U.S. Environmental Protection Agency
Hazard Characterization Document
September, 2014
SCREENING-LEVEL HAZARD CHARACTERIZATION
SPONSORED CHEMICAL
l,2-Dimethyl-4-nitrobenzene CASRN 99-51-4
SUPPORTING CHEMICALS
2,4-Dimethyl-l-nitrobenzene
CASRN 89-87-2
CASRN 88-72-2
CASRN 99-08-1
CASRN 99-99-0
2-Nitrotoluene
3-Nitrotoluene
4-Nitrotoluene
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 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 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.) and 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.
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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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.
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.
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Chemical Abstract Service Registry Number
(CASRN)
SDonsored Chemical
99-51-4
Supporting Chemical
89-87-2
88-72-2
99-08-1
99-99-0
Chemical Abstract Index Name
Sponsored Chemical
Benzene, l,2-dimethyl-4-nitro-
SuDDorting Chemical
Benzene, 2,4-dimethyl-l-nitro-
Benzene, l-methyl-2-nitro-
Benzene, l-methyl-3-nitro-
Benzene, l-methyl-4-nitro-
Structural Formula
See Appendix
Summary
l,2-Dimethyl-4-nitrobenzene is a pale, straw yellow-colored liquid with moderate vapor
pressure and moderate water solubility. It is expected to have moderate mobility in soil.
Although no biodegradation data are available for this compound, based on close structural
analogs (2,4-dimethyl-l -nitrobenzene, 2-nitrotoluene, 3-nitrotoluene and 4-nitrotoluene),
l,2-dimethyl-4-nitrobenzene is not expected to be readily biodegradable. Volatilization of
l,2-dimethyl-4-nitrobenzene is expected to be moderate. The rate of hydrolysis is negligible.
l,2-Dimethyl-4-nitrobenene is not expected to bioconcentrate in aquatic organisms based on
bioconcentration data measured in carp for 2-nitrotoluene. The rate of atmospheric
photooxidation is negligible. l,2-Dimethyl-4-nitrobenzene is expected to have low persistence
(PI) and low bioaccumulation potential (Bl).
The acute oral and dermal toxicity of l,2-dimethyl-4-nitrobenzene is low in rats and rabbits,
respectively. Dietary 13-week repeated-dose toxicity studies in rats with the supporting
chemicals, 2-, 3- and 4-nitrotoluene, revealed reduced body weight gain and toxic effects to the
liver, spleen and kidney (e.g., increased liver weights and clinical chemistry changes in these
organs) at 42- 47 mg/kg-bw/day; the NOAEL is not established. Impaired testicular function
(e.g., decreased sperm density and motility) and increased estrous cycle length were also seen at
>375 mg/kg-bw/day. Similar systemic effects were seen in rats at 300 mg/kg-bw/day in a 28-
day dietary study with the supporting chemical 2,4-dimethyl-4-nitrobenzene; the NOAEL is 60
mg/kg-bw/day. In mice, in 13-week dietary studies, decreased body weights were seen with
4-nitrotoluene at 813/1075 mg/kg-bw/day (males/females); the NOAEL is 439/625 mg/kg-
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bw/day (males/females). 3-Nitrotoluene showed effects on relative liver weights 101 mg/kg-
bw/day; the NOAEL was not established. In mice, 2-Nitrotoluene showed increased relative
liver weights in females and nasal cavity lesions in both sexes at 187 mg/kg-bw/day; the
NOAEL is 101 mg/kg-bw/day. No specific reproductive toxicity studies are available for 1,2-
dimethyl-4-nitrobenzene. An oral gavage combined reproductive/developmental toxicity
screening test in rats with the supporting chemical 4-nitrotoluene showed mortality in both sexes
at 400 mg/kg-day and reduced pup weights at 100 mg/kg-day; the NOAEL for reproductive and
developmental toxicity is 25 mg/kg-day. Based on decreasing body weights at all doses, the
NOAEL for maternal toxicity is not established. In the 13-week dietary repeated-dose studies in
rats, effects were observed on the reproductive systems of both sexes. Similar studies in mice
showed no effects. l,2-Dimethyl-4-nitrobenzene and the supporting chemical, 2,4-dimethyl-l-
nitrobenzene, induced gene mutations in bacteria in vitro and the supporting chemicals, 3- and
4-nitrotoluene, induced chromosomal aberrations in mammalian cells in vitro. 4-Nitrotoluene
induced sister chromatid exchange in vitro. The supporting chemicals, 2- and 4-nitrotoluene
increased the incidence of tumors in rats and mice. l,2-Dimethyl-4-nitrobenzene is not irritating
to rabbit eyes or skin.
The fish 96-h LCso for l,2-dimethyl-4-nitrobenzene ranges between 10-18 mg/L, based on the
supporting chemical 2,4-dimethyl-l-nitrobenzene. The aquatic invertebrate 48-h EC so for
l,2-dimethyl-4-nitrobenzene is 16 mg/L. The algal 72-h EC so for l,2-dimethyl-4-nitrobenzene
is 8.9 mg/L.
No data gaps were identified under the HPV Challenge Program.
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The sponsor, Arcadis G&M, Inc., submitted a test plan and robust summaries to EPA for 1,2-
dimethyl-4-nitrobenzene (CASRN 99-51-4; CA Index name: benzene, l,2-dimethyl-4-nitro-)
dated November 29, 2006. EPA posted the submission on the ChemRTK HPV Challenge Web
site on February 20, 2007
(http://www.epa.gov/chemrtk/pubs/summaries/12dim4nt/cl6493tc.htm). EPA comments on the
submission were posted to the website on June 26, 2009. Public comments were also received
and posted to the website. The sponsor also provided data on several proposed supporting
chemicals: 2,4 dimethyl-1-nitrobenzene (4-nitro-w-xylene, CASRN 89-87-2), 2-nitrotoluene
(CASRN 88-72-2), 3-nitrotoluene (CASRN 99-08-1) and 4-nitrotoluene (CASRN 99-99-0). In
October 2009, EPA received revised test plan and robust summary documents for 1,2-dimethyl-
4-nitrobenzene (CASRN 99-51-4) and its proposed supporting chemicals.
Justification for Supporting Chemicals
The sponsor proposed to use data for the following four supporting chemicals to address the
aquatic toxicity and most human health effects endpoints: 2,4-dimethyl-l -nitrobenzene (4-nitro-
w-xylene, CASRN 89-87-2), 2-nitrotoluene (CASRN 88-72-2), 3-nitrotoluene (CASRN 99-08-1)
and 4-nitrotoluene (CASRN 99-99-0). Based on available data, EPA agrees that use of the
supporting chemical data is appropriate to address particular endpoints. For ecological and health
effects endpoints, 2,4-dimethyl-l-nitrobenzene is the most appropriate analog. Additionally, for
health effects endpoints, EPA considers data for 2-, 3- and 4-nitrotoluene are also appropriate
based on close structural similarities.
For environmental fate endpoints, data for the close structural analogs, 2,4-dimethyl-l-
nitrobenzene, 2-nitrotoluene, 3-nitrotoluene and 4-nitrotoluene are considered appropriate for
the biodegradation endpoint.
For the aquatic toxicity endpoints, data for the sponsored chemical and the most appropriate
supporting chemical, 2,4-dimethyl-l-nitrobenzene, were available and are sufficient to
characterize these endpoints for the purposes of the HPV Challenge program. Therefore, aquatic
toxicity data supplied for the three other supporting chemicals (2-, 3- and 4-nitrotoluene) were
not considered in this hazard characterization.
4-Nitrotoluene has been assessed in the OECD HPV program and the data can be viewed at:
http://webnet.oecd.org/hpv/UI/handler.axd?id=4cdf8442-3ecb-44b8-8d66-411f4a3cel3c
2-Nitrotoluene has been assessed in the OECD HPV program and the European Union Risk
Assessment Report can be viewed at:
http://esis.irc.ec.europa.eu/doc/risk assessment/REPORT/2nitrotoluenereport403.pdf.
1. Chemical Identity
1.1 Identification and Purity
l,2-Dimethyl-4-nitrobenzene is a pale, straw yellow-colored liquid with moderate vapor pressure
and moderate water solubility.
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Hazard Characterization Document
1.2 Physical-Chemical Properties
The physical-chemical properties of l,2-dimethyl-4-nitrobenzene are summarized in Table 1.
Table 1. Physical-Chemical Properties of l,2-Dimethyl-4-nitrobenzene and Supporting
Chemicals1
Property
SPONSORED
CHEMICAL
l,2-Dimcthvl-4-
nitrobcn/cnc
SUPPORTING
CHEMICAL
2,4-Dimethyl-l-
nitrobenzene
SUPPORTING
CHEMICAL
2-Nitrotoluene
SUPPORTING
CHEMICAL
3-Nitrotoluene
SUPPORTING
CHEMICAL
4-Nitrotoluene
CASRN
99-51-4
89-87-2
88-72-2
99-08-1
99-99-0
Molecular Weight
151.17
151.17
137.14
137.14
137.14
Physical State
Pale, straw yellow
liquid
Liquid
Yellow liquid
Yellow liquid
Colorless to light
yellow solid
Melting Point
30.5°C
(measured)
9°C
(measured)
-10°C
(measured)
15.5°C
(measured)
51.6°C
(measured)
Boiling Point
251°C
(measured)
247.8°C
(measured)
222°C
(measured)
232°C
(measured)
238.3°C
(measured)
Vapor Pressure
3.59><10"3 mmHg
at 25°C
(measured)
50 mm Hg at
153.7°C
(measured);
0.042 mm Hg at
25 °C (estimated)2
0.12 mmHg at
20°C (measured)
7.5 mmHg at
89.7°C
(measured);
0.098 mmHg at
25°C (estimated)2
0.098 mmHg at
20°C (measured)
Dissociation
Constant
Not applicable
Not applicable
Not applicable
Not applicable
Not applicable
Henry's Law
Constant
5.1xl0"5atm-
m3/mole at 25°C
(estimated)3
8.6><10"5atm-
m3/mole at 25°C
(estimated)3
1.25><10"5atm-
m3/mole at 25°C
(measured)4
9.3xl06atm-
m3/mole at 25°C
(measured)4
5.4xl06atm-
m3/mole at 25°C
(measured)4
Water Solubility
111.8 mg/L at
25°C (estimated)3
133 mg/L at 20°C
(measured)
650 mg/L at 30°C
(measured)
500 mg/L at 30°C
(measured)
442 mg/L at 30°C
(measured)
Log Kow
2.91 (measured)
2.91 (estimated)3
2.3 (measured)
2.45 (measured)
2.37 (measured)
'BASF Corporation. 2009. Revised Test Plan and Robust Summary for l,2-dimethyl-4-nitrobenzene. Available
online at http://www.epa.gov/chemrtk/pubs/summaries/12dim4nt/cl6493tc.htm as of June 22, 2012.
2N0M05. 1987. Programs to Enhance PC-Gems Estimates of Physical Properties for Organic Compounds. The
Mitre Corp.
3U.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 0f June 22, 2012.
4SRC. 2012. The Physical Properties Database (PHYSPROP). Syracuse, NY: Syracuse Research Corporation.
Available online at http://www.svrres.com/esc/phvsprop.htm as of June 22, 2012.
2. General Information on Exposure
2.1 Production Volume and Use
CASRN 99-51-4 had an aggregated production and/or import volume in the United States
between 10 to 50 million pounds during calendar year 2005.
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Non-confidential information in the IUR indicated that the industrial processing and uses of the
chemical include pesticide and other agricultural chemical manufacturing as intermediates. No
commercial and consumer uses were reported for the chemical.
2.2 Environmental Exposure and Fate
The environmental fate characteristics of l,2-dimethyl-4-nitrobenzene are summarized in Table
2.
l,2-Dimethyl-4-nitrobenzene is expected to possess moderate mobility in soil. No
biodegradation studies were available for this compound; however, data from several structural
analogs indicates that it should not be persistent in the environment. 4-Nitrotoluene (CASRN
99-99-0) was not readily biodegradable using an activated sludge inoculum and the modified
MITI (OECD 301C) test and it did not degrade over the course of 20 days using a method similar
to the closed bottle (OECD 301D) test, but it was completely degraded in 21 days (10-day
acclimation period) and 98% degraded within 5 days using a method similar to the Zahn-Wellens
(OECD 302B) test. Therefore, it is reasonable to conclude this substance is not readily
biodegradable but is inherently biodegradable. 3-Nitrotoluene (CASRN 99-08-1) achieved 2%
of its theoretical biochemical oxygen demand (BOD) after 14 days using an activated sludge
inoculum and the MITI test. However, it degraded 93% over the course of 28 days using mixed
microbial cultures amended with the sludge in a semi-continuous activated sludge (SCAS)
system. 2-Nitrotoluene (CASRN 88-72-2) did not pass a MITI test degrading, <1% after
14 days; however, it was readily biodegradable (>70% loss after 20 days) using an adapted
sludge as inoculum and the closed bottle (OECD 301D) test. 2,4-Dimethyl-l-nitrobenzene
(CASRN 89-87-2) was reported to be inherently biodegradable as measured by the Zahn-
Wellens test, although apparently there was significant loss due to air stripping during the test.
The data from the structural analogs suggest that l,2-dimethyl-4-nitrobenzene may not be readily
biodegradable, but is likely to be inherently biodegradable. Volatilization is expected to be
moderate for l,2-dimethyl-4-nitrobenzene given its Henry's Law constant. The rate of
hydrolysis of l,2-dimethyl-4-nitrobenzene is negligible. The rate of atmospheric photooxidation
is negligible. No measured bioconcentration data are available for l,2-dimethyl-4-nitrobenzene;
however, structural analog 2-nitrotoluene had measured BCF values of <100 measured in carp at
2 different concentrations. The overall weight of evidence suggests that l,2-dimethyl-4-
nitrobenzene is expected to have low persistence (PI) and low bioaccumulation potential (Bl).
Table 2. Environmental Fate Properties of l,2-Dimethyl-4-nitrobenzene and Supporting
Chemicals'
Property
SPONSORED
CHEMICAL
l,2-Dimethyl-4-
nitrobenzene
SUPPORTING
CHEMICAL
2,4-Dimethyl-l-
nitrobenzene
SUPPORTING
CHEMICAL
2-Nitrotoluene
SUPPORTING
CHEMICAL
3-Nitrotoluene
SUPPORTING
CHEMICAL
4-Nitrotoluene
CASRN
99-51-4
89-87-2
88-72-2
99-08-1
99-99-0
Photodegradation
Half-life
10.2 days
(estimated)2
5.5 days
(estimated)2
13.8 days
(estimated)2
18.4 days
(estimated)2
13.8 days
(estimated)2
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Table 2. Environmental Fate Properties of l,2-Dimethyl-4-nitrobenzene and Supporting
Chemicals1
Property
SPONSORED
CHEMICAL
l,2-Dimcthvl-4-
nitrobenzene
SUPPORTING
CHEMICAL
2,4-Dimethyl-l-
nitrobenzene
SUPPORTING
CHEMICAL
2-Nitrotoluene
SUPPORTING
CHEMICAL
3-Nitrotoluene
SUPPORTING
CHEMICAL
4-Nitrotoluene
Hydrolysis Half-
life
Stable
Stable
Stable
Stable;
18.1% loss after
8 days at pH 7.4 and
25°C
Stable
Biodegradation
No data
95% after 27 days
(inherently
biodegradable)
>90% after 15 days
(inherently
biodegradable);
>70% after 20 days
(readily
biodegradable)5;
0.5-0.8% after
14 days
(not readily
biodegradable)3
2% after 14 days
(not readily
biodegradable);
93% after 28 days
(inherently
biodegradable)
0.8% after 14 days
(not readily
biodegradable);
100% after 20 days
(inherently
biodegradable);
98% after 5 days
(inherently
biodegradable);
0% after 20 days
(not readily
biodegradable)
Bioaccumulation
Factor
BAF = 51.4
(estimated)2
BAF = 50.9
(estimated)2
BCF = 12.5-29.9
(measured in carp
at 0.1 ppm)3;
BCF = 6.6-29.7
(measured in carp
at 0.01 ppm)3;
BAF = 18.3
(estimated)2
BAF = 19.8
(estimated)2
BAF = 14.4
(estimated)2
Log Koc
2.8 (estimated)2
2.8 (estimated)2
2.6 (estimated)2
2.6 (estimated)2
2.6 (estimated)2
Fugacity
(Level III
Model)2
Air (%)
Water (%)
Soil (%)
Sediment (%)
3.1
16.3
80.0
0.5
3.0
15.4
81.1
0.5
2.8
18.9
77.8
0.4
2.6
19.1
77.9
0.4
2.6
19.2
77.8
0.4
Persistence4
PI (low)
PI (low)
PI (low)
PI (low)
PI (low)
Bioaccumulation4
B1 (low)
B1 (low)
B1 (low)
B1 (low)
B1 (low)
1 BASF Corporation. 2009. Revised Test Plan and Robust Summary for 1,2-dimethyl-4-nitrobenzene. Available online at
http://www.epa.gov/chemrtk/pubs/summaries/12dim4nt/cl6493tc.htm as of June 22, 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 June
22, 2012.
3National Institute of Technology and Evaluation. 2002. Chemical Risk Information Platform (CHIRP). Available online
at http://www.safe.nite.go.ip/english/db.html as of June 22, 2012.
4Federal Register. 1999. Category for Persistent, Bioaccumulative, and Toxic New Chemical Substances. Federal Register
64, Number 213 (November 4, 1999) pp. 60194-60204.
5Used adapted sludge as inoculum.
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Conclusion: l,2-Dimethyl-4-nitrobenzene is a pale, straw yellow-colored liquid with moderate
vapor pressure and moderate water solubility. It is expected to have moderate mobility in soil.
Although no biodegradation data are available for this compound, based on close structural
analogs, (2,4-dimethyl-l -nitrobenzene, 2-nitrotoluene, 3-nitrotoluene and 4-nitrotoluene), 1,2-
dimethyl-4-nitrobenzene is not expected to be readily biodegradable. Volatilization of 1,2-
dimethyl-4-nitrobenzene is expected to be moderate. The rate of hydrolysis is negligible. 1,2-
Dimethyl-4-nitrobenene is not expected to bioconcentrate in aquatic organisms based on
bioconcentration data measured in carp for 2-nitrotoluene. The rate of atmospheric
photooxidation is negligible. l,2-Dimethyl-4-nitrobenzene is expected to have low persistence
(PI) and low bioaccumulation potential (Bl).
3. Human Health Hazard
A summary of health effects data for SIDS and other endpoints is provided in Table 3. The table
also indicates where data for the supporting chemicals are read-across (RA) to the sponsored
substance.
Acute Oral Toxicity
l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4)
Male Wistar rats (10/dose) were administered neat l,2-dimethyl-4-nitrobenzene orally at 712,
1424, 2848 or 5695 mg/kg (length of post-dosing observation period was not specified).
Mortality (9/10 rats) was observed at 5695 mg/kg within 24 hours. No mortality was observed at
712 mg/kg.
LDso = 2636 mg/kg
2,4-Dimethyl-l-nitrobenzene (CASRN 89-87-2, supporting chemical)
Wistar rats of both sexes (number not specified) were administered 2,4-dimethyl-l-nitrobenzene
(> 99% purity) orally at unspecified dose levels. No additional information provided.
LD50 = 1690/2240 mg/kg (male/female)
Acute Dermal Toxicity
l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4)
Five male New Zealand White rabbits received a single dermal application of an unspecified
amount of neat l,2-dimethyl-4-nitrobenzene (purity not provided) to clipped, intact skin for 24
hours under an occlusive wrap. Animals were observed for 14 days post-exposure. One animal
died.
LD50 = 5695 mg/kg
Repeated-Dose Toxicity
l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4)
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No Data.
2,4-Dimethyl-l-nitrobenzene (CASRN 89-87-2, supporting chemical)
In a 28-day dietary repeated-dose toxicity study, Wistar rats (5/sex/dose) were fed 0, 100, 600 or
3000 ppm (approximately 0, 10, 60 or 300 mg/kg-bw/day) 2,4-dimethyl-l-nitrobenzene (purity
not provided) daily and observed for 14 days post exposure. There were no effects on general
health, behavior, water consumption and on urinary parameters. At 3000 ppm, reduced weight
gain (significance not stated) was observed both during the treatment period and during the
recovery period. This reduction in body weight gain was accompanied by a reduction in feed
consumption. Hematological effects in the 3000 ppm group included a decreased concentration
of hemoglobin and red blood cells and an increase in platelet count. In addition, decreased serum
protein levels were observed. Increased liver and spleen weights were observed; however, no
test substance-related gross pathological or histopathological effects occurred. Additional
information not provided.
LOAEL ~ 300 mg/kg-bw/day (based on reduced body weight gain, hematological and clinical
chemistry effects, increased liver and spleen weights)
NOAEL ~ 60 mg/kg-bw/day
4-Nitrotoluene (CASRN 99-99-0, supporting chemical)
(1) In a 13-week National Toxicology Program (NTP) dietary study, F334/N rats
(10/sex/concentration) were administered 4-nitrotoluene (>96% purity) in feed at 0, 625, 1250,
2500, 5000 or 10,000 ppm [approximately 0, 42, 82, 165, 342 or 723 mg/kg-bw/day (males) or
0, 44, 82, 164, 335 or 680 mg/kg-bw/day (females)]. There were no mortalities. Decreased body
weights were seen at > 342/335 mg/kg-bw/day. There were many organ weight changes.
Absolute heart weight was reduced at > 82 mg/kg-bw/day (males) and 680 mg/kg-bw/day
(females). In males, absolute kidney weights were reduced at 723 mg/kg-bw/day and relative
kidney weights were increased at > 342 mg/kg-bw/day; in females, absolute kidney weights were
reduced at > 335 mg/kg-bw/day and relative kidney weights were increased at 680 mg/kg-
bw/day. Absolute liver weights were reduced in males at 723 mg/kg-bw/day and relative liver
weights were increased in males at > 342 mg/kg-bw/day and in females at 680 mg/kg-bw/day.
Absolute thymus weights were decreased in males at 723 mg/kg-bw/day and in females at > 335
mg/kg-bw/day. Absolute lung weights were reduced in both sexes at 723/680 mg/kg-bw/day;
relative lung weights were increased in males at > 342 mg/kg-bw/day. Absolute testis weights
were decreased in males at > 342 mg/kg-bw/day. Hyaline droplet nephropathy5 was seen in
males at all dose levels and karyomegaly was seen in both males (> 82 mg/kg-bw/day) and
females (all dose levels). Pigmentation in kidneys was also seen in males (723 mg/kg-bw/day)
and females (all dose levels). Splenic hematopoiesis, hemosiderin pigment and congestion were
seen in both sexes at all dose levels. Testicular degeneration was seen in males at 723 mg/kg-
bw/day. Changes in hematology and clinical chemistry were seen in both sexes and included
5 The presence of nephropathy in association with the hyaline droplet accumulation in male rats suggests that the
nephropathy in the males may be occurring by an alpha2U-globulin-mediated mechanism, which appears to be unique
to male rats and the response is probably not relevant to humans for purposes of risk assessment. EPA's Risk
Assessment Forum has outlined the key events and the data that are necessary to demonstrate this mode of action
(Alpha2u-Globulin: Association with Chemically Induced Renal Toxicity and Neoplasia in the Rat, EPA/625/3 -
91/019F). A key event, alpha2U-globulin accumulation, has been demonstrated in the NTP study.
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increased hematocrit in males (723 mg/kg-bw/day) and females (> 335 mg/kg-bw/day) at week 1
and decreased hematocrit in females at 680 mg/kg-bw/day at week 13; increased erythrocytes in
males (723 mg/kg-bw/day) and females (> 335 mg/kg-bw/day) at week 1 and decreased
erythrocytes in males (723 mg/kg-bw/day) and females (680 mg/kg-bw/day) at week 13;
decreased creatinine in both sexes at > 165/164 mg/kg-bw/day; and increased methemoglobin
and bile acid in both sexes at the high dose level.
LOAEL ~ 42/44 mg/kg-bw/day (based on karyomegaly in females, kidney pigmentation in
females, splenic hematopoiesis, hemosiderin pigment and congestion in both sexes)
NOAEL ~ Not established
(2)	In a 13-week NTP dietary study, B6C3F1 mice (10/sex/concentration) were administered
4-nitrotoluene (purity >96%) daily in feed at 0, 675, 1250, 2500, 5000 or 10,000 ppm
[approximately 0, 131, 212, 439, 813 or 1491 mg/kg-bw/day (males) or 0, 164, 320, 625, 1075 or
1634 mg/kg-bw/day (females)]. There were no mortalities. Decreased final body weights were
seen in males at > 813 mg/kg-bw/day and in females at 1634 mg/kg-bw/day. Increased relative
liver weights were seen at all dose levels in both sexes, but the summary indicated that since no
treatment-related gross lesions and no histopathological liver lesions were observed, this weight
increase was judged to be not treatment related. No effect on the reproductive system of either
sex was observed.
LOAEL ~ 813/1075 mg/kg-bw/day (male/female) (based on decreased body weights)
NOAEL ~ 439/625 mg/kg-bw/day (male/female)
(3)	See human health data at: http://webnet.oecd.org/hpv/UI/handler.axd?id=4cdf8442-3ecb-
44b8-8d66-411f4a3cel3c
3-Nitrotoluene (CASRN 99-08-1, supporting chemical)
(1) In a 13-week NTP dietary study, F334/N rats (10/sex/concentration) were administered
3-nitrotoluene (>96% purity) daily in feed at 0, 625, 1250, 2500, 5000 or 10,000 ppm
[approximately 0, 47, 94, 188, 375 or 750 mg/kg-bw/day]. There were no mortalities. Decreased
body weight gain and increased relative liver weights were seen in both sexes at 750 mg/kg-
bw/day. Increased relative kidney weights were seen at 750 mg/kg-bw/day in males and 375
mg/kg-bw/day in females. Other effects included increased bile acids in males at > 375 mg/kg-
bw/day and in females at 750 mg/kg-bw/day; a mild increase in ALT in females at > 188 mg/kg-
bw/day; hyaline droplet nephropathy6 in males at all dose levels; changes in hematology and
clinical chemistry in both sexes at all dose levels, including increased methemoglobin at 375
mg/kg-bw/day (males only) and 750 mg/kg-bw/day (both sexes); and splenic hemosiderin and/or
congestion in both sexes at > 375 mg/kg-bw/day. Testicular degeneration was seen in males at
750 mg/kg-bw/day, along with decreased epididymal sperm count and concentration. Estrous
cycle length was increased and the number of cycling animals was decreased at > 375 mg/kg-
6 The presence of nephropathy in association with the hyaline droplet accumulation in male rats suggests that the
nephropathy in the males may be occurring by an alpha2U-globulin-mediated mechanism, which appears to be unique
to male rats and the response is probably not relevant to humans for purposes of risk assessment. EPA's Risk
Assessment Forum has outlined the key events and the data that are necessary to demonstrate this mode of action
(Alpha2u-Globulin: Association with Chemically Induced Renal Toxicity and Neoplasia in the Rat, EPA/625/3 -
91/019F). A key event, alpha2U-globulin accumulation, has been demonstrated in the NTP study.
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bw/day. Additional information from NTP TR 23 available at:
http://ntp.niehs.nih.gov/ntp/htdocs/ST rpts/tox023.pdf
LOAEL ~ 47 mg/kg-bw/day (based on hematological and clinical chemistry changes in both
sexes and splenic hemosiderin and/or congestion in both sexes)
NOAEL ~ Not established
(2) In a 13-week NTP dietary study, male and female B6C3F1 mice (10/sex/group) were
administered 3-nitrotoluene (purity >96%) daily in feed at 0, 625, 1250, 2500, 5000 or 10,000
ppm [approximately 0, 101, 187, 375, 750 or 1500 mg/kg-bw/day]. There were no mortalities.
Decreased food consumption and body weight gain were seen in both sexes at > 750 mg/kg-
bw/day. Increased relative liver weights were seen in both sexes at all dose levels (there were no
gross or microscopic liver lesions). Increased relative lung weights were seen in both sexes at
750 mg/kg-bw/day. No effect on the reproductive system of either sex was observed. Additional
information from NTP TR 23 available at:
http://ntp.niehs.nih.gov/ntp/htdocs/ST rpts/tox023.pdf
LOAEL ~ 101 mg/kg-bw/day (based on increased relative liver weights in both sexes)
NOAEL ~ Not established
2-Nitrotoluene (CASRN 88-72-2, supporting chemical)
(1)	In a 13-week NTP dietary study, F334/N rats (10/sex/concentration) were administered 2-
nitrotoluene (>96% purity) daily in feed at 0, 625, 1250, 2500, 5000 or 10,000 ppm
[approximately 0, 47, 94, 188, 375 or 750 mg/kg-bw/day]. There were no mortalities. There
were decreases in final body weight at > 188 mg/kg-bw/day and food consumption at 750
mg/kg-bw/day. Other effects included increased relative liver weights in both sexes (all dose
levels); increased relative kidney weights (> 188 mg/kg-bw/day in males, > 94 mg/kg-bw/day in
females); toxic effects to the liver (cytoplasmic vacuolization and oval cell hyperplasia in males),
spleen (increase in splenic hematopoiesis; hemosiderin deposition and/or congestion) and kidney
(hyaline droplets in the renal tubular epithelial cells of males7) at > 94 mg/kg-bw/day; decreased
spermatid count and an increase in testicular lesions in males (> 375 mg/kg-bw/day); increased
estrous cycle length in females at 750 mg/kg-bw/day; and increases in mesothelial cell
hyperplasia and mesothelioma in males at > 375 mg/kg-bw/day. Additional information from
NTP TR 23 available at: http://ntp.niehs.nih.gov/ntp/htdocs/ST rpts/tox023.pdf
LOAEL ~ 47 mg/kg-bw/day (based on increased relative liver weights in both sexes)
NOAEL ~ Not established
(2)	In 13-week NTP study, B6C3F1 mice (10/sex/concentration) were administered 2-
nitrotoluene (>96% purity) daily in feed at 0, 625, 1250, 2500, 5000 or 10,000 ppm
[approximately 0, 101, 187, 375, 750 or 1500 mg/kg-bw/day]. There were no mortalities.
Lesions of the nasal cavity were seen at > 187 mg/kg-bw/day and decreased final body weights
were noted at >375 mg/kg-bw/day. Increased relative liver weights were also seen at > 375
mg/kg-bw/day in males and >187 mg/kg-bw/day in females. Males at 750 mg/kg-bw/day had
7 The presence of nephropathy in association with the hyaline droplet accumulation in male rats suggests that the
nephropathy in the males may be occurring by an alpha2U-globulin-mediated mechanism, which appears to be unique
to male rats and the response is probably not relevant to humans for purposes of risk assessment. EPA's Risk
Assessment Forum has outlined the key events and the data that are necessary to demonstrate this mode of action
(Alpha2u-Globulin: Association with Chemically Induced Renal Toxicity and Neoplasia in the Rat, EPA/625/3 -
91/019F). A key event, alpha2U-globulin accumulation, has been demonstrated in the NTP study.
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decreased epididymal sperm motility. Additional information from NTP TR 23 available at:
http://ntp.niehs.nih.gov/ntp/htdocs/ST rpts/tox023.pdf
LOAEL ~ 187 mg/kg-bw/day (based on increased relative liver weights in females and nasal
cavity lesions in both sexes)
NOAEL ~ 101 mg/kg-bw/day
(3) See human health data at:
http://esis.irc.ec.europa.eu/doc/risk assessment/REPORT/2nitrotoluenereport403.pdf.
Reproductive Toxicity
l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4)
No Data.
4-Nitrotoluene (CASRN 99-99-0, supporting chemical)
(1)	In an oral gavage reproductive/developmental toxicity screening test, Wistar rats
(12/sex/dose) were administered 0, 25, 100 or 400 mg/kg-day 4-nitrotoluene (purity 99%;
vehicle: 1% methylcellulose). Males were dosed for 2 weeks prior to mating, during mating and
up to day 36. Females were dosed for 2 weeks prior to mating, during mating and gestation and
up to day 4 or 5 postpartum. Additional investigations included histopathological examinations
of liver, spleen, kidney, pituitary gland, uterus, uterine cervix and vagina, mammary gland,
epididymides and prostate. Three males and five females at 400 mg/kg-day died during the
mating period and an additional female was sacrificed moribund on day 22. Significantly
decreased food consumption (P<0.05 - <0.01) was seen at 400 mg/kg-day in both sexes.
Females showed s significantly reduced (P< 0.01) body weight gain at 400 mg/kg-day during
gestation and lactation. Clinical signs seen in both sexes at 400 mg/kg-day included piloerection,
sunken flanks and increased water intake and urination. Mean absolute and relative liver
weights in males were significantly increased (P<0,05 - 0.01) at 100 and 400 mg/kg-day,
respectively. Mean absolute and relative spleen weights were significantly increased (P<0.01) at
400 mg/kg-day in both sexes. Splenic congestion was seen in males and females at 100 and 400
mg/kg-day. Histopathological findings included increased amounts of pigment in the spleen and
increased amount of periportal pigment at 400 mg/kg-day. In a single male at 400 mg/kg-day,
debris was observed in the epididymides together with exfoliation of spermatids. No histological
changes in male accessory sexual glands, ovaries, female mammae with mamillae and pituitary
gland were observed. Testicular atrophy and epididymal cellular debris were seen at 400 mg/kg-
day. There was no effect on fertility index. The gestation index, the number of corpora lutea
and mean number of pups delivered were decreased at 400 mg/kg-day; prenatal loss was
increased at 400 mg/kg-day. Mean pup weights were significantly reduced at 100 mg/kg-day
(P<0.05) and at 400 mg/kg-day (P<0.01). See human health data at:
http://webnet.oecd.org/hpv/UI/handler.axd?id=4cdf8442-3ecb-44b8-8d66-411f4a3cel3c
LOAEL (reproductive toxicity) = 100 mg/kg-day (based on reduced pup weights)
NOAEL (reproductive toxicity) = 25 mg/kg-day
(2)	In the NTP study in F334/N rats described above, absolute testis weights were decreased in
males at > 342 mg/kg-bw/day and testicular degeneration and decreased sperm count and
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motility were seen in males at 723 mg/kg-bw/day. Increased estrous cycle length and increased
proportion of rats in diestrus were seen in females at 680 mg/kg-bw/day.
(3)	In the NTP study in B6C3F1 mice described above, there were no effects on reproductive
parameters.
(4)	In a 13-week oral gavage study (frequency of treatment not stated), Fischer 344 rats
(10/sex/dose) were administered 0, 90, 180 or 360 mg/kg-day 4-nitrotoluene (vehicle: corn oil).
Sperm morphology and vaginal cytology examinations were performed. Males at 360 mg/kg-
day had decreased terminal body weights, decreased absolute cauda epididymis, epididymides
and testes weights and decreased relative epididymis weights. There were no effects on sperm
parameters. There were no effects on females.
(5)	In a 13-week oral gavage study (frequency of treatment not stated), B6C3F1 mice
(10/sex/dose) were administered 0, 40, 80 or 160 mg/kg-day 4-nitrotoluene (vehicle: corn oil).
Sperm morphology and vaginal cytology examinations were performed. There were no effects
on reproductive organ weights, sperm parameters or estrous cycle length.
(6)	See human health data at: http://webnet.oecd.org/hpv/UI/handler.axd?id=4cdf8442-3ecb-
44b8-8d66-411f4a3cel3c
3-Nitrotoluene (CASRN 99-08-1, supporting chemical)
(1)	In the NTP 13-week study in F334/N rats described previously, testicular degeneration was
seen in males at 750 mg/kg-bw/day, along with decreased epididymal sperm count and
concentration. Estrous cycle length was increased at > 375 mg/kg-bw/day.
(2)	In the NTP 13-week study in B3C6F1 mice described previously, there were no effects on
reproductive parameters.
2-Nitrotoluene (CASRN 88-72-2, supporting chemical)
(1)	In the 13-week NTP study in F334/N rats described previously, males at 375 and 750 mg/kg-
bw/day had marked degeneration of the seminiferous tubules and decreased epididymal sperm
motility and density. At 750 mg/kg-bw/day, an increased proportion of female rats were in
diestrus, and estrous cycle length was increased.
(2)	In the 13-week NTP study in B3C6F1 mice described previously, males at 750 mg/kg-
bw/day had decreased epididymal sperm motility. No effect on the estrous cycle was observed.
(3)	See human health data at:
http://esis.irc.ec.europa.eu/doc/risk assessment/REPORT/2nitrotoluenereport403.pdf.
Developmental Toxicity
l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4)
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No Data.
4-Nitrotoluene (CASRN 99-99-0, supporting chemical)
In the reproductive/developmental toxicity screening test described previously, Wistar rats
administered 4-nitrotoluene via gavage at 0, 25, 100 or 400 mg/kg-day
(vehicle: 1% methylcellulose) mean pup weights were reduced at 100 and 400 mg/kg-day. Clear
signs of maternal toxicity were observed at 400 mg/kg-day and included reduced feed intake,
severe body weight loss, hypoactivity, alteration of gaits, piloerection, respiratory sounds,
increased water intake and urination, sunken flanks, and reduced amount of feces with an
increased incidence of soft and light colored feces. Six females were sacrificed in a moribund
condition. Spleen weights were increased. At 100 and 25 mg/kg bw/day, feed intake and body
weight gain during lactation were marginally reduced. Mean pup weights were significantly
decreased during day 0-day 4 at 100 mg/kg-day (P<0.05) and 400 mg/kg-day (P<0.01). Two
pups from two litters at 400 mg/kg-day had hematomas, an effect not seen in any other groups.
See human health data at: http://webnet.oecd.org/hpv/UI/handler.axd?id=4cdf8442-3ecb-44b8-
8d66-411f4a3cel3c
LOAEL (maternal toxicity) = 25 mg/kg-day (based on decreased body weight gain)
NOAEL (maternal toxicity) = Not established
LOAEL (developmental toxicity) = 100 mg/kg-day (based on decreased pup weights)
NOAEL (developmental toxicity) = 25 mg/kg-day
Genetic Toxicity — Gene Mutation
l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4)
No Data.
In vitro
l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4)
(1)	In a National Toxicology Program (NTP) reverse mutation assay, Salmonella typhimurium
strains TA97, TA98, TA100 and TA1535 were treated with l,2-dimethyl-4-nitrobenzene purity
not provided) at concentrations of 0, 3, 10, 33, 100, 333, 1000 and 1666 |ig/plate with and
without metabolic activation. Positive and vehicle controls were included and responded
appropriately. Cytotoxicity was observed at > 333 |ig/plate. l,2-Dimethyl-4-nitrobenzene
caused reproducible positive responses with TA100 with activation. Additional information
from NTP website:
http://tools.niehs.nih.gov/ntp tox/index.cfm?fuseaction=salmonella.salmonellaData&studv no=
589230&cas no=99%2D5l%2D4&endpointlist=SA
l,2-Dimethyl-4-nitrobenzene was mutagenic in this assay.
(2)	In a reverse mutation assay, Salmonella typhimurium strains TA98 and TA100 were treated
with l,2-dimethyl-4-nitrobenzene (purity not provided) at unspecified concentrations with and
without metabolic activation. No information about controls or cytotoxic concentrations was
provided in the summary.
l,2-Dimethyl-4-nitrobenzene was not mutagenic in this assay.
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2,4-Dimethyl-l-nitrobenzene (CASRN 89-87-2, supporting chemical)
In a NTP reverse mutation assay, Salmonella typhimurium strains TA97, TA98, TA100 and
TA1535 were treated with 2,4-dimethyl-l-nitrobenzene (purity not provided) at concentrations
of 0, 3, 10, 33, 100, 333 and 1000 |ig/plate with and without metabolic activation. Positive and
vehicle controls were included and responded appropriately. Cytotoxicity was observed at > 100
|ig/plate. 2,4-Dimethyl-l-nitrobenzene caused reproducible positive responses with TA100 with
activation. Additional information from NTP website:
http://tools.niehs.nih.gov/ntp tox/index.cfm?fuseaction=salmonella.salmonellaData&studv no=
153433&cas no=89%2D87%2D2&endpointlist=SA
2,4-Dimethyl-l-nitrobenzene was mutagenic in this assay.
Genetic Toxicity — Chromosomal Aberrations
l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4)
No Data.
In vitro
4-Nitrotoluene (CASRN 99-99-0, supporting chemical)
In an in vitro test, Chinese hamster ovary (CHO) cells were treated with 4-nitrotoluene (> 96%
purity) with and without metabolic activation at concentrations ranging from 300 to 600 |ig/mL.
Positive and solvent controls were tested concurrently, but no information was provided on
whether they responded appropriately. Cytotoxicity was observed at > 500 |ag/m L;
chromosomal aberrations were induced both with and without metabolic activation.
4-Nitrotoluene induced chromosomal aberrations in this assay.
3-Nitrotoluene (CASRN 99-08-1, supporting chemical)
(1)	In an in vitro test, Chinese hamster ovary (CHO) cells were exposed to 3-nitrotoluene (>96%
purity) with and without metabolic activation at concentrations of 150, 300, 398, 437, 460 or 483
|ig/mL, Positive and negative controls were tested concurrently, but no information was
provided on whether they responded appropriately. It was not stated whether negative controls
were included. The high concentration was estimated to reduce cell growth by 50%.
3-Nitrotoluene did not induce chromosomal aberrations in this assay.
(2)	In an in vitro test, Chinese hamster lung cells were exposed to 3-nitrotoluene (purity not
stated) without metabolic activation at concentrations up to 250 |ig/mL. There was no
information on cytotoxicity or positive and negative controls.
3-Nitrotoluene did not induce chromosomal aberrations in this assay.
(3)	Human peripheral lymphocytes were exposed to 3-nitrotoluene (purity not stated) without
metabolic activation at concentrations of 0.002, 0.02, 0.1 and 0.5 mmol/L. There was no
information on cytotoxicity or positive and negative controls. The summary noted that 3-
nitrotoluene caused an increase in the ratio of the number of aberrant cells to the number of
metaphase cells scored.
3-Nitrotoluene induced chromosomal aberrations in this assay.
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Genetic Toxicity - Other
4-Nitrotoluene (CASRN 99-99-0, supporting chemical)
In an in vitro test, Chinese hamster ovary (CHO) cells were treated with 4-nitrotoluene (> 96%
purity) with and without metabolic activation at concentrations ranging from 300 to 600 |ig/mL.
Positive and solvent controls were tested concurrently, but no information was provided on
whether they responded appropriately. Cytotoxicity was observed at > 500 |ag/m L; sister
chromatid exchange were induced both with and without metabolic activation.
4-Nitrotoluene induced sister chromatid exchange in this assay.
Additional Information
Eye Irritation
l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4)
l,2-Dimethyl-4-nitrobenzene (0.1 mL) was instilled into the conjunctival sac of one eye of each
of six rabbits (strain and sex not indicated). The cornea, iris and conjunctivae were examined at
24, 48 and 72 hours post-instillation. No irritation was observed.
l,2-Dimethyl-4-nitrobenzene was not irritating to rabbit eyes in this study.
Dermal Irritation
l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4)
Six rabbits (sex and strain not specified) were exposed to 0.5 mL neat l,2-dimethyl-4-
nitrobenzene on shaved, intact or abraded skin under an impervious wrap for 24 hours. Skin
reactions were scored according to Draize for up to 72 hours after exposure. The primary
irritation score was 1.25 (no other results details were provided).
l,2-Dimethyl-4-nitrobenzene was not irritating to rabbit skin in this study.
Carcinogenicity
4-Nitrotoluene (CASRN 99-99-0, supporting chemical)
(1) In a 105-week NTP study, F344/N rats (50/sex/concentration) were fed 0, 1250, 2500 or 5000
ppm [approximately 0, 55, 110 or 240 mg/kg-bw/day (males) or 0, 60, 125 or 265 mg/kg-bw/day
(females)] 4-nitrotoluene (> 99% purity) in the diet for 105 - 106 weeks. In female rats,
increased incidence of clitoral gland neoplasms over the control was seen at 125 mg/kg-bw/day
and this also exceeded historical control ranges. The study authors indicated that there was no
increase in the highest dose group, possibly because of lower body weights in that group. In
male rats, incidences for fibromas and fibrosarcomas were seen at 55 and 110 mg/kg-bw/day, but
not at 240 mg/kg-bw/day; therefore, the study authors indicated that the increased incidences
were considered as an "uncertain finding." In male rats at 240 mg/kg-bw/day, incidences of
germinal epithelial atrophy of the testes were increased.
4-Nitrotoluene increased the incidence of tumors in rats in this study.
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(2) In a 105-week NTP study, B6C3F1 mice (50/sex/concentration) were fed 0, 1250, 2500 or
5000 ppm [approximately 0, 170, 345 or 690 mg/kg-bw/day (males) or 0, 155, 315 or 660
mg/kg-bw/day (females)] 4-nitrotoluene (> 99% purity) in the diet for 105 - 106 weeks. In male
mice at 690 mg/kg-bw/day, the incidence of alveolar/bronchiolar adenomas or carcinomas was
increased. In the liver, the incidences of hepatocyte syncytial alterations were increased in males
in all test groups. This change was considered to be preneoplastic by the study authors.
4-Nitrotoluene increased the incidence of tumors in male mice in this study.
2-Nitrotoluene (CASRN 88-72-2, supporting chemical)
(1)	In a 105-week NTP study, F344/N rats (60 - 70/sex/group) were fed 0, 1250, 2500 or 5000
ppm [approximately 0, 25, 50 or 90 mg/kg-bw/day (males) or 0, 30, 60 or 100 mg/kg-bw/day
(females)] 2-nitrotoluene (> 99% purity). In a 3-month, stop-exposure study, groups of 70 male
rats were fed 125 or 315 mg/kg-bw/day in the diet for 13 weeks followed by undosed feed for the
remainder of the study. Incidences of subcutaneous skin neoplasms were increased in males at
all treatment levels and the incidences of fibroma or fibrosarcoma (combined) were increased in
treated females. In all treated groups except 90 mg/kg-bw/day core study males, the incidences
of mammary gland fibroadenoma were increased. Increased incidences of mesothelioma, skin
neoplasms and mammary gland fibroadenoma were seen in the stop-exposure males. The
incidences of mammary gland hyperplasia were increased in females at 30 and 60 mg/kg-bw/day
(but not 100 mg/kg-bw/day). Liver weights of stop-exposure males at 315 mg/kg-bw/day were
greater than those of the controls at 3 months. The incidences of hepatocellular adenoma in
high-dose core study males and females and hepatocellular adenoma or carcinoma (combined) in
high-dose core study and high-dose stop-exposure males were increased. Cholangiocarcinoma
occurred in three high-dose stop-exposure males. The incidences of alveolar/bronchiolar
adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) were increased in high-
dose stop-exposure males, as were alveolar/bronchiolar hyperplasia in most exposed groups of
males and females.
2-Nitrotoluene increased the incidences of tumors in rats in this study.
(2)	In a 105-week NTP study, B6C3F1 mice (60/sex/group) were fed 0, 1250, 2500 or 5000 ppm
[approximately 0, 165, 360 or 700 mg/kg-bw/day (males) or 0, 150, 320 or 710 mg/kg-bw/day
(females)] 2-nitrotoluene (> 99% purity) in the diet. The incidences of hemangiosarcoma in all
exposed groups of males and in high-dose females were greater than those in the controls. Large
intestine carcinomas were observed in all exposed groups except high-dose males. The
incidences of hepatocellular neoplasms were increased in mid- and high-dose females.
2-Nitrotoluene increased the incidence of tumors in mice in this study.
Conclusion: The acute oral and dermal toxicity of l,2-dimethyl-4-nitrobenzene is low in rats
and rabbits, respectively. Dietary 13-week repeated-dose toxicity studies in rats with the
supporting chemicals, 2-, 3- and 4-nitrotoluene, revealed reduced body weight gain and toxic
effects to the liver, spleen and kidney (e.g., increased liver weights and clinical chemistry
changes in these organs) at 42- 47 mg/kg-bw/day; the NOAEL is not established. Impaired
testicular function (e.g., decreased sperm density and motility) and increased estrous cycle length
were also seen at >375 mg/kg-bw/day. Similar systemic effects were seen in rats at 300 mg/kg-
bw/day in a 28-day dietary study with the supporting chemical 2,4-dimethyl-4-nitrobenzene; the
NOAEL is 60 mg/kg-bw/day. In mice, in 13-week dietary studies, decreased body weights were
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seen with 4-nitrotoluene at 813/1075 mg/kg-bw/day (males/females); the NOAEL is 439/625
mg/kg-bw/day (males/females). 3-Nitrotoluene showed effects on relative liver weights 101
mg/kg-bw/day; the NOAEL was not established. In mice, 2-Nitrotoluene showed increased
relative liver weights in females and nasal cavity lesions in both sexes at 187 mg/kg-bw/day; the
NOAEL is 101 mg/kg-bw/day. No specific reproductive toxicity studies are available for 1,2-
dimethyl-4-nitrobenzene. An oral gavage combined reproductive/developmental toxicity
screening test in rats with the supporting chemical 4-nitrotoluene showed mortality in both sexes
at 400 mg/kg-day and reduced pup weights at 100 mg/kg-day; the NOAEL for reproductive and
developmental toxicity is 25 mg/kg-day. Based on decreasing body weights at all doses, the
NOAEL for maternal toxicity is not established. In the 13-week dietary repeated-dose studies in
rats, effects were observed on the reproductive systems of both sexes. Similar studies in mice
showed no effects. l,2-Dimethyl-4-nitrobenzene and the supporting chemical, 2,4-dimethyl-l-
nitrobenzene, induced gene mutations in bacteria in vitro and the supporting chemicals, 3- and 4-
nitrotoluene, induced chromosomal aberrations in mammalian cells in vitro. 4-Nitrotoluene
induced sister chromatid exchange in vitro. The supporting chemicals, 2- and 4-nitrotoluene
increased the incidence of tumors in rats and mice. l,2-Dimethyl-4-nitrobenzene is not irritating
to rabbit eyes or skin.
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September, 2014
Summary of the Screening Information Data Set as Submitted under the U.S. HPV Challenge Program -
Human Health Data
Endpoints
SPONSORED
CHEMICAL
1,2-Dimcthyl-
4-nitrobcnzcnc
(99-51-4)
SUPPORTING
CHEMICAL
2,4-Dimethylbenzene-
1-nitrobenzene
(89-87-2)
SUPPORTING
CHEMICAL
2-Nitrotoluene
(88-72-2)
SUPPORTING
CHEMICAL
3-Nitrotoluene
(99-08-1)
SUPPORTING
CHEMICAL
4-Nitrotoluene
(99-99-0)
Acute Oral Toxicity
LDso (mg/kg)
2636
1690-2240
-
-
-
Acute Dermal Toxicity
LDso (mg/kg)
5695
-
-
-
-
Repeated-Dose Toxicity
NOAEL/LOAEL
Oral (mg/kg-bw/day)
No Data
LOAEL = 42
NOAEL = Not
established
(RA)
LOAEL = 300
NOAEL = 60
LOAEL = 47
NOAEL = Not
established
LOAEL = 47
NOAEL = Not
established
LOAEL = 42/44 (m/f)
NOAEL = Not
established
Reproductive Toxicity
NOAEL/LOAEL
Oral (mg/kg-day)
Reproductive Toxicity
No Data
LOAEL = 100
NOAEL = 25
(RA)

Effects on male and
female reproductive
system in males and
females in 13-week
dietary studies in rats
and male mice
Effects on male and
female reproductive
system in males and
females in 13-week
dietary studies in rats
but not in mice
LOAEL = 100
NOAEL = 25
Developmental Toxicity
NOAEL/LOAEL
Oral (mg/kg-bw/day)
Maternal Toxicity
Developmental Toxicity
No Data
LOAEL = 25
NOAEL = Not
established
LOAEL = 100
NOAEL = 25
(RA)



LOAEL = 25
NOAEL = Not
established
LOAEL = 100
NOAEL = 25
Genetic Toxicity - Gene Mutation
In vitro
Positive
Positive
-
-
-

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U.S. Environmental Protection Agency
Hazard Characterization Document
September, 2014
Summary of the Screening Information Data Set as Submitted under the U.S. HPV Challenge Program -
Human Health Data
Endpoints
SPONSORED
CHEMICAL
1,2-Dimcthyl-
4-nitrobcnzcnc
(99-51-4)
SUPPORTING
CHEMICAL
2,4-Dimethylbenzene-
1-nitrobenzene
(89-87-2)
SUPPORTING
CHEMICAL
2-Nitrotoluene
(88-72-2)
SUPPORTING
CHEMICAL
3-Nitrotoluene
(99-08-1)
SUPPORTING
CHEMICAL
4-Nitrotoluene
(99-99-O)
Genetic Toxicity - Chromosomal
Aberrations
In vitro
No Data
Positive
(RA)


Positive
Positive
Additional Information
Eye Irritation
Skin Irritation
Carcinogenicity
Not irritating
Not irritating

Positive

Positive
Bold = measured data; RA = read across; - indicates endpoint not addressed for this chemical; m = male; f=female
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U.S. Environmental Protection Agency
Hazard Characterization Document
September, 2014
4. Hazard to the Environment
A summary of ecotoxicity data for SIDS and other endpoints is provided in Table 4. The table
also indicates where data for the supporting chemical are read-across (RA) to the sponsored
substances.
Since the submitted ecotoxicity studies were lacking relevant testing details, ECOSAR (v. 1.1)
was used to support the endpoint values.
Acute Toxicity to Fish
l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4)
The ECOSAR-predicted 96-hour LCso value for fish was 19.1 mg/L. A measured log Kow value
of 2.91 was input into the estimation program.
96-h LCso =19.1 mg/L (estimated)
2,4-Dimethyl-l-nitrobenzene (CASRN 89-87-2, supporting chemical)
Rainbow trout (Brachydanio rerio) were exposed to nominal concentrations of 0, 10, 18, and 25
mg/L of 2,4-dimethyl-l-nitrobenzene (> 99% purity) under static conditions for 96 hours. No
further methods or results details were provided.
96-h LCso =10-18 mg/L
Acute Toxicity to Aquatic Invertebrates
l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4)
(1)	Daphnia magna were exposed to unspecified measured concentrations of l,2-dimethyl-4-
nitrobenzene (> 99% purity, dissolved in dimethyl sulfoxide) under static conditions for 48
hours. No details concerning mortality/effects at each test concentration were provided.
48-h ECso = 16 mg/L
(2)	The ECOSAR-predicted 48-hour LC50 value for daphnids was 11.8 mg/L. A measured log
Kow value of 2.91 was input into the estimation program.
48-h LCso = 11.8 mg/L (estimated)
2,4-Dimethyl-l-nitrobenzene (CASRN 89-87-2, supporting chemical)
Daphnia magna were exposed to unspecified nominal concentrations of 2,4-dimethyl-l-
nitrobenzene (unspecified purity) under unspecified conditions for 48 hours. No further methods
or results details were provided.
48-h ECso = 15 mg/L
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U.S. Environmental Protection Agency
Hazard Characterization Document
September, 2014
Toxicity to Aquatic Plants
l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4)
(1)	Green algae (Chlorellapyrenoidosa) were exposed to unspecified nominal concentrations of
l,2-dimethyl-4-nitrobenzene (> 99% purity) for 96 hours. No details concerning biomass at each
test concentration were provided.
96-h ECso (biomass) = 8.9 mg/L
(2)	The ECOSAR-predicted 96-hour EC50 value for algae was 8.44 mg/L. A measured log Kow
value of 2.91 was input into the estimation program.
96-h EC50 = 11.2 mg/L (estimated)
Conclusion: The fish 96-h LC50 for l,2-dimethyl-4-nitrobenzene ranges between 10-18 mg/L,
based on the supporting chemical 2,4 dimethyl-1-nitrobenzene. The aquatic invertebrate 48-h
EC50 for l,2-dimethyl-4-nitrobenzene is 16 mg/L. The algal 72-h EC50 for 1,2-dimethyl-
4-nitrobenzene is 8.9 mg/L.
Summary of the Screening Information Data Set as Submitted
under the U.S. HPV Challenge Program - Aquatic Toxicity Data
Endpoints
SPONSORED CHEMICAL
l,2-Dimcthyl-4-nitrobcnzcnc
(99-51-4)
SUPPORTING CHEMICAL
2,4-Dimcthylbcnzcnc-
l-nitrobcnzcnc
(89-87-2)
Fish
96-h LCso
(mg/L)
No Data
10-18
(RA)
10-18
Aquatic Invertebrates
48-h ECso (mg/L)
16
15
Aquatic Plants
72-h ECso (mg/L)
(growth)
(biomass)
8.9
-
Bold = experimentally-derived test data; RA = read across; - indicates endpoint not addressed for
this chemical
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U.S. Environmental Protection Agency
Hazard Characterization Document
September, 2014
APPENDIX
Sponsored Chemical
Chemical Name
CASRN
Structure
l,2-Dimethyl-4-
nitrobenzene
99-51-4
CH,
6r
no2
SMILES: 0=N(=0)c(ccc(c 1 C)C)c 1
Supporting Chemicals
Chemical Name
CASRN
Structure
2,4-Dimethyl-l-
nitrobenzene
89-87-2
CH,
£
T CH3
no2
SMILES: 0=N(=0)c(c(cc(cl)C)C)c 1
2-Nitro toluene
88-72-2
ch3
cr
SMILES: N(=0)(=0)c(c(cccl)C)c 1
3-Nitro toluene
99-08-1
CH,
&
^no2
SMILES: N(=0)(=0)c(cccclC)c 1
4-Nitrotoluene
99-99-0
CH,
no2
SMILES: N(=0)(=0)c(ccc(cl)C)c 1
24

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