United States
Environmental Protection
1=1 m m Agency
EPA/690/R-07/025F
Final
9-19-2007
Provisional Peer Reviewed Toxicity Values for
N-Nitrosodiphenylamine
(CASRN 86-30-6)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

-------
Acronyms and Abbreviations
bw
body weight
cc
cubic centimeters
CD
Caesarean Delivered
CERCLA
Comprehensive Environmental Response, Compensation and Liability Act

of 1980
CNS
central nervous system
cu.m
cubic meter
DWEL
Drinking Water Equivalent Level
FEL
frank-effect level
FIFRA
Federal Insecticide, Fungicide, and Rodenticide Act
g
grams
GI
gastrointestinal
HEC
human equivalent concentration
Hgb
hemoglobin
i.m.
intramuscular
i.p.
intraperitoneal
IRIS
Integrated Risk Information System
IUR
inhalation unit risk
i.v.
intravenous
kg
kilogram
L
liter
LEL
lowest-effect level
LOAEL
lowest-observed-adverse-effect level
LOAEL(ADJ)
LOAEL adjusted to continuous exposure duration
LOAEL(HEC)
LOAEL adjusted for dosimetric differences across species to a human
m
meter
MCL
maximum contaminant level
MCLG
maximum contaminant level goal
MF
modifying factor
mg
milligram
mg/kg
milligrams per kilogram
mg/L
milligrams per liter
MRL
minimal risk level
MTD
maximum tolerated dose
MTL
median threshold limit
NAAQS
National Ambient Air Quality Standards
NOAEL
no-ob served-adverse-effect level
NOAEL(ADJ)
NOAEL adjusted to continuous exposure duration
NOAEL(HEC)
NOAEL adjusted for dosimetric differences across species to a human
NOEL
no-ob served-effect level
OSF
oral slope factor
p-IUR
provisional inhalation unit risk
p-OSF
provisional oral slope factor
p-RfC
provisional inhalation reference concentration
1

-------
p-RfD
provisional oral reference dose
PBPK
physiologically based pharmacokinetic
ppb
parts per billion
ppm
parts per million
PPRTV
Provisional Peer Reviewed Toxicity Value
RBC
red blood cell(s)
RCRA
Resource Conservation and Recovery Act
RDDR
Regional deposited dose ratio (for the indicated lung region)
REL
relative exposure level
RfC
inhalation reference concentration
RfD
oral reference dose
RGDR
Regional gas dose ratio (for the indicated lung region)
s.c.
subcutaneous
SCE
sister chromatid exchange
SDWA
Safe Drinking Water Act
sq.cm.
square centimeters
TSCA
Toxic Substances Control Act
UF
uncertainty factor
l^g
microgram
[j,mol
micromoles
voc
volatile organic compound
11

-------
9-19-2007
PROVISIONAL PEER REVIEWED TOXICITY VALUES FOR
N-NITROSODIPHENYLAMINE (CASRN 86-30-6)
Background
On December 5, 2003, the U.S. Environmental Protection Agency's (EPA's) Office of
Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human
health toxicity values for Superfund risk assessments, establishing the following three tiers as the
new hierarchy:
1.	EPA's Integrated Risk Information System (IRIS).
2.	Provisional Peer-Reviewed Toxicity Values (PPRTV) used in EPA's Superfund
Program.
3.	Other (peer-reviewed) toxicity values, including:
~	Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR),
~	California Environmental Protection Agency (CalEPA) values, and
~	EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in EPA's Integrated Risk Information System (IRIS). PPRTVs are
developed according to a Standard Operating Procedure (SOP) and are derived after a review of
the relevant scientific literature using the same methods, sources of data, and Agency guidance
for value derivation generally used by the EPA IRIS Program. All provisional toxicity values
receive internal review by two EPA scientists and external peer review by three independently
selected scientific experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the
multi-program consensus review provided for IRIS values. This is because IRIS values are
generally intended to be used in all EPA programs, while PPRTVs are developed specifically for
the Superfund Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a five-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV manuscripts conclude
that a PPRTV cannot be derived based on inadequate data.
Disclaimers
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and RCRA program offices are advised to carefully review the information provided
in this document to ensure that the PPRTVs used are appropriate for the types of exposures and
circumstances at the Superfund site or RCRA facility in question. PPRTVs are periodically
updated; therefore, users should ensure that the values contained in the PPRTV are current at the
time of use.
1

-------
9-19-2007
It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
users are strongly encouraged to read the entire PPRTV manuscript and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center for OSRTI. Other EPA programs or external parties who may
choose of their own initiative to use these PPRTVs are advised that Superfund resources will not
generally be used to respond to challenges of PPRTVs used in a context outside of the Superfund
Program.
Questions Regarding PPRTVs
Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on
chemicals not covered, or whether chemicals have pending IRIS toxicity values) may be directed
to the EPA Office of Research and Development's National Center for Environmental
Assessment, Superfund Health Risk Technical Support Center (513-569-7300), or OSRTI.
INTRODUCTION
IRIS (U.S. EPA, 1993) did not list a RfD or RfC for N-nitrosodiphenylamine. No RfD
for N-nitrosodiphenylamine was listed in the Drinking Water Regulations and Health Advisories
list (U.S. EPA, 2006). In addition, no RfD or RfC was listed in the HEAST (U.S. EPA, 1997).
The CARA database (U.S. EPA, 1991, 1994) listed two potentially relevant documents, a Health
and Environmental Effects Profile (HEEP) for nitrosamines (U.S. EPA, 1986), and a Health
Effects Assessment (HEA) for N-nitrosodiphenylamine (U.S. EPA, 1987). Neither document
calculated a RfD for N-nitrosodiphenylamine because of its oral carcinogenicity in rodents.
Similarly, an ATSDR (1993) Toxicological Profile did not derive a chronic oral MRL for N-
nitrosodiphenylamine because the critical noncancer endpoints identified by ATSDR (urinary
bladder hyperplasia and metaplasia) in a chronic rat bioassay (NCI, 1979) were considered to be
preneoplastic. Neither the HEEP nor the HEA contained information regarding inhalation
toxicity of N-nitrosodiphenylamine. ATSDR did not derive inhalation MRLs for N-
nitrosodiphenylamine due to the absence of reliable inhalation data. Standards or guidelines
relating to occupational inhalation exposure to N-nitrosodiphenylamine were not listed by the
ACGIH (2007), NIOSH (2005), or OSHA (2006). However, for the important metabolite,
diphenylamine, AGGIH listed a TLV-TWA of 10 mg/m3, based on liver, kidney, and blood
toxicity; and NIOSH recommended a REL-TWA of 10 mg/m3.
Both a cancer classification and an oral slope factor for N-nitrosodiphenylamine were
available on IRIS (U.S. EPA, 1993). The cancer assessment classified N-nitrosodiphenylamine
in category B2 (probable human carcinogen) under 1986 Guidelines for Carcinogen Assessment,
based on bladder tumors in rats of both genders, reticulum cell sarcomas in mice, and the
structural similarity of the chemical to other carcinogenic nitrosamines. IRIS (U.S. EPA, 1993)
also reported an oral slope factor of 4.9 E-3 per mg/kg-day based on transitional cell carcinomas
of the bladder in female Fischer 344 rats reported by NCI (1979). No quantitative estimate of
cancer risk from inhalation exposure was available on IRIS.
2

-------
9-19-2007
Other resources examined included an Environmental Health Criteria document on
nitroso compounds (WHO, 1978), an IARC (1982) monograph, and the NTP (2006) database.
No information regarding noncancer toxicity of N-nitrosodiphenylamine was located in Patty's
Industrial Hygiene and Toxicology (Lijinsky, 2001). Literature searches of TOXLINE (1981-
1993), TSCATS, RTECS, and HSDB were conducted and screened in May 1993; TOXLINE and
TSCATS were updated in November 2000, and updated literature searches of MedLine,
ToxLine, ToxCenter, TSCATS, CCRIS, DART/ETIC, GENETOX, RTECS, HSDB, and Current
Contents were conducted in January 2006.
REVIEW OF PERTINENT DATA
Human Studies
No data were located regarding the toxicity of N-nitrosodiphenylamine to humans
following chronic or subchronic exposure by any route.
Animal Studies
Oral Exposure
NCI (1979) conducted long-term carcinogenicity studies of N-nitrosodiphenylamine in
F344 rats and B6C3Fi mice that also examined some systemic toxicity endpoints, which were
considered for RfD derivation. No other studies of non-neoplastic endpoints were identified in
the literature, although the NCI data also were summarized in Cardy, et al., 1979.
In order to identify maximum tolerated doses (MTDs) for the chronic studies, NCI (1979)
administered N-nitrosodiphenylamine (98% pure) in the diet to F344 rats and B6C3F1 mice
(5/gender/species) for 8 weeks (male and female mice and female rats) or 11 weeks (male rats).
Concentrations of N-nitrosodiphenylamine in the diet ranged from 1000 to 46,000 ppm. NCI
(1979) did not report food consumption. Using default reference values for body weight and
food consumption (U.S. EPA, 1988), we estimated corresponding doses from 100 to 4600
mg/kg-day in rats and from 180 to 8300 mg/kg-day in mice. Body weights were recorded twice
each week; animals were sacrificed under carbon dioxide and necropsied upon study termination.
Details of the pathology examinations were not provided. Three of the five female rats exposed
to -1600 mg/kg-day died prior to scheduled sacrifice. All female rats treated with higher doses
also died. Terminal body weights were reduced by 10% or more in male and female rats
exposed to concentrations of at least -400 mg/kg-day , in male mice exposed to at least -1700
mg/kg-day, and in female mice exposed to either -3700 mg/kg-day or -8300 mg/kg-day.
Female mice treated at intermediate concentrations did not exhibit significantly reduced body
weights. NCI (1979) did not provide details of the pathology findings in the subchronic studies,
except to note that the only histopathological finding was trace pigmentation of Kupffer's cells in
the livers of male mice exposed to -8300 mg/kg-day N-nitrosodiphenylamine.
In the chronic studies, groups of 50 male and 50 female F344 rats were fed 1000 or 4000
ppm N-nitrosodiphenylamine (98% pure) in the diet for 100 weeks (NCI, 1979). Controls
3

-------
9-19-2007
consisted of 40 rats (20/gender) fed the basal diet. NCI did not report food consumption for this
study. Using default reference values for body weight and food consumption (U.S. EPA, 1988),
we estimated the administered doses to be 80 or 300 mg/kg-day for males and 90 or 400 mg/kg-
day for females. Animals were observed twice daily and weighed monthly, except during weeks
38-68 (reason not provided); moribund animals were sacrificed. Clinical examinations were
conducted monthly. Comprehensive macroscopic and microscopic examinations of major
organs and all gross lesions were conducted on animals that died and on surviving animals at
study termination.
In the male rats, survival at study termination was comparable between groups (NCI,
1979). Survival was 16/20 (80%), 44/50 (88%) and 43/50 (86%), for the control, low- and high-
dose groups, respectively. In female rats, there was a statistically significant decrease in survival
only in the high-dose group, with 18/20 (90%), 44/50 (88%) and 35/50 {10%) of the control,
low- and high-dose groups surviving until study termination. Nearly all deaths of high-dose
females occurred during the last 15 weeks of the study and might have reflected tumor-related
mortality.
From data presented graphically (NCI, 1979), a dose-related depression in body weight
was apparent in the high-dose male rats throughout the study and in low-dose males after week
70; weight was not measured for weeks 38-68. Body weights in treated males were
approximately 5% and 13% lower than controls during weeks 70-101 for the low- and high-dose
groups, respectively. Dose-related depression of body weight also became apparent in treated
females after week 70, with the low-dose group showing an approximate 7% decrease and the
high dose group showing about an 18% decrease.
NCI (1979) reported that corneal opacity was observed in 15/50 high-dose male rats
(0/20 in controls) and 16/50 low-dose females (1/20 in controls). NCI (1979) did not report
incidences at other doses, the wording of the document implied that it was not observed in low-
dose males or high-dose females. The report did not state when, during the course of the study,
these lesions were noted. The opacity was discussed as a "clinical sign" and presumably was
detected during the monthly clinical examinations. The eye was not included in the list of tissues
examined for histopathology. No information was presented as to the nature or severity of the
opacities seen. NCI (1979) reported that corneal opacity might have been related to N-
nitrosodiphenylamine treatment. However, the absence of corneal opacities in high-dose
females, despite the high incidence in low-dose females, suggested that these opacities were not
related to chemical treatment.
The only finding reported by NCI (1979) as non-neoplastic that was dose-related and not
generally found in untreated aging F344 rats was an increased incidence of epithelial hyperplasia
of the bladder in male (0/19, 2/46, 6/45) and female (0/18, 4/48, 7/49) rats, summarized in Table
1. Although pairwise comparisons of these data using Fisher's exact test (conducted for this
review) did not indicate a significant change from control values, a Cochran-Armitage trend test
for the male rat data was significant at p<0.05. The trend in females was marginally significant
(p<0.10). As described below and summarized in Table 1, similar rates of bladder epithelial
hyperplasia were observed in mice treated at much higher doses.
4

-------
9-19-2007
Table 1. Incidence of bladder epithelial hyperplasia in rodents fed N-Nitrosodiphenylamine in diet
(NCI 1979)
Rats
Mice

Control
(mg/kg-day)
Low dose
(mg/kg-day)
High dose
(mg/kg-day)
Control
(mg/kg-day)
Low dose
(mg/kg-day)
High dose
(mg/kg-day)
Males
0/19
(0)
2/46 (4%)
(80)
6/45 (13%)
(300)
0/18
(0)
2/49 (4%)
(1700)
7/46(15%)
(3400)
Females
0/18
(0)
4/48 (8%)
(90)
7/49 (14%)
(400)
0/18
(0)
3/49 (6%)
(-400)
5/38 (13%)
(-1000)
The US EPA National Center for Environmental Assessment commissioned reviews of
the NCI (1979) rat bladder epithelial hyperplasia data, by three independent pathologists, to
assist in determining the suitability of this endpoint for use in deriving a p-RfD (Sciences
International, 2007). In response to EPA questions, the three pathologists each concluded that
the rat bladder epithelial hyperplasia was preneoplastic, indicating the hyperplasia had preceded
and in some cases evolved into carcinoma. NCI (1979) descriptions of microscopic observations
of rat bladders specified two features of the lesions that had been seen only in malignant
neoplasms, anaplasia and invasion. In addition, previous population studies with rats chronically
administered the urothelial carcinogen N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)
demonstrated that hyperplastic lesions were highly prevalent with early exposure, prior to the
appearance of carcinomas (Akagi et al., 1973; Ito et al., 1969). The architecture of epithelial
hyperplasia in the NCI (1979) study could be subdivided into lesions that conformed to the
overall structure of the underlying bladder, flat lesions, and those that began to form connective
tissue stroma and appeared papilloma-like, papillary lesions (Sciences International, 2007). The
acinar type structures, noted in some cases, often have been identified in conjunction with
neoplastic changes in human bladders rather than reactive or inflammatory conditions. The
pathology reviewer concluded that at least the subset of flat lesions that contained these acinar-
type structures most likely were associated with early neoplastic transformation of the
urothelium and that the papillary-type lesions described in the NCI (1979) study appeared to
represent primary neoplasms of the bladder. In further support of this view, the degree of mitotic
activity, atypia, and sheet-like growth pattern suggested that at least a proportion of these lesions
conformed to current nomenclature for high-grade papillary urothelial carcinomas. In the rat
model, the increased rates of hyperplasia seen in the high-dose paradigm were paralleled by the
development of invasive urothelial carcinoma, which supported this conclusion. In contrast,
similar rates of urothelial hyperplasia were present in mice treated with N-nitrosodiphenylamine,
although only rare invasive urothelial carcinomas appeared to arise in this model system, which
may have represented differences in molecular mechanisms underlying tumor progression in
these model systems.
NCI (1979) reported neoplasms in rat bladder transitional cells and referred to the
hyperplasia as non-neoplastic lesions in the bladder epithelium. However, transitional cells form
the mucosal lining of the urinary bladder and serve as the bladder epithelium. NCI reported that
5

-------
9-19-2007
in high-dose groups of each gender of rats, "the entire spectrum from transitional-cell
hyperplasia to transitional-cell carcinoma was observed in the urinary bladder." Based on these
observations and conclusions of the pathologists who independently reviewed these data
(Sciences International, 2007), the bladder epithelial hyperplasia appeared to have been
preneoplastic rather than non-neoplastic.
Two of the three pathologist reviewers concluded that the NCI (1979) preneoplastic
hyperplasia was a non-threshold response resulting from mutagenicity; the third expert pathology
reviewer concluded the preneoplastic hyperplasia was a threshold response, resulting from
irritation with consequent regenerative proliferation, and was not due to mutagenicity. This
reviewer supported his conclusion that the bladder epithelial hyperplasia was a threshold
response by making the following points:
•	Bladder hyperplasia and neoplasia clearly were due to irritation with consequent
regenerative proliferation
o Irritation resulted from cytotoxic stimulus
o Cytotoxic stimulus probably was urinary tract calculi, or other solids
•	Mutagenic MOA was not possible based on chemistry & metabolism of N-
nitrosodiphenylamine & metabolites
o N-nitrosodiphenylamine does not have an alpha-carbon available for
hydroxylation
o Possibility of transnitrosation seemed highly unlikely
•	N-nitrosodiphenylamine dose required for hyperplasia or tumors was extremely high
compared to N-nitrosamines that have produced bladder cancer in rats, such as N-butyl-
N-(4-hydroxybutyl) nitrosamine (BBN)
o Hyperplastic lesions were observed in mice, without tumors, even though the dose
was much higher in mouse than in the rat
o Rat and mouse have appeared to be similarly susceptible to other N-nitrosamines,
as demonstrated with BBN, with the mouse possibly being somewhat more
susceptible
o Nonmutagenic chemicals that act via toxicity and regeneration tend to be more
active in rats than mice
•	Lack of evidence of an inflammatory reaction in rats might have been because the
inflammation might have resolved by the time of examination
•	Chronic inflammation or chronic infection with schistosomes appeared to contribute
directly to the pathogenesis of some bladder cancers, such as squamous cell carcinomas
A reviewer who concluded that the neoplastic urothelial hyperplasias most likely resulted
from a non-threshold mutagenic response also noted that in a subset of bladder cancers, such as
squamous cell carcinomas, chronic inflammation or chronic infection with schistosomes
appeared to contribute directly to the pathogenesis of these tumors. However, this reviewer also
pointed out that, although chronic submucosal inflammation was a striking finding in a large
number of mice treated with N-nitrosodiphenylamine (Table 2), few cases ultimately progressed
to invasive urothelial carcinoma. The reviewers who concluded the hyperplasia was a
nonthreshold response also supported their position with the following points:
6

-------
9-19-2007
•	Progressive molecular alterations arising from changes at the genetic level generally have
been understood to cause neoplastic changes independent of associated inflammation
(Wu, 2005)
•	Few cases of mouse submucosal inflammation progressed to invasive urothelial
carcinoma
•	Rates of urothelial hyperplasia did not vary tremendously between rat and mouse models,
despite markedly higher level of chronic inflammation in the mouse
•	Urothelium typically undergoes morphologic changes associated with chronic irritation,
most notably squamous metaplasia.
o Unlike hyperplastic changes, squamous metaplasia is a relatively stable change.
o Incidence of squamous metaplasia in the NCI study was extremely low (only 3
cases)
o Incidence of squamous metaplasia should be higher in a threshold response where
only a subset of chronically injured bladders would progress to carcinoma.
•	Scarring, chronic inflammation, & other signs of chronic injury were not mentioned as
features in the bladders of N-nitrosodiphenylamine treated rats
The pathologist reviewers (Sciences International, 2007) disagreed regarding the
likelihood that the rat bladder preneoplastic hyperplasia, reported by NCI (1979) was a threshold
response, resulting from inflammation, or a non-threshold response, resulting from mutagenicity.
Because of the uncertainty about the MOA leading to these lesions, the default view of a non-
threshold linear response was considered to apply to these data.
The 300 and 400 mg/kg-day doses in male and female rats were considered to be
LOAELs for decreased (13-18%) body weight, based on positive trend test (NCI, 1979). The
body weight depression observed in the rats was consistent with the reported depression of body
weight gain in dogs following chronic ingestion of diphenylamine, a metabolite of N-
nitrosodiphenylamine (Thomas et al., 1967; U.S. EPA, 2007). In rats, N-nitrosodiphenylamine
apparently is denitrosated to diphenylamine and nitric oxide (Appel et al., 1984). The low dose
of 80-90 mg/kg-day in male and female rats was a NOAEL for weight gain depression in this
study. Body weight changes at this dose were only 5-7%, the incidence of bladder hyperplasia
was 4-8%), and the lack of dose-response in corneal opacities suggested that the observed
opacities were not chemical-related.
NCI (1979) reported a variety of neoplastic lesions in the rats, most notably urinary
bladder transitional cell carcinomas that were observed in 16/45 high-dose males, and in 40/49
high-dose females; no equivalent carcinomas were observed in control or low-dose rats of either
gender. Among the high-dose rats exhibiting these carcinomas, one of the males and two of the
females had a squamous metaplasia. While only one carcinoma appeared to have metastasized,
the carcinoma growth patterns were described by NCI (1979) as "quite large", and might have
been the cause of death in many animals
In the mouse portion of the chronic bioassay (NCI, 1979), groups of 50 male B6C3Fi
mice were fed 10,000 or 20,000 ppm of N-nitrosodiphenylamine (98% pure) in the diet for 101
weeks. Female B6C3Fi mice (50/treatment group) initially were fed 5000 or 10,000 ppm in the
diet. This regimen in females was stopped at 38 weeks due to severely decreased body weight
7

-------
9-19-2007
gain relative to controls in both treated groups. At 41 weeks, dietary concentrations were
modified to 1000 or 4000 ppm and treatment continued for the following 60 weeks. NCI
calculated the time-weighted average dietary concentrations for the females to be 2315 or 5741
ppm for the experiment. Controls consisted of 20 mice/gender fed the basal diet for 101 weeks.
NCI (1979) did not report food consumption for this study, making interpretation of weight gain
data difficult. Using default reference values for body weight and food consumption (U.S. EPA,
1988), we estimated administered doses to be 1700 or 3400 mg/kg-day for the males, and an
average of 400 or 1000 mg/kg-day for the females. Animals were observed twice daily and
weighed monthly; moribund animals were sacrificed. Clinical examinations were conducted
monthly. Comprehensive macroscopic and microscopic examinations of major organs and all
gross lesions were conducted on animals that died and on surviving animals at study termination.
With the exception of high-dose females, mouse survival was comparable between the
control and treatment groups. In males, 18/20 (90%), 46/50 (92%) and 41/50 (82%) of the
controls, low- and high-dose animals, respectively, lived until the end of the study. Survival in
females at study termination was 16/20 (80%) for the control, 42/50 (84%) for the low-dose, and
31/50 (62%) for the high-dose groups. Tarone tests conducted by NCI (1979) demonstrated no
significant dose-related trend.
Mice of both genders showed a dose-related decrease in body weight that persisted
throughout the study, according to data presented only in graphical form (NCI, 1979). Body
weights were approximately 12% and 22% lower in treated males than in controls for weeks 50-
101 in the low- and high-dose groups, respectively. After week 50, treated females had gained
approximately 36% and 49% less weight than controls in the low-dose and high-dose groups,
respectively.
Bladder lesions occurred with higher frequency in treated mice of both genders than in
their respective controls. Chronic submucosal inflammation of the bladder was increased in a
dose-dependent fashion in both male and female mice (See Table 2). While there were
transitional cell carcinomas (one in each gender of the low-dose group), papillomas (one in each
low- and high-dose group of males), a hemangioma, and bladder epithelial hyperplasia (9 cases
in each gender of treated groups), NCI (1979) did not consider these treatment-related.
Perivascular lymphocytic cuffing in the kidney also was reported in both genders of mice, but the
incidence of these lesions was not dose-related and did not correlate with the changes in the
urinary bladder. The female mice were more susceptible to the adverse bladder effects of N-
nitrosodiphenylamine at high doses. A NOAEL was not identified in this study.
Several chronic studies of N-nitrosodiphenylamine in rodents focused on tumor induction
(BRL, 1968; Innes et al., 1969; Druckrey et al., 1967; Argus and Hoch-Ligeti, 1961).
Experimental details of systemic toxicity (e.g., body weight, non-neoplastic lesions) or of
controls in these studies either were not provided or were inadequate to assess noncancer
endpoints.
No data were identified regarding potential adverse developmental effects from exposure
to N-nitrosodiphenylamine. However, Crocker, et al. (1972) noted renal effects in rats following
8

-------
9-19-2007
Table 2. Incidence of Bladder Submucosal Inflammation in Mice Fed Diets Containing
N-Nitrosodiphenylamine (NCI, 1979)

Control
Low dose
High dose
Male
0/18a
(0 mg/kg-day)
12/49b (24%)
(1700 mg/kg-day)
31/50c (62%)
(3400 mg/kg-day)
Female
0/18a
(0 mg/kg-day)
31/47c (66%)
(-400 mg/kg-day)
3 0/3 8C (79%)
(-1000 mg/kg-day)
a pO.OOOl by Cochran-Armitage trend test performed for this review
b p<0.05 by Fisher Exact test performed for this review
0 pO.OOOl by Fisher Exact test performed for this review
maternal ingestion of the N-nitrosodiphenylamine metabolite, diphenylamine. Similar effects
have been observed in diphenylamine-dosed adult hamsters (Lenz et al., 1995), mice (Rohrbach
et al., 1993), chickens (Sorrentino et al., 1978), dogs (Thomas et al., 1967), rats, and gerbils
(Lenz and Carlton, 1990).
Inhalation Exposure
A 20-day rat inhalation study (Zhilova and Kasparov, 1966) was cited by ATSDR (1993).
However, ATSDR did not consider this study to be reliable due to inadequate reporting of
experimental methods and results. No other studies were located regarding inhalation toxicity of
N-nitrosodiphenylamine in animals.
FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC OR CHRONIC
ORAL RfD VALUES FOR N-NITROSODIPHENYLAMINE
No information regarding adverse health effects in humans following oral exposure to
N-nitrosodiphenylamine were available. The available subchronic toxicity study in animals was
not of adequate quality for deriving a subchronic p-RfD. Specifically, the subchronic portion of
the study conducted by NCI (1979) was aimed at identifying the maximum tolerance dose
(MTD). Small numbers of animals were used (5/gender/species) and few details of the
pathology examinations and findings were presented.
The NCI (1979) chronic dietary study in rats and mice was chosen as the critical study to
attempt to derive a chronic p-RfD. This study used adequate numbers of animals and provided
adequate dose-response information; however, the study was limited by a lack of data on
hematology and clinical chemistry, and incomplete reporting of data relating to noncancer
endpoints. The variation in dosing of the female mice was especially problematic, since these
animals seemed to be most sensitive to adverse bladder effects from ingestion of N-
nitrosodiphenylamine.
The NCI (1979) chronic rat study identified a LOAEL of 300-400 mg/kg-day for
decreased body weight gain (13-18%) in male and female rats, with a NOAEL of 80-90 mg/kg-
9

-------
9-19-2007
day. However, the validity of significantly decreased body weight gain as a critical effect was
questionable because food consumption was not reported; the weight deficits might have resulted
from reduced food consumption due to palatability. In female rats at the high dose of 400
mg/kg-day, there was a tumor-related reduction in survival. The low dose was considered a
minimal LOAEL for bladder epithelial hyperplasia in male and female rats. However, the
pathology review commissioned by NCEA (Sciences International, 2007) concluded these
lesions were preneoplastic. One reviewer specifically concluded that the hyperplasia was a
threshold response due to irritation with a consequent regenerative proliferation, and was not a
mutagenic response, although no data for N-nitrosodiphenylamine, per sc\ specifically supported
this view. However, the other two pathology reviewers concluded it probably was a non-
threshold response; one pathologist specifically indicated the response resulted from
mutagenicity.
Based on the unanimous conclusion that the hyperplasia was preneoplastic and the
disagreement regarding whether it was a threshold response, we concluded this endpoint was
inappropriate for use as a critical effect to derive a p-RfD. Thus, the only potential POD for
deriving a p-RfD from the NCI (1979) rat data was the NOAEL of 80-90 mg/kg-day for
depression of weight gain. However, this potential POD was questionable because it might have
resulted from reduced food consumption rather than toxicity.
In the mouse study, NCI (1979) reported large increases in the incidence of chronic
submucosal inflammation of the bladder (Table 2) at both dose levels and dose-related
depression of body weight gains in mice of both genders exposed to N-nitrosodiphenylamine in
the diet. Although similar effects were seen in both genders, the effective doses were much
lower and incidence of inflammation higher in females. The study identified no NOAEL for
bladder inflammation or depression of body weight in mice. These data indicated that bladder
submucosal inflammation in female mice was the most sensitive endpoint from which to derive a
potential p-RfD for N-nitrosodiphenylamine. However, the female mouse data were extremely
problematic. During the first 38 weeks of treatment, female mice were fed approximately 900 or
1800 mg/kg-day and exhibited severely depressed body weight gains, suggesting the maximum
tolerated dose (MTD) might have been exceeded. As a result, NCI (1979) apparently stopped
dosing these animals for three weeks and then resumed feeding at approximately 180 or 700
mg/kg-day for the remaining 60 weeks of the study. Based on NCI (1979) calculated average
concentrations in feed and default values for food ingestion and body weight (US EPA, 1988),
we estimated time-weighted average doses for the female mice to be approximately 400 or 1000
mg/kg-day. This resulted in a nominal LOAEL of approximately 400 mg/kg-day for bladder
submucosal inflammation in female mice. The very high response rate of 66% suggested that a
point of departure for this inflammatory effect should be substantially lower. Consequently, we
conducted benchmark dose modeling of these data (Appendix A), calculating a BMDio of 25
mg/kg-day and a BMDLio of 17 mg/kg-day.
All quantal models in U.S. EPA's Benchmark Dose Software (BMDS) were fit to the
incidence data for bladder submucosal inflammation in female mice. The default BMR of 10%
increase in extra risk was used for this analysis. Models were run using the default restrictions
on parameters built into the BMDS. Appendix A contains a summary of the modeling results
and a plot of the best fitting model. Adequate fits (goodness of fit p-value >0.10) were achieved
10

-------
9-19-2007
by multiple models, however, the log-logistic model provided the best fit, as indicated by lowest
Aikaike Information Criterion (AIC). However, the female mouse BMD of 25 mg/kg-day and
BMDL of 17 mg/kg-day appeared unreliable because of the questionable shape of the BMDR
curve and the fact that these values were based on a lowest incidence rate of 66%, resulting in a
BMDL 24 times lower than the lowest experimental data.
In addition to these concerns about the reliability of the BMD analysis, were concerns
resulting from the severe weight gain depression exhibited by the female mice during the early
high-dose period, suggesting that these doses might have approached or exceeded the maximum
tolerated dose. In addition, the dose calculations were extremely uncertain because of the lack of
food consumption data, variation in N-nitrosodiphenylamine feed concentrations, and cessation
of exposure for three weeks during which the female mouse treatment groups apparently
received no N-nitrosodiphenylamine.
Considering these problems, we concluded that confidence in the mouse data from the
critical chronic oral study (NCI, 1979) was too low to use for derivation of a p-RfD. While the
study used adequate numbers of animals, it did not include evaluation of hematology or clinical
chemistry, featured only limited reporting of noncarcinogenic endpoints, and identified neither a
NOAEL nor a reasonable LOAEL in mice. Especially problematic was the dosing regimen for
the most sensitive species, female mice, which was changed during the course of the study due to
significant effects on body weight gain. The rat body weight gain NOAEL of 80 - 90 mg/kg-day
also was considered an inappropriate POD, because it clearly was not the most sensitive endpoint
and might have resulted from reduced food consumption rather than toxicity. Finally, the
observation of corneal opacity in certain treatment groups of rats was an insufficient endpoint
because the effect apparently was not related to dose.
Consequently, the poor quality of the available data, the availability of an IRIS oral slope
factor addressing the cancer and hyperplasia risk observed in rats, and major uncertainties in
dosing and modeling results led us to conclude that data were insufficient to derive either a
chronic or a subchronic p-RfD for N-nitrosodiphenylamine.
FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC OR CHRONIC
INHALATION RfC VALUES FOR N-NITROSODIPHENYLAMINE
A provisional inhalation RfC could not be derived for N-nitrosodiphenylamine because
data on adverse health effects following inhalation exposure were lacking for humans and
animals. Without pharmacokinetic data and information to rule out portal-of-entry effects, there
was no basis to support a route-to-route extrapolation from the oral data, even if it were
otherwise considered sufficient.
11

-------
9-19-2007
PROVISIONAL CARCINOGENICITY ASSESSMENT FOR
N-NITRO SODIPHENYL AMINE
Weight-of-Evidence Descriptor
The IRIS cancer assessment (U.S. EPA, 1993), classified N-nitrosodiphenylamine in
category B2, probable human carcinogen, using the 1986 Guidelines for Carcinogen Assessment.
This classification was based on the NCI (1979) data on bladder tumors in rats (both genders),
reticulum cell sarcomas in mice, and the structural similarity of the chemical to other
carcinogenic nitrosamines. These data seemed consistent with the equivalent descriptor of
"Likely to be carcinogenic to humans" in the updated U.S. EPA (2005) guidance.
Quantitative Estimate of Carcinogenic Risk
IRIS (U.S. EPA, 1993) reported an oral slope factor of 4.9xl0"3 per mg/kg-day, based on
transitional cell carcinomas of the bladder in female F344 rats reported by NCI (1979). No
quantitative estimate of cancer risk from inhalation exposure was available on IRIS. None was
developed here due to lack of information.
REFERENCES
ACGIH (American Conference of Government Industrial Hygienists). 2007. Threshold limit
values (TLV) for chemical substances and physical agents, and biological exposure indices.
ACGIH, Cincinnati, OH.
Akagi, G., Akagi, A., Kimura, M., and Otsuka, H. (1973). Comparison of bladder tumors
induced in rats and mice with N-butyl-N-(4-hydroxybutyl)-nitrosoamine. Gann 64, 331-336.
Cited in Sciences International, 2007.
Appel, K.E., C.S. Ruhl, B. Mahr et al. 1984. Denitrosation of diphenylnitrosamine in vivo.
Toxicol. Lett. 23: 353-358.
Argus, M.F. and C. Hoch-Ligeti. 1961. Comparative study of the carcinogenic activity of
nitrosamines. J. Natl. Cancer Inst. 27:695-709.
ATSDR (Agency for Toxic Substances and Disease Registry). 1993. Toxicological Profile for
jV-Nitrosodiphenylamine. TP-92/15. Online, www.atsdr.cdc.gov/toxprofiles/tpl6.html
BRL (Bionetics Research Laboratory). 1968. Evaluation of carcinogenic, teratogenic and
mutagenic activities of selected pesticides and industrial chemicals. Vol 1. Carcinogenic study.
NTIS PB223-159.
Cardy, H.R., W. Lijinsky and P.K. Hildebrandt. 1979. Neoplastic and non-neoplastic urinary
bladder lesions induced in Fischer 344 rats and B6C3F1 hybrid mice by N-
nitrosodiphenylamine. Ecotoxicology and Environmental Safety, 3: 29-35.
12

-------
9-19-2007
Crocker, J.F., D.M. Brown, R.F. Borch and R.L. Vernier 1972. Renal cystic disease induced in
newborn rats by diphenylamine derivatives. Am. J. Pathol. 66: 343-350
Druckrey, H., R. Preussmann, S. Iuankovic and D. Schmaehl. 1967. Organotropic carcinogenic
effects of 65 different N-nitroso compounds on BD-rats. Z. Krebsforsch. 69(2): 103-201.
IARC (International Agency for Research on Cancer). 1982. A-Nitrosodi phenyl amine. IARC
monographs on the evaluation of the carcinogenic risk of chemicals to humans. Some aromatic
amines, anthraquinones and nitroso compounds, and inorganic fluorides used in drinking-water
and dental preparations. Vol. 27, p. 213-225.
Innes, J.R.M., B.M. Ulland, M.G. Valeria et al. 1969. Bioassay of pesticides and industrial
chemicals for tumorigenicity in mice: A preliminary note. J. Natl. Cancer Inst. 42: 1101-1114.
Ito, N., Hiasa, Y., Tamai, A., Okajima, E., and Kitamura, H. (1969). Histogenesis of urinary
bladder tumors induced by N-butyl-N-(4-hydroxybutyl)nitrosamine in rats. Gann 60, 401-410.
Cited in Sciences International, 2007.
Lenz, S.D. and W.W. Carlton. 1990. Diphenylamine-induced renal papillary necrosis and
necrosis of the pars recta in laboratory rodents. Vet. Pathol. 27: 171-178
Lenz, S.D., J.J. Turek and W.W. Carlton. 1995. Early ultrastructural lesions of diphenylamine-
induced renal papillary necrosis in Syrian hamsters. Exper. Toxicol. Pathol.
47: 447-452
Lijinsky, W. 2001. /V-Nitroso Compounds. In: Patty's Industrial Hygiene and Toxicology.
2005 Online Edition. Bingham, E., B. Cohrssen, and C.H. Powell, Eds. John Wiley and Sons,
New York.
NCI (National Cancer Institute). 1979. Bioassay of N-nitrosodiphenylamine for possible
carcinogenicity. NCI Carcinogenesis. Tech. Rep. Ser. No. 164. p. 106 (Also publ. as NIH 79-
1720 and NTIS PB298-275). Available at http://ntp.niehs.nih.gov/ntp/htdocs/LT rptsZtrl64.pdf
NIOSH (National Institute for Occupational Safety and Health). 2005. Pocket Guide to
Chemical Hazards. Index by CASRN. Available at http://www.cdc.gov/niosh/npg/npgdcas.html
NTP (National Toxicology Program). 2006. Management Status Report. Available at
http://ntp-server.niehs.nih.gov/
OSHA (Occupational Safety and Health Administration). 2006. OSHA Regulations. Available
at http://www.osha.gov/pls/oshaweb/owadisp.show document?p table=STANDARDS&p id=9992
Rohrbach, D.H., L.K. Robinson and V.A. Murrah. 1993. Loss of the basement membrane
matrix molecule, bamin, in diphenylamine-treated mice. Matrix. 13: 341-350.
13

-------
9-19-2007
Sax, N.I. and R.J. Lewis. 1989. Dangerous Properties of Industrial Materials. 7th Edition. Van
Nostrand Reinhold, New York.
Sciences International. 2007. Pathological opinion on NCI published paper, Bioassay of N-
nitrosodiphenylamine for possible carcinogenicity; contract no. GS-10F-0127K. Prepared by
Sciences International Inc, Alexandria VA for U.S. EPA, National Center for Environmental
Assessment, Cincinnati OH. Available from the Superfund Human Health Risk Technical
Support Center (STSC).
Sorrentino, F., A. Fella and A. Pota. 1978. Diphenylamine-induced renal lesions in the chicken.
Urolog. Res. 6: 71-75
Thomas, J.O., W.E. Ribelin, J.R. Woodward and F. Deeds. 1967. The chronic toxicity of
diphenylamine for dogs. Toxicol. Appl. Pharmacol. 11:184-194.
U.S. EPA. 1986. Health and Environmental Effects Profile for Nitrosamines. Prepared by
Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment,
Cincinnati, OH for Office of Solid Waste and Emergency Response, Washington, DC.
U.S. EPA. 1987. Health Effects Assessment for N-Nitrosodiphenylamine. Prepared by
Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment,
Cincinnati, OH for Office of Solid Waste and Emergency Response, Washington, DC.
U.S. EPA. 1988. Recommendations for and Documentation of Biological Values for use in Risk
Assessment. EPA 600/6-87/008, NTIS PB88-179874/AS, February 1988. Available at
http://cfpub. epa.gov/ncea/cfm/recordisplay. cfm?deid=34855
U.S. EPA. 1991. Chemical Assessments and Related Activities (CARA). Office of Health and
Environmental Assessment, Washington, DC. April 1991.
U.S. EPA. 1993. N-Nitrosodiphenylamine. Integrated Risk Information System (IRIS). Online.
Office of Research and Development. National Center for Environmental Assessment,
Washington, DC. Available at http://www.epa.gov/iris/subst/0178.htm
U.S. EPA. 1994. Chemical Assessments and Related Activities (CARA). Office of Health and
Environmental Assessment, Washington, DC. December 1994.
U.S. EPA. 1997. Health Effects Assessment Summary Tables (HEAST). Office of Research
and Development, Office of Emergency and Remedial Response, Washington, DC. July 1997.
EPA/540/R-97/036. NTIS PB 97-921199.
U.S. EPA. 2000. Benchmark Dose Technical Guidance Document: External Review Draft. Risk
Assessment Forum, U.S. EPA, Washington DC. EPA/63O/R-00/001. Available at
http://epa.gov/ncea/cfm/recordisplav.cfm?deid=20871
14

-------
9-19-2007
U.S. EPA. 2004. 2004 Edition of the Drinking Water Standards and Health Advisories. Office
of Water, Washington, DC. Winter 2004. EPA 822-R-02-038. Online.
http://www.epa.gov/waterscience/drinking/standards/dwstandards.pdf
U.S. EPA. 2005. Guidelines for carcinogen risk assessment. Risk Assessment Forum,
Washington, DC; EPA/630/P 03/001F. Federal Register 70(66): 17765-17817. Available online
at http://cfpub. epa.gov/ncea/raf/recordisplav. cfm?deid=l 16283
WHO (World Health Organization). 1978. Nitrates, nitrites and /V-nitroso compounds.
Environmental Health Criteria No. 5. Geneva, Switzerland. Available at
http://www.who.int/ipcs/publications/ehc/ehc alphabetical/en/index.html
Wu, X.R. 2005 Urothelial turnorigenisis: a tale of divergent pathways. Nat. Rev. Cancer.
5:713-725. Cited in Sciences International, 2007.
Zhilova, N.A. and A. A. Kasparov. 1966. [Comparative toxicological characteristics of
antiscorchings: phthalic anhydride and N-nitrosodiphenylamine (Vulkalent A).] Gig. Tr. Prof.
Zabol. 10:60-62. [inRus.] (Cited by ATSDR, 1993)
15

-------
9-19-2007
APPENDIX A
RESULTS OF BENCHMARK DOSE MODELING FOR CHRONIC ORAL DOSING
16

-------
9-19-2007
Table A-l. BMD Modeling Results for Female Mouse Submucosal Inflammation of Bladder
(NCI, 1979)
Model
Degrees
of
Freedom
x2
X2 Goodness
of Fit p-
Valuc"
AIC
BMD10
(mg/kg-day)
BMDL10
(mg/kg-day)
Log-logistic
(slope >1)
2
0.26
0.8783
101.65
25
17
Log-probit
(slope >1)
2
3.31
0.1912
104.44
90
70
Gamma (power >1)
2
4.14
0.1265
105.30
52
41
Multistage (degree=l)b
2
4.14
0.1265
105.30
52
41
Quantal Linear
2
4.14
0.1265
105.30
52
41
Weibull (power > 1)
2
4.14
0.1265
105.30
52
41
Logistic
1
11.64
0.0006
119.03
128
100
Probit
1
11.90
0.0006
119.34
128
104
Quantal Quadratic
1
17.23
0.0000
125.26
250
197
aAdequate fit indicated by p>0.10
bModel shown is lowest degree polynomial providing adequate fit. Betas restricted to > 0.
17

-------
9-19-2007
Log-Logistic Model with 0.95 Confidence Level
Log-Logistic
2 0.6
BMD.U BMD
0
200
400
600
800
1000
dose
19:48 05/05 2006
Figure A-l. Log-logistic BMDi0 Model Fit to Incidence Data for Submucosal Inflammation of
Bladder in Female Mouse (NCI, 1979)
18

-------