TECHNICAL REPORT DATA
(netuntd Instruction* on the reverse In fort eomplennt/
1. REPORT NO.
EPA/600/8-88/049
2.
3. RECIPIENT'S ACCESSION NQ.
PB88-178975
4. TITLE ANO SUBTITLE
S. REPORT DATE
Health Effects Assessment for Nitrobenzene
*. PERFORMING ORGANIZATION CODE
7. AUTMOR(S)
. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME ANO ADDRESS
10. PROGRAM ELEMENT
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
13. TYPE OF REPORT ANO PERIOD COVERED
Environmental Criteria and Assessment Office
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati. OH 45268
14. SPONSORING AGENCY CODE
EPA/600/22
15 SUPPLEMENTARY NOTES
16. ABSTRACT
This report summarizes and evaluates information relevant to a preliminary interim
assessment of adverse health effects associated with specific chemicals or compounds..
The Office of Emergency and Remedial Response (Superfund) .uses these documents in
preparing cost-benefit analyses under Executive Order 12991 for decision-making under
CERCLA. All estimates of acceptable intakes and carcinogenic potency presented in
this document should be considered as preliminary and reflect limited resources
allocated to this project. The intent in these assessments is to suggest acceptable
exposure levels whenever sufficient data are available. The interim values presented
reflect the relative degree of hazard associated with exposure or risk to the
chemical(s) addressed. Whenever possible, two categories of values have been
estimated for systemic toxicants (toxicants for which cancer is not the endpoint of
concern). The first, RfD$ or subchronic reference dose, is an estimate of an exposure
level that would not be expected to cause adverse effects when exposure occurs during
a limited time interval. The RfD is an estimate of an exposure level that would not
be expected to cause adverse effects when exposure occurs for a significant portion
of the lifespan. For compounds for which there is sufficient evidence of
carcinogenicity, QI*S have been computed, if appropriate, based on oral and
inhalation data if available.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Croup
a. DISTRIBUTION STATEMENT
Public
19. SECURITY CLASS (This Re fort I
Unclassified
21. NO. OF PAGES
20. SECURITY CLASS (Tl\ilpat*l
Unclassified
22. PRICE
fun* 2220-1 (*.'. 4-77) PHCVIOU* COITION i* oa.toi.CTC
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EPA/600/8-88/049
Hay, 1987
HEALTH EFFECTS ASSESSMENT
FOR NITROBENZENE
ENVIRONMENTAL CRITERIA AND ASSESSMENT OFFICE
OFFICE OF HEALTH AND ENVIRONMENTAL ASSESSMENT
OFFICE Of RESEARCH AND-DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OH 45268
U.S. Envirciunental Protectl-.m Agency
T\H'-'-i- 5, Library (5?!.-::)
:'?-.; 5. Dearborn Street, Ho;;'i 1!:'70
Cnicago, IL 60504
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DISCLAIMER
This document has been reviewed In accordance with the U.S.
Environmental Protection Agency's peer and administrative review policies
and approved for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
11
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PREFACE
This report summarizes and evaluates Information relevant to a prelimi-
nary Interim assessment of adverse health effects associated with nitro-
benzene. All estimates of acceptable Intakes and carcinogenic potency
presented In this document should be considered as preliminary reflecting
limited resources allocated to this project. Pertinent toxlcologlc and
environmental data were located through on-line literature searches of the
TOXLINE, CANCERLINE and the CHEMFATE/DATALOG data bases. The basic litera-
ture searched supporting this document Is current up to May, 1986. Secon-
dary sources of Information have also been relied upon In the preparation of
this report and represent large scale health assessment efforts that entail
extensive peer and Agency review. The following Office of Health and
Environmental Assessment (OHEA) sources have been extensively utilized:
U.S. EPA. 1980a. Ambient Water Quality Criteria for
Nitrobenzene. Prepared by the Office of Health and Environmental
Assessment, Environmental Criteria and Assessment Office,
Cincinnati, OH for the Office of Water Regulations and Standards,
Washington, DC. EPA-440/5-80-061. NTIS PB81-117723.
U.S. EPA. 1983a. Reportable Quantity Document for Benzene,
NHro. Prepared by the Office of Health and Environmental
Assessment, Environmental Criteria and Assessment Office,
Cincinnati, OH for the Office of Emergency and Remedial Response,
Washington, DC.
U.S. EPA. 1985. Health and EnvlronmentaT Effects Profile for
Nitrobenzene. Prepared by the Office of Health and Environmental
Assessment, Environmental Criteria and Assessment Office.
Cincinnati, OH for the Office of Solid Waste and Emergency
Response, Washington, DC.
The Intent In these assessments 1s to suggest acceptable exposure levels
for noncardnogens and 'risk cancer potency estimates for carcinogens
whenever sufficient data were available. Values were not derived or larger
uncertainty factors were employed when the variable data were limited In
scope tending to generate conservative (I.e., protective) estimates.
Nevertheless, the Interim values presented reflect the relative degree of
hazard or risk associated with exposure to the chemlcal(s) addressed.
Whenever possible, two categories of values have been estimated for
systemic toxicants (toxicants for which cancer Is not the endpolnt of
concern). The first, RfDs (formerly AIS) or subchronlc reference dose, Is
an estimate of an exposure level that would not be expected to cause adverse
effects when exposure occurs during a limited time Interval (I.e., for an
Interval that does not constitute a significant portion of the llfespan).
in
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This type of exposure estimate has not been extensively used, or rigorously
defined, as previous risk assessment efforts have been primarily directed
towards exposures from toxicants 1n ambient air or water where lifetime
exposure 1s assumed. Animal data used for RFD$ estimates generally
Include exposures with durations of 30-90 days. Subchronlc human data are
rarely available. Reported exposures are usually from chronic occupational
exposure situations or from reports of acute accidental exposure. These
values are developed for both Inhalation (RfD$i) and oral (RfD$rj)
exposures.
The RfO (formerly AIC) Is similar In concept and addresses chronic
exposure. It 1s an estimate of an exposure level that would not be expected
to cause adverse effects when exposure occurs for a significant portion of
the Hfespan [see U.S. EPA (1980b) for a discussion of this concept]. The
RfD 1s route-specific and estimates acceptable exposure for either oral
(RfDrj) or Inhalation (RfOj) with the Implicit assumption that exposure
by other routes Is Insignificant.
Composite scores (CSs) for noncardnogens have also been calculated
where data permitted. These values are used for Identifying reportable
quantities and the methodology for their development Is explained 1n U.S.
EPA (1983).
For compounds for which there 1s sufficient evidence of cardnogenlcHy
RfO$ and RfD values are not derived. For a discussion of risk assessment
methodology for carcinogens refer to U.S. EPA (1980b). Since cancer 1s a
process that 1s not characterized by a threshold, any exposure contributes
an Increment of risk. For carcinogens, q-|*s have been computed, 1f appro-
priate, based on oral and Inhalation data If available.
1v
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ABSTRACT
»
In order to place the risk assessment evaluation In proper context,
refer to the preface of this document. The preface outlines limitations
applicable to all documents of this series as well as the appropriate
Interpretation and use of the quantitative estimates presented.
The major toxldty study with nitrobenzene was a 90-day Inhalation study
using rats and mice (CUT, 1984) In which hemolytlc anemia and testlcular
lesions occurred at all levels (5, 16 or 50 ppm equivalent to 25, 81 or 252
mg/m3) 1n rats, and vacuollzatlon of the adrenal cortex occurred at all
levels (same as rats) 1n female mice. From the LOAEL of 25 mg/m3 In mice,
an RfD$i and RfDj of 0.4 and 0.04 mg/day, respectively, were derived. A
CS of 37.6 associated with testlcular effects In rats was also calculated
for 25 mg/m3.
An RfD$o and RfDfl of °-3 and °-03 "ig/day, respectively, were also
derived from the LOAEL of 25 mg/m3 In mice, associated with adrenal
changes.
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ACKNOWLEDGEMENTS
The Initial draft of this report was prepared by Syracuse Research
Corporation under Contract No. 68-03-3112 for EPA's Environmental Criteria
and Assessment Office, Cincinnati, OH. Dr. Christopher DeRosa and Karen
Blackburn were the Technical Project Monitors and John Helms (Office of
Toxic Substances) was the Project Officer. The final documents In this
series were prepared for the Office of Emergency and Remedial Response,
Washington, DC.
Scientists from the following U.S. EPA offices provided review comments
for this document series:
Environmental Criteria and Assessment Office, Cincinnati, OH
Carcinogen Assessment Group
Office of A1r Quality Planning and Standards
Office of Solid Waste
Office of Toxic Substances
Office of Drinking Water
Editorial review for the document series was provided by the following:
Judith Olsen and Erma Durden
Environmental Criteria and Assessment Office
Cincinnati, OH
Technical support services for the document series was provided by the
following:
Bette Zwayer, Jacky Bohanon and Kim Davidson
Environmental Criteria and Assessment Office
Cincinnati, OH
v1
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TABLE OF CONTENTS
1.
2.
3.
4.
5.
ENVIRONMENTAL CHEMISTRY AND FATE
ABSORPTION FACTORS IN HUMANS AND EXPERIMENTAL ANIMALS . . . ,
2.1.
2.2.
ORAL
INHALATION
TOXICITY IN HUMANS AND EXPERIMENTAL ANIMALS
3.1.
3.2.
3.3.
3.4.
SUBCHRONIC
3.1.1. Oral
3.1.2. Inhalation ,
CHRONIC ,
3.2.1. Oral
3.2.2. Inhalation
TERATOGENICITY AND OTHER REPRODUCTIVE EFFECTS. . . .
3.3.1. Oral
3.3.2. Inhalation
TOXICANT INTERACTIONS
CARCINOGENICITY ,
4.1.
4.2.
4.3.
4.4.
HUMAN DATA . ,
4.1.1. Oral
4.1.2. Inhalation
BIOASSAYS
4.2.1. Oral
4.2.2. Inhalation
OTHER RELEVANT DATA
WEIGHT OF EVIDENCE
REGULATORY STANDARDS AND CRITERIA . .
Page
1
. . . 3
. . . 3
. . . 3
. . . 4
4
. . . 4
, , , 4
5
. . . 5
5
. . . 5
. . . 5
6
. . . 9
. . . 10
. . . 10
. . . 10
10
. . . 10
. . . 10
10
. . . 11
11
. . . 12
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TABLE OF CONTENTS
Page
6. RISK ASSESSMENT 13
6.1. SUBCHRONIC REFERENCE DOSE (RfOs) 13
6.1.1. Oral (RfOso) 13
6.1.2. Inhalation (RfDSi) 13
6.2. REFERENCE DOSE (RfO) 15
6.2.1. Oral (RfD0) 15
6.2.2. Inhalation (RfOi) 15
7. REFERENCES 17
APPENDIX 23
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LIST OF ABBREVIATIONS
ADI Acceptable dally Intake
CS Composite score
DNA DeoxyMbonuclelc acid
LOAEL Lowest-observed-adverse-effect lev«l
MED Minimum effective dose
MTD Maximum tolerated dose
NOAEL No-observed-adverse-effect level
ppm Parts per million
RfD Reference dose
RfDj Inhalation reference dose
RfDo Oral reference dose
RfD$ Subchronlc reference dose
RfD$j Subchronlc Inhalation reference dose
RfD$o Subchronlc oral reference dose
RVd Dose-rating value
RVe Effect-rating value
TLV Threshold limit value
TWA Time-weighted average
1x
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1. ENVIRONMENTAL CHEMISTRY AND FATE
The relevant physical and chemical properties and environmental fate of
nitrobenzene are presented In Table 1-1. Synonyms for. nitrobenzene are
nltrobenzol, essence of mlrbane and oil of mlrbane.
Reaction with HO radical and photolysis appear to be the significant
fate mechanisms In the ambient atmosphere. Based on an estimated reaction
rate constant of 0.06x10~12 cmVmolecule-sec and an assumed atmospheric
HO radical concentration of 10* molecule/cm3, the HO radical reaction
half-life has been calculated to be -133 days (U.S. EPA, 1985). In
moderately polluted air, the half-life may decrease by a factor of 10 (U.S.
EPA, 1985).
The aquatic half-life of nitrobenzene listed 1n Table 1-1 Is based on an
Investigation by Zoeteman et al. (1980). In the aquatic environment,
photolysis, volatilization and blodegradatlon are potentially significant
fate processes. It Is estimated that <3X of the nitrobenzene 1n rivers,
lakes and ponds will remain In the sediments. Nitrobenzene should not
bloaccumulate 1n aquatic organisms or ecologically magnify (U.S. EPA, 1985).
The soil half-life listed In Table 1-1 1s the volatilization half-life
of nitrobenzene obtained from a soil screening model. Potential exists for
nitrobenzene contamination of groundwater, since this compound appears to be
susceptible to significant leaching (U.S. EPA, 1985).
0073h -1- 05/14/87-
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TABLE 1-1
Selected Physical and Chemical Properties
and Half-Lives for Nitrobenzene
Property
Value
Reference
CAS number:
Chemical class:
Chemical formula:
Molecular weight:
Melting point:
Boiling point:
Vapor pressure:
at 20°C
at 25°C
at 30°C
Water solubility:
at 20°C
at 25°C
Log octanol/water
partition coefficient:
Bloconcentratlon factor:
Soil/sediment adsorption
coefficient:
Half-lives:
Air
Water
Soil
98-95-3
nltroaromatlc hydrocarbon
0.15 mm Hg
0.27 mm Hg
0.35 mm Hg
1900 mg/l
2090 mg/t
1.85
.<10, golden orfe
(Leuclscus Idus);
carp (Cyprlnus carplo)
24, green algae
(Chlorella fusca)
15, fathead minnow
(Plmephales promelas)
36-650 (estimated)
NR
0.3-3 days (estimated)
NR
Wlndholz, 1983
Wlndholz, 1983
U.S. EPA, 1985
U.S. EPA, 1985
Hansch and Leo,
1985
U.S. EPA, 1985
U.S. EPA, 1985
U.S. EPA, 1985
NR = Not reported
0073h
-2-
12/05/86
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2. ABSORPTION FACTORS IN HUMANS AND EXPERIMENTAL ANIMALS
2.1. ORAL
Rats that were treated with single 22.5 or 225 mg/kg doses of
[14C]-n1trobenzene by gavage eliminated -61-66, 12-21 and 1-3% of the
administered radioactivity In the urine, feces and expired air, respective-
ly, 1n 72 hours (Rlckert et al., 1983). Mice that were similarly treated
with 225 mg/kg [l4C]-n1trobenzene eliminated a significantly lower
percentage (-35%) of the administered radioactivity In the urine; similar
amounts were eliminated In the feces and expired air. B1le collected from
rats for 12 hours after administration of 225 mg/kg contained -2-4X of the
administered radioactivity; data after 72 hours were not reported, but It
was Indicated that biliary elimination of radioactivity was not fast enough
to account for all of the fecal radioactivity found. This Information
Indicates that gastrointestinal absorption of nitrobenzene Is likely to be
high (>80X), but that species differences 1n distribution or metabolism may
exist as Indicated by the mouse data.
2.2. INHALATION
Human pharmacoklnetlc data of Plotrowskl (1967, 1977) and Salmowa et al.
(1963JF Indicate that there 1s 80% retention of nitrobenzene during Inhala-
tion (U.S. EPA, 1980a; Beauchamp et al.. 1982). Nitrobenzene, however, can
be absorbed simultaneously through the skin; the ratio of dermal-to-
1nhalat1on absorption was reported to be 7:18 In an Inductlonal setting
(Plotrowskl, 1967).
0073h -3- 12/05/86
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3. TOXICITY IN HUMANS AND EXPERIMENTAL ANIMALS
3.1. SUBCHRONIC
3.1.1. Oral. Pertinent data regarding the toxic effects of nitrobenzene
from oral exposure could not be located 1n the available literature.
3.1.2. Inhalation. Groups of 10 male and 10 female Fischer 344 rats,
Sprague-Oawley CD rats and B6C3F1 mice were exposed by Inhalation to nitro-
benzene at concentrations of 0. 5, 16 or 50 ppm (0, 25, 81 or 252 mg/m3)
for 6 hours/day, 5 days/week for 90 days (CUT, 1984). Increased methemo-
globln levels were observed at >25 mg/m3 In Fischer rats,, at >81 mg/m3
In CD rats and at 252 mg/m3 1n the mice. Evidence of hematopolesls and
hemolytlc anemia, Including retlculocytosls, was observed at >81 mg/m3 In
Fischer 344 rats and at 252 mg/m3 1n the male CD rats, but not 1n the
mice. Increased extramedullary hematopolesls and hemoslderosls In the
spleens were observed In both F344 and CD rats at >25 mg/ma, especially at
the highest exposure level. Increased hemoslderosls also occurred 1n ml.ce
at >25 mg/m3. A significant Increase 1n splenic weight was. observed 1n
rats exposed to >81 mg/m3 and 1n mice exposed to 252 mg/m3.
A "minimum or very slight degree" of toxic nephrosls was observed 1n
-50% of CD and Fischer 344 rats exposed to 25 mg/m3 (CUT, 1984). Both
the Intensity and Incidence of toxic nephrosls Increased with Increasing
exposure levels In the rats. At 252 mg/m3, CD rats had Increased kidney
weights. Toxic nephrosls was not seen In the mice.
Hepatic lesions also occurred In the rats and mice. These Included
Increased Incidences of focal centrllobular necrosis at 252 mg/m3 1n the
F344 rats, hepatocellular hypertrophy and Kupffer cell pigmentation at >25
ng/n>3 In the CD rats, perlportal basophllla and enlarged nucleoll at >81
0073h -4- 08/14/86
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mg/nt3 In the CD rats and centrllobular hepatocellular hyperplasla at >81
mg/m3 In the mice. The Incidence and Intensity of these effects generally
Increased with Increased exposure levels.
Severe degeneration of the spermatogenlc epithelium coupled with signif-
icantly decreased testlcular weight and an absence of mature sperm In the
epldldymls was observed 1n 9/10 CO and 10/10 F344 rats that were exposed to
252 mg/m3 of nitrobenzene (CUT, 1984). Severe spermatogenlc epithelium
degeneration with testlcular atrophy occurred In 1/10 CD rats exposed to 25
mg/m3, but only very slight spermatogenlc epithelial loss occurred 1n 2/10
males at 81 mg/m3. Testlcular alterations were not observed In any of the
mice.
Vacuollzatlon of the zona retlcularls was observed In the adrenal glands
of all female B6C3F1 mice exposed to nitrobenzene at all levels (CUT,
1984). Although the adrenal lesion Increased In Intensity with Increased
exposure levels, the clinical significance of adrenal gland vacuollzatlon In
the retlcular zone Is unknown:
3.2. CHRONIC
3.2.1. Oral. Pertinent data regarding the toxic effects of nitrobenzene
from oral exposure could not be located 1n the available literature.
3.2.2. Inhalation. Pertinent data regarding the toxic effects of nitro-
benzene from Inhalation exposure could not be located In the available
literature. However, a 2-year Inhalation carc1nogen1c1ty study of nitro-
benzene, based on the findings of the 90-day study summarized 1n Section
3.1.2., Is currently being conducted by CUT (Section 4.2.2.).
3.3. TERATOGENICITY AND OTHER REPRODUCTIVE EFFECTS
3.3.1. Oral. Fischer 344 rats that were given single oral doses of 300
or 400 mg/kg nitrobenzene In corn oil developed necrotlc primary and
0073h -5- 12/05/86
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secondary spermatocytes with multlnucleated giant cells In the seminiferous
tubules after 1-5 days (Bond et al.t 1981). Necrotlc debris and decreased
numbers of spermatozoa were observed In the epldldymls within 3 days of
treatment. Groups containing three animals were evaluated In this study,
but these effects were not evident at lower doses (50-200 mg/kg).
3.3.2. Inhalation. Bio/Dynamics Inc. (1983) studied the fetotoxlclty of
nitrobenzene following Inhalation exposure In New Zealand white rabbits. In
this range finding study, pregnant rabbits were exposed to nitrobenzene at
0, 10, 40 or 80 ppm (0, 50, 200 or 400 mg/m3) for 6 hours/day on days 7-19
of gestation. Methemoglobin levels were measured 1n five rabbits of each
exposure group on gestation days 13 and 19 and In all rabbits on gestation
day 20. On gestation day 20, the rabbits were sacrificed, a gross post-
mortem examination was conducted, liver and kidney weights were recorded and
the numbers of live fetuses, dead fetuses, resorptlons and Implantation
sites were determined. Fetuses were not examined for malformations.
No adverse maternal effects on mortality, body weight, gross examination
at necropsy, or kidney and liver weights were noted. Methemoglobin levels
In rabbits exposed at 80 ppm were significantly higher than controls
throughout the study. Methemoglobln levels In rabbits from the lower
exposure group also tended to be elevated compared to controls, but the only
significant difference was the 40 ppm exposed group on gestation day 20. No
differences 1n the numbers of live fetuses, dead fetuses,, resorptlons and
Implantation sites were noted.
In the definitive developmental toxlclty study with nitrobenzene 1n
rabbits (B1o/Dynam1cs Inc., 1984), groups of 19-21 females were exposed to
0, 10, 40 or 100 ppm (0, 50, 200 or 500 mg/m3) according to the same
protocol described In the B1o/Dynam1cs Inc. (1983) study and were sacrificed
0073h -6- 12/05/86
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on gestation day 30. The only evidence of maternal toxldty was slight
elevations In relative liver weight and 40-60% Increases In blood methemo-
globin concentrations at 10 and 100 ppm. A slight but not statistically
significant Increase In the Incidence of embryonal absorptions was observed
at 100 ppm. There were no effects on fetal body weight or crown-rump length
and no treatment-related Increase In the Incidence of malformations or
developmental variations.
Tyl (1984) exposed groups of 26 mated CD rats to nitrobenzene at 0, 1.0,
10.0 or 40.0 ppm (0, 5.0, 50.3 or 201 mg/m3) for 6 hours/day on days 6-15
of gestation; gestation day 0 was the day that a copulatory plug was found.
Dams were sacrificed on gestation day 21. Parameters of maternal toxldty
(clinical signs, body weight, gross necropsy, selected organ weights) and
parameters of developmental toxlclty (fetal body weights, and external,
visceral and skeletal malformations) were examined. Maternal toxldty was
manifested In the high dose group by reduced rate of body weight gain during
the exposure period, but there were no differences 1n terminal body weights
at sacrifice. Effects on body weight gain were not observed at 1.0 or 10.0
ppm. Rats at 10 and 40 ppm had a dose-related Increase In absolute and
relative spleen weights. Exposure to nitrobenzene had no effect on repro-
ductive parameters or fetotoxldty. There was no significant Increase at
any exposure concentration In the number of Utters containing one or more
fetuses with malformations or variations. There was an Increase, however.
1n the Incidence of total malformations at 1.0, but not at 10 or 40 ppm.
The Investigator concluded that nitrobenzene was not teratogenlc In this
study.
Dodd and Klntlgh (1985) Investigated the effects of Inhaled nitrobenzene
In a 2-generat1on reproductive study In CD rats. Groups of 30 male and 30
0073h . -7- 12/05/86
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female rats were exposed to 0, 1, 10 or 40 ppm (0, 5, 50 or 200 mg/m3), 6
hours/day, 5 days/week for 10 weeks before mating. Exposure continued 6
hours/day, 7 days/week through a 2-week mating period. Mated females
(mating evidenced by a dropped vaginal plug) were exposed 6 hours/day
through day 19 of gestation and allowed to deliver. Exposure of nursing
dams resumed for 6 hours/day, 7 days/week starting on postpartum day 5 and
continuing through postpartum day 20. FQ males were necropsled at the end
of the mating period, f. rats were selected and continued on the same
exposure regimen as their parents. The F, groups were adjusted to 30
rats/sex. F~ rats were sacrificed at weaning. The usual reproductive
Indices were monitored.
There were no effects on survival or lactation Indices at any exposure
level. Body weights of F, rats of both sexes at 40 ppm were slightly
lower than controls at weaning. Fertility was significantly (p<0.001)
decreased only at 40 ppm. The-fertility Indices In the FQ generation were
53.3 and 10.0%, respectively, compared with 100.OX for F. and F,
controls. The reduction In fertility was attributed to effects on the
reproductive tracts of the males. At necropsy, F_ males had reduced-
relative testlcular and epldldymal weights, testes that were grossly reduced
In size and hlstologlcally observed seminiferous tubular atrophy and
spermatocyte degeneration. These effects were noted only at 40 ppm; there
were no treatment-related effects In females.
In order to Investigate the reversibility of nitrobenzene-Induced
effects on the reproductive organs of the male, control and 40 ppm F,
males were allowed a 9-week recovery period after which they were mated with
unexposed virgin females. The fertility Index after the recovery period had
Increased to 46.7X, which the Investigator Interpreted as evidence for the
0073H -8- 12/05/86
-------
reversibility of the nitrobenzene-Induced lesions 1n the testes of exposed
rats. At necropsy, F, males exposed to 40 ppm and allowed a 9-week
recovery period had reductions In relative testlcular and epldldymal weights
greater than those of the F_ males. Lesions observed microscopically were
similar to those observed 1n the F_ males, but the degeneration of
spermatocytes appeared to be less active.
3.4. TOXICANT INTERACTIONS
Human clinical and animal experimental evidence Indicates that alcohol
has a synerglstlc effect on nitrobenzene poisoning (DoMgan and Hushon,
1976; Rejsek, 1947; Smyth et al., 1969). Dorlgan and Hushon (1976) cited a
case In which Ingestlon of an alcoholic beverage triggered symptoms, Includ-
ing coma, of acute nitrobenzene poisoning In a worker who had apparently
recovered from the effects of chronic nitrobenzene poisoning. Rejsek (1947)
concluded that Ingestlon of one beer can precipitate an acute crisis In
Individuals suffering from subchronlc nitrobenzene poisoning as long as 6
weeks after the disappearance of symptoms.
0073h -9- 12/05/86
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4. CARCINOGENICITY
4.1. HUMAN DATA
4.1.1. Oral. Pertinent data regarding the carcinogenic effects of oral
exposure to nitrobenzene 1n humans could not be located In the available
literature.
4.1.2. Inhalation. Pertinent data regarding the carcinogenic effects of
Inhalation exposure to nitrobenzene 1n humans could not be located In the
available literature.
4.2. BIOASSAYS
4.2.1. Oral. Pertinent data regarding the carcinogenic effects of oral
exposure to nitrobenzene 1n animals could not be located In the available
literature.
4.2.2. Inhalation. Nitrobenzene was recommended for a carcinogen
bloassay to the National Cancer Institute (Helmes et al., 1982), based on
the suspicion that nitrobenzene Is carcinogenic since H 1s the nltro analog
of aniline and because of the substantial potential for human exposure.
Nitrobenzene Is not Included, however, on the most current 11st of chemicals
scheduled for testing by the National Toxicology Program (NTP, 1986).
CUT 1s currsntly conducting a 2-year Inhalation cardnogenlclty study
In which Fischer 344 and Sprague-Oawley rats are exposed to 0, 1, 5 and 25
ppm nitrobenzene (0, 5, 25 and 126 mg/m3, respectively), and B6C3F1 mice
are exposed to 0, 5, 25 or 50 ppm nitrobenzene (0, 25, 126 or 252 mg/m3,
respectively). This bloassay 1s based on the results of the 90-day
Inhalation study summarized 1n Section 3.1.2.
0073h -10- 12/05/86
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4.3. OTHER RELEVANT DATA
Nitrobenzene has been tested for mutagenldty In the Ames assay with
Salmonella typhlmuMum strains TA92, TA94, TA97, TA98, TA100, TA1535, TA1537
and TA1538 with negative results (Garner and Nutman, 1977; Chlu et al.,
1978; Shlmlzu et al., 1983; Ho et al., 1981; Haworth et al., 1983; Anderson
and Styles, 1978; Mlyata et al., 1981). These assays were conducted with
and without added exogenous metabolic activation preparations by plate
Incorporation, spot test or. In one study (Hughes et al., 1984) by vapor
exposure methods. Nitrobenzene was mutagenlc 1n S. typhlmurlum TA98 but not
TA 100 In the presence of metabolic activation and norharman, which Is found
In tobacco smoke (Suzuki et al., 1983).
Nitrobenzene did not cause an Increase In unscheduled ONA synthesis 1n
hepatocytes from gavage-treated rats (Mlrsalls et al., 1982). It 1s report-
ed In the abstract of a Russian study that nitrobenzene did not produce
mlcronuclel or chromosome aberrations In bone marrow cells or dominant
lethal mutations In mice following unspecified 1ntragastr1c administration
(Fel'dt, 1985).
4.4. WEIGHT OF EVIDENCE
The carclnogenlclty of nitrobenzene has not been adequately tested In
animals or sufficiently evaluated 1n humans. Pending the results of the
current 2-year CUT Inhalation study, nitrobenzene should be classified as
an IARC Group 3 and CAG Group D chemical, reflecting the Inability to
classify the chemical because of lack of data.
0073h -11- 12/05/86
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5. REGULATORY STANDARDS AND CRITERIA
ACGIH (1986) currently recommends a TWA-TLV of 5 mg/m3 (1 ppm) for an
8-hour nitrobenzene exposure, with a caution that cutaneous exposure can
significantly contribute to total exposure. The TLV Is based on occupation-
al exposures that did not result In the development of headaches, methemo-
globlnemla or anemia In workers. OSHA (1985) proposed a TWA permissible
exposure limit of 5 mg/m3 (1 ppm) for nitrobenzene.
U.S. EPA (1980a) recommended an ambient water quality criterion of 30
ug/l based on organoleptlc effects. An ADI of 0.032 mg/day was calcu-
lated from a subchronlc Inhalation LOAEL of 25 mg/m3 (CUT, 1984), which
reflects hepatic, renal and testlcular effects In rodents.
0073h -12- 12/05/86
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6. RISK ASSESSMENT
6.1. SUBCHRONIC REFERENCE DOSE (RfD$)
6.1.1. Oral (RfD-J. Subchronlc oral toxlclty studies of nitrobenzene
could not be located 1n the available literature. An RfDSQ can be
calculated, however, from the subchronlc Inhalation LOAEL of 25 mg/m3 from
the CUT (1984) study (Section 6.1.2.).
Route-to-route extrapolation for nitrobenzene Is appropriate because the
extent of oral and Inhalation absorption appears to be comparable (see
Chapter 2) and because similar toxic effects (e.g., methemogloblnemla and
liver and testls alterations) are produced by oral (Bond et al., 1981;
Goldstein et al., 1984) and Inhalation (CUT, 1984) exposures. Using the
same approach as In Section 6.1.2., but using an Inhalation absorption
factor of 0.8 (see Section 2.2.), the dose absorbed by mice exposed to 25
mg/m3 1s estimated to be 4.64 mg/kg/day and 1s considered to be a LOAEL.
Using an uncertainty factor of 1000, as In Section 6.1.2., the RfD-Q 1s
calculated to be Q.0046 mg/kg/day or 0.3 mg/day for a 70 kg human.
6.1.2. Inhalation (RfO..). An RfO.. can be derived from the CUT
(1984) study 1n which groups of 10 Fischer 344 and CO rats and B6C3F1
mice/sex were exposed to 0, 5, 16 or 50 ppm (0, 25, 81 or 252 mg/m3,
respectively) nitrobenzene for 6 hours/day, 5 days/week for 90 days. The
effects associated with .the exposure are detailed 1n Section 3.1.2. In
rats, hemolytlc anemia and lesions of the spleen (extramedullary hemato-
polesls), kidney (toxic nephrosls) and Hver (hepatocellular hypertrophy and
Kupffer cell pigmentation) occurred at 25 mg/m3. The Incidences of these
lesions were similar to controls and Intensity was minimal at 25 mg/m3,
but Incidences and severity Increased with Increasing exposure levels. One
of 10 male CD rats exposed to 25 mg/m3 had severe testlcular epithelial
0073h -13- 05/14/87
-------
mg/m3), the rats and mice were exposed to 2.84 and 5.80 mg/kg/day, respec-
tively. Using the higher LOAEL of 5.80 mg/kg/day and an uncertainty factor
of 1000 (10 for Interspedes extrapolation, 10 to estimate a NOAEL from a
LOAEL and 10 to protect the most sensitive humans), the RfD$I 1s calcu-
lated to be 0.0058 mg/kg/day or 0.4 mg/day for a 70 kg human.
6.2. REFERENCE DOSE (RfD)
6.2.1. Oral (RfDQ). Chronic oral toxldty data for nitrobenzene could
not be located 1n the available literature. An RfDQ for nitrobenzene can
be calculated, however, from the subchronlc Inhalation LOAEL of 25 mg/m3
(CUT, 1984) by the approach used to calculate the RfO_Q. By using the
absorbed dose for mice estimated 1n Section 6.1.1. (4.64 mg/kg/day) with an
additional uncertainty factor of 10 to approximate chronic exposure (total
uncertainty factor of 10,000), the RfD. Is calculated to be 0.0005
mg/kg/day or 0.03 mg/day for a 70 kg man (U.S. EPA, 1986).
6.2.2. Inhalation (RfD,). Chronic Inhalation toxldty data for- nitro-
benzene could not be located 1n the available literature. Pending the out-
come of the current CUT chronic toxldty study, an RfD. for nitrobenzene
can be calculated from the subchronlc Inhalation LOAEL of 25 mg/m3 (CUT,
1984) by using an approach Identical to that 1n Section 6.2.1. WHh an
additional uncertainty factor of 10 to approximate chronic exposure, the
RfDj 1s calculated to be 0.0006 mg/kg/day or 0.04 mg/kg/day for a 70 kg
human.
CSs for nitrobenzene can be calculated from the LOAEL (25 mg/m3)
associated with the hemolytlc, liver, kidney and testlcular effects (252
mg/m3) Identified 1n the CUT (1984) subchronlc Inhalation rat study
(Section 6.1.2.). Equivalent animal doses were estimated from the exposure
concentration by multiplying by 6 hours/24 hours and 5 days/7 days to adjust
0073h ' -15- 05/14/87
-------
degeneration and atrophy; degeneration was minimal and without atrophy In
2/10 CO rats at 81 mg/m3 and severe with atrophy and lack of mature sperm
In the ep1d1dym1s 1n 9/10 CD rats and 10/10 Fischer 344 rats at 252 mg/m3.
Increased methemoglobln levels were also observed at >25 mg/m3 1n Fischer
rats. In mice. Increased hemos1deros1s 1n the spleen and vacuollzatlon of
the zona retlcularls of the adrenal gland In females occurred at >25
mg/m3; the Incidence and Intensity of these effects Increased with higher
exposure concentrations. No liver, kidney or testlcular effects were
observed In the mice at 25 mg/ma.
The 25 mg/m3 exposure level 1s judged to be the LOAEL 1n rats because
of the hemolytlc anemia and spleen, kidney, liver and testls alterations.
The testlcular alterations at 25 mg/m3 (and 81 mg/m3) are treatment-
related 1n a particularly sensitive animal In the more sensitive rat strain
(CO). Although the Fischer rats also had Increased serum methemoglobln
levels and evidence of hemolytlc anemia at 25 mg/m3, this effect 1n the
rat may be an Inappropriate basis for deriving an RfDSIt since the TLV of
5 mg/m3 (ACGIH, 1986} 1s designed to be protective against methemoglobln-
emla and hematologlcal effects In humans. This exposure level (25 mg/m3)
1s also considered to be the LOAEL 1n mice because of the spleen and adrenal
cortex alterations; however, the toxlcologlcal significance of the adrenal
effect (Increased vacuollzatlon of the cortex) 1s unknown.
The dally doses to which the animals were exposed are estimated by
multiplying the exposure level by 6 hours/24 hours and 5 days/7 days to
adjust to continuous exposure and by the Inhalation rates (assumed to be
0.223 mVday for rats and 0.039 mVday for mice) (U.S. EPA, 1980b) and
by dividing by the reference body weights (assumed to be 0.35 kg for rats
and 0.03 kg for mice). These calculations show that at the LOAEL (25
0073h -14- 05/14/87
-------
to continuous exposure, by the rat Inhalation rate of 0.223 mVday (U.S.
EPA, 1980b) and by dividing by the reference body weight of the rats (0.35
kg). The dose was then divided by an uncertainty factor of 10 to approxi-
mate chronic exposure. HEDs were calculated by multiplying the equivalent
animal dose by the cube root of the ratio of reference animal body weight to
human body weight (70 kg) and by the human body weight to express the MEO 1n
units of mg/day.
Since the MED Is the same for all the effects In rats In this study,
differences In CSs would reflect differences In severities of the effects
(I.e., In the RV assigned). The testlcular alterations are the most
serious effects and warrant an RV of 8, associated with decreased repro-
ductive capacity; although absence of mature sperm 1n the epldldymls was
reported only at 252 mg/m3, this manifestation Is consistent with the
effects reported at 25 mg/m3. A CS of 37.6 Is based on the testlcular
effects In rats.
The CS calculated for the testlcular effects (37.6) differs slightly
from that (25.6) calculated by U.S. EPA (1985). The U.S. EPA (1985) deriva-
tion calculated the MED from the 81 mg/m3 exposure level, which Is not the
LOAEL Identified In the risk assessment section of the same report. The
LOAEL for testlcular effects was determined to be 252 mg/m3 by U.S. EPA
(1985), but additional consideration Indicates that the testlcular effects
at 25 mg/m3 are not anomalous, as Indicated 1n Section 6.1.2.
0073h -16- 12/05/86
-------
7. REFERENCES
ACGIH (American Conference of Governmental Industrial Hyglenlsts). 1986.
Documentation of the Threshold Limit values and Biological Exposure Indices,
5th ed. Cincinnati, OH. p. 431.
Anderson, D. and J.A. Styles. 1978. An evaluation of 6 short-term tests
for detecting organic chemical carcinogens. Appendix II. The bacterial
mutation test. Br. J. Cancer. 37: 924-930.
Beauchamp, Jr., R.O., R.D. Irons, O.E. Rlckert, D.B. Couch and T.E. Harm,
Jr. 1982. A critical review of the literature on nitrobenzene toxldty.
CRC CMt. Rev. Toxlcol. 11(1): 33-84.
B1o/Dynam1cs Inc. 1983. A range finding study to evaluate the toxldty of
nitrobenzene In the pregnant rabbit. OPTS, U.S. EPA, Washington, DC.
Public Files. Microfiche No. 0730509345.
Bio/Dynamics Inc. 1984. An Inhalation teratology study In rabbits with
nitrobenzene. Final Report. Project No. 83-2725. Unpublished data prepared
for Nitrobenzene Association Toxicology Task Group. Submitted to OTS, U.S.
EPA, Washington, DC.
Bond, J.H., J.P; Chlsm, D.E. Rlckert and J.P. Papp. 1981. Production of
hepatic and testlcular lesions In Fischer 344 rats by single oral doses of
nitrobenzene. Fund. Appl. Toxlcol. 1: 389-394.
0073h -17- 12/05/86
-------
Chlu, C.W., H.L. Lanfong, C.Y. Hang and G.T. Bryan. 1978. Mutagenldty of
some commercially available nltro compounds for Salmonella typhlmurlum.
Mutat. Res. 58: 11-22.
CUT (Chemical Industry Institute of Toxicology). 1984. Ninety-day Inhala-
tion study of nitrobenzene 1n F-344 rats, CO rats and B6C3F1 mice with cover
letter dated 6/24/84 and EPA response dated 8/06/84. Unpublished study.
FYI-OTS-0784-0333 and computer print-out of pathology finding.
Dodd, O.E. and W.J. K1nt1gh. 1985. Potential effects of nitrobenzene
Inhalation on reproduction and fertility 1n rats. Unpublished Report No.
47-524. Bushey Run Research Center for ICI Americas, Inc. Submitted to
OTS, U.S. EPA, Washington DC.
Oorlgan, J. and J. Hushon. 1976. Air pollution assessment of nitrobenzene.
NTIS PB257-776. (Cited In U.S. EPA, 1980a)
Fel'dt, E.G. 1985. Evaluation of the ntutagenlc hazards of benzene and some
of Us derivatives. G1g. Sanlt. 7: 21-23. (Rus.) (CA 103:09930h)
Garner, R. and C.A. Nutman. 1977. Testing of some azo dyes and their
reduction products for mutagenldty using Salmonella typhlmurlum TA1538.
Mutat. Res. 44: 9-19.
Goldstein, R.S., J.P. Chlsm, J.M. SherMll a"d T.E. Hamm, Jr. 1984. Influ-
ence of dietary pectin on Intestinal mlcrofloral metabolism and toxlclty of
nitrobenzene. Toxlcol. Appl. Pharmacol. 75(3): 547-553.
0073h , -18- 12/05/86
-------
Hansch, C. and A.J. Leo. 198S. Medchem Project. Issue No. 26. Pomona
College, Claremont, CA.
Haworth, S., T. Lawlor, K. Mortelmans, W. Speck and E. Zelger. 1983.
Salmonella mutagenlclty test results for 250 chemicals. Environ. Mutagen.
Suppl. 1: 3-142.
Helmes. C.T.. V.A. Fung. B. lewln, K.E. HcCaleb, S. Malko and A.M. Pawlovlch.
1982. A study of aromatic nltro compounds for the selection of candidates
for carcinogen bloassay. J. Environ. Scl. Health, Part A. A17(l): 75-128.
Ho, C.H., B.R. Clark, M.R. Guerln, B.O. Barkenbus. T.K. Rao and J.L. Epler.
1981. Analytical and biological analyses of test materials from the
synthetic fuel technologies. IV. Studies of chemical structure-mutagenlc
activity relationships of aromatic nitrogen compounds relevant to synfuels.
Mutat. Res. 85(5): 335-345.
Hughes, T.J., C. Sparaclno and S. Frazler. 1984. Validation of chemical
and biological techniques for evaluation of vapors In ambient alr/mutagenlc-
1ty testing of twelve (12) vapor-phase compounds. U.S. EPA, RTP, NC. EPA
600/1-84-005. NTIS PB84-164210.
MlrsaHs, J.C., C.K. Tyson and B.E. Butterworth. 1982. Detection of
genotoxlc carcinogens In the |n. vlvo-ln vitro hepatocyte DNA repair assay.
Environ. Mutagen. 4: 553-562.
0073h -19- 12/05/86
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Miyata, R., T. Nohml. K. Yoshlkawa and M. Ishldate. 1981. Metabolic
activation of p-nltrotoluene and trlchloroethylene by rat Hver S9 or mouse
liver S9 fractions 1n Salmonella typhlmuMum strains. Elsel Shlkensho
Hokoku. 99: 60-65. (Jap.) (CA 96:117264n)
NTP (National Toxicology Program). 1986. Management Status Report dated
09/05/86.
OSHA (Occupational Safety and Health Administration). 1985. Code of
Federal Regulations. 29: 1910.1000.
Plotrowskl, J. 1967. Further Investigations on the evaluation of exposure
to nitrobenzene. Br. J. Ind. Med. 24(1): 60. (Cited In Beauchamp et al.,
1982)
Plotrowskl, J. 1977. Exposure tests for organic compounds 1n Industrial
toxicology. U.S. DHEW. NIOSH 77-144. (Cited In U.S. EPA, 1980a)
Rejsek, K. 1947. m-DlnUrobenzene poisoning. Mobilization b> alcohol and
sunlight. Acta Med. Scan. 127: 179. (Cited 1n U.S. EPA, 1980a)
Rlckert, D.E., J.A. Bond, R.M. Long and J.P. Chlsm. 1983. Metabolism and
excretion of nitrobenzene by rats and mice. Toxlcol. Appl. I'haYmacol.
67(2): 206-214.
Salmowa, J., J. Plotrowskl and U. Neuhorn. 1963. Evaluation of exposure to
nitrobenzene. Br. J. Ind. Med. 10: 41. (Cited In Beauchamp et al., 1982;
U.S. EPA, 1980a)
0073h -20- 12/05/86
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Sh1m1zu, M., Y. Yasul and N. Matsumoto. 1983. Structural specificity of
aromatic compounds with special reference to mutagenlc activity In Salmo-
nella typhlmurlum -- A series of chloro- or fluoro-nltrobenzene derivatives.
Mutat. Res. 116: 217-238.
Smyth, Jr., H.F., et al. 1969. An exploration of Joint toxic action:
Twenty-seven Industrial chemicals Intubated In rats In all possible pairs.
Toxlcol. Appl. Pharmacol. 14: 340. (Cited In U.S. EPA, 1980a)
Suzuki, J., T. Koyaml and S. Suzuki. 1983. Mutagenlcltles of mono-nitro-
benzene derivatives In the presence of norharman. Mutat. Res. 120: 105-110.
Tyl, R.W. 1984. Teratogen1c1ty evaluation of Inhaled nitrobenzene In .the
CD rat. Unpublished Project Report 47-522. Bushey Run Research Center for
the Nitrobenzene Association. Submitted to OTS, U.S. EPA, Washington, DC.
U.S. EPA. 1980a. Ambient Water Quality Criteria for Nitrobenzene.
Prepared by the Office of Health and Environmental Assessment, Environmental
Criteria and Assessment Office, Cincinnati, OH for the Office of Water
Regulations* and Standards, Washington, DC. EPA 440/5-80-061. NTIS
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U.S. EPA. 1980b. Guidelines and Methodology for the Preparation of Health
Effect Assessment Chapters of the Ambient Water Quality Documents. Federal
Register. 45(231): 79347-79357.
0073h -21- 06/16/87
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U.S. EPA. 1983a. Reportable Quantity Document for Benzene, NHro.
Prepared by the Office of Health and Environmental Assessment, Environmental
Criteria and Assessment Office, Cincinnati, OH for the Office of Emergency
and Remedial Response, Washington, DC.
U.S. EPA. 1983b. Methodology and Guidelines for the Reportable Quantity
Determinations Based on Chronic Toxldty Data. Prepared by the Office of
Health and Environmental Assessment, Environmental Criteria and Assessment
Office, Cincinnati, OH, OHEA for the Office of Solid Waste and Emergency
Response, Washington, DC.
U.S. EPA. 1985. Health and Environmental Effects Profile for Nitrobenzene.
Prepared by the Office of Health and Environmental Assessment, Environmental
Criteria and Assessment Office, Cincinnati, OH for the Office of Solid Waste
and Emergency Response, Washington, DC.
U.S. EPA. 1986. Integrated Risk Information System (IRIS). Reference Dose
(RfD) for Oral Exposure for Nitrobenzene. Online (verification date 7/8/85).
Office of Health and Environmental Assessment, Environmental Criteria and
Assessment Office, Cincinnati, OH.
Wlndholz, M. 1983. The Merck Index, ??? ed. Merck and Co., Inc., Rahway,
*J. p. 945.
Zoeteman, B.C.J., K. Harmsen, J.B.H.J. Llnders, C.F.H. Morra and W. Slooff.
1980. Persistent organic pollutants 1n river water and ground water of The
Netherlands. Chemosphere. 9: 231-249.
0073h -22- 06/16/87
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