United States
Environmental Protection
1=1 m m Agency
EPA/690/R-12/023F
Final
12-20-2012
Provisional Peer-Reviewed Toxicity Values for
Di-?7-octyl Phthalate
(CASRN 117-84-0)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Dan D. Petersen, PhD, DABT
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
ICF International
9300 Lee Highway
Fairfax, VA 22031
PRIMARY INTERNAL REVIEWERS
Audrey Galizia, DrPH
National Center for Environmental Assessment, Washington, DC
Suryanarayana V. Vulimiri, BVSc, PhD, DABT
National Center for Environmental Assessment, Washington, DC
This document was externally peer reviewed under contract to
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421-3136
Questions regarding the contents of this document may be directed to the U.S. EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center (513-569-7300).
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TABLE OF CONTENTS
TABLE OF CONTENTS	iii
COMMONLY USED ABBREVIATIONS	iv
BACKGROUND	1
DISCLAIMERS	1
QUESTIONS REGARDING PPRTVS	1
INTRODUCTION	2
REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER)	4
HUMAN STUDIES	10
Oral Exposures	10
Inhalation Exposures	10
ANIMAL STUDIES	12
Oral Exposures	12
Inhalation Exposure	19
Other Data (Short-Term Tests, Other Examinations)	20
Tests Evaluating Carcinogenicity, Genotoxicity, and/or Mutagenicity	30
Other Toxicity Studies (Exposures Other Than Oral or Inhalation)	30
DERIVATION OI PROVISIONAL VALUES	36
DERIVATION OF ORAL REFERENCE DOSES	36
Derivation of Subchronic Provisional RfD (Subchronic p-RfD)	36
Derivation of Chronic Provisional RfD (Chronic p-RfD)	38
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS	40
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR	40
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES	40
APPENDIX A. PROVISIONAL SCREENING VALUES	41
APPENDIX B. DATA TABLES	42
APPENDIX C. BMD OUTPUTS	58
APPENDIX D. REFERENCES	59
in
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COMMONLY USED ABBREVIATIONS
BMC
benchmark concentration
BMCL
benchmark concentration lower bound 95% confidence interval
BMD
benchmark dose
BMDL
benchmark dose lower bound 95% confidence interval
HEC
human equivalent concentration
HED
human equivalent dose
IUR
inhalation unit risk
LOAEL
lowest-observed-adverse-effect level
LOAELadj
LOAEL adjusted to continuous exposure duration
LOAELhec
LOAEL adjusted for dosimetric differences across species to a human
NOAEL
no-ob served-adverse-effect level
NOAELadj
NOAEL adjusted to continuous exposure duration
NOAELhec
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
POD
point of departure
p-OSF
provisional oral slope factor
p-RfC
provisional reference concentration (inhalation)
p-RfD
provisional reference dose (oral)
RfC
reference concentration (inhalation)
RfD
reference dose (oral)
UF
uncertainty factor
UFa
animal-to-human uncertainty factor
UFC
composite uncertainty factor
UFd
incomplete-to-complete database uncertainty factor
UFh
interhuman uncertainty factor
UFl
LOAEL-to-NOAEL uncertainty factor
UFS
subchronic-to-chronic uncertainty factor
WOE
weight of evidence
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
DI-n-OCTYL PHTHALATE (CASRN 117-84-0)
BACKGROUND
A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value
derived for use in the Superfund Program. PPRTVs are derived after a review of the relevant
scientific literature using established Agency guidance on human health toxicity value
derivations. All PPRTV assessments receive internal review by a standing panel of National
Center for Environment Assessment (NCEA) scientists and an independent external peer review
by three scientific experts.
The purpose of this document is to provide support for the hazard and dose-response
assessment pertaining to chronic and subchronic exposures to substances of concern, to present
the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to
characterize the overall confidence in these conclusions and toxicity values. It is not intended to
be a comprehensive treatise on the chemical or toxicological nature of this substance.
The PPRTV review process provides needed toxicity values in a quick turnaround
timeframe while maintaining scientific quality. PPRTV assessments are updated approximately
on a 5-year cycle for new data or methodologies that might impact the toxicity values or
characterization of potential for adverse human health effects and are revised as appropriate. It is
important to utilize the PPRTV database flittp://hhpprtv.ornl.gov) to obtain the current
information available. When a final Integrated Risk Information System (IRIS) assessment is
made publicly available on the Internet (www.epa.eov/iris). the respective PPRTVs are removed
from the database.
DISCLAIMERS
The PPRTV document provides toxicity values and information about the adverse effects
of the chemical and the evidence on which the value is based, including the strengths and
limitations of the data. All users are advised to review the information provided in this
document to ensure that the PPRTV used is appropriate for the types of exposures and
circumstances at the site in question and the risk management decision that would be supported
by the risk assessment.
Other U.S. Environmental Protection Agency (EPA) programs or external parties who
may choose to use PPRTVs are advised that Superfund resources will not generally be used to
respond to challenges, if any, of PPRTVs used in a context outside of the Superfund program.
QUESTIONS REGARDING PPRTVS
Questions regarding the contents and appropriate use of this PPRTV assessment should
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).
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INTRODUCTION
Di-«-octyl phthalate (DNOP), CAS No. 117-84-0, is an odorless, colorless, oily liquid. A
table of physicochemical properties for DNOP is provided below (see Table 1). DNOP has a
low vapor pressure and, therefore, does not evaporate easily. DNOP is also characterized by its
low solubility, high boiling point, and low melting point, indicating that DNOP is stable at room
temperature and usually occurs as a liquid (ATSDR, 1997). The empirical formula for DNOP is
C24H38O4 (see Figure 1). Phthalate esters, as a class, are most often mixed with polyvinyl
chloride (PVC) formulations for the production of flexible PVC materials. There are no known
commercial sources of pure DNOP; it comprises approximately 20% of the C6-10 phthalate
material, of which 50 million pounds were produced in the 1990s.
O
C— O — CoH
C— O — C8H
II
o
Figure 1. DNOP Structure
Table 1. Physicochemical Properties of DNOP (CASRN 117-84-0)a
Property (unit)
Value
Boiling point (°C at 760 mmHg)
390° C
Melting point (°C)
-25
Density (g/mL at 25°C)
0.978
Vapor pressure (mmHg at 25°C)
1.44 x 10~4
pH (unitless)
No data
Solubility in water (mg/L at 25°C)
Essentially Insoluble (0.5 ug/L)
Relative vapor density (air =1)
No data
Molecular weight (g/mol)
390.54
Log Kow
8.06
"Source: ATSDR (1997).
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The European Chemicals Agency (ECHA, 2010) and Agency for Toxic Substances and
Disease Registry (ATSDR, 1997) confirm that there is confusion between the chemical name
"di-/7-octyl phthalate (DNOP)" and the more general term "di-octyl phthalate (DOP)," which is
considered a synonym of bis(2-ethylhexyl)phthalate (DEHP). Studies occasionally use
ambiguous phthalate nomenclature, which can lead to confusion regarding the identity of the
chemical being described. Although studies sometimes use the term "di-octyl phthalate" to
indicate di-/7-octyl phthalate, ATSDR concluded that almost all references to "di-octyl phthalate"
were in fact referring to DEHP (ATSDR, 1997).
In an evaluation of DNOP, ECHA (2010) included references that specifically used the
chemical name "di-/7-octyl phthalate." If a study used the term "di-octyl phthalate (DOP)," it
was considered to refer to di -/7-octyl phthalate if the CAS number (117-84-0) for di -/v-octyl
phthalate was provided. This rule is applied in this review in order to limit the possibility of
including data on incorrect phthalate esters. In addition, several of the retrieved studies used the
acronym "DOP" but also included the full chemical name, "di-/7-octyl phthalate." In these
instances, it was assumed that the chemical of interest was di-/7-octyl phthalate, and these
references are considered in this review.
No reference dose (RfD), reference concentration (RfC), or cancer assessment for DNOP
is included on the EPA IRIS database (U.S. EPA, 2010) or on the Drinking Water Standards and
Health Advisories List (U.S. EPA, 2009). No RfD or RfC values are reported in the Health
Effects Assessment Summary Tables (HEAST) (U.S. EPA, 2010). The Chemical Assessments
and Related Activities (CARA) list includes a Health and Environmental Effects Profile (HEEP)
for DNOP that declines to derive the potential carcinogenicity or noncancer toxicity values due
to inadequate noncancer data on this chemical (U.S. EPA, 1987, 1994). The toxicity of DNOP
following oral exposure has been reviewed by ATSDR (1997). ATSDR has determined that the
liver is the target organ following acute oral exposure based on the reduction in ethoxycoumarin
(9-deethylase (ECOD) activity and increased relative liver weight in rats fed DNOP for 14 days.
An acute oral minimal risk level (MRL) of 3 mg/kg-day is determined based on a LOAEL of
1000 mg/kg-day that is established in rats by Lake et al. (1986). For intermediate-duration
exposures (exact duration not specified), ATSDR has determined an MRL of 0.4 mg/kg-day
using a NOAEL of 40.8 mg/kg-day that is based on a statistically significant increase in hepatic
ethoxyresorufin-O-deethylase (EROD) activity and histological changes that were observed in
the livers of male and female rats (Poon et al., 1997). Thyroid toxicity was also observed at the
doses specified by Poon et al. (1997). ATSDR did not derive a chronic MRL.
Neither the World Health Organization (WHO, 2010) nor The California Environmental
Protection Agency (CalEPA, 2008, 2009) has derived toxicity values for exposure to DNOP. No
occupational exposure limits for DNOP have been derived by the American Conference of
Governmental Industrial Hygienists (ACGIH, 2010), the National Institute of Occupational
Safety and Health (NIOSH, 2010), nor the Occupational Safety and Health Administration
(OSHA, 2010).
The HEAST (U.S. EPA, 2010) does not report any toxicity values or an oral slope factor
(OSF) for DNOP. The International Agency for Research on Cancer (IARC, 2010) has not
reviewed the carcinogenic potential of DNOP. DNOP is not included in the 12th Report on
Carcinogens (NTP, 2011). CalEPA (2009) has not prepared a quantitative estimate of the
carcinogenic potential for DNOP.
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Literature searches were conducted on sources published from 1900 through
September 2011 for studies relevant to the derivation of provisional toxicity values for di-/7-octyl
phthalate, CAS No. 117-84-0. Searches were conducted using U.S. EPA's Health and
Environmental Research Online (HERO) database of scientific literature. HERO searches the
following databases: AGRICOLA; American Chemical Society; BioOne; Cochrane Library;
DOE: Energy Information Administration, Information Bridge, and Energy Citations Database;
EBSCO: Academic Search Complete; GeoRef Preview; GPO: Government Printing Office;
Informaworld; IngentaConnect; J-STAGE: Japan Science & Technology; JSTOR: Mathematics
& Statistics and Life Sciences; NSCEP/NEPIS (U.S. EPA publications available through the
National Service Center for Environmental Publications [NSCEP] and National Environmental
Publications Internet Site [NEPIS] database); PubMed: MEDLINE and CANCERLIT databases;
SAGE; Science Direct; Scirus; Scitopia; SpringerLink; TOXNET (Toxicology Data Network):
ANEUPL, CCRIS, ChemlDplus, CIS, CRISP, DART, EMIC, EPIDEM, ETICBACK, FEDRIP,
GENE-TOX, HAPAB, HEEP, HMTC, HSDB, IRIS, ITER, LactMed, Multi-Database Search,
NIOSH, NTIS, PESTAB, PPBIB, RISKLINE, TRI; and TSCATS; Virtual Health Library; Web
of Science (searches Current Content database among others); World Health Organization; and
Worldwide Science. The following databases outside of HERO were also searched for relevant
health information: ACGM, AT SDR, CalEPA, EPA IRIS, EPA HEAST, EPA HEEP, EPA OW,
EPA TSCATS/TSCATS2, NIOSH, NTP, OSHA, and RTECS.
REVIEW OF POTENTIALLY RELEVANT DATA
(CANCER AND NONCANCER)
Table 2 provides an overview of the relevant databases on DNOP and includes all
potentially relevant and repeated short-term-, subchronic-, and chronic-duration studies. The
principal studies are identified. The phrase, "statistical significance," as used throughout the
document, indicates ap-walue of <0.05.
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Table 2. Summary of Potentially Relevant Data for DNOP (CASRN 117-84-0)
Category
Number of
Male/Female, Species,
Study Type, Study
Duration
Dosimetry3
Critical Effects
NO A EL'
BMDL/
BMCLa
LOAEL'
Reference
Notesb
Human
1. Oral (mg/kg-d)a
Subchronic
ND
Chronic
ND
Developmental
ND
Reproductive
ND
Carcinogenic
ND
2. Inhalation (mg/m3)a
Subchronic
ND
Chronic
ND
Developmental
ND
Reproductive
0/49 with endometriosis,
38 female controls with
other causes of infertility
other than endometriosis,
case-control study. A
second fertile control
group of 21 was also used.
Concentrations of
phthalates were
measured by gas
chromatographic
analysis of blood.
Women with endometriosis had
statistically significant higher
blood concentrations of DNOP
and other phthalate esters
compared with controls;
correlation between DNOP and
endometriosis: r = +0.57
(p< 0.0001)
NDr
NDr
NDr
Reddy et al. (2006)
PR
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Table 2. Summary of Potentially Relevant Data for DNOP (CASRN 117-84-0)
Category
Number of
Male/Female, Species,
Study Type, Study
Duration
Dosimetry"
Critical Effects
NOAEL"
BMDL/
BMCLa
LOAEL'
Reference
Notesb
Neurological
54/77 exposed (lived near
plant that reprocessed
used motor oil and
chemical waste), 29/37
unexposed controls,
adults (aged 15-65),
matched cohort design,
average exposure time
9.3 yr
Direct exposure not
measured; 770-ppb
DNOP measured in
sludge; 960-ppb
DNOP measured in
sludge and soil.
Exposure to water and
air plumes from the
facility may have
occurred.
Exposed subjects had
significantly impaired body
balance (e.g., sway speed),
reaction times, and cognitive and
perceptual motor functions;
increased signs of depression
NDr
NDr
NDr
Kilburn and
Warshaw (1995)
PR
Carcinogenic
ND
Animal
1. Oral (mg/kg-d)a
Subchronic
5/0, F344 rat, diet,
7 d/wk, 4 wk
0, 100, 1000 (adjusted)
Significant increases in relative
liver weights and peroxisomal
beta-oxidation (PBOX) activities
at 2 wk, but not 4 wk, and
elevated periportal DNA
synthesis at 2 and 4 wk observed
in the 1000 mg/kg-d group
100
NDr
1000
Smith et al. (2000)
PR
10/10, Sprague-Dawley
rat, diet, 7 d/wk, 13 wk
0, 0.4, 3.5, 36.8,350.1
(males); 0,0.4,4.1,
40.8,402.9 (females)
(adjusted)
Mild to moderate cytoplasmic
vacuolation in the liver at the
highest dose (males and
females); 3-fold (males) and
2-fold (females) increases in
liver EROD activity at the
highest dose.
36.8
NDr
350.1
Poon et al. (1997)
PR, PS
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Table 2. Summary of Potentially Relevant Data for DNOP (CASRN 117-84-0)
Category
Number of
Male/Female, Species,
Study Type, Study
Duration
Dosimetry"
Critical Effects
NOAEL"
BMDL/
BMCLa
LOAEL'
Reference
Notesb
Subchronic
5/0, B6C3F, mouse, diet,
7 d/wk, 4 wk
0, 90, 1804 (adjusted)
Elevated levels of peroxisomal
beta-oxidation activity (PBOX)
at 4 wk in animals administered
90 mg/kg-d; elevated levels of
PBOX at 2 and 4 wk at
1804 mg/kg-d; effects were not
considered adverse
NDr
NDr
90
Smith et al. (2000)
PR
Chronic
Unspecified number,
males, F344 rat, diet,
unspecified frequency,
65 wk
0, 789.5 (adjusted)
Increased \-acctyl-
(3-glucosaminidase,
(3-galactosidase, a-mannosidase,
aryl sulfatase, cathepsin D, and
(^-glucuronidase levels
NDrd
NDr
NDrd
Carter etal. (1989)
(abstract only)
PR
12-18/0, F344 rat, diet,
7 d/wk, 60-65 wk
0, 395, 789.5 (initiated
with 30-mg/kg
diethylnitrosamine
(DEN) (adjusted)
No noncancer effects reported
NDrd
NDr
NDrd
DeAngelo et al.
(1989) [conference
proceeding]
PR
Reproductive
10/10, CD-I mouse, diet,
7 d/wk, 7 d prior to and
for 98 d of cohabitation
F0: 0, 1820, 3620,
7460
(adjusted)0
Fl: 0, 8640
(adjusted)0
F0: No effects on reproductive or
clinical parameters in any
animals at any dose
Fl: Highest dose of DNOP
resulted in significant increases
in liver and seminal vesicle
weights in males and kidney and
liver weights in females; no
effects on reproductive indices
or pup outcomes
F0: 7460°
Fl: NDr
NDr
F0: NDr
Fl: 8640
Heindel et al.
(1989); Morrisey et
al. (1989).
Heideletal. (1989)
is the original
report, Morrisey et
al. (1989) is a
meta-analysis of 48
RACB
reproductive
studies including
the Heindel et al.
(1989) study
PR
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Table 2. Summary of Potentially Relevant Data for DNOP (CASRN 117-84-0)
Category
Number of
Male/Female, Species,
Study Type, Study
Duration
Dosimetry"
Critical Effects
NOAEL"
BMDL/
BMCLa
LOAEL'
Reference
Notesb
Reproductive
F0: 20/20 (treated), 40/40
(control), CD-I mouse,
diet, 7 d/wk, 18 wk
Fl: 20/20, CD-I mouse,
diet, 7 d/wk, 16 wk
F0 males: 0, 1820,
3620, 7460 (adjusted)
F0 females: 0, 1699,
3411,7120 (adjusted)
Fl males: 0, 8101
(adjusted)
Fl females: 0, 9438
(adjusted)
F0: No significant effects on
reproductive or clinical
parameters examined
Fl: Significant increases in
absolute and relative kidney and
liver weights in males and
females; significant decrease in
seminal vesicle weight in Fl
males
F0: 7120e
Fl: NDr

F0: NDr
Fl: 8101
NTP (1985).
This study reports
the same data as
Heinel et al. (1989)
and Morrisey et al.
(1989)
PR

SD rats, 7 d per wk by
gavage, 500 mg/kg-d,
4 wk. Number of animals
not reported.
500 mg/kg-d
Significant changes in sperm
counts and sperm motility
NDr
NDr
500
Kwack et al. (2009)
PR
Carcinogenic
Unspecified number,
males, F344 rat, diet,
unspecified frequency,
65 wk
0,214
Increase in observed liver
nodules
NDrd
NDr
NDrd
Carter etal. (1989)
(abstract only)
PR

12-18/0, F344 rat, diet,
7 d/wk, 60-65 wk
0, 107, 214
1/13 (8%) with liver carcinoma
and 3/13 (23%) with adenoma in
the 214-mg/kg-d group
NDrd
NDr
NDrd
DeAngelo et al.
(1989) [conference
proceeding]
(abstract only)
PR
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Table 2. Summary of Potentially Relevant Data for DNOP (CASRN 117-84-0)
Category
Number of
Male/Female, Species,
Study Type, Study
Duration
Dosimetry"
Critical Effects
NOAEL3
BMDL/
BMCLa
LOAEL3
Reference
Notesb
2. Inhalation (mg/m3)a
Subchronic
ND
Chronic
20/0, ICR rat, inhalation,
2 hr/d, 3 d/wk, 4-16 wk
NR
No effects reported
NDr
NDr
NDr
Lawrence et al.
(1975)
PR
Developmental
ND
Reproductive
ND
Carcinogenic
ND
""Dosimetry: NOAEL, BMDL/BMCL, and LOAEL values are converted to an adjusted daily dose (ADD in mg/kg-d) for oral noncancer effects and a human equivalent
concentration (HEC in mg/m3) for inhalation noncancer effects. Values are converted to a human equivalent dose (HED in mg/kg-d) for oral carcinogenic effects. All
long-term exposure values (4 wk and longer) are converted from a discontinuous to a continuous (weekly) exposure. Values from animal developmental studies are not
adjusted to a continuous exposure.
Doses were converted from percentage of food to ppm by multiplying by 10,000 (1% = 10,000 ppm) and then converted from ppm to mg/kg-day using the following
equation: DoseADj= Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
bNotes: IRIS = utilized by IRIS, date of last update; PS = principal study; PR = peer reviewed; NPR = not peer reviewed.
°Study author-adjusted doses (converted from % in food to mg/kg-d); all other adjusted doses were calculated for this PPRTV document.
dNOAELs/LOAELs are not identified because only abstract of study was available.
eThis value is a NOEL (no-observed-effect level) rather than a NOAEL.
NA = not applicable; ND = no data; NDr = not determinable; NR = not reported; NR/Dr = not reported in study but determined from data.
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HUMAN STUDIES
Oral Exposures
The effects of oral exposure to DNOP in humans have not been evaluated in any
subchronic, chronic, developmental, reproductive, or carcinogenic studies.
Inhalation Exposures
The effects of inhalation exposure to DNOP in humans have been evaluated in one
case-control reproductive study of endometriosis (Reddy et al., 2006) and one neurological
cohort study involving persons proximal to a petroleum processing facility (Kilburn and
Warshaw, 1995). These studies are summarized below. No additional subchronic, chronic,
developmental, or carcinogenic human studies were identified.
Reproductive Studies
Reddy et al. (2006)
In a case-control study, Reddy et al. (2006) investigated the association between
phthalate exposure and endometriosis in Indian women. The study group consisted of 49
infertile women who were diagnosed with endometriosis using laparoscopy and were recruited
from a hospital and research center serving the region of Andhra Pradesh, India. There were two
control groups used in this study. Control Group 1 consisted of 38 women who were attending
the same hospital for other gynecological conditions (but were confirmed negative for
endometriosis by laparoscopy). All women in Control Group 1 were infertile and 17% reported
dyspareunia (painful intercourse), 26% complained of mild dysmenorrhea (pain during
menstruation), and 6% complained of severe dysmenorrhea. Control Group 2 consisted of
21 women who had visited the same hospital for laparoscopic tubal sterilization. These women
were fertile and had no evidence of endometriosis or any other gynecological conditions. The
authors reported that all of the women in the case and control groups had no history of
occupational exposure to reproductive toxicants, lived in urban areas, were nonsmokers, and did
not consume alcohol.
The authors collected and analyzed blood samples using gas chromatography to
determine the concentrations of di-//-butyl phthalate (DNBP), di-//-octyl phthalate (DNOP), butyl
benzyl phthalate (BBP), and diethyl hexyl phthalate (DEHP). When the authors compared the
fertility histories of the three study groups, they found that the groups had comparable ages of
menarche, durations of infertility (exposed and Control Group 1), ages, and body mass index
(BMI). Only pain during intercourse differed among these three groups, with women with
endometriosis more commonly reporting this problem (34% versus 17% in Control Group 1).
The results showed that there were significant differences in the blood concentrations of
phthalate esters in women with endometriosis compared with women without the condition. The
mean blood concentration of DNBP was 0.44 [ig/mL in cases diagnosed with endometriosis
compared with 0.08 [j,g/mL in Control Group 1 and 0.15 p,g/mL in Control Group 2; mean
concentrations of BBP were 0.66 [j,g/mL in cases diagnosed with endometriosis compared with
0.12 [j,g/mL in Control Group 1 and 0.11 ng/mL in Control Group 2; mean concentrations of
DNOP were 3.32 [j,g/mL in cases diagnosed with endometriosis compared with 0.00 [j,g/mL in
Control Group 1 and 0.00 [j.g/mL in Control Group 2; and mean concentrations of DEHP were
2.44 [j,g/mL in cases diagnosed with endometriosis compared with 0.50 [ig/mL in Control
Group 1 and 0.45 [ig/mL in Control Group 2. The correlation between phthalate concentration
and severity of endometriosis was statistically significant and strong for all of the phthalates
examined (r = +0.57 andp < 0.0001 for DNOP).
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The authors concluded that this study suggests an association between phthalate ester
exposure (exposure route unknown) and endometriosis in Indian women. The authors also noted
that DNOP was found at the highest concentration of all of the phthalates examined, followed by
DEHP. The results of this study support a study performed by Cobellis et al. (2003) that
reported that women with endometriosis had higher serum concentrations of DEHP compared
with those without this condition. However, Cobellis et al. (2003) did not measure levels of
DNOP. Although this study is limited by its inability to separate the effects of the individual
phthalate esters, it is a well-conducted epidemiologic study that lends support to the literature
that indicates that DNOP causes reproductive effects in animals.
Neurological Studies
Kilburn and War show (1995)
In a cohort study conducted in Louisiana, Kilburn and Warshaw (1995) investigated
neurobehavioral endpoints in residents living near a motor oil and chemical reprocessing plant
that was in operation from 1966-1983. The study included those individuals living beyond the
plant's dispersion and drainage areas. A large number of chemicals were identified at the
combustion site, including methylene chloride, chloroform, trichloroethylene, polychlorinated
biphenyls (PCBs), toluene, styrene, chlorobenzene, arsenic, and DNOP. No measurements taken
during the time of the plant's operation were available. The study group consisted of 77 women
and 54 men between the ages of 15 and 65 years old who were living near the plant and were
identified during the course of a class action law suit against the plant (thus recall bias may
complicate the interpretation of self-reported symptoms). This group had resided near the site
for an average of 9.3 years during the plant's operation. A randomly-selected reference group of
37 women and 29 men was identified from a nearby town and matched for sex and age.
Self-administered surveys were designed to assess the demographics, occupational histories,
toxic exposures, and neurological and medical histories of the treatment and reference groups.
Exposure was not measured directly, but the maximum concentrations of DNOP in the sludge
and soil were measured to be 770 ppb and 960 ppb, respectively. Duration of residence and
distance from the plant were used as surrogates for exposure measurements. Trained health
professionals, blind to the subjects' status, administered a neurophysiological (blink, balance,
reaction time, and color discrimination) and neuropsychological (recall; intelligence; visual
attention and integrative capacity; constructional, interpretative, and integrative capacity;
decision making; peripheral sensation; and discrimination) test batteries. The results of the tests
were adjusted for a 1.4-year average difference in educational attainment between the exposed
and reference groups.
The results showed that there were significant differences in simple and choice reaction
times, body balance, and cognitive and perceptual motor functions between the cohort and
reference groups. The effects remained significant after adjusting for age and education. Blink
reflex latency and eye closure speed were reported to be normal in both groups. Differences in
recall and memory were not significant. Self-reported symptom frequencies and scores for
depression, anger, confusion, tension, and fatigue were elevated in the exposed group, which the
study authors reported as indicators of depression. Confounding from other diagnosed disorders
or occupational disorders were reported as minimal. None of the surrogates for exposure were
found to be correlated with the effects identified within the exposed population; however, the
range of surrogate values within the group was limited.
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The authors concluded that this study suggests an association between exposure to the
combustion products and the neurological effects that were observed in the local residents. A
large number of chemicals were found at this site, some of which are known toxins. While
DNOP was identified on the site, no conclusion regarding the toxicity of DNOP can be made
from the available study data.
Carcinogenicity
The carcinogenic effects of inhalation exposure to DNOP have not been evaluated in
humans.
ANIMAL STUDIES
Oral Exposures
The effects of oral exposure to DNOP in animals have been evaluated in two subchronic
(Smith et al., 2000 and Poon et al., 1997), two chronic (Carter et al., 1989; DeAngelo et al.,
1989), and three reproductive (Heindel et al., 1989; NTP, 1985; Kwack et al., 2009) studies. No
developmental studies were identified. Two carcinogenicity studies were identified
(Carter et al., 1989; DeAngelo et al., 1989), but only the abstracts are available for these studies.
A number of short-term toxicity studies are available and are presented in the "Other Data"
section and summarized in Table 3B.
Subchronic Studies
Smith et al. (2000)
In the rat component of a peer-reviewed subchronic-duration study, Smith et al. (2000)
administered doses of 0-, 1000-, or 10,000-ppm DNOP (>99% pure) in the diet to groups of 5
male Fisher 344 (F344) rats for 2 or 4 weeks. Adjusted daily doses are calculated as 0, 100, or
1000 mg/kg-day, respectively, using standard body weight and food consumption rates (0.18 kg
and 0.018 kg/day, respectively) because experimental data were not available (U.S. EPA, 1988).
Rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Researchers followed the
NIH Guide for the Care and Use of Laboratory Animals (U.S. DHEW, 1978). Animals were
housed in polycarbonate cages, and the room was kept on a 12-hour light/dark cycle. Diet
consisted of NIH-07 pelletized feed and deionized water ad libitum. The authors did not report
whether this study was Good Laboratory Practice (GLP) compliant.
Smith et al. (2000) sacrificed, weighed, and necropsied the animals following treatment.
Blood was collected, and the livers were removed. The authors recorded the relative liver
weights and measured liver samples for gap junctional intercellular communication (GJIC),
replicative DNA synthesis, and peroxisomal beta-oxidation (PBOX) activity. The authors used
two-way analysis of variance (ANOVA) followed by a Dunnett's test to evalute the statistical
differences (p < 0.05) between the groups.
Smith et al. (2000) presented the results in graphs, which were digitized, and the
information is presented in Table B. 1. The authors noted significant increases in the relative
liver weights (113% of controls) and PBOX activities in animals treated with 1000 mg/kg-day
compared with controls at 2 weeks. However, at 4 weeks, there were no significant differences
between the groups. The authors also noted elevated periportal DNA synthesis in rats
administered 1000 mg/kg-day at 2 and 4 weeks (421% and 1370%) of controls, respectively).
The authors concluded that, because the chronic data are limited for DNOP, it is difficult to
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understand the exact significance of the studied endpoints. Based on the effects observed in the
liver (increased relative liver weight and elevated periportal DNA synthesis), a NOAEL of
100 mg/kg-day and a LOAEL of 1000 mg/kg-day are identified.
Poon et al. (1997)
Poon et al. (1997) is selected as the principal study for the derivation of subchronic
and chronic p-RfD values. Poon et al. (1997) investigated the oral toxicity of DNOP
(99% pure; in diet and 4% corn oil) in a 13-week, peer-reviewed study in Sprague-Dawley rats.
Young male rats (105-130 g) and female (93-111 g) were obtained from Charles River
Laboratories. It is unknown if the study was conducted in compliance with GLP. The study
authors administered 0-, 5-, 50-, 500-, or 5000-ppm DNOP daily via diet to groups of 10 animals
per sex per dose. The study authors calculated average daily doses of 0, 0.4, 3.5, 36.8, and
350.1 mg/kg-day for males and 0, 0.4, 4.1, 40.8, and 402.9 mg/kg-day for females. The study
authors measured animal body weights and food consumption weekly throughout the course of
the study. Clinical observations were made daily. At sacrifice, hematology (hematocrit,
hemoglobin, red blood cell, platelet, and total and differential white blood cell counts) and serum
biochemistry (alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase
activities, and albumin, calcium, cholesterol, glucose, inorganic phosphate, potassium, sodium,
bilirubin, uric acid, creatinine, blood urea nitrogen, and total protein levels) were measured. The
testes, epididymides, adrenal, aorta, bone marrow, brain, esophagus, eyes, heart, intestinal tract,
kidneys, liver, mammary glands, mandibular and mesenteric lymph nodes, ovaries, pancreas,
pituitary, prostate, salivary glands, sciatic nerve, seminal vesicles, skeletal muscle, skin, spleen,
stomach, trachea and lungs, thyroid and parathyroid, tongue, urinary bladder, and uterus were
fixed, sectioned, and stained. It is unclear if histopathologic evaluation was conducted in a
blinded fashion. The authors performed a one-way ANOVA and Duncan's multiple range tests.
Poon et al. (1997) reported no signs of clinical toxicity or changes in food consumption in
any of the animals exposed to DNOP. Similarly, no significant changes in organ weights were
reported in either male or female animals (see Table B.2). Hematology and serum biochemistry
measurements were not significantly altered, with the exception of increased calcium levels in
males exposed to 350.1 mg/kg-day (117% of controls) and increased inorganic phosphate levels
in females exposed to 4.1 mg/kg-day (see Table B.3). Hepatic EROD levels were 3-fold higher
(308%) of controls) in males and 2-fold higher (212% of controls) in females exposed to the
high-dose levels (see Table B.4). No increase in peroxisome proliferation was observed
(observational, no quantitative data presented). The levels of DNOP in the livers of the animals
of all treatment groups were very low or below the detection limit. However, concentrations of
DNOP were 3- to 6-fold higher in the adipose tissue compared with the liver of the high-dose
animals (see Table B.5).
Poon et al. (1997) observed "mild microscopic changes" in the livers and thyroids of both
sexes of treated animals (see Tables B.6 and B.7). All animals in the high-dose group (10/10 in
both sexes) showed a moderate increase in zonation of the liver. Many of the animals in this
group also showed mild-to-moderate increases in the perivenous cytoplasmic vacuolization
(9/10 males; 5/10 females) in addition to increased perivenous cytoplasmic volume. Mild
nuclear prominence was observed in the liver interstitium of a number of rats in the high-dose
group (7/10 males; 10/10 females). The study authors also reported mild-to-moderate,
dose-dependent anisokaryosis, nuclear hyperchromicity, and vesiculation in treated male rats
(observational, no quantitative data presented). Follicles in the thyroid were found to be reduced
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in size, and small decreases in colloid density were reported in animals in the high-dose group.
None of the other examined endpoints were significantly altered, including morphological effects
on male reproductive organs.
Poon et al. (1997) concluded that DNOP is not a peroxisome proliferator at the levels
tested in this subchronic study. Despite not observing an effect on liver weight, the study authors
concluded that the observed increase in EROD activity indicates that DNOP is a
3-methylcholanthrene-type enzyme inducer. The study authors identified a NOAEL of
36.8 mg/kg-day based on "all gross, histopathological, and biochemical changes" (the study
authors reported a NOAEL of 36.6 mg/kg-day; however, this is believed to be a typo, and the
correct NOAEL is 36.8 mg/kg-day). Mild-to-moderate cytoplasmic vacuolation accompanied by
other hepatic histological changes, thyroid histopathology, and increased EROD activity support
the identification of a LOAEL of 350.1 mg/kg-day and a corresponding NOAEL of
36.8 mg/kg-day.
Smith et al. (2000)
In the mouse component of the previously summarized peer-reviewed subchronic study,
Smith et al. (2000) administered 0-, 500-, or 10,000-ppm DNOP to groups of 5 male B6C3Fi
mice via diet for 2 or 4 weeks. The adjusted daily doses are calculated as 0, 90, and
1804 mg/kg-day, respectively, based on standard body weight and food consumption rates
(0.0316 kg and 0.0057 kg/day, respectively) because no experimental data were available
(U.S. EPA, 1988). The mice were kept under the same conditions as the rats in this study (see
Smith et al., 2000). The authors did not report whether this study was GLP compliant.
Smith et al. (2000) conducted a similar analysis as was performed previously in rats.
Again, the results were presented graphically, which were digitized, and the information is
presented in Table B.8. The authors found elevated levels of PBOX at both 2 and 4 weeks in
mice administered 1804 mg/kg-day (control values not reported). PBOX levels were elevated at
4 weeks in mice administered 90 mg/kg-day (see Table B.8). No other significant changes were
noted. The authors concluded that, because chronic data are limited for DNOP, it is difficult to
understand the exact significance of the studied endpoints, and may not be a negative health
effect. Based on the observed changes in PBOX levels, a LOAEL of 90 mg/kg-day is identified.
The LOAEL is the lowest dose administered, preventing the identification of a NOAEL.
Chronic Studies
Carter et al. (1989)
In an abstract, Carter et al. (1989) describe a carcinogenicity study in which 0 or
1% DNOP was administered via the diet to groups (number unreported) of male F344 rats for
65 weeks. The adjusted daily dose for 1% DNOP is 789.5 mg/kg-day, and the human equivalent
dose is 214 mg/kg-day, calculated using 0.38 kg as the standard body weight and 0.03 kg/day as
the food consumption rate based on values presented by U.S. EPA (1988). The authors reported
"numerous liver nodules" in DNOP-treated rats; however, the full study report could not be
obtained. In the abstract, the authors reported 3-fold increases in hepatic A-acetyl-
P-glucosaminidase, P-galactosidase, a-mannosidase, and aryl sulfatase levels. Additionally,
cathepsin D and P-glucuronidase levels were increased. The abstract authors concluded that the
upregulation of glycosidases may cause sublethal autolysis, and they proposed that this activity
may result in tumor induction. These data support the previously summarized study by
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Poon et al. (1997) in the identification of enzyme induction and pathologies in the liver. Because
only the abstract of this study was available, further evaluation of the carcinogenicity of DNOP
could not be accomplished.
DeAngelo et al. (1989)
In the abstract of another carcinogenicity study, DeAngelo et al. (1989) completed a
partial hepatectomy and administered a single dose of 30-mg/kg diethylnitrosamine (DEN; a
complete carcinogen) to groups of 12-18 male F344 rats and then dosed them with DNOP (0.5%
or 1.0%) or DEHP (0.1%, 0.5%, or 2.0%) via the feed for 60-65 weeks. The authors dosed
comparable groups that were not initiated with DEN. A control group received the diet and DEN
alone, and a positive control group was administered 0.05% phenobarbital (PB; 11 mg/kg-day) in
the drinking water. The adjusted daily doses of 395 and 789.5 mg/kg-day and the human
equivalent doses of 107 and 214 mg/kg-day DNOP are calculated based on the standard body
weight of 0.38 kg and food consumption rate of 0.03 kg/day (U.S. EPA, 1988) because
experimental data were not available. It is unclear if this study was GLP-compliant because only
an abstract from the Proceedings of the American Association for Cancer Research could be
obtained.
After sacrifice, the animals were examined for liver tumors (no other tumor types were
examined). Rats that were given only DEN had a 6% incidence of carcinomas (1/18 animals),
while the positive control group that received PB had a 94% incidence of carcinomas
(16/17 animals). The authors reported that DEHP did not increase the incidence of carcinomas at
any dose in the DEN-initiated rats, whereas DNOP did increase the carcinoma incidence to 54%
(7/13) at 107-mg/kg-day DNOP and 61% (11/18) at 214-mg/kg-day DNOP. In the animals that
were not initiated with DEN, only the group dosed at 214-mg/kg-day DNOP developed liver
neoplasms (1/13 [8%] with carcinomas; 3/13 [23%] with adenomas). The authors concluded that
DNOP can promote liver carcinomas in rats initiated with DEN through a mechanism other than
peroxisome proliferation. Furthermore, DNOP may be carcinogenic without an initiator
chemical. This summary was published in a conference proceeding; upon further research, it
appears that this study was never published in full. Because only the abstract of this study was
available, further evaluation of the carcinogenicity of DNOP could not be accomplished.
Reproductive Studies
Heindel et al. (1989)
In a peer-reviewed, continuous breeding, reproductive toxicity study, Heindel et al.
(1989) administered diets containing 0, 1.25, 2.5, or 5.0% DNOP (Midwest Research Institute;
>99% pure and stable in feed at room temperature for up to 2 weeks) to male and female CD-I
mice (20 pairs per group) for 7 days prior to and during a 98-day cohabitation period. The
authors calculated average daily doses of 0, 1820, 3620, and 7460 mg/kg-day, respectively
(calculated by Morrissey et al., 1989). A group consisting of 40 pairs of control animals
received feed only. The study was conducted using the Reproductive Assessment by Continuous
Breeding Protocol (RACB). This protocol describes four possible tasks: (1) a 14-day
dose-setting study with 5 doses and a control group (8 animals per sex per group); (2) a
continuous breeding phase with a control group of 40 breeding pairs and 3 dose groups with
20 pairs per group; (3) a 1-week crossover mating trial after the Task 2 litter is weaned using
3 groups of 20 pairs (conducted if Task 2 is positive for reproductive effects); and (4) offspring
assessment (conducted if Task 2 is negative for reproductive effects).
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Heindel et al. (1989) obtained mice from Charles River Breeding Laboratories, Inc.
(Kingston, NY) that were 11 weeks old when the breeding phase began. All animals were
quarantined for at least 2 weeks. Animals were initially housed in groups that were segregated
by sex during the quarantine and premating periods and then were subsequently housed as
breeding pairs or individually. The cages were solid-bottom polypropylene or polycarbonate
with stainless steel wire lids. Rodent chow (NIH-07) and deionized/filtered water were provided
ad libitum. The room was kept at 23 ± 2°C (humidity unreported) and maintained on a 14-hour
light/10-hour dark cycle. Although this study does not include a GLP certificate, according to
Morrisey et al. (1989), all RACB protocol studies are GLP compliant. This study is an
acceptable reproductive study based on the reported methodology. However, analysis of the
developmental endpoints is limited in the RACB protocol because the offspring are not
examined for skeletal or soft tissue anomalies; thus, this study is not considered an acceptable
developmental study.
After 98 days of cohabitation in Task 2, Heindel et al. (1989) housed animals
individually, and dosing was continued. Any litters born after the continuous breeding phase
were weaned and then provided with the dosed feed. These animals were then used for Task 4.
The endpoints that were evaluated included clinical signs, parental body weight, fertility (number
of adults producing a litter/number of breeding pairs), litters per pair, live pups per litter,
proportion of pups born alive, sex of the live pups, pup body weights within 18 hours of birth,
and food and water consumption. At the end of the task, the authors necropsied the F0 animals
and measured their body weights; organ weights; epididymal sperm motility, morphology, and
number; and estrous cyclicity (monitored for 7 days using vaginal lavage). Organs selected for
microscopic evaluation were fixed in 10% neutral buffered formalin (Bouin's fixative for the
testes), embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Task 3 was
not completed for DNOP because the results of Task 2 were not positive for reproductive effects.
Task 4 however, was completed because Task 2 did not indicate reproductive toxicity.
The control and high-dose F1 animals were reared, weaned, and dosed until sexually mature
(74 ±10 days) using the same concentrations of DNOP in the diet as their parents. These
animals were then cohabitated for 7 days and dosed until necropsy at 95 days. The adjusted
daily doses were calculated as 0 and 8640 mg/kg-day (calculated by Morrissey et al., 1989). The
endpoints that were examined following mating and necropsy of the F1 mice were the same as
those examined in the F0 mice, which included an examination of the F2 offspring for the
following: live pups per litter, proportion of pups born alive, sex of the live pups, and pup body
weights within 18 hours of birth.
In order to test for dose-related trends (Tasks 2 and 4), Heindel et al. (1989) employed the
Cochran-Armitage test. The authors used the Fisher's exact test to make pairwise comparisons
between the control and DNOP-dosed groups. The number of litters and live pup indices were
calculated for each fertile pair, and group means were calculated for each dose level. The live
pup index was calculated as the number of pups born alive divided by the total number of pups
produced by each pair. The sex ratio was calculated by dividing the number of male pups born
alive by the total number of live pups produced by each pair. The authors used the
Kruskal-Wallis test to determine the overall differences between the treatment group means and
the Jonckheere's test to determine the ordered differences. The authors used the
Wilcoxon-Mann-Whitney I /-test to make pairwise comparisons of the treatment group means.
In order to adjust for any effect that the number of pups per litter had on the average pup weight,
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the authors performed analysis of covariance (ANCOVA) using the average litter size as the
covariate (live and dead pups). The authors then used the /'-test and /-test to evaluate for overall
equality and pairwise equality, respectively, for least-squares estimates of dose-group means.
Analyses were performed for males, females, and both sexes in order to account for any sex
differences within the parameters that were examined. Organ weights were adjusted for total
body weight using ANCOVA. The Kruskal-Wallis and Wilcoxon-Mann-Whitney I /-tests were
used to analyze unadjusted body and organ weights. Finally, the authors used Jonckheere's test
to detect any dose-related trends (Tasks 2 and 4).
Heindel et al. (1989) reported that they did not find any treatment-related alterations in
physical appearance, body-weight gain, or food consumption in the Task 2 animals. In addition,
data indicated that there were no significant treatment-related effects on fertility or reproductive
performance in the parental animals. Because there were no reproductive effects observed in
Task 2, the F1 animals were cohabitated and mated. The mating indices (percentage of
plug-positive animals divided by the number of cohabitated animals) were 80% for the control
mice and 95% for the treated F1 mice, and the fertility indices were 75% and 90%, respectively.
The number of pups born alive, pup sex, and pup weights were not affected in the F2 generation.
However, treatment with 8640-mg/kg-day DNOP significantly increased (by >10% over control)
the liver weight in both sexes of the F1 animals, kidney weight in F1 females, and the seminal
vesicle weight in F1 males (see Tables B.9 and B. 10). Other reproductive parameters, terminal
body weights, and reproductive organ weights of the F1 animals of both sexes were not
significantly affected by the highest dose of DNOP.
Heindel et al. (1989) concluded that DNOP is not a reproductive toxicant at
concentrations of up to 7460 mg/kg-day in CD-I mice. The authors compared DNOP with other
phthalates of varying alkyl chain lengths and concluded that phthalates with side chains
containing 3-7 carbons (DNOP has a side chain of 8 carbons) are the phthalates that are toxic to
the reproductive system. Based on the lack of any kind of effects in the F0 generation, a
no-observed-effect level (NOEL) of 7460 mg/kg-day was determined; a LOAEL could not be
identified. Although DNOP did not cause reproductive effects in either generation, a LOAEL of
8640 mg/kg-day is identified based on decreased liver, kidney, and seminal vesicle weights
observed at this dose level in the F1 generation. The identification of a NOAEL for the
F1 generation is precluded because the only dose identified is the LOAEL.
NTP (1985)
NTP (1985) published a GLP-compliant, multigenerational reproductive toxicity study
that exposed male and female CD-I mice (Charles River Laboratories) to DNOP (Midwest
Research Institute, >98% pure) in feed. This study, like Heindel et al. (1989), was completed
under the RACB. The F0 generation of mice (20 per sex per group) was administered 1.25, 2.50,
or 5.0%) DNOP in the diet. A control group consisting of 40 males and 40 females received feed
only. Adjusted daily doses of 1820, 3620, and 7460 mg/kg-day, respectively, for the F0 males
are calculated using average food consumption and body-weight data that were reported by the
study authors. Adjusted daily doses of 1699, 3411, and 7120 mg/kg-day, respectively, are
calculated for the F0 females using time-weighted average food consumption and body-weight
values that were reported by the study authors. Food and water were provided ad libitum.
F0 animals were exposed to DNOP for 1 week prior to mating, 14 weeks of cohabitation, and
3 weeks after cohabitation (18 weeks in total). The authors measured the body weight, number
of litters produced, number of live and dead pups per litter, mean male and female pup body
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weights per litter, percentage of infertile pairs, and number of abnormal pups along with a brief
description of any abnormalities. Litters born during the 14 weeks of cohabitation were
examined and immediately sacrificed. Pups born during the 3 weeks after cohabitation were
allowed to remain with their mothers. The authors did not report the sacrifice or necropsy of the
F0 generation after the delivery of the pups. NTP (1985) is an acceptable reproductive study
based on the reported methodology. However, the developmental endpoints are limited in the
RACB protocol because the offspring are not examined for skeletal or soft tissue anomalies;
therefore, this is not considered an acceptable developmental study.
F1 pups from pairs in the high-dose group and the control group were allowed to mature
for approximately 70 days after weaning. Pups from the high-dose group received DNOP
treatment through lactation until weaning at 3 weeks and then received 5% DNOP in the feed for
approximately 13 weeks (16 weeks of total exposure time). The adjusted daily doses were
calculated using average body weight and food consumption data that were reported in the study
for Weeks 27-31 (from the time of mating until necropsy). The authors reported body weights
at weaning (Week 19) but did not record body weights again until mating (Week 27); therefore,
an adjusted daily dose based on the time-weighted average body weight would be skewed
towards a greatly reduced body weight. The adjusted doses based on averages from the final
5 weeks are much more conservative estimates. These values are 8101 mg/kg-day for males and
9438 mg/kg-day for females. After weaning, 20 males and 20 females from each group were
cohabitated for up to 7 days. Mating continued for the full 7 days or until a copulatory plug was
found. The authors then examined the same reproductive parameters described above for litters
born to the F1 generation. F1 pups were weighed at 3 weeks, on the first day of cohabitation,
and once a week thereafter. F1 food consumption was monitored during the week of
cohabitation and once per week thereafter. F1 pups were necropsied, and absolute and body
weight-adjusted liver, kidney, right epididymis, right cauda, right testis, seminal vesicles, and
prostate gland weights were recorded.
The authors used the Kruskal-Wallis test for trend analysis and the Jonckheere's test for
ANOVA. Comparisons of proportions were made using the Mann-Whitney t/-test, chi-squared
test, and the Cochran-Armitage test. Data from pairs in which one or both partners died were
excluded from statistical analyses.
NTP (1985) reported the death of 3 mice (2 females and 1 male) in the control group and
2 mice (1 female and 1 male) of the F0 generation in the 3620-mg/kg-day dose group
(3411 mg/kg-day for females). The authors did not report any effects on body weight (see
Tables B.ll and B.12) or food consumption (see Tables B.13 and B.14) in F0 males or females.
Treatment had no significant effects on the number of litters delivered per mated pair, number of
live pups per litter, sex ratio, or average or adjusted live pup weights of pups born to the
F0 generation.
For the F1 generation, successful mating and fertility ratios of the treated mice were
comparable to the controls. There were no significant differences in parental body weight (see
Tables B.15 and B.16), food consumption, number of litters delivered per mated pair, number of
live pups per litter, sex ratio, or average or adjusted live pup weights (F2 generation).
Significantly increased absolute and relative liver weights (123% and 128%, respectively) and
significantly decreased absolute and relative weights of the seminal vesicles (87% and 89%,
respectively) were reported in male mice treated with 8101 mg/kg-day (see Table B.17). No
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other significant organ-weight differences were reported in treated males. Necropsy revealed
significantly increased absolute and relative kidney weights (111% and 110%, respectively) and
absolute and relative liver weights (124% and 122%, respectively) in female mice treated with
9438 mg/kg-day (see Table B.18).
The authors concluded that DNOP did not affect fertility or reproduction in adult or
second generation CD-I mice. Because there were no effects of any kind reported in the
F0 generation, a NOEL of 7120 mg/kg-day is identified; no LOAEL could be identified. Based
on the increased liver weights in F1 male and female mice, a LOAEL of 8101 mg/kg-day was
identified. Because F1 mice were only treated at one dose level, the establishment of a NOAEL
for the F1 generation is precluded.
Kwacketal. (2009)
In a third peer-reviewed, reproductive study, Kwack et al. (2009) examined the systemic
and sperm toxicity of several phthalate esters including DNOP (purity unreported) when
administered to SD rats by gavage. The doses were 250 mg/kg-day for the monoesters and
500 mg/kg-day for the phthalate diesters for 4 weeks to male rats (number of animals not
reported) from 6 to 10 weeks of age.
DNOP did not cause systemic changes including increases in food consumption, body
weight, or organ weight (thymus, heart, liver, spleen, kidney, adrenal, testis, and epididymis
examined). No changes in red blood cell counts or hematocrit, hemoglobin, platelets, or mean
corpuscular hemoglobin were observed. The only serum chemistry parameter significantly
altered was alkaline phosphatase (ALP), which was increased 3.5-fold. DNOP significantly
lowered sperm counts and sperm motility in epididymal sperm. DNOP was intermediate among
the phthalates for potency in this regard.
DNOP caused a significant decrease (38% of control) in sperm counts and in sperm
motility (31% of control). Other sperm motility indicators including linearity, straightness, beat
cross frequency, amplitude of head displacement, curvilinear velocity, straight-line velocity, and
average path velocity were unchanged.
Because only one dose was used, a NOAEL cannot be established; however, a LOAEL of
500 mg/kg-day is established for sperm counts and sperm motility.
Inhalation Exposure
The effects of inhalation exposure to DNOP have been evaluated in one chronic animal
study (Lawrence et al., 1975). Studies evaluating DNOP inhalation exposure have not been
evaluated in subchronic, developmental, reproductive, or carcinogenicity studies.
Short-Term Studies
The short-term effects of inhalation exposure to DNOP have not been evaluated in
animals.
Subchronic Studies
The subchronic effects of inhalation exposure to DNOP have not been evaluated in
animals.
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Chronic Studies
Lawrence etal. (1975)
Lawrence et al. (1975) investigated the inhalation toxicity of DNOP (purity unreported)
in a 16-week study on male ICR rats. The study authors saturated the air with DNOP vapors and
exposed groups of 20 animals to the vapors for 2 hours per day, 3 days per week, for
4-16 weeks. The actual exposure period could be considered less than chronic; however, the
duration of the study was chronic. The exposure cannot be adjusted to continuous exposure
because the authors did not measure the concentration used in the study. Following 4, 8, 12, and
16 weeks, 5 mice were sacrificed. It is unknown if the study was conducted in compliance with
GLP. The lungs and other unspecified tissues were removed and preserved for histopathology.
The study authors reported no effects associated with exposure to DNOP, although data
supporting this conclusion were not presented in the study report.
The lack of any exposure measurements and descriptions of the methods and results
limits the usefulness of this study. For these reasons, neither a NOAEL nor a LOAEL can be
identified. This study does not support the derivation of a subchronic RfC value due to the lack
of reported methods and data.
Developmental Studies
The developmental effects of inhalation exposure to DNOP have not been evaluated in
animals.
Reproductive Studies
The reproductive effects of inhalation exposure to DNOP have not been evaluated in
animals.
Carcinogenecity Studies
The carcinogenic effects of inhalation exposure to DNOP have not been evaluated in
animals.
Other Data (Short-Term Tests, Other Examinations)
Several studies are identified and presented in Table 3 A that report the genotoxic activity
of DNOP in prokaryotic organisms (Goodyear Tire and Rubber Company, 1982a,b; Zeiger et al.,
1982, 1985; Sato et al., 1994; Shibamoto and Wei, 1986; Seed, 1982). Other types of
genotoxicity studies are not identified. Table 3B presents summaries of DNOP tumor promoter
carcinogenicity studies (Carter et al., 1992; DeAngelo et al., 1986), short-term studies (NTP,
1985; Oishi and Hiraga, 1980, 1982; Hinton et al., 1986; Mann et al, 1985; Lake et al., 1984,
1986; Jones et al., 1993; Oishi, 1990; Foster et al., 1980), metabolism/toxicokinetic studies
(Albro and Moore, 1974; Calafat et al., 2006; Silva et al., 2005), mode-of-action/mechanistic
studies (Mann et al., 1985; Gray et al., 1983; Hinton et al., 1986; Zacharewski et al., 1998), an in
vitro neurotoxicity study (Teranishi and Kasuya, 1980), in vitro reproductive studies
(Fredricsson et al., 1993; Gray and Beamand, 1984), and an in vitro cytotoxicity study
(Jones et al., 1975).
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Table 3A. Summary of DNOP Genotoxicity Studies



Resultsb


Endpoint
Test System
Dose
Concentration"
Without
Activation
With
Activation0
Comments
References
Genotoxicity studies in prokaryotic organisms
Reverse mutation
Liquid suspension assay with
Escherichia coli strains W3100 and
P3478
10-2000 ng
DNOP/plate


Not mutagenic to Escherichia
coli
Goodyear Tire and
Rubber Company
(1982a)

Ames assay with Salmonella
typhimurium strains TA98, TA1535,
TA1537, and TA100 with and without
S9 activation
10-3200 ng
DNOP/plate


Not mutagenic to Salmonella
typhimurium
Goodyear Tire and
Rubber Company
(1982b)

Preincubation modification of the Ames
assay with ,V. typhimurium strains TA98,
TA100, TA1535, TA1537; incubated at
37°C for 2 d
100-10,000 ng
DNOP/plate
(5 doses)


Not mutagenic to Salmonella
typhimurium
Zeiger et al.
(1982); Zeiger et
al. (1985)

Preincubation procedure with
Salmonella typhimurium strain TA98
0.25-500 nmol
DNOP/plate
-
-
Not mutagenic to Salmonella
typhimurium
Sato et al. (1994)

Modified Ames assay with Salmonella
typhimurium strains TA98 and TA100
with and without S9 activation
NR


Not mutagenic to Salmonella
typhimurium; concentrations of
20-300 (ig/plate reported for
synthetic rubber extract, but
unclear if same concentration
range used for DNOP
Shibamoto and
Wei (1986)

Salmonella typhimurium strain TA100;
mutation to 8-azaguanine resistance and
histidine prototrophy assessed
NR


Concentrations ranged up to
10 mM for other chemicals
tested; no DNOP concentration
reported
Seed (1982)
SOS repair induction
SOS chromotest with Escherichia coli
PQ37
0.025-50 nmol
DNOP/plate
-
-
Mutagenicity of Trp-1 slightly
suppressed by DNOP
Sato et al. (1994)
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Table 3A. Summary of DNOP Genotoxicity Studies
Endpoint
Test System
Dose
Concentration"
Resultsb
Comments
References
Without With
Activation Activation0
Genotoxicity studies in nonmammalian eukaryotic organisms
Mutation
ND
Recombination
induction
ND
Chromosomal aberration
ND
Chromosomal
missegregation
ND
Mitotic arrest
ND
Genotoxicity studies in mammalian cells—in vitro
Mutation
ND
Chromosomal
aberrations
ND
Sister chromatid
exchange (SCE)
ND
DNA damage
ND
DNA adducts
ND
Genotoxicity studies in mammals—in vivo
Chromosomal
aberrations
ND
Sister chromatid
exchange (SCE)
ND
DNA damage
ND
DNA adducts
ND
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Table 3A. Summary of DNOP Genotoxicity Studies
Endpoint
Test System
Dose
Concentration"
Resultsb
Comments
References
Without With
Activation Activation0
Mouse biochemical or
visible specific locus
test
ND
Dominant lethal
ND
Genotoxicity studies in subcellular systems
DNA binding
ND
"Low est effective dose for positive results; highest dose tested for negative results.
b+ = positive; ± = equivocal or weakly positive; - = negative; T = cytotoxicity; NA = not applicable; ND = no data; NDr = not determined; NR = not reported;
NR/Dr = not reported by the study author, but determined from data.
°S9 from rat liver induced with phenobarbital, aroclor, or 5,6-benzoflavone.
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Table 3B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Carcinogenicity
studies other than
oral/inhalation
Male F344 rat; 2/3 partial
hepatectomy and single injection of
30 mg/kg of the initiator DEN; 10 d
later, 0.5% or 1.0% DNOP
administered in diet for 26 wk;
immunohistochemistry and
histochemistry for neoplasms
(gamma-glutamyl transpeptidase
[GGT] and glutathione ^-transferase
[GST-P])
Increased GGT levels compared with
controls treated with DEN; GGT and GST-P
expression increased in liver compared with
controls treated with DEN, although this
could not be definitively localized to foci or
nodules; absolute liver weight not affected by
DNOP although slight nonsignificant
increase in relative liver weight
Acts as promoter under study
conditions
Carter etal. (1992)

Male Sprague-Dawley rat (5/group);
2/3 partial hepatectomy and single
injection of 30-mg/kg DEN
(initiator); treated with 0 or 1%
DNOP in diet for 7 d/wk for 10 wk;
hepatotoxicity evaluated
Significant increase (p < 0.05) in GGT+ foci
and GGT activity at 1% DNOP compared
with controls and animals administered
bis(2-ethylhexyl) phthalate (DEHP), MEHP,
or 2-ethylhexanol (2-EH); carnitine
acetyltransferase (CAT) activity increased in
animals treated with DNOP
No concurrent liver enlargement in
animals treated with DNOP; DNOP
may be a promoter of carcinogenic
activity
DeAngelo et al.
(1986)
Short-term studies
Male Wister rat (young);
administered diets containing
2% mono-«-octyl phthalate (MNOP)
for 1 wk; serum levels evaluated
Significantly increased levels of serum
nonesterified fatty acids; decreased levels of
triglycerides and total cholesterol; increased
percentage of oleic acids in serum
triglycerides; free cholesterol and serum
lipoperoxide not significantly altered in
treated rats; enlarged liver
Possible hepatotoxicity along with
serum lipid effects
Oishi and Hiraga
(1982)
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Table 3B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Short-term studies
Male Wistar rat (6 rats in control,
4 rats in exposure group);
administered via diet; 0 or
2000 mg/kg-d dose for 3, 10, or 21 d
Accumulation of fat in the centrilobular zone
of the liver and fatty necrosis observed at 10
and 21 d; relative liver weight significantly
increased at 10 and 21 d; peroxisome
proliferation and hepatomegaly at 21 d;
smooth endoplasmic proliferation and loss of
rough endoplasmic reticulum (unclear at
which sacrifice time this effect was
observed); serum thyroxine levels decreased
at 21 d with ultrastructural changes in thyroid
Indications of liver and thyroid
toxicity with severity increasing
with duration of exposure
Hintonetal. (1986);
Mannetal. (1985)

Male (unspecified number)
Sprague-Dawley rat; 1000 mg/kg-d
DNOP by gastric intubation for 14 d;
relative liver weight evaluated and
hepatic microsomal activities
measured
Significantly higher relative liver weight
(4.2 ±0.1 g/100 g-bw) compared with control
(3.6 ±0.1 g/100 g-bw); 7-ethoxycoumarin
O-deethylase (ECOD) activity significantly
lower than controls
Acute hepatotoxic effects at
1000 mg/kg-d; less correlation
between in vivo and in vitro effects
for DNOP than for other phthalate
esters
Lake et al. (1984,
1986)

Male Wistar rat (unspecified
number); 2% DNOP administered via
diet for 1 wk; body weight and food
consumption, relative organ weights,
serum levels, and zinc concentrations
in tissue assessed
Relative liver weight significantly higher
than control; serum levels of testosterone and
dihydrotestosterone not significantly
affected; zinc concentrations significantly
decreased in the testes
2% DNOP does not cause
significant testicular atrophy, but
has hepatic effects
Oishi and Hiraga
(1980)

Wistar rat (3/group; 2-g/kg DNOP
administered via gavage for 2 d;
testicular tissue examined for changes
in Leydig cells
No changes observed to the seminiferous
tubular structure or Leydig cell morphology
No testicular toxicity
Jones et al. (1993)

Male Wistar rat; in vitro;
mitochondrial fraction of testes
incubated with DNOP for 2 min;
mitochondrial oxygen consumption
measured
Significant decrease in mitochondrial oxygen
consumption at 1.3 |imol/mL
May induce testicular atrophy under
the experimental conditions
Oishi (1990)
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Table 3B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Short-term studies
Male Wistar rat (5/group); single dose
of 2 mg/kg DNOP administered via
gavage and animals sacrificed 6 hr
later; mitochondrial fraction of testes
prepared and mitochondrial oxygen
consumption measured
Significant decrease in the respiration control
ratio and oxygen consumption
May induce testicular atrophy under
the experimental conditions. DNOP
causes decreased mitochondrial
function with potential fertility
effects in male rats
Oishi (1990)

Male Sprague-Dawley rat (12/group);
2800 mg/kg-d administered via
gavage for 4 d; testes removed,
weighed, fixed, sectioned, and stained
No significant effects observed
No testicular toxicity
Foster etal. (1980)

Male and female (8/sex) CD-I
mouse; 0.0, 0.50, 1.25, 2.50, 5.0, or
10.0% DNOP administered via diet
for 2 wk; animals observed for
clinical toxicity
No effect on daily food consumption or body
weight; significant number of males (6/8) and
females (4/8) in the 10.0% dose group
displayed rough coats
DNOP may cause clinical toxicity in
CD-I mice at 10.0% DNOP.
NTP (1985)
Metabolism/
toxicokinetic
Male CD rat; 0.2-mL DNOP
administered via gavage at 24-hr
intervals; urine collected for 48 hr
following initial dose
Urine contained 31.0% of the phthalate
moiety; alkyl side chain permitted a series of
a- and -oxidations of the
carboxyl-terminated metabolites; phthalic
acid is a minor metabolite
Indicates oral absorption; high
occurrence of oxidative metabolites
Albro and Moore
(1974)

Female Sprague-Dawley rat; single
gavage dose of 300-mg/kg DNOP;
24-hr urine samples collected and
analyzed for metabolites; concurrent
human samples taken from a random
population without documented
exposure to phthalates
Urinary levels of mono-(3-carboxypropyl)
phthalate (MCPP) highest (225 |ig/mg
creatinine) and higher than mono-«-octyl
phthalate (MNOP) (0.4 |ig/mg creatinine);
MCPP also detected in 86% of human
samples with a mean of 1.4 ng/mL; however,
MCPP can also result from other phthalate
esters
MCPP more abundant in urine than
MNOP, indicating primary
metabolism to MNOP followed by
oxidative metabolism to compounds
such as MCPP
Calafat et al. (2006)
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Table 3B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Metabolism/
toxicokinetic
Female Sprague-Dawley rat
(2/group); single oral dose of
300-mg/kg DNOP; urinary metabolite
levels measured
MNOP, MCPP, and parent compound
detected, with MCPP considered the major
metabolite; biphasic excretion pattern; levels
decreased significantly after the first day;
MCPP, mono-(7-carboxy-«-heptyl) phthalate
(MCHpP), mo no - hy d ro xy -«-o c t v 1 phthalate
(MHOP), and mono-oxo-«-octyl phthalate
(MOOP) still detectable after 4 d
Authors concluded that metabolism
of DNOP in rats results in a high
percentage of oxidative metabolic
products in urine; MCPP and select
MNOP oxidation products present
in urine at higher levels than MNOP
Silva et al. (2005)
Mode-of-action/
mechanistic
Male Wistar rat; 4/time period (6 in
control); 2% DNOP administered via
diet; animals sacrificed after 3, 10, or
21 d; peroxisomal enzyme and
hepatic enzyme activities assessed
Increased activity of cyanide-insensitive
palmitoyl CoA oxidation observed in animals
sacrificed at Day 10 and Day 21; percentage
of catalase activity as part of the liver
homogenate significantly increased at
Days 10 and 21; 5'-nucleotidase,
glucose-6-phosphatase, and succinate
dehydrogenase activities significantly
decreased at 21 d. and smooth endoplasmic
reticulum proliferation and the loss of rough
endoplasmic reticulum beginning at 3 d.
DNOP causes hepatotoxic effects in
this protocol
Mannetal. (1985);
Hintonetal. (1986)

Primary hepatocytes from male
Sprague-Dawley rat; 0.2-mM DNOP
or MNOP administered for 48-72 hr
DNOP: carnitine acetyltransferase activity
202% of control;
MNOP: carnitine acetyltransferase activity
660% of control; carnitine
palmitoyltransferase activity 234% of
control; no effect on peroxisome levels
DNOP: slowly hydrolyzed; few
effects compared with 2-ethylhexyl
ester;
MNOP: most potent of
straight-chained monoesters;
increased enzyme levels due to
effects other than peroxisome
proliferation
Gray et al. (1983)
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Table 3B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Mode-of-action/
mechanistic
In vitro rat hepatocytes; incubated
with 0.05-, 0.1-, or0.25-mM DNOP;
DNA, protein, and enzyme (including
cytochrome P450 and cytochrome b5)
estimation conducted; fat metabolism
investigated in a separate assay
Signs of systemic toxicity such as blebbing
and vacuolation at 0.25 mM; increased lipid
accumulation in all treated groups; fat
metabolism assay showed that isolated
hepatocytes in rats fasted in the early or late
afternoon had increased incorporation of
l-14C-palmitate into triglyceride and
cholesterol esters and an increase in fatty acid
oxidation
Results suggest connection between
the observed early hepatic changes
and subsequent liver tumor
formation in rats
Hintonetal. (1986)

17(3-estradiol (E2)-dependent
recombinant Saccharomyces
cervevisae strain PL3 incubated with
10 |im DNOP at 30°C and
photographed every 24 hr
Did not support estrogen receptor-mediated
growth of PL3
No estrogen receptor-mediated
growth
Zacharewski et al.
(1998)

Uterine tissues from 22-d-old
Sprague-Dawley rat collected,
weighed, homogenized on ice, and
centrifuged to separate the cytosol;
cytosol incubated with 1 nM [3H]E2
and 1-1000 DNOP for 30 min at
30°C and then cooled to 4°C; specific
binding of [3H]E2 measured
Did not compete with [3H]E2 for binding
with estrogen receptor at any concentration
tested
DNOP does not bind with the
estrogen receptor
Zacharewski et al.
(1998)

Mature, ovariectomised female
Sprague-Dawley rat (10/group);
orally dosed with 20, 200, or
2000 mg/kg DNOP in sesame oil for
4 d; vaginal lavages performed once
per day to assess vaginal cornification
Statistically significant, but not
dose-dependent, decreases in body weight;
no significant differences in uterine wet
weights; no significant induction of vaginal
cornification at any concentration tested
DNOP does not display estrogen
receptor-mediated estrogenic
activity in vivo
Zacharewski et al.
(1998)
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Table 3B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Mode-of-action/
mechanistic
Transiently transfected MCF-7
human breast cancer estrogen
receptor-positive cells and stably
transfected HeLa cells; concentrations
of 0.1, 1, or 10 |iIVI phthalates;
incubated for 24 hr and assayed for
luciferase activity
No significant induction of luciferase activity
at any concentration
DNOP does not bind with the
estrogen receptor
Zacharewski et al.
(1998)
Immunotoxicity
Adjuvant effects of di -/7-buty l -.
di-«-octyl-. di-iso-nonyl- and
di-iso-decyl phthalate were studied
Adjuvant effect was accepted to be present if
a statistical increase in antibody production
occurred in a test group as compared to an
ovalbumin control group together with the
fulfillment of dose-response relationships
Phthalates with 8 or 9 carbon atoms
in the alkyl side chains were the
stronger adjuvants, whereas
phthalates with shorter or longer
alkyl side chains possessed less
adjuvant activity.
Larson et al (2003)
Neurotoxicity
In vitro test; fibroblasts from newborn
rat cerebellum in primary culture;
concentrations of 1.3 x 10
7.5 x 10~4, or 12.5 x 10~4M
Granulation at 1.3 x 10 4 M and slightly
depressed outgrowth at 7.5 and
12.5 x 10 4 M; results not significant
DNOP the least toxic to fibroblasts
out of the five esters tested (DNOP,
dimethyl phthalate [DMP], diethyl
phthalate [DEP], di-«-butyl
phthalate [DNBP], and di-«-heptyl
phthalate [DNHP])
Teranishi and
Kasuya (1980)
Reproductive
toxicity
In vitro test on human spermatozoa
transferred to a defined medium by
the swim-up procedure or separation
by the Percoll gradient;
concentrations of 0-640 |iIVI DNOP
Significant, dose-response decrease in
motility; DNOP did not affect linearity, mean
amplitude of lateral displacement of the
sperm head, or velocity
DNOP the least toxic to sperm
compared with other phthalate esters
tested (dibutyl phthalate [DBP],
diethyl phthalate [DEP], DMP, and
DEHP)
Fredricsson et al.
(1993)
Cultures of adhered rat Sertoli cells
with germ cells (spermatocytes and
spermatogonia); treated with
10~6-10~4 M DNOP for 24-48 hr
Dose-dependent increase in germ cell
detachment after treatment with DNOP;
marked detachment at 10 4 M and disruption
of Sertoli-cell monolayer
Positive for toxicity in male
reproductive cells
Gray and Beamand
(1984)
Other
Human WI-38 diploid cells; exposed
to unreported DNOP concentration
for 22 hr
170 iiM caused 50% inhibition of growth
DNOP has mid-level ID 50
compared with other phthalate esters
Jones et al. (1975)
ND = no data.
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Tests Evaluating Carcinogenicity, Genotoxicity, and/or Mutagenicity
All studies evaluating DNOP for mutagenic activities were negative (see Table 3A).
DNOP has been tested using reverse mutation and SOS repair induction assays in Salmonella
typhimurium and Escherichia coli. The results of these studies did not indicate that DNOP has
mutagenicity ratios that are significantly different from the negative controls, with or without
metabolic activation (Goodyear Tire and Rubber Company, 1982a,b; Zeiger et al., 1982, 1985;
Sato et al., 1994; Shibamoto and Wei, 1986; Seed, 1982). In vivo and in vitro genotoxicity
studies performed on mammalian eukaryotic organisms, mammalian cells, mammals, or
subcellular models were not identified.
Other Toxicity Studies (Exposures Other Than Oral or Inhalation)
Carcinogenicity
Two studies investigated the ability of DNOP to act as a liver tumor promoter using
surgical procedures and the administration of DEN as an initiating agent.
Carter et al. (1992)
Carter et al. (1992) performed a 2/3 partial hepatectomy on male F344 rats (unspecified
number of animals; control group included) and dosed them with 30-mg/kg DEN to initiate
carcinogenesis. Ten days later, the authors administered 0, 0.5%, or 1.0% DNOP via the diet for
26 weeks. The authors evaluated neoplastic activity by measuring gamma-glutamyl
transpeptidase (GGT) and glutathione S-transferase (GST-P) enzyme levels in the liver.
Immunohistochemical methods detected a significant increase in the percentage of the liver
expressing GGT after the administration of both concentrations of DNOP. The volume
percentage of liver expressing GST-P was significantly greater in animals treated with both DEN
and 1.0% DNOP than those treated with DEN alone. No changes in absolute liver weight were
reported; however, the authors did note a slight increase in the relative liver weight after DNOP
treatment. The authors concluded that DNOP acted as a tumor promoter in conjunction with
DEN as an initiator.
DeAngelo et al. (1986)
DeAngelo et al. (1986) performed a 2/3 partial hepatectomy on male Sprague Dawley
rats (five animals per goup; control group included) and dosed them with 30-mg/kg DEN to
initiate carcinogenesis prior to the administration of DNOP. Animals were treated with 0 or
1.0%) of DNOP via the diet for 7 days per week for 10 weeks. After sacrifice by carbon dioxide
asphyxiation, the authors measured the body and liver wet weights, performed gross
examinations of the livers, measured enzyme levels, and stained and counted the number of
GGT+ foci. The authors found a significant increase in GGT+ foci and GGT activity in animals
administered 1.0% DNOP compared with the controls and in animals that were administered
DEHP, MEHP, or 2-EH. In addition, carnitine acetyltransferase (CAT) activity was increased in
animals treated with DNOP compared with controls, although no concurrent liver enlargement
was observed in these animals. These results suggest that DNOP is a promoter of carcinogenic
activity. However, neither of these carcinogenicity studies can be considered for reference
derivation due to the lack of methodology and data presented in the abstracts.
Short-Term Studies
Oishi and Hiraga (1982)
In a short-term study, Oishi and Hiraga (1982) administered diets containing
2% mono-/7-octyl phthalate (MNOP), a metabolite of DNOP, to young male Wistar rats (number
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not specified) and evaluated serum parameters after 1 week of administration. The authors
reported significant increases in nonesterified fatty acids in the serum, decreases in triglycerides
and total cholesterol levels, an increased percentage of oleic acids in serum triglyceride levels,
and enlarged livers. These results indicate that MNOP significantly alters serum lipid
components and may be hepatotoxic.
Hinton et al. (1986)
Hinton et al. (1986) administered concentrations of 2000-mg/kg-day DNOP to male
Wistar rats (6 rats in control group; 4 per exposure group) via the diet for 3, 10, or 21 days. Rats
were then sacrificed, blood was drawn, and organs were removed for analysis. Administration of
DNOP led to the accumulation of fat in the centrilobular zone of the liver and fatty necrosis in
those animals dosed for 10 and 21 days. Liver weight was also increased at 10 and 21 days, and
peroxisome proliferation and hepatomegaly was noted at 21 days. The authors posited that these
hepatic changes may be indicative of subsequent tumor formation. Effects on the thyroid were
also reported. Serum thyroxine (T4) reportedly decreased on Days 3, 10, and 21 (47, 59, and
76% of the control, respectively), while serum triiodothyronine (T3) increased to 133% of the
control on Day 21. The study authors also reported ultrastructural changes in the thyroid that
included an increase in the number and size of the lysosomes, enlarged Golgi apparatus, and
damaged mitochondria. The results suggest a connection between the observed early hepatic
changes and subsequent liver tumor formation in rats
Lake et al. (1984, 1986)
In another study, Lake et al. (1984, 1986) administered 1000-mg/kg-day DNOP by
gavage to an unspecified number of male Sprague-Dawley rats for 14 days. The authors
measured the relative liver weight and activity of the hepatic microsomes. Relative liver weight
was significantly increased (117%) in treated animals compared with controls. The authors also
reported increased palmitoyl-CoA oxidation (125%), enoyl-CoA hydratase heat labile activity
(165%>), carnitine acetyltransferase activity (305%), lauric acid hydroxylation (125%), and
ethylmorphine A-demethylase activity (125%) as well as reduced D-amino acid oxidase activity
(55%>), EROD activity (55%>), and ECOD activity (70%>). While the liver appeared to be the
target organ for DNOP toxicity, peroxisome proliferation was not observed.
Oishi and Hiraga (1980)
To investigate testicular effects of DNOP in male rats, Oishi and Hiraga (1980)
administered 2.0%> DNOP via the diet for 1 week and then measured food consumption, relative
organ weights, serum levels of hormones, and zinc concentrations in the testicular tissue (zinc
deficiency can cause atrophy and retarded growth). The relative liver weight was significantly
increased in the treated animals compared with the controls. Zinc concentrations in the testes
were significantly decreased (91%>) in the treated animals compared with the controls; however,
serum levels of testosterone and dihydrotestosterone were not significantly affected by treatment
with DNOP. The authors concluded that, while the dietary administration of 2.0% DNOP did
not result in testicular atrophy, it did produce hepatic toxicity.
Jones et al. (1993)
Jones et al. (1993) administered 2-g/kg DNOP via gavage to 3 male Wistar rats for 2 days
and examined the testicular tissue for changes to Leydig cells. No changes were observed in the
structure of the seminiferous tubules or Leydig cell morphology of the treated rats; thus, the
authors concluded that DNOP is not a testicular toxin.
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Oishi (1990)
In an in vivo experiment, Oishi (1990) administered a single dose of 2-g/kg DNOP via
gavage to male Wistar rats (5 per group). Six hours later, the rats were sacrificed, and the
mitochondrial function of the testes was examined. As in the in vitro experiment, there was a
significant decrease in the mitochondrial oxygen consumption as well as a significant decrease in
the respiratory control ratio in the treated rats. In another experiment, Oishi (1990) incubated the
mitochondrial fraction from the testes of male Wistar rats with DNOP for 2 minutes and
measured mitochondrial oxygen consumption. DNOP did not affect oxygen consumption up to a
concentration of 0.65 |imol/mL, but there were significant decreases in oxygen consumption at
1.3 |imol/mL, The effects seen in this study suggest that DNOP causes decreased mitochondrial
function with potential fertility effects in male rats.
Foster et al. (1980)
In another in vivo test of testicular toxicity, Foster et al. (1980) administered
2800-mg/kg-day DNOP via gavage to male Sprague-Dawley rats (12 per group) for 4 days.
After sacrifice, the testes were removed, weighed, fixed, sectioned, stained, and examined. The
treated animals did not display any clinical effects (body weight or food consumption changes).
Testicular weights were unaffected, and there were no histological indications of toxicity in the
testes. The testicular zinc content was not significantly affected by treatment. The results of this
study and the Oishi and Hiragi (1980) in vivo study do not support the results of the in vitro
assays (Oishi, 1990; Jones et al., 1993) that suggest DNOP may be toxic to the testes of male
rats.
NTP (1985)
NTP (1985) administered 0.0, 0.50, 1.25, 2.50, 5.0, or 10.0% DNOP to male and female
CD-I mice (8 per sex) via the diet for 2 weeks. All animals were observed for clinical toxicity.
The authors reported that a significant number of males (6/8) and females (4/8) in the
10.0%-dose group had rough coats; no other effects were noted.
Metabolism/Toxicokinetic Studies
Several studies have examined the metabolism and toxicokinetics of DNOP.
Albro andMoore (1974)
Albro and Moore (1974) administered 0.2-mL DNOP neat via gavage to male CD rats at
24-hour intervals. Urine testing indicated that 31.0% of the phthalate moiety remained, that
there were many oxidative metabolites present, and that phthalic acid was a relatively minor
metabolite. The study authors concluded that DNOP is initially metabolized to
carboxyl-terminated metabolites and then a- and P-oxidations of the alkyl side chains produce a
high incidence of oxidative metabolites.
Calafat et al. (2006)
Calafat et al. (2006) found similar results. The authors administered a single dose of
300-mg/kg DNOP via gavage to female Sprague-Dawley rats and analyzed 24-hour urine
samples for metabolites. The study authors also analyzed human urine samples collected from a
random population with no documented exposure to phthalates and measured phthalate
metabolites. Mono-(3-carboxypropyl) phthalate (MCPP) was the most abundant metabolite
detected in the experimental rats (average of 255 |ig/mg creatinine). In contrast, MNOP was
detected at an average of 0.4 |ig/mg creatinine. MCPP was detected in 86% of the human urine
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samples (mean: 1.4 ng MCPP/mL). While MCPP is a primary metabolite of DNOP, it is also a
minor metabolite of other phthalates (e.g., DBP). However, other phthalates produce much
smaller quantities of MCPP than DNOP (e.g., 0.6 |ig/mg creatinine MCPP from DNOP
metabolism and 11.6 |ig/mg creatinine MCPP from DBP). The observed production of MCPP,
as compared with the monoester, suggests that DNOP is primarily metabolized through an
oxidative pathway.
Silva et al. (2005)
In another metabolic study, Silva et al. (2005) administered single oral doses of
300-mg/kg DNOP to 2 female Sprague-Dawley rats and measured urinary metabolite levels. As
in the Calafat et al. (2006) study, MCPP was considered the major metabolite. MNOP and the
parent compound were detected at much smaller concentrations. The authors observed a
biphasic excretion pattern with metabolite levels decreasing significantly after the first day.
Other DNOP oxidative metabolites identified included mono-carboxymethyl phthalate (MCMP),
mono-(7-carboxy-//-heptyl) phthalate (MCHpP), mono-(5-carboxy-//-pentyl) phthalate (MCPeP),
and isomers of mono-hydroxy-/7-octyl phthalate (MHOP), and mono-oxo-/7-octyl phthalate
(MOOP), all of which remained detectable in the urine 4 days after administration.
Mode-of-Action/Mechanistic Studies
Mann et al. (1985)
Mann et al. (1985) examined the mechanisms of liver toxicity that were observed in rats
in a previously described study (Hinton et al., 1986) in which the authors administered
2.0% DNOP via the diet for 3, 10, or 21 days. Mann et al. (1985) noted peroxisome proliferation
and hepatomegaly at 21 days and smooth endoplasmic reticulum proliferation and the loss of
rough endoplasmic reticulum beginning at 3 days, which remained apparent through 21 days.
The percentage of catalase activity as part of the liver homogenate was significantly increased at
10 and 21 days, and 5'-nucleotidase, glucose-6-phosphatase, and succinate dehydrogenase were
significantly decreased at 21 days. There was also a slight, significant increase in
cyanide-insensitive palmitoyl Coenzyme A oxidation at both 10 and 21 days.
Gray et al. (1983)
Using an in vitro assay, Gray et al. (1983) exposed primary male Sprague-Dawley rat
hepatocytes to 0.2-mM DNOP or MNOP for 48-72 hours. DNOP produced a 202% increase
(significant when compared with controls) in carnitine acetyltransferase activity. The
administration of MNOP resulted in a 660% increase in carnitine acetyltransferase activity and a
234% increase in carnitine palmitoyltransferase activity. Peroxisome numbers were unaffected
by 48-hour treatment with 0.2-mM MNOP. The authors concluded that MNOP was the most
potent straight-chained monoester examined and that it is much more potent than the parent
compound, DNOP.
Hinton et al. (1986)
Using a similar in vitro assay, Hinton et al. (1986) incubated rat hepatocytes with 0.05-,
0.1-, or 0.25-mM DNOP and measured protein and enzyme levels and fat metabolism. In
agreement with Gray et al. (1983), the authors did not observe any increase in
cyanide-insensitive palmitoyl Coenzyme A oxidation. Systemic toxicity was seen at 0.25 mM,
which included signs such as blebbing and vacuolation, but no increase in cell death. All treated
groups showed increased lipid accumulation. In a separate fat metabolism assay, the authors
noted that hepatocytes isolated from fasted rats or those fed ad libitum in the afternoon showed
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increased incorporation of l-14C-pamitate into triglyceride and cholesterol esters and an increase
in fatty acid oxidation. Rats fed ad libitum in the morning showed minor changes.
Zacharewski et al. (1998)
Zacharewski et al. (1998) utilized an estrogen receptor-mediated yeast assay in which
17P-estradiol (E2)-dependent recombinant Saccharomyces cervevisae strain PL3 was incubated
with 10 |im DNOP on a selective medium at 30°C and photographed every 24 hours. The results
indicated that DNOP does not exhibit ER-mediated growth of PL3, suggesting it is not
estrogenic in vitro.
Due to evidence of the estrogenic activities of other phthalate esters, a series of
experiments on the estrogenicity of DNOP were conducted by Zacharewski et al. (1998). In an
in vitro competitive ligand-binding assay, uterine tissues were collected from 22-day-old
Sprague-Dawley rats, weighed, homogenized, and centrifuged in order to separate the cytosol.
The cytosol was then incubated with 1 nM [3H]E2 and 1-1000 |iM DNOP for 30 minutes at
30°C and then cooled to 4°C. The results suggest that DNOP is not estrogenic because DNOP
"3
did not compete with [ H]E2 for binding to the estrogen receptor at any of the concentrations
tested.
Using another assay, Zacharewski et al. (1998) administered to mature, ovariectomized
female Sprague-Dawley rats (10 per group) oral doses of 20, 200, or 2000 mg/kg DNOP in
sesame oil over 4 days. The authors performed vaginal lavages once per day to access vaginal
cornification. Signs of clinical toxicity were also investigated. Statistically significant, but not
dose-dependent, decreases in body weight were observed at all three doses. Uterine wet weight
was not affected by treatment with DNOP, and vaginal cornification was not found to be
significantly increased in the treated animals. These results indicate that DNOP does not have
estrogen receptor-mediated estrogenic activity in vivo.
Using a gene expression in vitro assay, Zacharewski et al. (1998) utilized MCF-7 human
breast cancer estrogen receptor-positive cells and HeLa cells transfected with a chimeric
receptor/reporter system to assess binding of DNOP to the human estrogen receptor. These cells
were exposed to concentrations of 0.1, 1, or 10 |iM DNOP for 24 hours and then assayed for
luciferase activity as an indicator of estrogen receptor binding. DNOP did not cause significant
induction of luciferase activity at any concentration, providing further support that DNOP is not
estrogenic in vitro.
Immunotoxicity
There are several studies by Larsen and colleagues that address immunological effects of
phthalates. In the Larsen et al. (2003) study, the adjuvant effects of di-//-butyl-, di-//-octyl-,
di-iso-nonyl- and di-iso-decyl phthalate are studied in a screening model. Ovalbumin, used as
the model antigen, was injected subcutaneously in the neck region of BALB/cJ mice with the
selected phthalate in concentrations from 2-2000 [j,g/ml. Additionally, the mice were boosted
once or twice with ovalbumin alone. Immunization with ovalbumin alone, the ovalbumin
control group, served as the baseline for antibody production, whereas aluminium hydroxide
served as the positive control. The levels of ovalbumin-specific IgE, IgGl, and IgG2a antibodies
in sera were determined. Adjuvant effect was accepted to be present if a statistical increase in
antibody production occurred in a test group as compared to an ovalbumin control group together
with the fulfillment of dose-response relationships. Adjuvant effect varied strongly between the
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phthalates investigated. Phthalates with 8 or 9 carbon atoms in the alkyl side chains were the
stronger adjuvants, whereas phthalates with shorter or longer alkyl side chains possessed less
adjuvant activity. Adjuvant effects were apparent either from the IgE or the IgGl response or
both, whereas no effect was seen on the IgG2a response.
Neurotoxicity
Teranishi andKasuya (1980)
Using an in vitro assay, Teranishi and Kasuya (1980) incubated primary fibroblasts from
newborn rat cerebellum with concentrations of 1.3 x 10 4, 7.5 x 10 4, or 12.5 x 10 4 M DNOP.
Although the results were not significant, the authors noted granulation in the fibroblasts exposed
to 1.3 x 10 4 M DNOP and slightly depressed outgrowth of cells exposed to 7.5 x 10 4and
12.5 x 10 4 M DNOP. DNOP was the least toxic phthalate ester to fibroblasts of the esters
studied (DNOP, DMP, DEP, DNBP, and DNHP).
Reproductive Toxicity
Two studies investigated the in vitro reproductive toxicity of DNOP in human males.
Fredricsson et al. (1993)
Fredricsson et al. (1993) transferred human spermatozoa to a defined medium by either
the swim-up procedure or separation by Percoll gradient. The spermatozoa were then exposed to
concentrations of 0-640 |iM DNOP and examined for effects to their motility and morphology.
The results indicated that there was a significant, dose-response decrease in motility. However,
DNOP treatment did not affect linearity, mean amplitude of lateral displacement of the sperm
head, or velocity at any concentration. Although this indicates that DNOP may slightly affect
male reproduction, the authors considered it to be the least toxic chemical to sperm when
compared with DBP, DEP, DMP, and DEHP.
Gray and Beamand (1984)
Gray and Beamand (1984) treated cultures of rat germ cells (spermatocytes and
spermatogonia) that were adhered to Sertoli cells in concentrations of 10 6—10 4 M DNOP for
24-48 hours. The authors reported a dose-dependent increase in germ cell detachment after
treatment with DNOP and marked detachment and disruption of the Sertoli-cell monolayer at
10 4 M DNOP. Although these two in vitro studies suggest that DNOP treatment may cause
male reproductive effects, there is very little in vivo data to support this hypothesis
(Heindel et al., 1989; NTP, 1985).
Other Studies
Jones et al. (1975)
In order to examine the cytotoxicity of DNOP that could potentially leach out of certain
medical devices, Jones et al. (1975) exposed human Wl-diploid cells to an unreported
concentration of DNOP for 22 hours. A concentration of 170-|iM DNOP caused a
50% inhibition in the growth of cells. Compared with other phthalate esters, DNOP was in the
middle of the range of calculated ID50 values (i.e., dose causing 50% inhibition in growth).
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DERIVATION OF PROVISIONAL VALUES
Tables 4 and 5 present a summary of the noncancer reference and cancer values,
respectively. IRIS data are indicated in the table, if available.
Table 4. Summary of Noncancer Reference Values for DNOP (CASRN 117-84-0)
Toxicity Type
(units)
Species/
Sex
Critical Effect
p-Reference
Value
POD
Method
POD
UFC
Principal Study
Subchronic p-RfD
(mg/kg-d)
Rat/M
Mild-to-moderate
cytoplasmic vacuolation in
the liver
l x ict1
NOAEL
36.8
300
Poon et al. (1997)
Chronic p-RfD
(mg/kg-d)
Rat/M
Mild-to-moderate
cytoplasmic vacuolation in
the liver
1 x 1(T2
NOAEL
36.8
3000
Poon et al. (1997)
Subchronic p-RfC
(mg/m3)
NDr
Chronic p-RfC
(mg/m3)
NDr
NDr = not determined
Table 5. Summary of Cancer Values for DNOP (CASRN 117-84-0)
Toxicity Type
Species/Sex
Tumor Type
Cancer Value
Principal Study
p-OSF
NDr
p-IUR
NDr
NDr = not determined
DERIVATION OF ORAL REFERENCE DOSES
Derivation of Subchronic Provisional RfD (Subchronic p-RfD)
Liver effects (changes in histopathology, enzyme activity, and weight) were observed in
subchronic (Smith et al., 2000; Poon et al., 1997), chronic (Carter et al., 1989; DeAngelo et al.,
1989), and reproductive studies (F1 generation—Heindel et al., 1989; NTP, 1985). Sperm
effects were also noted (Kwack et al., 2009) but occurred at higher doses than those eliciting
effects on the liver. The Poon et al. (1997) study is selected as the principal study for the
derivation of the subchronic p-RfD value. It is a published, peer-reviewed study that provides
sufficient information in the materials and methods section. Details of the Poon et al. (1997)
study are provided in the "Review of Potentially Relevant Data" section of this document. This
study employed the lowest doses of any studies found in the database, and provides the most
sensitive indication of toxicity. The critical effect selected from this study is mild-to-moderate
cytoplasmic vacuolation observed in the liver of rats. This effect was supported by mild-to-
moderate accentuation of zonation, endothelial prominence, anisokaryosis, and nuclear
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hyperchromicity. Cytoplasmic vacuolation had a high incidence (9/10 males; 5/10 females) at
the highest dose (350.1 mg/kg-day in males and 402.9 mg/kg-day in females), corresponding to a
NOAEL of 36.8 mg/kg-day in males and 40.8 mg/kg-day in females. These histopathological
changes were the most sensitive endpoint identified in this study or any other study found in the
database. Enzymatic induction also occurred; hepatic EROD was increased 3-fold in high-dose
males and 2-fold in high-dose females. Benchmark dose (BMD) analysis of the data from
Poon et al. (1997) do not show adequate model fits in the low-dose region of the curve (i.e., none
of the models showed a goodness of fit greater than 0.1).
Other oral in vivo studies support the identification of the liver as the target organ of
toxicity for DNOP. Several studies have reported changes in liver weight or liver enzymes
(NTP, 1985; Heindel et al., 1989; Smith, 2000; Carter et al., 1989). Absolute and relative liver
weights were significantly increased in male mice (23% and 28%, respectively) dosed with
8101-mg/kg-day DNOP in feed and in female mice (24% and 22%, respectively) dosed with
9438-mg/kg-day DNOP in feed (NTP, 1985). Decreased liver weights were reported in
Heindel et al. (1989) at 8640-mg/kg-day DNOP in the diet. Enzymatic changes in the liver were
supported by Smith et al. (2000) who found elevated levels of PBOX in mice administered
1804-mg/kg-day DNOP in the feed at both 2 and 4 weeks and in mice administered
90-mg/kg-day DNOP in the feed at 4 weeks. Carter et al. (1989) reported a 3-fold increase in
hepatic A-acetyl-P-glucosaminidase, P-galactosidase, a-mannosidase, and aryl sulfatase levels
and an increase in cathepsin D and P-glucuronidase levels in mice dosed with 789.5-mg/kg-day
DNOP. Two chronic studies also reported increases in liver nodules or liver tumors following
DNOP administration, although the full text of these studies could not be obtained (Carter et al.,
1989; DeAngelo et al., 1989). As a result, these chronic studies were not suitable for use in the
derivation of a chronic RfD.
The histopathological and enzymatic liver effects described by Poon et al. (1997) were
not accompanied by any significant changes in liver weight. Thoolen et al. (2010) discussed this
issue and indicated that hepatocellular hypertrophy is characterized by the enlargement of the
hepatocyte cytoplasm and other alterations in cytosolic protein and/or organelle numbers that can
be considered an "adaptive response" to chemical stress. However, excessive hypertrophy can
lead to hepatocellular degeneration and necrosis. The high incidence of numerous
histopathological changes and the consistency across the Poon et al. (1997) study and other
studies suggest that DNOP ultimately overcomes homeostatic mechanisms in the liver.
Therefore, given the weight of evidence of liver effects described in the principal and supporting
studies as well as the high incidence of the critical effect (mild-to-moderate cytoplasmic liver
vacuolation in 9/10 male and 5/10 female rats that is accompanied by the accentuation of
zonation, endothelial prominence, anisokaryosis, and nuclear hyperchromicity), this effect is
considered the most sensitive for the derivation of a subchronic p-RfD. The NOAELadj of
36.8 mg/kg-day in male rats is chosen as the POD to derive the subchronic p-RfD.
Subchronic p-RfD = NOAELadj ^ UFc
= 36.8 mg/kg-day300
= 1 x 10-1 mg/kg-day
Table 6 summarizes the uncertainty factors for the subchronic p-RfD for DNOP.
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Table 6. Uncertainty Factors for Subchronic p-RfD for DNOP
UF
Value
Justification
UFa
10
A UFa of 10 is applied for interspecies extrapolation to account for potential toxicokinetic
and toxicodynamic differences between rats and humans.
ufd
3
A UFd of 3 is applied because the database includes two acceptable two-generation
reproduction studies in mice (Heindel et al., 1989; NTP, 1985), but there are no acceptable
developmental studies.
UFh
10
A UFh of 10 is applied for intraspecies differences to account for potentially susceptible
individuals in the absence of information on the variability of response in humans.
ufl
1
A UFl of 1 is applied because the POD was developed using a NOAEL.
UFS
1
A UFS of 1 is applied because a subchronic-duration study was utilized.
UFC <3000
300

The confidence of the subchronic p-RfD for DNOP is medium, as explained in Table 7
below.
Table 7. Confidence Descriptors for Subchronic p-RfD for DNOP
Confidence Categories
Designation"
Discussion
Confidence in study
M
Confidence in the key study is medium. Poon et al. (1997) is peer
reviewed, but it is unknown if the study was conducted in
compliance with GLP. The critical effect of cytoplasmic
vacuolation of the liver is supported by other liver effects observed
in this study as well as a number of other studies that reported
significant liver alterations.
Confidence in database
M
The database includes subchronic toxicity studies in two species (rat
and mouse), two chronic toxicity studies in rats, and two
two-generation reproductive studies in mice but no developmental
toxicity studies.
Confidence in subchronic
p-RfDb
M
The overall confidence in the subchronic p-RfD is medium.
aL = low; M = medium; H = high.
bThe overall confidence cannot be greater than lowest entry in table.
Derivation of Chronic Provisional RfD (Chronic p-RfD)
Because the data from the two chronic-duration studies on oral DNOP exposure
(Carter et al., 1989; DeAngelo et al., 1989) was only available in abstract form and a thorough
evaluation of the chronic toxicity of DNOP could not be performed, the Poon et al. (1997) study
was also selected as the critical study for derivation of the chronic p-RfD value. The same
critical effect (mild-to-moderate cytoplasmic vacuolation in the liver that is supported by mild-
to-moderate accentuation of zonation, endothelial prominence, anisokaryosis, and nuclear
hyperchromicity) as that used to derive the subchronic p-RfD value was used to derive the
chronic p-RfD, and the NOAELadj of 36.8 mg/kg-day in male rats is chosen as the POD. The
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subchronic p-RfD derivation section of this document further describes the justification for the
selection of this study as the principal study and provides a summary of the critical effects.
Chronic p-RfD = NOAELadj ^ UFc
= 36.8 mg/kg-day3000
= 1 x 10~2 mg/kg-day
Table 8 summarizes the uncertainty factors for the chronic p-RfD for DNOP.
Table 8. Uncertainty Factors for the Chronic p-RfD for DNOP
UF
Value
Justification
ufa
10
A UFa of 10 is applied to interspecies extrapolations to account for potential toxicokinetic
and toxicodynamic differences between rats and humans.
ufd
3
A UFd of 3 is applied because the database includes two acceptable two-generation
reproduction studies in mice (Heindel et al., 1989; NTP, 1985), but there are no acceptable
developmental studies.
UFh
10
A UFh of 10 is applied for intraspecies differences to account for potentially susceptible
individuals in the absence of information on the variability of response in humans.
ufl
1
A UFl of 1 is applied because the POD was developed using a NOAEL.
UFS
10
A UFS of 10 is applied for using data from a subchronic-duration study to assess potential
effects from chronic-duration exposure because data for evaluating response from
chronic-duration exposure are unavailable or insufficient.
UFC <3000
3000

The confidence of the chronic p-RfD value is medium, as explained in Table 9 below.
Table 9. Confidence Descriptors for the Chronic p-RfD for DNOP
Confidence Categories
Designation"
Discussion
Confidence in study
M
Confidence in the key study is medium. Poon et al. (1997) is peer
reviewed, but it is unknown if the study was conducted in
compliance with GLP. The critical effect of cytoplasmic
vacuolation of the liver is supported by other liver effects observed
in this study as well as a number of other studies that reported
significant liver alterations.
Confidence in database
M
The database includes subchronic toxicity studies in two species
(rat and mouse), two chronic toxicity studies in rats, and two
two-generation reproductive studies in mice but no developmental
toxicity studies.
Confidence in subchronic
p-RfDb
M
The overall confidence in the chronic p-RfD value is medium.
aL = low; M = medium; H = high.
bThe overall confidence cannot be greater than lowest entry in table.
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DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
No subchronic or chronic p-RfC values can be derived because no adequate,
well-described studies are available. There is only one study available on inhalation exposure to
DNOP in animals (Lawrence et al., 1975). The study authors exposed animals 3 days a week for
16 weeks. However, the authors did not provide any exposure measurements or descriptions of
the methods and results. Therefore, this study cannot be used for the derivation of an RfC value.
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR
Table 10 identifies the cancer weight-of-evidence descriptor for DNOP. The abstracts of
two chronic bioassays with evidence of DNOP-induced liver tumors (or preneoplastic lesions)
are available (DeAngelo et al., 1989; Carter et al., 1989), although the complete studies could not
be obtained. In particular, DeAngelo et al. (1989) indicated that, without DEN initiation or
surgical alteration, the administration of 1% DNOP (214 mg/kg-day) in the diet increases the
incidence of hepatic tumors in male F344 rats. However, no complete carcinogenicity bioassays
were located; thus, the available information is not adequate to assess the carcinogenic potential
of DNOP.
Table 10. Cancer WOE Descriptor for DNOP
Possible WOE
Descriptor
Designation
Route of Entry
(Oral, Inhalation, or
Both)
Comments
"Carcinogenic to
Humans "
Not selected
NA
No human cancer studies are available.
"Likely to Be
Carcinogenic to
Humans "
Not selected
NA
No strong animal cancer data are available.
"Suggestive Evidence
of Carcinogenic
Potential"
Not selected
NA
No statistically significant increases in cancer
incidence were found in the scientific literature
for any animal, at any site, or in any gender.
"Inadequate
Information to Assess
Carcinogenic
Potential"
Selected
Both
Adequate information is not available to
assess carcinogenic potential. Available
data in abstract form did not contain
sufficient information to make a
determination.
"Not Likely to Be
Carcinogenic to
Humans "
Not selected
NA
No strong evidence of noncarcinogenicity in
humans or animals is available.
NA = not applicable.
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
The lack of data on the carcinogenicity of DNOP precludes the derivation of quantitative
estimates for either oral (p-OSF) or inhalation (p-IUR) exposure. Although two abstracts of
chronic bioassays examined the carcinogenic potential of DNOP, there are no full reports
available that adequately describe the methodology or present complete data sets; therefore, no
carcinogenicity values can be derived.
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APPENDIX A. PROVISIONAL SCREENING VALUES
No screening values were calculated.
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APPENDIX B. DATA TABLES
Table B.l. DNA Synthesis, PBOX Activities, and Relative Liver Weights of Fisher Rats
Exposed to DNOP for 2 or 4 Weeks"
Parameter0'"1
Exposure Group, ppm (Adjusted Daily Dose, mg/kg-d)b
0
1000 (100)
10,000 (1000)
2 wk
DNA synthesis (hepatic
labeling index, %)
2.08 ±0.79
2.00 ± 0.43 (96)
8.75 ±6.16 (421)*
PBOX activity (-fold
increase)
NR
1.13 ±0.07
3.12 ±0.28*
Relative liver weight (% of
body weight)
4.29 ±0.20
4.43 ±0.20 (103)
4.86 ±0.30 (113)*
4 wk
DNA synthesis (hepatic
labeling index, %)
1.20 ±0.50
0.96 ± 0.64 (80)
16.45 ± 1.93 (1370)*
PBOX activity (-fold
increase)
NR
1.32 ±0.07
1.25 ±0.14
Relative liver weight (% of
body weight)
4.49 ±0.21
4.42 ± 0.48 (98)
4.79 ±0.28 (107)
aSource: Smith et al. (2000).
bDoses converted from ppm to mg/kg-day using the following equation: Doscadj = Dose x Food Consumption per
Day x (1 -f- Body Weight) x (Days Dosed ^ Total Days).
°Values expressed as mean ± SD (% of control determined by independent calculations).
dAll data digitized using GetData Graph Digitizer.
* Significantly different from control (p < 0.05); data analyzed using two-way ANOVA followed by Dunnett's test.
NR = not reported.
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Table B.2. Organ Weights in Sprague-Dawley Rats After Oral
Exposure to DNOP for 13 Weeks"
Parameter0
Exposure Group, ppm (Adjusted Daily Dose, mg/kg-d)b
Male
0
5 (0.4)
50 (3.5)
500 (36.8)
5000 (350.1)
Sample size
10
10
10
10
10
Final body weight (g)
518 ± 36
540 ± 43 (104)
543 ±43 (105)
530 ±61 (102)
534 ± 38 (103)
Liver
Weight (g)
17.1 ± 1.7
18.5 ±2.1 (108)
18.4 ±2.1 (108)
18.6 ±3.4 (109)
18.6 ± 1.5 (109)
% Body
weight
3.31 ±0.26
3.42 ±0.38
(103)
3.39 ±0.27
(102)
3.49 ±0.25
(105)
3.49 ±0.15
(105)
Kidney
Weight (g)
1.6 ±0.1
1.66 ±0.17
(104)
1.62 ±0.13
(101)
1.7 ±0.27 (106)
1.69 ±0.15
(106)
% Body
weight
0.31 ±0.02
0.31 ±0.03
(100)
0.3 ± 0.02 (97)
0.32 ±0.03
(103)
0.32 ±0.03
(103)
Testis
Weight (g)
3.46 ±0.23
3.25 ±0.42 (94)
3.3 ±0.19 (95)
3.3 ±0.26 (95)
3.5 ±0.23 (101)
% Body
weight
0.67 ±0.05
0.6 ± 0.08 (90)
0.61 ±0.05 (91)
0.62 ± 0.07 (93)
0.65 ± 0.05 (97)
Female
0
5 (0.4)
50 (4.1)
500 (40.8)
5000 (402.9)
Sample size
10
10
10
10
10
Final body weight (g)
296 ±31
307 ± 33 (104)
302 ±21 (102)
320 ±23 (108)
292 ± 32 (99)
Liver
Weight (g)
9.83 ± 1.04
9.66 ± 1.31 (98)
9.83 ±0.88
(100)
10.25 ± 1.16
(104)
10.3 ± 1.36
(105)
% Body
weight
3.32 ±0.23
3.15 ±0.26 (95)
3.26 ±0.21 (98)
3.2 ±0.17 (96)
3.52 ±0.17
(106)
Kidney
Weight (g)
1.02 ±0.06
1.06 ±0.18
(104)
0.96 ±0.17 (94)
1.07 ±0.1 (105)
1.06 ±0.07
(104)
% Body
weight
0.35 ±0.03
0.35 ±0.05
(100)
0.32 ±0.07 (91)
0.33 ± 0.02 (94)
0.37 ±0.03
(106)
aSource: Poon et al. (1997).
bDoses were converted to adjusted daily doses by the study authors.
°Values for weight expressed as mean ± SD (% of control determined by independent calculations).
43
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Table B.3. Hematology and Serum Biochemistry of Sprague-Dawley Rats After Oral
Exposure to DNOP for 13 Weeks"
Parameter
Exposure Group, ppm (Adjusted Daily Dose, mg/kg-d)b
Male
0
5 (0.4)
50 (3.5)
500 (36.8)
5000 (350.1)
Sample size
10
10
10
10
10
Hematology0
White blood cells
(io3)
5.14 ± 1.23
5.4 ±2.03
(105)
5.54 ±2.06
(108)
4.31 ± 1.54
(84)
4.29 ± 1.22
(83)
Mean corpuscular
hemoglobin (pg)
17.64 ±0.8
17.83 ±0.82
(101)
17.49 ±0.6
(99)
17.53 ±0.64
(99)
17.37 ±0.69
(98)
Mean corpuscular
volume
52.41 ±2.19
52.74 ±2.56
(101)
51.8 ± 1.93
(99)
51.9 ± 1.4
(99)
51.27 ± 1.8
(98)
Serum
biochemistry0
Platelet count (103)
911 ± 63
922 ± 81
(101)
895 ± 110
(98)
891 ±71
(98)
852 ± 72
(94)
Albumin (g/dL)
3.53 ±0.22
3.68 ±0.32
(104)
3.49 ±0.27
(99)
3.47 ±0.14
(98)
3.46 ±0.16
(98)
Calcium (mg/dL)
8.09 ± 1.56
9.17 ±0.98
(113)
8.68 ± 1.18
(107)
8.87 ±0.92
(110)
9.46 ± 1.06
(117)*
Inorganic
phosphate (mg/dL)
6.39 ±0.569
6.62 ± 1.99
(104)
7.05 ± 0.43
(110)
7.02 ±0.77
(110)
7.20 ±0.49
(113)
Female
0
5 (0.4)
50 (4.1)
500 (40.8)
5000 (402.9)
Sample size
10
10
10
10
10
Hematology0
White blood cells0
(io3)
3.58 ± 1.51
4.83 ± 1.47
(135)
4.42 ± 1
(123)
4.5 ±1
(126)
3.91 ± 1.3
(109)
Mean corpuscular
hemoglobin (pg)
18.66 ±0.6
18.47 ±0.48
(99)
18.56 ±0.33
(99)
18.3 ±0.39
(98)
18.44 ±0.6
(99)
Mean corpuscular
volume (|im/m3)
54.43 ± 1.3
54.54 ± 1.52
(100)
54.92 ± 1.9
(101)
53.7 ± 1.1
(99)
53.7 ± 1.4
(99)
Serum
biochemistry0
Platelet count (103)
836 ±99
923 ± 104
(110)
876 ± 96
(105)
836 ± 85
(100)
817 ±64
(98)
Albumin (g/dL)
3.92 ±0.41
3.83 ±0.29
(98)
3.91 ±0.21
(100)
3.92 ±0.21
(100)
4.08 ±0.24
(104)
Calcium (mg/dL)
9.44 ±0.92
10.05 ± 1.89
(106)
9.65 ± 1.59
(102)
9.79 ± 1.62
(104)
9.33 ± 1.94
(99)
Inorganic
phosphate (mg/dL)
7.22 ± 1.54
7.99 ±0.68
(HI)
8.52 ± 1.14
(118)*
7.64 ±0.89
(106)
7.96 ± 1.08
(110)
aSource: Poon et al. (1997).
bDoses were converted to adjusted daily doses by the study authors.
°Values are expressed as the mean ± SD (% of control determined by independent calculations).
*Significant (p < 0.05) using one-way ANOVA, /-test, or Duncan's Multiple Range test.
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12-20-2012
Table B.4. Ethoxyresorufin-O-deethylase Activity in Sprague-Dawley Rats After Oral
Exposure to DNOP for 13 Weeks"
Parameter0'"1
Exposure Group, ppm (Adjusted Daily Dose, mg/kg-d)b
Male
0
5 (0.4)
50 (3.5)
500 (36.8)
5000 (350.1)
Ethoxyresorufin-O-deethylase activity
0.12 ±0.01
0.13 ±0.02
(106)
0.14 ±0.03
(HI)
0.15 ±0.03
(125)
0.38 ±0.20
(308)*
Parameter
Exposure Group, ppm (Adjusted Daily Dose, mg/kg-d)b
Female
0
5 (0.4)
50 (4.1)
500 (40.8)
5000 (402.9)
Ethoxyresorufin-O-deethylase activity
0.16 ±0.03
0.17 ±0.03
(103)
0.17 ±0.04
(103)
0.20 ±0.04
(122)
0.35 ±0.06
(212)*
"Source: Poon et al. (1997).
bDoses were converted to adjusted daily doses by the study authors.
°Values expressed as the mean ± SD, nmol/min/mg protein (% of control determined by independent calculations).
dData were extracted from the study graph(s) using GetData Graph Digitizer, Graph Digitizer Software version 2.24.
*Significant (p < 0.05) using one-way ANOVA, /-test, or Duncan's Multiple Range test.
Table B.5. Liver and Adipose Tissue Residues in Sprague-Dawley Rats After Oral
Exposure to DNOP for 13 Weeks"
Parameter0
Exposure Group, ppm (Adjusted Daily Dose, mg/kg-d)b
Male
0
5 (0.4)
50 (3.5)
500 (36.8)
5000 (350.1)
Liver
<3
<3
<3
<3
5 ± 4
Adipose tissue
<3
<3
4 ± 2
7 ± 7
15 ±4
Female
0
5 (0.4)
50 (4.1)
500 (40.8)
5000 (402.9)
Liver
<3
<3
4 ± 2
5 ± 3
4 ± 2
Adipose tissue
<3
7 ± 5
<3
<3
25 ±7
"Source: Poon et al. (1997).
bDoses were converted to adjusted daily doses by the study authors.
°Values expressed as the mean ± SD, ppm wet weight.
45
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12-20-2012
Table B.6. Histopathology of Male Sprague-Dawley Rats After Oral
Exposure to DNOP for 13 Weeks"


Exposure Group, ppm (Adjusted Daily Dose, mg/kg-d)b
Parameter
0
5 (0.4)
50 (3.5)
500 (36.8)
5000 (350.1)
Peroxisomes (% cell area)
4.53
1.50
2.59
1.75
5.50
Liver0
Accentuation of
zonation
1/10 (10)
[0.1]
2/10 (20)
[0.2]
1/10 (10)
[0.1]
1/10 (10)
[0.1]
10/10 (100)
[3.1]

Anisokaryosis
1/10 (10)
[0.1]
0/10
4/10 (40)
[0.3]
5/10 (50)
[0.4]
9/10 (90)
[1.9]

Nuclear
hyperchromicity
0/10
0/10
2/10 (20)
[0.3]
3/10 (30)
[0.4]
5/10 (50)
[1.0]

Perivenous
cytoplasmic
vacuolation
0/10
0/10
0/10
0/10
9/10 (90)
[2.7]

Endothelial
prominence
0/10
0/10
0/10
0/10
7/10 (70)
[1.1]
Thyroid0
Reduced follicle size
4/10 (40)
[0.4]
5/10 (50)
[0.6]
6/10 (60)
[1.1]
6/10 (60)
[1.0]
5/10 (50)
[0.8]

Decreased colloid
density
0/10
0/10
3/10 (30)
[0.3]
5/10 (50)
[0.2]
6/10 (60)
[0.4]
Testis0
Seminiferous tubule
atrophy
7/10 (70)
[0.8]
5/10 (50)
[0.6]
6/10 (60)
[0.5]
6/10 (60)
[0.4]
5/10 (50)
[0.4]
Epididymis0
Bilateral reduction in
sperm density
0/10
0/10
0/10
0/10
0/10
aSource: Poon et al. (1997).
bDoses were converted to adjusted daily doses by the study authors.
°Values expressed as the number of lesions observed/number examined (% incidence) [severity score]; percentage
was calculated. Values in brackets denote the average severity score where 1 = minimal, 2 = mild, 3 = moderate,
and 4 = severe. For tissue changes that were focal, locally extensive, and multiple, a score of less than the integer is
assigned. These scores are as follows: minimally focal = 0.25; minimal, locally extensive = 0.5; minimal,
multifocal = 0.75; mild, focal = 1.25; mild, locally extensive = 1.50; mild, multifocal = 1.75; etc.
46
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12-20-2012
Table B.7. Histopathology of Female Sprague-Dawley Rats After Oral
Exposure to DNOP for 13 Weeks"


Exposure Group, ppm (Adjusted Daily Dose, mg/kg-d)b
Parameter
0
5 (0.4)
50 (4.1)
500 (40.8)
5000 (402.9)
Peroxisomes (% cell area)
3.69
ND
ND
ND
4.15
Liver0
Accentuation of
zonation
5/10 (50)
[0.4]
6/10 (60)
[0.4]
9/10 (90)
[0.7]
10/10 (100)
[0.8]
10/10 (100)
[1.6]

Anisokaryosis
9/10 (90)
[1.5]
10/10 (100)
[2.0]
10/10 (100)
[2.3]
10/10 (100)
[2.5]
10/10 (100)
[3.0]

Nuclear
hyperchromicity
3/10 (30)
[0.6]
10/10 (100)
[2.1]
9/10 (90)
[1.6]
10/10 (100)
[1.9]
10/10 (100)
[2.0]

Perivenous
cytoplasmic
vacuolation
0/10
0/10
0/10
0/10
5/10 (50)
[1.2]

Endothelial
prominence
0/10
0/10
5/10 (50)
[0.5]
9/10 (90)
[0.9]
10/10 (100)
[1.5]
Thyroid0
Reduced follicle size
4/10 (40)
[0.4]
6/10 (60)
[0.7]
6/10 (60)
[0.6]
5/10 (50)
[1.0]
8/10 (80)
[1.6]

Decreased colloid
density
2/10 (20)
[0.1]
0/10
1/10 (10)
[0.1]
5/10 (50)
[0.3]
4/10 (40)
[0.2]
aSource: Poon et al. (1997).
bDoses were converted to adjusted daily doses by the study authors.
°Values expressed as the number of lesions observed/number examined (% incidence) [severity score]; percentage
was calculated. Values in brackets denote the average severity score where 1 = minimal, 2 = mild, 3 = moderate,
and 4 = severe. For tissue changes that were focal, locally extensive, and multiple a score of less than the integer is
assigned. These scores are as follows: minimally focal = 0.25; minimal, locally extensive = 0.5; minimal, multifocal
= 0.75; mild, focal = 1.25; mild, locally extensive = 1.50; mild, multifocal = 1.75; etc.
ND = not determinable.
47
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12-20-2012
Table B.8. DNA Synthesis, PBOX Activities, and Relative Liver Weights of Mice
Exposed to DNOP for 2 or 4 Weeks3
Parameter0'"1
Exposure Group, ppm (Adjusted Daily Dose, mg/kg-d)b
0
500 (90)
10,000 (1804)
2 wk
DNA synthesis (hepatic
labeling index, %)
2.80 ± 1.16
2.95 ±0.85 (105)
3.64 ±0.31 (130)
PBOX activity (-fold
increase)
NR
0.90 ±0.15
1.12 ± 0.15*
Relative liver weight (% of
body weight)
5.52 ±0.51
5.21 ±0.31 (94)
5.91 ±0.54 (107)
4 wk
DNA synthesis (hepatic
labeling index, %)
2.16 ±0.69
2.24 ±0.62 (104)
2.85 ± 1.54 (132)
PBOX activity (-fold
increase)
NR
1.73 ±0.08*
2.03 ±0.23*
Relative liver weight (% of
body weight)
5.82 ±0.42
5.40 ±0.23 (93)
5.71 ±0.42 (98)
aSource: Smith et al. (2000).
bDoses converted from ppm to mg/kg-day using the following equation: Doscadj = Dose x Food Consumption per
Day x (1 -f- Body Weight) x (Days Dosed ^ Total Days).
°Values expressed as the mean ± SD (% of control determined by independent calculations).
dAll data digitized using GetData Graph Digitizer.
* Significantly different from control (p < 0.05); determined using two-way ANOVA followed by Dunnett's test.
NR = not reported.
48
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12-20-2012
Table B.9. Mean Body Weights, Organ Weights, and Sperm Parameters
in Male CD-I Mice After Dietary Exposure to DNOP Using
a Continuous Breeding Protocol (F1 Generation)3
Parameter
Exposure Group, % (Adjusted Daily Dose, mg/kg-d)b
0
5 (8640)
No. of animals
20
20
Weight0
Body (g)
36.69 ±0.86
35.5 ±0.86 (97)
Liver (g)
1.96 ±0.06
2.42 ±0.08 (123)*
Kidneys (g)
0.70 ±0.02
0.70 ±0.02 (100)
Right epididymis (mg)
49 ± 1
50 ± 1.6(102)
Right cauda
epididymis (mg)
19.1 ±0.7
18.3 ±0.4 (96)
Right testis (mg)
131 ± 5
131 ±6 (100)
Seminal vesicles (mg)
429 ±2
374 ± 9 (87)*
Prostate (mg)
27 ±2
28 ± 2 (104)
Sperm parameters0
Concentration
(106sperm/g)
1118 ± 64
1239 ±78 (111)
% Motile
94 ± 1
94 ± 1 (100)
% Abnormal sperm
5.0 ±0.6
3.5 ±0.4 (70)
aSource: Heindel et al. (1989).
bConverted by authors based on average feed consumption.
°A11 parameters expressed as the mean ± SE (% of control determined by independent calculations).
* Significantly different from control (p < 0.05).
49
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12-20-2012
Table B.10. Mean Body Weights, Organ Weights, and Estrous Cycle Length
in Female CD-I Mice After Dietary Exposure to DNOP Using
a Continuous Breeding Protocol (F1 Generation)"
Parameter
Exposure Group, % (Adjusted Daily Dose, mg/kg-d)b
0
5 (8640)
No. of animals
20
20
Weight0
Body (g)
30.03 ±0.82
30.61 ±0.54 (102)
Liver (g)
1.88 ±0.06
2.34 ±0.05 (124)*
Kidneys (g)
0.479 ±0.014
0.533 ±0.011 (111)*
Estrous cycle length (days)
4.63 ±0.1
4.83 ± 0.2 (104)
aSource: Heindel et al. (1989).
bConverted by authors based on average feed consumption.
°A11 parameters expressed as the mean ± SE (% of control determined by independent calculations).
* Significantly different from control (p < 0.01).
50
Di-//-octyl phthalate

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FINAL
12-20-2012
Table B.ll. Body Weights of F0 Male CD-I Mice After Oral Exposure to DNOP for 18
Weeks in a Reproductive Study"
Parameter0
Exposure Group, % food (Adjusted Daily Dose, mg/kg-d)b
0
1.25 (1820)
2.5 (3620)
5.0 (7460)
Wk 1
Sample size
40
20
20
20
Body weight (g)
35.3 ±0.30
34.9 ±0.52 (99)
34.6 ±0.38 (98)
34.4 ± 0.45 (97)
Wk 2
Sample size
40
20
20
20
Body weight (g)
36.4 ±0.30
35.9 ±0.54 (99)
35.7 ±0.45 (98)
35.4 ±0.49 (97)
Wk 3
Sample size
40
20
20
20
Body weight (g)
35.4 ±0.30
35.2 ±0.51 (99)
35.0 ±0.46 (99)
34.6 ± 0.46 (98)
Wk 6
Sample size
40
20
20
20
Body weight (g)
37.4 ±0.47
36.7 ±0.73 (98)
36.3 ±0.51 (97)
36.6 ±0.65 (98)
Wk 10
Sample size
40
20
20
20
Body weight (g)
39.9 ±0.65
38.9 ±0.79 (97)
38.0 ±0.61 (95)
39.0 ±0.88 (98)
Wk 14d
Sample size
39e
20
19e
20
Body weight (g)
40.3 ± 0.72
39.8 ±0.92 (99)
38.8 ±0.75 (96)
39.5 ± 1.07 (98)
"Source: NTP (1985).
bStudy authors estimated the administered dose in mg/kg-day based on food consumption and body-weight data
gathered over the duration of the study.
°Values expressed as the mean ± SE (% of control determined by independent calculations).
dAll animals were sacrificed during Week 17 of the study.
eOne male died during Week 12 of the study.
51
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12-20-2012
Table B.12. Body Weights of F0 Female CD-I Mice After Oral Exposure to DNOP for 18
Weeks in a Reproductive Study3
Parameter0
Exposure Group, % food (Adjusted Daily Dose, mg/kg-d)b
0
1.25 (1699)
2.5 (3411)
5.0 (7120)
Wk 1
Sample size
40
20
20
20
Body weight (g)
27.8 ±0.24
27.7 ±0.50 (100)
27.0 ± 0.47 (97)
27.4 ± 0.44 (99)
Wk 2
Sample size
40
20
20
20
Body weight (g)
28.9 ±0.28
28.6 ±0.53 (99)
29.0 ±0.62 (100)
29.4 ±0.45 (102)
Wk 3
Sample size
40
20
20
20
Body weight (g)
31.5 ±0.27
32.0 ±0.50 (102)
30.9 ±0.45 (98)
31.6 ±0.47 (100)
Wk 6
Sample size
40
20
20
20
Body weight (g)
36.3 ±0.43
37.7 ±0.84 (104)
36.6 ±0.66 (101)
36.3 ± 0.60 (100)
Wk 10
Sample size
38d
20
20
20
Body weight (g)
49.3 ± 1.07
49.2 ± 1.75 (100)
47.0 ± 1.84 (95)
49.2 ± 1.65 (100)
Wk 14e
Sample size
38
20
19f
20
Body weight (g)
42.2 ±0.89
44.0 ± 1.35 (104)
43.2 ± 1.9 (102)
44.8 ± 1.77(106)
"Source: NTP (1985).
bDoses were converted from percentage of food to ppm by multiplying by 10,000 (1% = 10,000 ppm), then from
ppm to mg/kg-day using the following equation: Doscadi = Dose x Time-Weighted Average Food Consumption per
Day x (1 Time-Weighted Average Body Weight) x (Days Dosed Total Days); Time-Weighted Average Food
Consumption per Day and Time-Weighted Average Body Weight were calculated from recorded data.
°Values expressed as the mean ± SE (% of control determined by independent calculations).
dTwo females died after Week 6 of the study.
eAll animals were sacrificed during Week 17 of the study except for pregnant or nursing animals.
fOne female died during Week 13 of the study.
52
Di-//-octyl phthalate

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12-20-2012
Table B.13. Food Consumption by F0 Male CD-I Mice After Oral Exposure to DNOP for
18 Weeks in a Reproductive Study"
Parameter0
Exposure Group, % food (Adjusted Daily Dose, mg/kg-d)b
0
1.25 (1820)
2.5 (3620)
5.0 (7460)
Wk 1
Sample size
40
20
20
20
Food consumption (g/d)
5.42 ±0.06
5.32 ±0.13 (98)
5.42 ±0.12 (100)
4.85 ±0.08 (89)
Wk 2
Sample size
40
20
20
20
Food consumption (g/d)
5.59 ±0.09
5.73 ±0.23 (103)
5.75 ±0.15 (103)
5.98 ±0.17 (107)
Wk 6
Sample size
40
20
20
20
Food consumption (g/d)
5.39 ±0.09
5.33 ±0.10 (99)
5.21 ±0.12 (97)
5.87 ±0.17 (109)
Wk 10
Sample size
40
20
20
20
Food consumption (g/d)
5.07 ±0.09
5.24 ±0.12 (103)
5.04 ±0.11 (99)
5.38 ±0.12 (106)
Wk 14d
Sample size
39e
20
19e
20
Food consumption (g/d)
5.30 ±0.10
5.25 ±0.15 (99)
4.91 ±0.11 (93)
5.20 ±0.15 (98)
"Source: NTP (1985).
bStudy authors estimated the dose in mg/kg-day using food consumption and body-weight data gathered over the
duration of the study.
°Values expressed as the mean ± SE (% of control determined by independent calculations).
dAll animals were sacrificed during Week 17 of the study.
eOne male died during Week 12 of the study.
53
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12-20-2012
Table B.14. Food Consumption by F0 Female CD-I Mice After Oral Exposure to DNOP
for 18 Weeks in a Reproductive Study"
Parameter0
Exposure Group, % food (Adjusted Daily Dose, mg/kg-d)b
0
1.25 (1699)
2.5 (3411)
5.0 (7120)
Wk 1
Sample size
40
20
20
20
Food consumption (g/d)
5.52 ±0.08
5.65 ±0.19 (102)
5.96 ±0.14 (108)
6.09 ±0.16 (110)
Wk 2
Sample size
40
20
20
20
Food consumption (g/d)
5.59 ±0.09
5.73 ±0.23 (103)
5.75 ±0.15 (103)
5.98 ±0.17 (107)
Wk 6
Sample size
40
20
20
20
Food consumption (g/d)
5.39 ±0.09
5.33 ±0.10 (99)
5.21 ±0.12 (97)
5.87 ±0.17 (109)
Wk 10
Sample size
38d
20
20
20
Food consumption (g/d)
5.10 ±0.09
5.24 ±0.12 (103)
5.04 ±0.11 (99)
5.38 ±0.12 (105)
Wk 14e
Sample size
38
20
19f
20
Food consumption (g/d)
5.34 ±0.10
5.25 ±0.15 (98)
4.88 ±0.11 (91)
5.20 ±0.15 (97)
"Source: NTP (1985).
bDoses were converted from percentage of food to ppm by multiplying by 10,000 (1% = 10,000 ppm), then from
ppm to mg/kg-day using the following equation: Doscadj = Dose x Time-Weighted Average Food Consumption per
Day x (1 Time-Weighted Average Body Weight) x (Days Dosed Total Days); Time-Weighted Average Food
Consumption per Day and Time-Weighted Average Body Weight are calculated from recorded data.
°Values expressed as the mean ± SE (% of control determined by independent calculations).
dTwo females died after Week 6 of the study.
eAll animals were sacrificed during Week 17 of the study except for pregnant or nursing animals.
fOne female died during Week 13 of the study.
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Table B.15. Body Weights of F1 Male CD-I Mice After Oral Exposure to DNOP for 16
Weeks in a Reproductive Study3
Parameter0
Exposure Group, % food (Adjusted Daily Dose, mg/kg-d)b
0
5.0 (8101)
Sample size
20
20
Body weight (g)
Wk 19-20d
12.5 ±0.70
11.4 ±0.60 (91)
Wk 27
34.3 ±0.74
33.1 ±0.67 (97)
Wk 28
32.9 ±0.69
31.9 ±0.70 (97)
Wk 29
33.9 ±0.69
32.8 ± 0.74 (97)
Wk 30
34.6 ±0.70
33.7 ±0.77 (97)
Wk 31
35.5 ±0.78
34.6 ± 0.84 (97)
"Source: NTP (1985).
bDoses are converted from percentage of food to ppm by multiplying by 10,000 (1% = 10,000 ppm), then from ppm
to mg/kg-day using the following equation: Dosc adj = Dose x Average Food Consumption per Day x (1 -f- Time-
Average Body Weight) x (Days Dosed Total Days); Average Food Consumption per Day and Average Body
Weights were calculated from recorded data.
°Values expressed as the mean ± SE (% of control determined by independent calculations).
Represents weight at weaning; pups were weaned during Weeks 19-20 of the study.
Table B.16. Body Weights of F1 Female CD-I Mice After Oral Exposure to DNOP for 14
Weeks in a Reproductive Study"
Parameter0
Exposure Group, % food (Adjusted Daily Dose, mg/kg-d)b
0
5.0 (9438)
Sample size
20
20
Body weight (g)
Wk 19-20d
11.1 ±0.44
11.0 ±0.38 (99)
Wk 27
27.0 ±0.76
26.1 ±0.56 (97)
Wk 28
28.5 ±0.77
28.4 ±0.55 (100)
Wk 29
33.6 ± 1.15
34.8 ±0.88 (104)
Wk 30
39.6 ±2.60
41.9 ±2.65 (106)
Wk 31
29.7 ±0.71
29.9 ±0.59 (101)
"Source: NTP (1985).
bDoses are converted from percentage of food to ppm by multiplying by 10,000 (1% = 10,000 ppm), then from ppm
to mg/kg-day using the following equation: DoseADj = Dose x Average Food Consumption per Day x (1 -f- Time-
Average Body Weight) x (Days Dosed Total Days); Average Food Consumption per Day and Average Body
Weight are calculated from recorded data.
°Values expressed as the mean ± SE (% of control determined by independent calculations).
Represents weight at weaning; pups were weaned during Weeks 19-20 of the study.
55
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Table B.17. Terminal Body Weights and Absolute and Relative Organ Weights of F1 Male
CD-I Mice After Oral Exposure to DNOP for 14 Weeks in a Reproductive Study"
Parameter0'"1
Exposure Group, % food (Adjusted Daily Dose, mg/kg-d)b
0
5.0 (8101)
Sample size
20
20
Terminal body weight (g)
36.690 ±0.859
35.540 ±0.857 (97)
Liver
Absolute weight (g)
1.962 ±0.058
2.422 ±0.082 (123)***
Adjusted weight (g)
1.923 ±0.042
2.461 ±0.042 (128)****
Kidney
Absolute weight (g)
0.702 ±0.021
0.697 ±0.018 (99)
Adjusted weight (g)
0.695 ±0.017
0.705 ±0.017 (101)
Right
epididymis
Absolute weight (mg)
49.225 ± 1.471
50.365 ± 1.623 (102)
Adjusted weight (mg)
48.609 ± 1.271
50.981 ± 1.271 (105)
Right cauda
Absolute weight (mg)
19.050 ±0.655
18.305 ±0.438 (96)
Adjusted weight (mg)
18.864 ±0.493
18.491 ±0.493 (98)
Right testis
Absolute weight (g)
0.131 ±0.005
0.131 ±0.006 (100)
Adjusted weight (g)
0.128 ±0.005
0.134 ±0.005 (105)
Seminal
vesicles
Absolute weight (g)
0.429 ±0.017
0.374 ± 0.009 (87)*
Adjusted weight (g)
0.425 ±0.013
0.377 ±0.013 (89)**
Prostate gland
Absolute weight (mg)
27.035 ± 1.782
27.795 ± 1.839 (103)
Adjusted weight (mg)
27.014 ± 1.846
27.816 ± 1.846 (103)
"Source: NTP (1985).
bDoses are converted from percentage of food to ppm by multiplying by 10,000 (1% = 10,000 ppm), then from ppm
to mg/kg-day using the following equation: Dosc adj = Dose x Average Food Consumption per Day x (1 -f- Time-
Average Body Weight) x (Days Dosed Total Days); Average Food Consumption per Day and Average Body
Weight are calculated from recorded data.
°Values expressed as the mean ± SE (% of control determined by independent calculations).
dEquation used for adjusting organ weights to body weight was not provided.
* Significantly different from control (p < 0.05); Wilcoxon rank-sum test was used.
**Significantly different from control (p < 0.05); ANCOVA F-test was used.
***Significantly different from control (p < 0.01); Wilcoxon rank-sum test was used.
****Significantly different from control (p < 0.01); ANCOVA F-test was used.
56
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Table B.18. Terminal Body Weights and Absolute and Relative Organ Weights of F1
Female CD-I Mice After Oral Exposure to DNOP for 14 Weeks in a Reproductive Study"
Parameter0'"1
Exposure Group, % food (Adjusted Daily Dose, mg/kg-d)b
0
5.0 (9438)
Sample size
20
20
Terminal body weight (g)
30.030 ±0.824
30.610 ±0.544 (102)
Liver
Absolute weight (g)
1.883 ±0.059
2.339 ±0.054 (124)*
Adjusted weight (g)
1.899 ±0.043
2.323 ±0.043 (122)**
Kidney
Absolute weight (g)
0.479 ±0.014
0.533 ±0.011 (111)*
Adjusted weight (g)
0.482 ±0.012
0.531 ±0.012 (110)**
"Source: NTP (1985).
bDoses are converted from percentage of food to ppm by multiplying by 10,000 (1% = 10,000 ppm), then from ppm
to mg/kg-day using the following equation: Dosc adj = Dose x Average Food Consumption per Day x
(1 Time-Average Body Weight) x (Days Dosed Total Days); Average Food Consumption per Day and Average
Body Weight are calculated from recorded data.
°Values expressed as the mean ± SE (% of control determined by independent calculations).
dEquation for adjusting organ weights to body weight was not provided.
* Significantly different from control (p < 0.01); Wilcoxon rank-sum test was used.
**Significantly different from control (p < 0.01); ANCOVA F-test was used.
57
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APPENDIX C. BMD OUTPUTS
BMDS provided no adequate model fits to the Poon et al. (1997) data sets.
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