resulted in reduced pregnancy rate and male fertility index. In developmental toxicity studies,
maternal effects (reduced body weight gains and reduced food consumption) were seen at oral doses > 300 mg/kg-
bw/day. Developmental effects included reduced fetal body weights, delayed ossification and skeletal and soft
tissue malformation at the highest tested dose (250 mg/kg-bw/day). Positive test results were seen in one tested
strain of Salmonella typhimurium (TA98) with 4-nitrobenzeneamine in the absence of metabolic activation. Neither
chemical increased micronuclei or induced chromosomal aberrations when tested in vivo.
The potential health hazard of mononitroanilines category members is high based on repeated-dose, reproductive
and developmental toxicity. Available data suggest the members of the mononitroanilines category have the
potential to be genotoxic.
5. Data Gaps
No data gaps have been identified under the HPV Challenge Program.
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Appendix
Mononitroanilincs
CAS No.
Chemical Name
Structure
SPONSORED CHEMICALS
88-74-4
2-Nitrobenzeneamine
Q-C
nh2
C6H6n2o2
100-01-6
4-Nitrobenzeneamine
C6H6n2o2
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