if!;	United States
Environmental Protectioi
if % Agency
EPA/690/R-11/007F
Final
2-17-2011
Provisional Peer-Reviewed Toxicity Values for
9,10-Anthraquinone
(CASRN 84-65-1)
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
Jeff Swartout
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
ICF International
9300 Lee Highway
Fairfax, VA 22031
PRIMARY INTERNAL REVIEWERS
Paul G. Reinhart, PhD, DABT
National Center for Environmental Assessment, Research Triangle Park, NC
Audrey Galizia, DrPH
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
COMMONLY USED ABBREVIATIONS	ii
BACKGROUND	1
HISTORY	1
DISCLAIMERS	1
QUESTIONS REGARDING PPRTVS	2
INTRODUCTION	2
REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER)	4
HUMAN STUDIES	9
Oral Exposures	9
Inhalation Exposures	9
ANIMAL STUDIES	10
Oral Exposures	10
Subchronic Studies	11
Chronic/Carcinogenicity Studies	13
Inhalation Exposures	16
Chronic Studies	16
Reproductive and Developmental Studies	16
OTHER DATA	16
DERIVATION 01 PROVISIONAL VALUES	23
DERIVATION OF ORAL REFERENCE DOSES	23
Derivation of Subchronic Provisional RfD (Subchronic p-RfD)	23
Derivation of Chronic Provisional RfD (Chronic p-RfD)	23
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS	23
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR (WOE)	25
MODE-OF-ACTION DISCUSSION	26
Mutagenic Mode of Action	26
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES	26
Derivation of Provisional Oral Slope Factor (p-OSF)	26
Derivation of Provisional Inhalation Unit Risk (p-IUR)	28
APPENDIX A. PROVISIONAL SCREENING VALUES	29
APPENDIX B. DATA TABLES	32
APPENDIX C. BMDS OUTPUTS	44
APPENDIX D. REFERENCES	48
l
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COMMONLY USED ABBREVIATIONS
BMC
benchmark concentration
BMD
benchmark dose
BMCL
benchmark concentration lower bound 95% confidence interval
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
p-OSF
provisional oral slope factor
p-RfC
provisional reference concentration (inhalation)
p-RfD
provisional reference dose (oral)
POD
point of departure
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
9,10-ANTHRAQUINONE (CASRN 84-65-1)
BACKGROUND
HISTORY
On December 5, 2003, the U.S. Environmental Protection Agency's (EPA) Office of
Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human
health toxicity values for Superfund risk assessments, establishing the following three tiers as the
new hierarchy:
1)	EPA's Integrated Risk Information System (IRIS)
2)	Provisional Peer-Reviewed Toxicity Values (PPRTVs) used in EPA's Superfund
Program
3)	Other (peer-reviewed) toxicity values, including
~	Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR);
~	California Environmental Protection Agency (CalEPA) values; and
~	EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in EPA's IRIS. PPRTVs are developed according to a Standard
Operating Procedure (SOP) and are derived after a review of the relevant scientific literature
using the same methods, sources of data, and Agency guidance for value derivation generally
used by the EPA IRIS Program. All provisional toxicity values receive internal review by a
panel of six EPA scientists and external peer review by three independently selected scientific
experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the multiprogram
consensus review provided for IRIS values. This is because IRIS values are generally intended
to be used in all EPA programs, while PPRTVs are developed specifically for the Superfund
Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a 5-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV documents conclude that
a PPRTV cannot be derived based on inadequate data.
DISCLAIMERS
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and Resource Conservation and Recovery Act (RCRA) program offices are advised to
carefully review the information provided in this document to ensure that the PPRTVs used are
appropriate for the types of exposures and circumstances at the Superfund site or RCRA facility
in question. PPRTVs are periodically updated; therefore, users should ensure that the values
contained in the PPRTV are current at the time of use.
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It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
users are strongly encouraged to read the entire PPRTV document and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center for OSRTI. Other EPA programs or external parties who may
choose of their own initiative to use these PPRTVs are advised that Superfund resources will not
generally be used to respond to challenges of PPRTVs used in a context outside of the Superfund
Program.
QUESTIONS REGARDING PPRTVS
Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on
chemicals not covered, or whether chemicals have pending IRIS toxicity values) may be directed
to the EPA Office of Research and Development's National Center for Environmental
Assessment, Superfund Health Risk Technical Support Center (513-569-7300), or OSRTI.
9,10-Anthraquinone is a natural component of some plants (U.S. EPA, 1994a).
Additionally, 9,10-Anthraquinone is an intermediate in the production of dyes and pigments, and
it can also be formed during the kraft-pulping process conducted by the pulp and paper industry
(Doi et al., 2005). It is also used as a catalyst in the isomerization of vegetable oils, as an
accelerant in nickel electroplating (Doi et al., 2005), and as a bird repellant (Butterworth et al.,
2001; Doi et al., 2005). 9,10-Anthraquinone is produced by three different methods: oxidation of
anthracene, Diels-Adler reaction between 1,4-naphthoquinone and 1,3-butadiene, and
Friedel-Craft reaction between benzene and phthalic anhydride with further treatment with
concentrated sulfuric acid (Butterworth et al., 2004). Because anthracene is generally obtained
from distilling coal tar, 9,10-anthraquinone obtained from oxidation of anthracene can contain
varying amounts of polycyclic aromatic hydrocarbons (PAHs) (Butterworth et al., 2004). The
empirical formula for 9,10-anthraquinone is C14H8O2 (see Figure 1). A table of physicochemical
properties is provided below (see Table 1). In this document, unless otherwise noted,
"statistically significant" denotes a/>value < 0.05.
INTRODUCTION
O
o
FIGURE 1. 9,10-Anthraquinone Structure
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Table 1. Physicochemical Properties Table for
9,10-Anthraquinone (CASRN 84-65-l)a
Property (unit)
Value
Boiling point (°C)
377
Melting point (°C)
286
Density (g/cm3)
-
Vapor pressure (Pa at 20°C)
7.21 x 1(T9
pH (unitless)
-
Solubility in water (g/100 mL at 25°C)
Estimated 3 * 10 7
Relative vapor density (air =1)
-
Molecular weight (g/mol)
208
Flash point (°C)
185
Log Octanol/water partition coefficient (unitless)
3.39
"Source: U.S. EPA (1994a)
No reference dose (RfD) or reference concentration (RfC) for 9,10-anthraquinone is
included in the EPA IRIS database (U.S. EPA, 2010) or on the Drinking Water Standards and
Health Advisories List (U.S. EPA, 2006). No RfD or RfC values have been reported in the
HEAST (U.S. EPA, 2010). The Chemical Assessments and Related Activities (CARA) list
(U.S. EPA, 1993, 1994b) does not include a Health and Environmental Effects Profile (HEEP)
for 9,10-anthraquinone. Neither the Agency for Toxic Substances and Disease Registry
(ATSDR, 2008) nor the World Health Organization (WHO, 2010) has reviewed the toxicity of
9,10-anthraquinone. CalEPA (2008a,b) has not derived toxicity values for exposure to
9,10-anthraquinone. No occupational exposure limits for 9,10-anthraquinone have been derived
by the American Conference of Governmental Industrial Hygienists (ACGIH, 2010), the
National Institute of Occupational Safety and Health (NIOSH, 2005), or the Occupational Safety
and Health Administration (OSHA, 2010).
The EPA IRIS database does not include a cancer assessment for 9,10-anthraquinone
(U.S. EPA, 2010) nor are cancer values included on the Drinking Water Standards and Health
Advisories List (U.S. EPA, 2006). The International Agency for Research on Cancer (IARC,
2010) has not reviewed the carcinogenic potential of 9,10-anthraquinone. 9,10-Anthraquinone is
not included in the 11th Report on Carcinogens (National Toxicology Program [NTP], 2005a).
However, NTP (2005b) has concluded that there is clear evidence of carcinogenicity in female
F344/N rats and both male and female B6C3Fi mice and some evidence of carcinogenicity in
male F344/N rats. CalEPA (2008b) has not prepared a quantitative estimate of carcinogenic
potential for anthraquinone. However, CalEPA's Office of Environmental Health Hazard
(OEHHA), listed anthraquinone as a known carcinogen under Proposition 65, as of September
28, 2007 (CalEPA, 2007).
Literature searches were conducted on sources published from the 1950s through
December 2010 for studies relevant to the derivation of provisional toxicity values for
9,10-anthraquinone, CAS No. 84-65-1. Searches were conducted using EPA's Health and
Environmental Research Online (HERO) database of scientific literature. HERO searches the
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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 (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 searched for toxicity
values: 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 information for all of the potentially relevant toxicity studies. Entries
for the principal studies are bolded and identified by the marking "PS."
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Table 2. Summary of Potentially Relevant Data for 9,10-Anthraquinone (CASRN 84-65-1)
Notes3
Category
Number of
Male/Female, Species,
Study Type, Study
Duration
Exposure
Levelsb
Critical Effects
NOAELb
BMDL/
BMCLb
LOAELbc
Reference
(Comments)
Human
1. Oral (mg/kg-day)b
None
2. Inhalation (mg/m3)b

Acute
Number not specified,
occupational exposure to
anthraquinone dust,
duration not specified
2-10
55-840
1650
Headache, general weakness, and
skin and eye irritation (no
quantitative information available).



Volodchenko et al.
(1971)

Subchronic
None

Chronic
Cause-specific mortality
in 3266 (2859 men and
407 women) workers in a
dye and resin
manufacturing plant in
New Jersey with at least
6 months employment
Not reported
Mortality, overall mortality was 10%
lower than the general population.



Sathiakumar and
Delzell (2000)

Developmental
None

Reproductive
None

Carcinogenic
Cause-specific mortality
in 3266 (2859 men and
407 women) workers in a
dye and resin
manufacturing plant in
New Jersey with at least
6 months employment
Not reported
No difference in mortality due to all
cancer; statistically significant
increase in lung cancers in workers
with anthraquinone dyes (South Dye
workers, standardized mortality ratio
[SMR] = 168; 95% CI: 115-237),
but the study did not control for
smoking or possible occupational
exposure to other lung carcinogens
(e.g., asbestos).



Sathiakumar and
Delzell (2000)
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Table 2. Summary of Potentially Relevant Data for 9,10-Anthraquinone (CASRN 84-65-1)
Notes3
Category
Number of
Male/Female, Species,
Study Type, Study
Duration
Exposure
Levelsb
Critical Effects
NOAELb
BMDL/
BMCLb
LOAELbc
Reference
(Comments)


1975 males, retrospective
cohort, at least 6 months
of employment in a dye
factory in Scotland
Not reported
Cancer-related mortality was
observed (not increased).



Gardiner et al.
(1982)
Stated to be
exposed to
substituted
anthraquinones

51 Caucasian male lung
cancer cases (dye
workers),
102 age-matched controls
Not reported
Lung cancer was observed (odds
ratio [OR] of 2.4; 95% confidence
interval [CI] of 1.1-5.2).



Barbone et al.
(1992)
Animal
1. Oral (mg/kg-day)b
PS
Subchronic
10 Male/10 Female,
F344 rat, dietary,
14 weeks
0,135, 275, 555,
1130, or 2350d
Decreased body-weight gain
(females), anemia (hematology
changes), changes in clinical
chemistry, increased estrous cycle,
increased relative right kidney and
liver weight, increased incidence of
histopathological lesions in the
liver, kidney, spleen, bone marrow,
and thyroid, and urinary bladder
effects were observed.
None
Not
conducted
135
NTP (2005b)
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Table 2. Summary of Potentially Relevant Data for 9,10-Anthraquinone (CASRN 84-65-1)
Notes3
Category
Number of
Male/Female, Species,
Study Type, Study
Duration
Exposure
Levelsb
Critical Effects
NOAELb
BMDL/
BMCLb
LOAELbc
Reference
(Comments)


10 Male/10 Female,
B6C3Fi Mouse, dietary,
14 weeks
0, 250, 500,
1050, 2150, or
4300 in males 0,
300, 640, 1260,
2600, or 5300 in
females d
Anemia (hematology changes),
increased relative right kidney
(males) and liver weight, increased
incidence of histopathological
lesions in the liver and spleen, and
urinary bladder.
None
Not
conducted
250
NTP (2005b)
PS
Chronic
50 Male/50 Female,
F344 rat, dietary, 2
years (105 weeks)
0,20, 45,90, or
180 in males
0,25,50,100,
or 200 in
females'1
Decreased body weight, increased
incidence of nonneoplastic lesions
in the kidney, liver, and spleen,
and bone marrow.
None
Not
conducted
20
NTP (2005b)

50 Male/50 Female,
B6C3F, mouse, dietary, 2
years (105 weeks)
0, 90, 265, or
825 in males
0, 80, 235, or
745 in femalesd
Decreased body weight, increased
incidence of nonneoplastic lesions in
the liver and spleen, and urinary
bladder.
None
Not
conducted
80
NTP (2005b)

Developmental
None

Reproductive
None

Carcinogenic
50 Male/50 Female, F344
rat, dietary, 2 years (105
weeks)
0, 5.4, 12.2,
24.4, or 48.9 in
males
0,6.0, 12.0,
23.9, or 47.8 in
females6
Increased incidence of kidney and
liver, and urinary bladder tumors
were observed.
NA
None
NA
NTP (2005b)f
PS
50 Male/50 Female,
B6C3Fi mouse, dietary,
2 years (105 weeks)
0,13.7, 40.3, or
125.3 in males
0,12.0, 35.2, or
111.6 in
females6
Increased incidence of liver and
thyroid tumors, males also had
increased number of animals with
malignant neoplasms.
NA
2.6
NA
NTP (2005b)f
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Table 2. Summary of Potentially Relevant Data for 9,10-Anthraquinone (CASRN 84-65-1)
Notes3
Category
Number of
Male/Female, Species,
Study Type, Study
Duration
Exposure
Levelsb
Critical Effects
NOAELb
BMDL/
BMCLb
LOAELbc
Reference
(Comments)


18/18 (C57BL/6 x
C3H/Anf)Fl and
(C57BL/6 x AKR)F1
mouse, gavage followed
by dietary, 19 months
464s
Tumors were investigated (study was
negative).
NA
Not
conducted
NA
Innes et al. (1969)
2. Inhalation (mg/m3)b

Subchronic
Number and sex not
reported, mouse (strain
not reported), inhalation,
4 months
0,5.2 or 12.1
mg/m3h
Reduced body weight, hemoglobin,
erythrocytes, vitamin C, relative
reticulopenia, and increased
incidence of lung lesions.
ND1
Not
conducted
ND1
Volodchenko et al.
(1971)

Chronic
None

Developmental
None

Reproductive
None

Carcinogenic
None
aNotes: PS = Principal study
bDosimetry: All long-term exposure values (4 weeks and longer) are converted from a discontinuous to a continuous (weekly) exposure. Values for inhalation (cancer
and noncancer), and oral (cancer only) are further converted to an HEC/D. Values from animal developmental studies are not adjusted to a continuous exposure.
°Not reported by the study author but determined from data
dDoses were reported in the study in mg/kg-day, presumably based on the concentrations in the diet (also provided in the study report), body weights, and food
consumption (both of which were routinely measured)
"These doses are human equivalent doses (HED) that were calculated as follows: average daily dose in mg/kg-day x (average animal body weight average human body
weight)1'4 e.g., 180 mg/kg-day x (0.38 kg [male rat] ^ 70 kg)1/4= 48.9 mg/kg-day
fButterworth et al. (2001) raised concern that the contaminant 9-nitroanthracene (9-NA) is responsible for the carcinogenicity observed in the NTP (2005b) studies. The
NTP subcommittee stated that it was unlikely that 9-NA contributed to the carcinogenicity because of the low exposure levels, bioavailability, and relative mutagenicity.
NTP concluded that there was clear evidence of carcinogenicity in female rats and mice of both genders
8Study states that animals were administered 464 mg/kg-day from 7 days of age until weaning at 28 days of age (dose was not adjusted for increase in body weight) via
gavage followed by 1206 ppm in the diet (which was selected based on the maximum tolerated dose of 464 mg/kg-day)
hThese exposure values are not adjusted to an HEC due to incomplete information in the report
'Not determined; insufficient detail
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HUMAN STUDIES
Oral Exposures
No studies investigating the effects of subchronic or chronic oral exposure to
9,10-anthraquinone in humans have been identified.
Inhalation Exposures
Little information is available regarding occupational exposure of humans to
9,10-anthraquinone, but a small number of epidemiology studies analyzing outcomes of workers
exposed during manufacturing are available and are presented below. Although the majority of
occupational exposure is likely via inhalation, dermal and oral exposures may have occurred.
ICI Americas (1985) provided a copy of Volodchenko et al. (1971) in the original
Russian, as well as an English translation. In a production area where anthraquinone was
produced by contact methods (specifics were not stated), the job area was stated to be
contaminated with anthraquinone dust. The study authors reported the levels of dust in three
areas as follows: 2-10 mg/m3 near the contact apparatus, 55-840 mg/m3 near scales, and as high
"3
as 1650 mg/m when cleaning the gas conduits, with the majority (70%) of the dust reported to
be 3.2 microns. No other specifics regarding any measurements were provided in the study
report. The workers were reported to have complained of headache, general weakness, and
irritation of skin (unprotected area) and eyes, though these effects were not analyzed
quantitatively.
Sathiakumar and Delzell (2000) examined the cause-specific mortality rates in
3266 workers (2859 men and 407 women) who had worked for at least 6 months between 1952
to 1996 at a dye and resin manufacturing plant in New Jersey. Subjects were followed up for an
average of 27 years, and the overall mortality rates were compared to the general population of
New Jersey. Subjects were selected based on availability of dates of employment and Social
Security numbers. Information was obtained from plant records and previous epidemiology
investigations. In the plant, the South Dyes area produced anthraquinone dyes and intermediates,
and the North Dyes area produced azo dyes and intermediates, as well as plastics and additives.
Employment information was used to classify subjects according to eight major work areas
(production, laboratories, maintenance, energy, waste treatment, warehouses, engineering, and
administration) and four production subgroups (south dyes, north dyes, plastics, and additives,
and other production). The overall mortality rate of the cohort was 10% lower than the general
population of New Jersey. There was no difference in the overall cancer mortality. Although the
overall cohort had slightly increased mortality from cancers of the colon,1 the results were not
statistically significant. There was, however, a statistically significant increase in deaths from
lung cancers in South Dye workers (SMR: 168, 95% CI: 115-237), and North Dye workers had
a statistically significant increase in deaths from stomach (SMR: 386, 95% CI: 125-901),
bladder (SMR: 515, 95% CI: 140-1318), and central nervous system cancer (SMR: 517,
95% CI: 168-1206). The relative risk for lung cancer in South Dye workers compared to other
workers was 1.7 (95% CI: 1.1-2.4). However, the results lacked a duration response, with
increases noted in workers with <5 years and those with 20+ years but not in workers with
interim years of employment. With regard to the increased incidence of bladder malignancies,
1 Standardized mortality ratio [SMR]: 118, 95% CI: 78-172), lung (SMR: 122, 95% CI: 98-150), liver (SMR: 143,
95% CI: 53-311), bladder (SMR: 139, 95% CI: 60-273), central nervous system (SMR: 134, 95% CI: 58-264), and
lymphosarcoma (SMR: 121, 95% CI: 25-354).
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the study authors established that some employees had previously worked at the Cincinnati
Chemical Works (CCW) in Ohio, which both produced and used benzidine, a known bladder
carcinogen. In addition, the current study did not control for smoking and did not have
information on occupational exposure to other possible carcinogens (e.g., asbestos).
Gardiner et al. (1982) conducted a retrospective cohort in 1975 workers of a dye factory
in Scotland who had worked there for at least 6 months from January 1, 1956, to December 31,
1965. Mortality of these subjects was evaluated through June 30, 1980. Workers were stated to
be exposed to substituted anthraquinones. All but 11 of the initial subjects were accounted for
through June 30, 1980. There were a total of 470 deaths of these subjects. The age-adjusted
mortality rate in Scotland as a whole was used as the control. There was no increase in overall
mortality (SMR: 76.9; 470 observed 611.5 expected) or in mortality from all malignant
neoplasms (SMR: 86.2; 129 observed ^ 149.7 expected). An excess of esophageal cancer was
noted in the engineering department (SMR: 139.5; 6 observed ^ 4.3 expected), but no common
cause could be identified. The study authors concluded that there was no increase in total or
cancer-related mortality based on the age-adjusted SMRs.
Barbone et al. (1992) conducted a case-control study using 51 lung cancer cases and
102 controls from a cohort of Caucasian males employed at a dye and resin manufacturing plant
in New Jersey. Workers in these cases developed lung cancer prior to October 1, 1988. Two
controls meeting the following two criteria were selected for each case: (1) each control had the
same birth year as the case; and (2) each control was still alive at the time of death or diagnosis
of the case. Work histories were used to classify subjects by six work areas. Interviews were
conducted to determine exposure assessment. The odds ratio (OR) for lung cancer in workers in
the anthraquinone dye and epichlorohydrin production was 2.4 (95% CI: 1.1-5.2). There was no
trend with duration of exposure. The ORs varied by building, with ORs of 12 (95% CI: 1.4-99)
in the anthraquinone production building, 1.8 (95% CI: 0.6-5.1) in the anthraquinone
intermediate dye building, 1.2 (95% CI: 0.5-2.9) in the anthraquinone dye synthesis building,
and 3.3 (95% CI: 1.0-11) in the anthraquinone dye standardization building. The study was
complicated because subjects had visited the infirmary for acute exposure to 48 different
chemicals. Smoking histories were not available for about 20% of the subjects, but smoking was
found to be a major cause of lung cancer among workers at this plant. In four of the six cases,
workers employed in the anthraquinone dye and epichlorohydrin production area were also
exposed to chlorine, which also had an elevated OR.
ANIMAL STUDIES
Oral Exposures
The effects of oral exposure of animals to anthraquinone have been evaluated in
subchronic (NTP, 2005b), chronic (NTP, 2005b), and cancer (NTP, 2005b; Innes et al., 1969)
studies. The National Toxicology Program (NTP, 2005b) conducted 14-week and 2-year
chronic/carcinogenicity dietary studies in rats and mice. Because there are four studies (and
genotoxicity studies discussed later), when discussing separate studies, the studies will be
designated with different letters such that the subchronic rat study is NTP (2005b), etc., as is
indicated in Tables 2 and 3, and when referring to the whole report throughout the document, it
will be cited as NTP (2005b). The NTP (2005b) studies were conducted according to Good
Laboratory Practice (GLP) regulations and were peer reviewed. There were no additional
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subchronic or short-term oral studies with 9,10-anthraquinone. However, there was an additional
chronic carcinogenicity study (Innes et al., 1969) using two strains of mice.
Subchronic Studies
The 14-week rat study by NTP (2005b) is selected as the principal study for
derivation of a screening subchronic p-RfD. The NTP (2005b) administered
9,10-anthraquinone (99.8% pure) to male and female F344 rats (10/sex/treatment group) at
dietary concentrations of 0, 1875, 3750, 7500, 15,000, or 30,000 ppm for 14 weeks. These
concentrations were stated to be equivalent to average daily doses of 0, 135, 275, 555, 1130, and
2350 mg/kg-day, respectively, for both males and females. Dose formulations were prepared
every 4 weeks by mixing 9,10-anthraquinone into the diet. Homogeneity analysis was conducted
on 1875- and 30,000-ppm dose formulations, and stability analysis was conducted on a 230-ppm
dose formulation. Dose formulations were found to be homogeneous and were stable for at least
35 days when stored at room temperature and protected from the air and light. Dose
formulations were analyzed at the beginning and end of the 14-week study. Dose concentrations
were within 10% of the nominal concentration. Animals were weighed, and clinical signs were
recorded weekly. Food consumption was recorded twice a week (five animals were housed to a
cage) and served as the basis for calculating the daily rates of compound consumption. Blood
was collected on Days 4 and 22 and at study termination for hematology (erythrocyte, platelet,
and leukocyte counts; hematocrit; hemoglobin, mean cell hemoglobin [MCH], mean cell volume
[MCV], and mean cell hemoglobin concentration [MCHC]; erythrocyte and platelet morphology;
and differential leukocyte counts) and clinical chemistry (urea nitrogen, creatinine, total protein,
albumin, alanine aminotransferase [ALT], alkaline phosphatase [ALP], creatine kinase, sorbitol
dehydrogenase [SDH], and bile salts). Urinalysis (creatinine, glucose, total protein, aspartate
aminotransferase, A'-acetyl -[3- D-glucosaminidase, y-glutamyltransferase, total volume, and
specific gravity) was conducted on urine collected in metabolism cages on Days 8, 26, and 89.
Rats (10/sex/treatment group) specified for interim hematology, clinical chemistry, and
urinalysis were administered the same concentrations and were euthanized without necropsy
after urine collection on Day 26. At the end of the study, samples were collected from males for
analysis of the reproductive organs (spermatid heads per testis and per gram testis, spermatid
counts, and epididymal spermatozoal motility and concentration; left cauda epididymis, left
epididymis, and left testis weight) and from females for vaginal morphology (estrous cycle
length and relative frequency of the estrous stages) in controls and the three highest
concentrations. Soluble protein and a2u-globulin were measured in the kidney homogenates at
study termination in males. Necropsies were performed at terminal sacrifice and organs (heart,
right kidney, liver, lungs, right testis, and thymus) were weighed. Complete histopathology was
conducted on control and high-dose animals. Histopathology of the bone marrow, liver, kidney,
spleen, thyroid gland, and urinary bladder (females only) were routinely examined in all groups.
All animals survived until study termination (NTP, 2005b). There were no changes in
overall body-weight gain in males (weekly data were not provided). Although there was an
initial decrease in food consumption in 275-mg/kg-day males, the food consumption was equal
to or greater than the controls during the final week of treatment (see Table B. 1). In female rats,
there was a dose-dependent decrease in overall body-weight gain that was statistically different
from the control even in the lowest treatment group. Although food consumption during the first
week of treatment decreased in a dose-dependent manner, food consumption during the final
week of treatment was 18—24% greater in all treatment groups compared to the control.
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However, the study authors stated that the increased food consumption may have been due to
scattering of the food and that the reduced food consumption was likely due to reduced
palatability. There were no clinical signs related to treatment reported. By Day 4, animals had
increased hematocrit, hemoglobin, and erythrocyte counts with doses >275 mg/kg-day,
indicating erythrocytosis. However, this was followed by anemia that began on Day 22 and
persisted through study termination (as measured by decreased hemoglobin, erythrocyte counts,
and hematocrit). Hematology results were not related to dose, and while the decrease in
hemoglobin and erythrocyte counts were statistically reduced in all treatment groups in both
sexes (see Table B.2), hematocrit values were not. The lack of a decrease in hematocrit is likely
due to the increase in mean cell volume. The statistically significant increase in reticulocytes in
all treatment groups in both sexes indicates an erythropoietic response to the anemia. There were
statistically significant increases in platelets, creatinine, total protein, and albumin at all doses
generally in both sexes (see Table B.2). Urea nitrogen levels were generally increased but were
only statistically significant in males with doses >555 mg/kg-day and in 275-mg/kg-day females.
There were statistically significant decreases in ALP (dose dependent, both sexes) and bile salts
(males only) and transient changes observed in the urine. Although there were no changes in
sperm parameters or reproductive organs in the males, females administered 1130 or
2350 mg/kg-day had longer estrous cycles. There was a statistically significant increase in
a2u-globulin (jug/g soluble protein) in the kidney homogenates in all treatment groups (only
males evaluated) at study termination. There was no dose-response, with the increase similar in
all treatment groups.
Necropsy body weight was significantly decreased in 2350-mg/kg-day males and in
females treated with >275 mg/kg-day (NTP, 2005b). There was a dose-dependent increase in the
relative weight of the right kidney and the liver with statistically significant increases observed at
all doses in both sexes (see Table B.3). Histopathology of the liver revealed hypertrophy in all
treated males and females, but none in the controls. In males, there was a dose-dependent
increase in severity that was not observed in females. Histopathology of the kidney revealed
hyaline droplet accumulation in all treated animals (both males and females) but not in the
controls. Although all male rats (including the controls) had nephropathy, the severity was
increased in treated rats. There was a statistically significant increase in nephropathy in females
treated with >1130 mg/kg-day. Other histopathological findings included congestion (all treated
rats), hematopoietic cell proliferation (all but one treated rat), and pigmentation (all treated rats)
in the spleen; hyperplasia in the bone marrow (statistically significant increase in all groups
except the lowest dose group in male rats); follicular cell hypertrophy in the thyroid (all rats
exposed to >275 mg/kg-day); and inflammation and transitional epithelium hyperplasia in the
urinary bladder (statistically significant increase in the highest female group only; males not
examined) (see Table B.4). As all animals (i.e., 10/10) were affected in one endpoint or another
of those listed in Table B.4, and at all doses including the lowest dose, no NOAEL can be
derived from the data. These levels of response also preclude any meaningful modeling of these
or of the organ-weight data (see Table B.3). However, a LOAEL of 135 mg/kg-day, the lowest
dose tested, is established from the data, based on numerous changes in hematology, clinical
chemistry, organ weights, body-weight gain (females), and histopathology.
In a separate study, NTP (2005b) administered 9,10-anthraquinone (99.8% pure) to male
and female B6C3Fi mice (10/sex/treatment group) at dietary concentrations of 0, 1875, 3750,
7500, 15,000, or 30,000 ppm for 14 weeks. These concentrations were stated to be equivalent to
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average daily doses of 0, 250, 500, 1050, 2150, and 4300 mg/kg-day, respectively, in males and
0, 300, 640, 1260, 2600, and 5300 mg/kg-day, respectively, in females. Dose formulations were
analyzed as in the rat study detailed above. Dose concentrations were within 10% of the nominal
concentration. Methods were the same as those used in the rat study detailed above except male
mice were individually housed, hematology and clinical chemistry were only conducted at study
termination, and urinalysis was not conducted.
All animals survived until study termination (NTP, 2005b). There were no changes in
body weight, body-weight gain, or food consumption in any of the treatment groups in either sex
compared to the control group. There were no clinical signs related to treatment reported.
Anemia, as measured by decreased hemoglobin, erythrocyte counts, and hematocrit, was noted at
study termination with results more pronounced in female mice compared to male mice (see
Table B.5). This was also associated with increases in mean cell volume and mean cell
hemoglobin concentration. The increase in reticulocytes in all treatment groups in both sexes
indicates an erythropoietic response to the anemia. There was also a statistically significant
increase in platelets (see Table B.5). Although the methods indicate that clinical chemistry was
conducted in the mice, there were no data reported. There were no changes in sperm parameters
and reproductive organs in the males, and there were no effects on the estrous cycles in females.
Necropsy body weight was not statistically significantly affected in either gender (NTP,
2005b). There was a dose-dependent increase in the relative weight of the liver with statistically
significant increases observed at all doses in both sexes (see Table B.6). High-dose males had a
statistically significant increase in relative kidney weight. Histopathology of the liver revealed
centrilobular hypertrophy in both sexes with statistically significant increases observed with
doses >500 mg/kg-day. Severity increased in a dose-dependent manner in both genders. Other
histopathological findings included (see Table B.7) hematopoietic cell proliferation (nearly all
treated males), and pigmentation (all but one treated male) in the spleen; and transitional
epithelium, cytoplasmic alteration in the urinary bladder (all treated mice) with a dose-dependent
increase in severity in both genders. This study was used to determine concentrations for the
2-year study (NTP, 2005b); therefore, no NOAEL or LOAEL was reported. As with rats, all
animals (10/10) were affected in at least one endpoint of those listed in Table B.7, and effects
were observed at all doses including the lowest dose. These levels of response also preclude any
meaningful modeling of these or of the organ-weight data (see Table B.6). No NOAEL can be
derived from the data. However, a LOAEL of 250 mg/kg-day in male mice and 300 mg/kg-day
in female mice is available from the data, based on numerous changes in hematology, organ
weights, and histopathology.
Chronic/Carcinogenicity Studies
The 2-year rat study by NTP (2005b) is selected as the principal study for derivation
of the screening chronic p-RfD. NTP (2005b) administered 9,10-anthraquinone (99.8% pure)
to male and female F344 rats (50-60/sex/treatment group) at dietary concentrations of 0, 469,
938, 1875, or 3750 ppm for 2 years. These concentrations were stated to be equivalent to
average daily doses of 0, 20, 45, 90, and 180 mg/kg-day, respectively, in males and 25, 50, 100,
and 200 mg/kg-day, respectively, in females. Dose formulations were analyzed for homogeneity
and stability as detailed above in the subchronic rat study. Dose formulations were analyzed for
concentration every 8 or 12 weeks. Dose concentrations were within 10% of the nominal
concentration. Food consumption was measured every four weeks (three males per cage and five
females per cage) and served as the basis for calculating the daily rates of compound
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consumption. Animals were examined twice daily with clinical signs recorded every four weeks.
Body weight was recorded on Day 8, every 4 weeks, and at study termination. Five rats per sex
in the control and high-dose groups were sacrificed for evaluation at 3 and 12 months. All
animals were necropsied. Kidneys and livers were weighed at 12 months. Soluble protein and
a2u-globulin were measured in the kidneys at 3 months for both males and females. At terminal
sacrifice, animals were necropsied and a complete histopathological examination was conducted.
The survival in all exposed groups of male rats was similar to that of the controls (NTP,
2005b). Survival of all exposed groups of female rats was statistically significantly greater than
that of the controls. Body weights were generally lower in both males and females during the
later part of the study. Treated male rats had overall body-weight gains 5-10% lower than the
controls, but the decrease was not correlated with increasing dose (see Table B.8). However, in
females, there was a dose-dependent decrease in overall body-weight gain (see Table B.9) that
ranged from 16-30% lower than the controls. a2u-Globulin (soluble protein) in kidney
supernatant was reported for the control and high-dose group at 3 months with results indicating
an increase in treated males and a decrease in treated females. There were extensive
nonneoplastic lesions in the kidney, liver, spleen, and bone marrow (see Tables B.8 and B.9) as
well as neoplastic lesions in the kidney, liver, and urinary bladder (see Tables B. 10 and B. 11) in
both male and female rats. There was a dose-dependent increase in renal tubule adenoma or
carcinoma in female rats—but not in male rats. Although there were increases in hepatocellular
adenomas in female rats, their increase was not related to dose, and only 50 mg/kg-day females
had a statistically significant increase above the controls. There was a statistically significant
increasing trend for transitional epithelial papilloma or carcinoma in the urinary bladder, but the
increase was small. The increase in males was greater, achieving a statistically significant
increase in males administered 90 mg/kg-day, but there was only an apparent trend if high-dose
males were dropped. Large fractions of treated animals were affected in one endpoint or the
other, which were statistically significantly increased even at the lowest dose examined.
However, the results generally were consistent across treatment groups and did not increase with
increasing doses. The nature of the dose-response precludes any meaningful modeling of the
data. Although there were some increases in lesions of the parathyroid gland, bone, stomach,
and lung (mainly in the males), the study authors considered these to be secondary to the
impaired renal function due to perturbations in calcium homeostasis, which is commonly
observed in rats with severe nephropathy. The study authors did not specify a NOAEL or
LOAEL but stated that there is some evidence for carcinogenicity in male rats and clear evidence
for carcinogenicity in female rats. No NOAEL can be derived from the data; however, the
LOAEL is 20 mg/kg-day in males and 25 mg/kg-day in females, based on the nonneoplastic
lesions in the kidney, liver, and spleen in both male and female rats.
The 2-year mouse study by NTP (2005b) is selected as the principal study for
derivation of the p-OSF. NTP (2005b) administered 9,10-anthraquinone (99.8% pure) to male
and female B6C3Fi mice (50/sex/treatment group) at dietary concentrations of 0, 833, 2500, or
7500 ppm for 2 years. The study authors stated these concentrations to be equivalent to average
daily doses of 0, 90, 265, and 825 mg/kg-day, respectively, in males and 0, 80, 235, and
745 mg/kg-day, respectively, in females. Dose formulations were analyzed as detailed in the
2-year NTP (2005b) rat study. Dose concentrations were within 10% of the nominal
concentration, food consumption was measured every 4 weeks (one male per cage and five
females per cage), which served as the basis for calculating the daily rates of compound
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consumption. Animals were examined twice daily with clinical signs recorded every 4 weeks
Body weight was recorded on Day 8, every 4 weeks, and at study termination. At terminal
sacrifice, animals were necropsied and complete histopathological examinations were conducted.
There was a statistically significant (p < 0.001) decrease in survival relative to controls in
high-dose male mice that also followed a significant (p < 0.001) trend, but there was no
difference in survival relative to the controls noted in female mice (NTP, 2005b). Body weights
were generally lower in high-dose males and females during the later part of the study. Treated
male mice had dose-dependent decreases in overall body weight, with gains 6-38% lower than
the controls. However, in females, there were decreases in overall body-weight gain (see
Table B.12) in only the mid- (235 mg/kg-day) and high-dose groups (745 mg/kg-day) compared
to the controls. There were extensive nonneoplastic lesions in the liver, spleen, urinary bladder,
and thyroid gland (see Tables B. 12 and B. 13) and neoplastic lesions in the liver and thyroid
gland (see Tables B.14 and B.15) of both male and female rats. Statistically significant increases
in nonneoplastic lesions included centrilobular hypertrophy in the liver (all groups in males and
females); focal, fatty degeneration in the liver (low- and mid-dose males and high-dose females);
erythrophagocytosis in the hepatocytes (all groups in males and females); eosinophilic focus in
the liver (mid- and high-dose males and high-dose females); focal necrosis (high-dose males and
females); hematopoietic cell proliferation in the spleen (high-dose males and females); follicular
cell hyperplasia in the thyroid gland (high-dose males), and intracytoplasmic inclusion body in
the urinary bladder (all treatment groups in both sexes). There was a dose-dependent increase in
hepatocellular adenoma, carcinoma, and/or hepatoblastoma in both sexes with significant
increases noted even with the low dose. There was also an increasing trend for follicular cell
adenoma or carcinoma in the thyroid in males and females, but the results did not achieve
statistical significance. However, male mice had a statistically significant increase in total
malignant neoplasms at all doses. The study authors did not specify a NOAEL or LOAEL but
stated that there is clear evidence for carcinogenicity in male and female mice. No NOAEL can
be derived from the data; however, the LOAEL is 90 mg/kg-day in males and 80 mg/kg-day in
females, based on the nonneoplastic lesions in the liver and bladder in both male and female rats.
Innes et al. (1969) present data on bioassays of 120 different chemicals including
9,10-anthraquinone. Female C57BL/6 mice were mated to C3H/Anf or AKR male mice to
produce (C57BL/6 x C3H/Anf)Fl and (C57BL/6 x AKR)F1 mice. The maximum tolerated dose
of 464 mg/kg was determined through a series of preliminary short-term studies that found no
mortality after a single dose, 6 daily doses, and then 19 daily doses. Subsequently, starting at
7 days of age, 464 mg/kg 9,10-anthraquinone (purity not specified) in 0.5% gelatin was
administered by daily gavage until weaning at 28 days of age. Although the dose was presented
as mg/kg of body weight, the dose was not adjusted for weight gain during the 3-week period.
At weaning, 18 mice of each sex per strain were retained and exposed to 1206 ppm mixed
directly into the diet (this was stated to be based on the food consumption rate and body weight
at weaning so that the animals continued to receive the maximum tolerated dose) for
approximately 18 months. Ethyl carbamate was used as a positive control. Animals were
sacrificed after 18 months (a range of termination times were noted due to the large number of
animals in the ongoing study). Animals were necropsied and all major organs and gross lesions
were examined histologically. 9,10-Anthraquinone was stated not to cause a significant increase
in tumors, but specific results were not provided.
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Inhalation Exposures
There is only a single subchronic inhalation study available (Volodchenko et al., 1971),
and no chronic inhalation studies were available. ICI Americas (1985) provided a copy of the
Volodchenko et al. (1971) Russian publication, as well as an English translation. Little useful
information about the methods, the subjects, or the results is available from the report or the
translation (reported exposure levels and effects given in Table 2). This study is not considered
further for derivation of a p-RfC.
Chronic Studies
No studies investigating the effects of chronic inhalation of 9,10-anthraquinone in
animals have been identified.
Reproductive and Developmental Studies
There are no formal developmental or reproductive studies available for
9,10-anthraquinone or its metabolites. However, some information for these endpoints is
available from the NTP (2005b) studies. At the end of the 14-week study in rats (NTP, 2005b)
and mice (NTP, 2005b), sperm samples were collected from all core study male rats and mice in
the 0-, 7500-, 15,000-, and 30,000-ppm groups for sperm count and motility evaluations. The
following parameters were evaluated: spermatid heads per testis and per gram testis, spermatid
counts, and epididymal spermatozoal motility and concentration. The left cauda epididymis, left
epididymis, and left testis were weighed. Vaginal samples were collected for up to 12
consecutive days prior to the end of the studies from the core study female rats and mice in the
0-, 7500-, 15,000-, and 30,000-ppm dose groups. Vaginal cytology evaluations were used to
evaluate estrous cycle length and relative frequency of the estrous cycle stages (i.e., diestrus,
proestrus, estrus, and metestrus).
No differences in epididymal spermatozoal measurements were observed between
exposed and control groups of rats (NTP, 2005b) or mice (NTP, 2005b). Estrous cycle lengths
(in days) were longer in 15,000- and 30,000-ppm female rats compared to the controls. Results
were also dose dependent with cycle lengths of 4.55 ± 0.17 days, 4.90 ± 0.15 days,
5.40 ± 0.31 days (p < 0.05), and 6.15 ± 0.33 days (p < 0.01) reported in the control, 7500-ppm,
15,000-ppm, and 30,000-ppm groups, respectively. No significant changes were observed in
female mice.
OTHER DATA
Table 3 summarizes the toxicokinetic, mutagenicity, and genotoxicity studies conducted
with 9,10-anthraquinone. Of particular note is the NTP (2005b) mutagenicity assays and the
Butterworth et al. (2001) assessment of the mutagenicity of 2-nitroanthracene (2-NA), a reported
contaminant of the anthraquinone formulation used in the NTP (2005b) bioassay.
Butterworth et al. (2001) maintained that the turnorigenicity of 9,10-anthraquinone observed in
the NTP (2005b)2 bioassay was entirely due to the mutagenic 2-NA contaminant. NTP (2005b),
however, found that the mutagenicity of 2-hydroxyanthracene, a major metabolite of
9,10-anthraquinone, was 7 times as mutagenic as 2-NA and would be a much more likely
2The results were apparently first reported in NTP Technical Report 494 (1999), which was not found on the NTP
Web site. The bioassay was conducted even earlier, with results reported in Zeiger et al. (1988). The report was
apparently updated in NTP, 2005b.
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candidate for the causative agent, if mutagenicity was involved in the mode of action (a
mutagenic MOA has not been established; see cancer oral descriptor section following).
A physiologically based pharmacokinetic (PBPK) model was developed to characterize
rat tissue concentrations of 9,10-anthraquinone resulting from oral exposure in rats (NTP,
2005b). Plasma 9,10-anthraquinone concentrations estimated from this model may serve as a
surrogate dosimeter for evaluating exposure-response data. However, the model is for rats only
and cannot be extended to mice or humans for purposes of dosimetric adjustment.
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Table 3. Other Studies for 9,10-Anthraquinone
Test
Materials and Methods
Results
Conclusions
Reference
Toxicokinetics
Blood was obtained on Day 8, and at 3, 6, 12,
and 18 months (2-3 animals per time point)
from male and female rats administered 3750
ppm during the core study.
Plasma concentrations were twice as high in
females as males.
N/A
NTP (2005b)
Toxicokinetics
Male and female F344 rats and B6C3Fi were
administered 149,10-anthraquinone as a single
administration either intravenously (2 mg/kg
in rats, 4 mg/kg in mice) or via gavage (40,
100, or 400 mg/kg in rats and 100, 200, or
800 mg/kg in mice). Plasma concentrations
were measured at various times.
After intravenous injection, plasma
concentrations peaked initially and then
decreased over time. After oral exposures,
plasma concentrations peaked at around 8-12
hours in rats and at about 4 hours in mice.
Although the plasma concentrations increased
with dose, the plasma concentrations did not
increase in a linear manner.
The data indicated first order
absorption and elimination in a
two-compartment open model.
NTP (2005b)
Toxicokinetics
24-hour urine samples were collected from
male F344 rats administered different types
of 9,10-anthraquinone samples; specifics on
the test were unclear.
2-Hydroxyanthraquinone was the major
metabolite excreted in the urine followed by
1-hydroxyanthraquinone. It was estimated
that 2-hydroxyanthraquinone was present at a
level 5.8 times greater than was theoretically
possible for 9-NA.
2-Hydroxyanthraquinone is a
major metabolite of 9,10-
anthraquinone.
NTP (2005b)
Mutagenicity
Tested for reverse mutation in Salmonella
typhimurium (preincubation assay) strains
TA98 and TA100, with and without
metabolic activation, at doses of 0 (DMSO
only), 33, 100, 333, 1000, or 2500 ng/plate.
9,10-Anthraquinone caused an increase in
reverse mutations, both in the presence and
absence of metabolic activation, with the
number greater than observed with the positive
control.
9,10-Anthraquinone was
considered mutagenic.
Zeiger et al. (1988)
Mutagenicity
Tested for reverse mutation in Salmonella
typhimurium (Ames assay) strains TA98,
TA100, TA1535, and TA1538, with
metabolic activation, at doses of 0 (DMSO
only), 4, 20, 100, 500, or 2500 ng/plate.
9,10-Anthraquinone was reported as negative.
9,10-Anthraquinone was not
considered mutagenic.
Anderson and Styles
(1978)
Mutagenicity
Tested for reverse mutation in Salmonella
typhimurium (Ames assay) strains TA97,
TA98, and TA100, with and without
metabolic activation, at doses of 0 (DMSO
only), 5, 10, 50, or 200 |ig/platc.
There was no increase in the mutation
frequency.
9,10-Anthraquinone was not
considered mutagenic.
Sakai et al. (1985)
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Table 3. Other Studies for 9,10-Anthraquinone
Test
Materials and Methods
Results
Conclusions
Reference
Mutagenicity
Tested for reverse mutation in Salmonella
typhimurium (Ames assay) TA97, TA100,
and TA2637 with metabolic activation with a
maximum dose of 100 |ig/plate: exact
concentrations tested not reported.
9,10-Anthraquinone was reported as negative.
9,10-Anthraquinone was not
considered mutagenic.
Tikkanen etal. (1983)
Mutagenicity
Tested for reverse mutation in Salmonella
typhimurium (Ames assay) strains TA98,
TA100, TA1535, TA1537, and TA1538, with
and without metabolic activation, at doses of
0 (DMSO only), 0.2, 2, 10, or 20 |ig/platc.
There was an increase in mutation frequency
with TA98, TA1537, and TA1538, without
metabolic activation.
9,10-Anthraquinone was
considered mutagenic.
Liberman etal. (1982)
Mutagenicity
Tested for reverse mutation in Salmonella
typhimurium (Ames assay) strains TA102
and TA1537, with and without metabolic
activation, at doses of 0, 0.1, 0.3, 1, 3, 10, 30,
100, or 300 ng/plate.
There was no increase in mutation frequency.
9,10-Anthraquinone was not
considered mutagenic.
Krivobok etal. (1992)
Mutagenicity
Tested for reverse mutation in Salmonella
typhimurium (Ames assay) strains TA98,
TA1537, and TA138, with and without
metabolic activation, at doses of 0 (not
specified if this was vehicle or untreated), 10,
or 20 ng/plate.
Hydroxylated anthraquinones (which included
9,10-anthraquinone) were considered positive
in the TA1537 strain, with and without
metabolic activation at both concentrations,
but not in the other two strains. However, the
report did not specifically state results for
9,10-anthraquinone.
No conclusion is made.
However, in a review, Brown
(1980) stated that 9,10-
anthraquinone was negative in
all strains (TA98, TA100,
TA1535, TA1537, and
TA1538).
Brown and Brown
(1976)
Mutagenicity
Tested for reverse mutation in Salmonella
typhimurium (Ames assay) strains TA98 and
TA100, with and without metabolic
activation, at doses of 0, 33, 100, 333, 1000,
or 2500 ng/plate of 97% pure
9,10-anthraquinone.
There was an increase in the number of
revertants with and without metabolic
activation.
The results were considered
positive.
NTP (2005b)a
Mutagenicity
Tested for reverse mutation in Salmonella
typhimurium (Ames assay) strains TA98,
TA100, and TA102, with and without
metabolic activation, at doses of 0, 100, 333,
1000, 3333, or 10,000 ng/plate of 100% pure
9,10-anthraquinone.
There was no increase in the number of
revertants with and without metabolic
activation.
The results were considered
negative.
NTP (2005b)'1
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Table 3. Other Studies for 9,10-Anthraquinone
Test
Materials and Methods
Results
Conclusions
Reference
Mutagenicity
2-Hydroxyanthraquinone tested for reverse
mutation in Salmonella typhimurium (Ames
assay) strain TA98, with and without
metabolic activation.
There was an increase in the number of
revertants (2.2 per plate).
The results were considered
positive.
NTP (2005b)a
Mutagenicity
2-NA tested for reverse mutation in
Salmonella typhimurium (Ames assay) strain
TA98, with and without metabolic activation.
There was an increase in the number of
revertants (0.37 per plate).
The results were considered
positive.
NTP (2005b)a
Mutagenicity
Tested for reverse mutation in Salmonella
typhimurium (Ames assay) strains TA98,
TA100, TA1535, and TA1537, and Escheria
coli WP2uvrA, with and without metabolic
activation, at doses of 0, 30, 60, 125, 250,
500, 1000 ,or 2000 |ig/platc of 99% pure
9,10-anthraquinone derived from the
oxidation of anthracene (this was the same
anthracene used in the NTP, 2005b studies).
There was an increase in revertants without
metabolic activation with the TA98, TA100,
and TA1537 strains and in TA98 with
metabolic activation.
It was concluded that the
mutagenicity of the
9,10-anthraquinone used in the
NTP (2005b)b study was due to
the 9-NA contaminant, which
was estimated to range from
0.04 to 2.4 ng/plate with the
concentrations of 9,10-
anthraquinone used.
Butterworth et al. (2001)
Mutagenicity
Tested for reverse mutation in Salmonella
typhimurium (Ames assay) TA98, TA100,
TA1535, and TA1537, sad Escheria coli
WP2uvrA, with and without metabolic
activation, at doses of 0, 30, 60, 125, 250,
500, 1000, or 2000 |ig/platc of purified
9,10-anthraquinone (the sample used in the
NTP, 2005b studies was purified to remove
contaminants).
There was no increase in revertants with or
without metabolic activation in any of the
strains at any of the concentrations.
It was concluded that the
mutagenicity of the
9,10-anthraquinone used in the
NTP (2005b)b study was due to
the 9-NA contaminant, which
was estimated to range from
0.04 to 2.4 ng/plate with the
concentrations of 9,10-
anthraquinone used.
Butterworth et al. (2001)
Mutagenicity
Tested for reverse mutation in Salmonella
typhimurium (Ames assay) TA98, TA100,
TA1535, and TA1537, and Escheria coli
WP2uvrA, with and without metabolic
activation, at doses of 0, 30, 60, 125, 250,
500, 1000, or 2000 |ig/platc of 99% pure
9,10-anthraquinone derived using the
Friedel-Crafts technology.
There was no increase in revertants in any of
the strains tested.
9,10-Anthraquinone was
considered negative for
mutagenicity.
Butterworth etal.
(2001)
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Table 3. Other Studies for 9,10-Anthraquinone
Test
Materials and Methods
Results
Conclusions
Reference
Mutagenicity
Tested for reverse mutation in Salmonella
typhimurium (Ames assay) TA98, TA100,
TA1535, and TA1537, and Escheria coli
WP2uvrA, with and without metabolic
activation, at doses of 0, 30, 60, 125, 250,
500, 1000, or 2000 |ig/platc of 99% pure
9,10-anthraquinone derived using Diels-
Alder chemistry.
There was no increase in revertants in any of
the strains tested.
9,10-Anthraquinone was
considered negative for
mutagenicity.
Butterworth et al. (2001)
Mutagenicity
L5178Y thymidine kinase (TK) +/- mouse
lymphoma forward mutation assay was
conducted, with or without metabolic
activation, at concentrations of 0, 1.57 (with
metabolic activation only), 3.13, 6.25, 12.5,
25.0, 37.5, or 50 |ig/ml using 99% pure
9,10-anthraquinone derived using Diels-
Alder chemistry.
There was no increase in the mutation
frequency.
9,10-Anthraquinone was
considered negative for
mutagenicity.
Butterworth et al. (2001)
Genotoxicity
Chromosomal aberrations were evaluated in
Chinese Hamster Ovary (CHO) cells after 20
and 44 hours of exposure to concentrations of
0, 12.5, 25, 37.5, or 50 ng/ml of 99% pure
9,10-anthraquinone derived using Diels-
Alder chemistry with and without metabolic
activation.
There was no increase in chromosomal
aberrations.
The study was considered
negative.
Butterworth et al. (2001)
Genotoxicity
The in vivo bone marrow mouse micronuclei
assay was used. Crl:CD-l (ICR)BR mice
were administered 1250, 2500, or 5000
mg/kg 99% pure 9,10-anthraquinone derived
using Diels-Alder chemistry in corn oil via
gavage, and five males and five females were
sacrificed at 24, 48, or 72 hours.
There was no increase in the micronucleated
polychromatic erythrocytes in the bone
marrow at any time point.
The study was considered
negative.
Butterworth et al. (2001)
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Table 3. Other Studies for 9,10-Anthraquinone
Test
Materials and Methods
Results
Conclusions
Reference
Genotoxicity
Male and female B6C3F, mice were fed
9,10-anthraquinone (99.8% pure) for 14
weeks at concentrations of
1875-30,000 ppm. Peripheral blood was
collected and evaluated for micronucleated
normochromatic erythrocytes.
In males, there was a statistically significant
increase in micronucleated normochromatic
erythrocytes in the 30,000-ppm group only but
females had a statistically significant increase
in all but the lowest dose group.
Results were considered
positive.
NTP (2005b)
Genotoxicity
Male B6C3Fi mice were injected i.p. with
three doses of 9,10-anthraquinone 24 hours
apart at doses of 500, 1000, or 2000 mg/kg,
and micronucleated polychromatic
erythrocytes in the bone marrow were
evaluated.
There was no increase in micronucleated cells.
The results were considered
negative.
NTP (2005b)
Genotoxicity
Syrian hamster embryo (SHE) cell
transformation assay was conducted. Cells
were exposed to 9,10-anthraquinone for 24
hours at concentrations of 0.5, 1, 3, 5, 6, 7, 8,
or 9 ng/ml, then plated for 7 days or exposed
to anthraquinone for 7 days at concentrations
of 4.25, 4.5, 5, or 5.25 ng/ml and
immediately evaluated.
After 24 hours of exposure, there were no
effects on the relative plating efficiency, and
there were no increases in morphological
transformations. After 7 days of exposure,
there was a dose-dependent decrease in
relative plating efficiency with the highest
dose 68% lower than the controls. However,
there still was no increase in transformations.
Results were considered
negative.
Kerckaert etal. (1996)
"It was stated in the NTP report that these results were also presented in Zeiger et al. (1988).
bResults apparently first reported in NTP Technical Report 494 (1999), which was not found; apparently superseded by NTP, 2005b.
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DERIVATION OF PROVISIONAL VALUES
Tables 4 and 5 below present a summary of noncancer and cancer reference values,
respectively.
DERIVATION OF ORAL REFERENCE DOSES
Derivation of Subchronic Provisional RfD (Subchronic p-RfD)
No subchronic p-RfD can be derived because doing so would require the application of a
composite uncertainty factor of 10,000. However, a screening subchronic p-RfD is provided in
Appendix A. NTP (2005b) studies in rats and mice were the only studies available to consider
for derivation of the subchronic p-RfD. The study in rats (NTP, 2005b) is used for derivation of
the screening subchronic p-RfD because the intake of test compound was lower in the rats, and
both studies produced a LOAEL with the lowest dose tested.
Derivation of Chronic Provisional RfD (Chronic p-RfD)
No chronic p-RfD can be derived because doing so would require the application of a
composite uncertainty factor of 10,000. However, a screening chronic p-RfD is provided in
Appendix A. NTP (2005b) studies in rats and mice were the only studies available to consider
for derivation of the chronic p-RfD. The study in rats (NTP, 2005b) is used for derivation of the
screening chronic p-RfD because the intake of test compound was lower in the rats, and both
studies produced a LOAEL with the lowest dose tested.
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
The available data do not support derivation of any inhalation toxicity values. No
subchronic or chronic p-RfC values can be derived for the following two reasons: there are no
adequate animal inhalation studies, and the epidemiology studies do not provide any
concentrations for 9,10-anthraquinone or demonstrate any definite relationship between
9,10-anthraquinone exposure and any toxic effect. The only inhalation animal study available
was Volodchenko et al. (1971); a foreign publication with an English translation provided by
ICI Americas (1985). Insufficient details on the study were provided (e.g., there were no
specifics on the strain, sex, or number of animals used, the compound details, or the methods of
inhalation exposure) to propose use of this study as a basis for derivation of any provisional
reference value.
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Table 4. Summary of Noncancer Reference Values for 9,10-Anthraquinone (CASRN 84-65-1)
Toxicity Type (units)
Species/Sex
Critical Effect
p-Reference
Value
PODa
Method
Pod
UFc
Principal Study
Screening subchronic
p-RfD
Rat/M+F
Liver, kidney, and
spleen lesions
0.01 mg/kg-day
NOAEL/
LOAEL
135
10,000
NTP (2005b)
Screening chronic p-RfD
(mg/kg-day)
Rat/M
Liver, kidney, and
spleen lesions
0.002
mg/kg-day
NOAEL/
LOAEL
20
10,000
NTP (2005b)
Subchronic p-RfC
(mg/m3)
None
None
None
None
None
None
None
Chronic p-RfC (mg/m3)
None
None
None
None
None
None
None
aPOD = point of departure.
Table 5. Summary of Cancer Values for 9,10-Anthraquinone (CASRN 84-65-1)
Toxicity Type
Species/Sex
Tumor Type
Cancer Value
Principal Study
p-OSF
Mouse/M
Hepatocellular adenoma, carcinoma,
or hepatoblastoma
0.04 (mg/kg-day) 1
NTP (2005b)
p-IUR
None
None
None
None
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CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR (WOE)
Table 6 identifies the cancer weight-of-evidence (WOE) descriptor for
9,10-anthraquinone.
Table 6. Cancer WOE Descriptor for 9,10-Anthraquinone
Possible WOE
Descriptor
Designation
Route of Entry
(Oral, Inhalation,
or Both)
Comments
"Carcinogenic to
Humans "
N/A
N/A
Two occupational studies (Sathiakumar and
Delzell, 2000; Barbone et al., 1992) found
statistically significant increases in lung cancer in
workers exposed to anthraquinone or
anthraquinone dyes, but the results did not
establish any definitive link with exposures, and
the studies did not evaluate smoking status in the
workers.
"Likely to Be
Carcinogenic to
Humans "
X
Oral
Under the Guidelines for Carcinogen Risk
Assessment (U.S. EPA, 2005), there is enough
evidence to suggest that 9,10-anthraquinone is
likely to be carcinogenic to humans based on
evidence of carcinogenicity in rats and mice in the
NTP (2005b) oral bioassays. There was some
controversy over the possibility that the
contaminant 9-NA was responsible for the
observed carcinogenicity in the NTP (2005b)
studies. However, the NTP subcommittee did not
believe the contaminant would be at a high enough
concentration to account for the increase in tumors
noted. In addition, there are no carcinogenicity
studies available for 9-NA, and the primary
metabolite, 2-hydroxyanthraquinone, was also
found to be mutagenic. Occupational studies
suggest carcinogenic potential via inhalation,
although the doses and routes were not controlled
or measured and other confounding factors were
not considered.
"Suggestive Evidence
of Carcinogenic
Potential"
N/A
N/A

"Inadequate
Information to Assess
Carcinogenic
Potential"
N/A
N/A

"Not Likely to Be
Carcinogenic to
Humans "
N/A
N/A
Positive cancer bioassay data exist.
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MODE-OF-ACTION DISCUSSION
The Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005) define mode of action
(MO A) as a sequence of key events and processes starting with the interaction of an agent with a
cell, proceeding through operational and anatomical changes, and resulting in cancer formation.
Examples of possible modes of carcinogenic action include mutagenic, mitogenic, antiapoptotic
(inhibition of programmed cell death), cytotoxic with reparative cell proliferation, and immune
suppression. The MO A of 9,10-anthraquinone-induced carcinogenicity has not yet been
determined.
Mutagenic Mode of Action
The majority of data on 9,10-anthraquinone indicate that 9,10-anthraquinone is not
mutagenic. 9,10-Anthraquinone may be clastogenic after longer-term exposure.
2-Hydroxyanthraquinone, a major metabolite of 9,10-anthraquinone, was found to be mutagenic
in an Ames assay, but cannot be established as the causative agent for the tumorigenicity
reported by NTP (2005b).
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
Because the mode of action of 9,10-anthraquinone is not known, the default linear
quantitative methodology was applied under the Guidelines for Carcinogen Risk Assessment
(U.S. EPA, 2005).
Derivation of Provisional Oral Slope Factor (p-OSF)
The carcinogenicity mouse study by NTP (2005b) is selected as the principal study for
derivation of the p-OSF. The cancer endpoint is the incidence of hepatocellular adenoma,
carcinoma, or hepatoblastoma in male mice. This study is well conducted, and the data from this
study are able to support a quantitative cancer dose-response assessment. This study is a
peer-reviewed technical report from the NTP, has been performed according to GLP principles,
and otherwise meets the standards of study design and performance with numbers of animals,
examination of carcinogenicity endpoints, and presentation of information. Details are provided
in the "Selection of Potentially Relevant Studies" section. Although there were concerns raised
about the presence of 9-NA and its possible contribution to the observed tumor incidence, the
NTP subcommittee concluded that the levels of 9-NA exposure, bioavailability, and relative
mutagenicity were not sufficient to cause the tumor incidence observed. The subcommittee also
stated that 2-hydroxyanthraquinone, a major metabolite of 9,10-anthraquinone, could account for
the pattern of tumorigenicity; 2-hydroxyanthraquinone was found to have a 7-fold greater
mutagenicity than 9-NA and occurred at a greater level in the urine than was theoretically
possible for 9-NA (NTP, 2005b). Therefore, NTP (2005b) concluded that the tumorigenicity
observed in the study was a result of 9,10-anthraquinone exposure, rather than the 2-NA
contaminant. The results from NTP (2005b) are used to derive the p-OSF.
NTP (2005b) provides the only acceptable carcinogenicity studies with increases in a
number of different tumor endpoints in male and female rats and mice. All relevant tumor
endpoints were modeled using EPA Benchmark Dose Software (BMDS version 2.1.1; U.S. EPA,
2009). The increase in hepatocellular adenoma, carcinoma, or hepatoblastoma in male mice
provided the lowest POD (BMDLio = 2.61 mg/kg-day).
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The following dosimetric adjustments were made for dietary treatment in adjusting doses
for oral cancer analysis:
HEDbmdl
Body-weight adjustment
BWh
BW
A
Body-weight adjustment
HEDbmdl
BMDLio x body-weight adjustment
(BWa - BWh)1 4
70 kg (human reference body weight) (U.S. EPA,
1997)
0.0373 kg (average body weight for male mice in
chronic study) (U.S. EPA, 1988)
(0.0373 -70)1/4 = 0.152
0.395 mg/kg-day (2.61 x 0.152)
Table 7 presents BMD input data for incidence of hepatocellular adenoma, carcinoma, or
hepatoblastoma in male mice administered 9,10-anthraquinone in the diet for 2 years.
Table 7. BMD Input for Incidence of Hepatocellular Adenoma, Carcinoma, or
Hepatoblastoma in Male Mice Exposed to Dietary 9,10-Anthraquinone for 2 Years"
Administered Dose
(mg/kg-day)
HEDb
(mg/kg-day)
Number of Animals
Response0
0
0
50
26 (52)
90
13.7
50
35 (70)*
265
40.3
50
43 (86)**
825
125.3
49
48 (98)**
aNTP (2005b)
bHuman equivalent dose (administered dose x 0.152)
°Number of rats with tumors, ()= percentage of rats with tumors
*p < 0.05, **p < 0.01.
Table 8 shows the modeling results for hepatocellular adenoma, carcinoma, or
hepatoblastoma in male mice. Table C.l provides BMD modeling results for all cancer
endpoints examined. Adequate model fit is obtained for the hepatocellular adenoma, carcinoma,
or hepatoblastoma incidence data using the multistage-cancer model. A benchmark response of
10% extra risk above the control mean was used to estimate the benchmark dose (BMD), as
recommended by EPA (2009). The BMD modeling results with 10% extra risk for
hepatocellular adenoma, carcinoma, or hepatoblastoma in male mice yield a BMDio of
3.8 mg/kg-day and a BMDLio of 2.6 mg/kg-day (see Table 8).
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Table 8. BMD Values for Cancer Data from Dichotomous Multistage-Cancer
BMD Model for 9,10-Anthraquinone for Derivation of the p-OSFa
Tumor Type
Species
Sex
BMDio
(mg/kg-day)
BMDL10
(mg/kg-day)
Goodness-of-Fit
p-Valueb
Conclusions
Hepatocellular
adenoma,
carcinoma, or
hepatoblastoma
Mouse/M
3.76
2.61
0.8591
Selected as POD
for p-OSF
aNTP (2005b)
bValues <0.10 fail to meet conventional goodness-of-fit criteria
Notes: BMD = benchmark dose; BMDL lower confidence limit (95%) on the benchmark dose; BMD10 and BMDL10
= BMD and BMDL response rate of 10% incidence, extra risk
The BMDS output details for the selected model are provided in Appendix C. The
BMDLio of 2.61 mg/kg-day from the 1-degree multistage model fit has been selected as the
POD.
p-OSF = 0.1 BMDLiohed
= 0.1^-2.6 mg/kg-day
= 0.0385 (mg/kg-day) 1 or 3.85 x 10 2 per (mg/kg-day) 1
The p-OSF rounds to 0.039 (mg/kg-day)-1 or 4 x 10~2 per (mg/kg-day)-1.
Derivation of Provisional Inhalation Unit Risk (p-IUR)
No human or animal studies investigating the carcinogenicity of 9,10-anthraquinone
following inhalation exposure have been identified. Therefore, derivation of a p-IUR is
precluded.
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APPENDIX A. PROVISIONAL SCREENING VALUES
For reasons noted in the main PPRTV document, it is inappropriate to derive a
provisional subchronic or chronic p-RfD for 9,10-anthraquinone. However, information is
available for this chemical which, although insufficient to support derivation of a provisional
toxicity value, under current guidelines, may be of limited use to risk assessors. In such cases,
the Superfund Health Risk Technical Support Center summarizes available information in an
Appendix and develops a "screening value." Appendices receive the same level of internal and
external scientific peer review as the PPRTV documents to ensure their appropriateness within
the limitations detailed in the document. Users of screening toxicity values in an appendix to a
PPRTV assessment should understand that there is considerably more uncertainty associated
with the derivation of an appendix screening toxicity value than for a value presented in the body
of the assessment. Questions or concerns about the appropriate use of screening values should
be directed to the Superfund Health Risk Technical Support Center.
DERIVATION OF SCREENING CHRONIC AND SUBCHRONIC ORAL REFERENCE
DOSES
Derivation of Screening Subchronic Provisional RfD (Subchronic p-RfD)
The subchronic study in rats by NTP (2005b) is selected as the principal study for
derivation of the screening subchronic p-RfD. The critical endpoint is pathology in several
organs, including the liver, kidney, and spleen, in male and female F344 rats. The lesions were
described as of mild to moderate severity at the LOAEL. The presence of a 100% response in
each of these organs at the lowest dose precludes conducting BMD modeling. This study is peer
reviewed and performed according to GLP principles and otherwise meets the standards of study
design and performance with numbers of animals, examination of potential toxic endpoints, and
presentation of information. Details are provided in the "Review of Potentially Relevant Data"
section. Among the available, acceptable studies, this study represents the lowest POD for
developing a subchronic p-RfD.
The POD in this study is a LOAEL of 135 mg/kg-day for liver, kidney, and spleen lesions
in male and female F344 rats.
No dosimetric adjustments were made for the dose in the principal study for dietary
treatment because the study authors report the average daily doses, and no animal-to-human
body-weight adjustment is used for oral noncancer assessments.
A screening subchronic p-RfD for 9,10-anthraquinone, based on 135 mg/kg-day in male
and female rats, is derived as follows:
Screening Subchronic p-RfD = LOAELadj ^ UFc
= 135 mg/kg-day ^ 10,000
= 0.01 mg/kg-day
The screening subchronic p-RfD rounds to 0.01 mg/kg-day or 1 x 10 per mg/kg-day.
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Table A. 1 summarizes the uncertainty factors for the screening subchronic p-RfD for
9,10-anthraquinone.
Table A.l. Uncertainty Factors for Screening Subchronic p-RfD
of 9,10-Anthraquinone
UF
Value
Justification
Notes
ufa
10
A UFa of 10 is applied for interspecies extrapolation to
account for potential toxicokinetic and toxicodynamic
differences between rats and humans. There are no data to
determine whether humans are more or less sensitive than
rats to 9,10-anthraquinone.

ufd
10
A UFd of 10 is selected because there are no acceptable
two-generation reproduction studies or developmental
studies. The available data do not suggest that additional
studies may reveal sensitive effects not yet characterized.
NTP (2005b) examined
and reported a lack of
effects on various sperm
parameters but some
effects on the female
estrous cycle.
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 to
humans.

ufl
10
A UFl of 10 is applied for using a POD based on a
LOAEL because a NOAEL cannot be determined from the
available database.
The doses employed in all
relevant animal studies
appear to be too high
because lesions are
present at high incidences
at the lowest dose tested.
UFC
<3000
10,000


Derivation of Screening Chronic Provisional RfD (Chronic p-RfD)
The study by NTP (2005b) is selected as the principal study for derivation of the
screening chronic p-RfD. The critical endpoint is pathology in several organs, including the
liver, kidney, and spleen, in male and female F344 rats. The presence of a near 100% response
in each of these organs at the lowest dose and the absence of biological response data at lower
doses preclude conducting a sensitivity analysis to distinguish dose-related responses in these
various organs. This study is peer reviewed and performed according to GLP principles and
otherwise meets the standards of study design and performance with numbers of animals,
examination of potential toxic endpoints, and presentation of information. A LOAEL is evident
at the lowest dose, with lesions noted in several organs of all treatment groups. Details are
provided in the "Review of Potentially Relevant Data section." The available response levels,
with near maximal response at the lowest dose tested for a number of systemic endpoints,
precludes BMD dose-response modeling.
The POD in this study is a LOAEL of 20 mg/kg-day in male rats (the LOAEL in female
rats is 25 mg/kg-day).
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No dosimetric adjustments were made for the dose in the principal study for dietary
treatment because the study authors reported the average daily doses, and no animal-to-human
body-weight adjustment is used for oral noncancer assessments.
The screening chronic p-RfD for 9,10-anthraquinone, based on 20 mg/kg-day in male
rats, is derived as follows:
Screening Chronic p-RfD = LOAELadj UFc
= 20 mg/kg-day ^ 10,000
= 0.002 mg/kg-day or 2 x 10~3 mg/kg-day
Table A.2 summarizes the uncertainty factors for the screening chronic p-RfD for
9,10-anthraquinone.
Table A.2. Uncertainty Factors for Screening Chronic p-RfD
of 9,10-Anthraquinone
UF
Value
Justification
Notes
ufa
10
A UFa of 10 is applied for interspecies extrapolation
to account for potential toxicokinetic and
toxicodynamic differences between rats and humans.
There are no data to determine whether humans are
more or less sensitive than rats to 9,10-anthraquinone.

ufd
10
A UFd of 10 is selected because there are no
acceptable two-generation reproduction studies or
developmental studies. The available data do not
suggest that additional studies may reveal sensitive
effects not yet characterized.
NTP (2005b) examined
and reported a lack of
effects on various sperm
parameters but some
effects on the female
estrous cycle.
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
to humans.

ufl
10
A UFl of 10 is applied for using a POD based on a
LOAEL because a NOAEL cannot be determined
from the available database.
The doses employed in all
relevant animal studies
appear to be too high
because lesions are
present at high incidences
at the lowest dose tested.
UFS
1
A UFS of 1 is applied because a chronic study was
utilized as the principal study.

UFC
<3000
10,000


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APPENDIX B. DATA TABLES
Table B.l. Body-Weight Gain and Food Consumption in Male and Female
F344/N Rats Exposed to Oral (in Feed) 9,10-Anthraquinone for 14 Weeks3
Endpoint
Average Daily Dose (mg/kg-day)
0
135
275
555
1130
2350
Males
Number of animals
10
10
10
10
10
10
Body-weight gain (g)
205 ± 4b
219 ±3
222 ±6
207 ±5
212 ±5
193 ±5
Food consumption Week 1 (g/animal/day)
15.0
14.7
13.9
12.6
12.9
12.2
Food consumption Week 14 (g/animal/day)
15.8
16.0
15.9
16.7
16.8
16.9
Females
Number of animals
10
10
10
10
10
10
Body-weight gain (g)
99 ±2
90 ±3**
80 ±2**
74 ± 2**
74 ±3**
65 ±2**
Food consumption Week 1 (g/animal/day)
10.9
9.4
8.6
7.3
6.1
6.0
Food consumption Week 14 (g/animal/day)
8.2
9.8
9.8
9.7
10.1
10.2
aNTP (2005b)
bMean ± standard error
**p < 0.01 (Williams orDunnett's test)
32
9,10-Anthraquinone

-------
FINAL
2-17-2011
Table B.2. Selected Hematology and Clinical Chemistry Parameters in Male and Female
F344/N Rats Exposed to Oral (in Feed) 9,10-Anthraquinone for 14 Weeks"
Endpoint
Average Daily Dose (mg/kg-day)
0
135
275
555
1130
2350
Males
Number of animals
10
10
10
10
10
10
Hemoglobin (g/dL)
16.3 ± 0.1b
14.9 ±0.2**
15.1 ±0.1**
14.7 ±0.2**
14.7 ±0.2**
14.9 ±0.2**
Erythrocytes (10'/|iL)
9.13 ± 0.10
8.02 ±0.09**
8.12 ± 0.11**
8.00 ±0.09**
8.03 ±0.15 **
8.29 ±0.10*
Reticulocytes (10'/|iL)
0.09 ±0.01
0.15 ±0.01**
0.15 ±0.01**
0.15 ± 0.01**
0.15 ±0.01**
0.17 ±0.02**
Mean cell volume (fL)
53.7 ±0.3
55.7 ±0.2**
56.2 ±0.3**
56.3 ±0.2**
56.0 ±0.4**
55.8 ±0.3**
Mean cell hemoglobin
concentration (g/dL)
33.3 ±0.2
33.4 ±0.2
33.1 ±0.2
32.8 ±0.2
32.9 ±0.2
32.3 ±0.1**
Platelets (107|iL)
668.3 ± 16.4
769.3 ±21.4
**
789.9 ±9.4
**
763.9 ± 15.1
**
824.4 ± 20.0
**
806.5 ±24.5
**
Urea nitrogen (mg/dL)
20.3 ±0.4
21.1 ± 0.5
21.3 ±0.3
22.1 ±0.6**
22.3 ±0.4**
22.4 ±0.5**
Creatinine (mg/dL)
0.67 ±0.02
0.71 ±0.01*
0.73 ±0.02**
0.73 ±0.02**
0.76 ±0.02**
0.74 ±0.02**
Total protein (g/dL)
6.7 ±0.1
7.2 ±0.1**
7.3 ±0.1**
7.4 ±0.1**
7.5 ±0.2**
7.8 ± 0.1**
Albumin (g/dL)
4.7 ±0.1
5.0 ±0.1**
5.1 ±0.1**
5.2 ± 0.1**
5.2 ±0.1**
5.3 ±0.1**
Alkaline phosphatase (IU/L)
624 ± 20
502±16**
478±14**
455±19**
442 ±21**
445 ±17**
Bile salts (|imolc/L)
20.3 ±0.8
13.5 ± 1.1**
10.7 ±0.5**
13.5 ±0.9**
11.6 ±0.8**
14.7 ±2.7**
Females
Number of animals
10
10
10
10
10
10
Hemoglobin (g/dL)
15.4 ±0.2
14.1 ±0.2**
14.4 ±0.1**
14.3 ±0.2**
14.3 ±0.1**
13.9 ±0.2**
Erythrocytes (106/|iL)
7.92 ±0.10
7.06 ±0.12**
7.41 ±0.04
7.39 ±0.07*
7.41 ±0.07*
7.24 ±0.11**
Reticulocytes (10'7|.iL)
0.10 ±0.01
0.20 ±0.02**
0.25 ±0.02**
0.26 ±0.02**
0.23 ±0.01**
0.25 ±0.02**
Mean cell volume (fL)
57.7 ±0.03
60.5 ±0.2**
59.3 ±0.2*
59.5 ±0.2*
59.6 ±0.3**
59.4 ±0.2**
Mean cell hemoglobin
concentration (g/dL)
33.7 ±0.3
33.0 ±0.1
32.7 ±0.1**
32.5 ±0.1**
32.4 ±0.2**
32.3 ±0.2**
Platelets (10:V|iL)
740.6 ± 11.7
804.6 ± 12.0
**
848.4 ± 14.6
**
839.9 ± 10.7
**
870.4 ± 11.2
**
874.9 ± 18.8
**
Urea nitrogen (mg/dL)
18.6 ±0.5
19.7 ±0.6
20.6 ±0.4*
18.5 ±0.5
18.9 ±0.4
19.5 ±0.6
Creatinine (mg/dL)
0.66 ±0.02
0.68 ±0.01
0.70 ±0.00*
0.72 ±0.01**
0.70 ±0.02*
0.71 ±0.02*
Total protein (g/dL)
6.5 ±0.1
7.2 ±0.1**
7.5 ±0.1**
7.9 ± 0.1**
7.9 ±0.1**
8.1 ±0.0**
Albumin (g/dL)
4.7 ±0.1
5.1 ± 0.1**
5.4 ±0.1**
5.5 ± 0.1**
5.6 ±0.1**
5.7 ±0.0**
Alkaline phosphatase (IU/L)
403 ± 20
330±10**
321±16**
293 ±17**
282±13**
274 ±12**
aNTP (2005b)
bValues are presented as mean ± standard errors
*p < 0.05 (Dunn's or Shirley's test)
**p < 0.01
33
9,10-Anthraquinone

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FINAL
2-17-2011
Table B.3. Selected Relative Organ Weights in Male and Female
F344/N Rats Exposed to Oral (in Feed) 9,10-Anthraquinone for 14-Weeksa'b
Endpoint
Average Daily Dose (mg/kg-day)
0
135
275
555
1130
2350
Males
Number of animals
10
10
10
10
10
10
Necropsy body
weight
338 ±5
349 ±3
347 ±5
331 ± 6
336 ±6
322 ±4*
Right kidney0
3.660 ±0.055
3.966 ±0.068
**
3.979 ±0.087
**
4.203 ± 0.057
**
4.324 ±0.092
**
4.537 ±0.088
**
Liver0
39.712 ±0.942
48.219 ± 1.008
**
53.535 ± 1.136
**
57.071 ±0.982
**
62.268 ± 1.053
**
69.315 ±0.755
**
Right testis0
4.335 ±0.039
4.396 ±0.066
4.501 ±0.039
4.636 ±0.092
**
4.605 ± 0.077
**
4.948 ± 0.075
**
Females
Number of animals
10
10
10
10
10
10
Necropsy body
weight
204 ±3
198 ±4
186 ±3**
182 ±2**
183 ±3**
174±1**
Right kidney0
3.476 ± 0.040
4.074 ± 0.048
**
4.378 ±0.032
**
4.347 ±0.068
**
4.526 ±0.071
**
4.891 ±0.076
**
Liver0
31.569 ±0.612
45.272 ±0.855
**
54.202 ± 1.282
**
60.189 ±0.842
**
62.101 ± 1.221
**
74.840 ± 1.011
**
aNTP (2005b)
bValues are presented as mean ± standard error
°Relative organ weights are presented as mg organ weight/g body weight
*p < 0.05 (Williams or Dunnett's test)
**p < 0.01
34
9,10-Anthraquinone

-------
FINAL
2-17-2011
Table B.4. Incidence of Selected Nonneoplastic Lesions in Male and Female
F344/N Rats Exposed to Oral (in Feed) 9,10-Anthraquinone for 14 Weeks3
Endpoint
Average Daily Dose (mg/kg-day)
0
135
275
555
1130
2350
Males
Liver
Hypertrophy
0/10
10/10**
10/10**
10/10**
10/10**
10/10**
Severity13
0
1.0
1.8
2.0
2.0
2.9
Kidney
Hyaline droplet accumulation
0/10
10/10**
10/10**
10/10**
10/10**
10/10**
Nephropathy severity13'0
1.0
1.7
1.6
1.7
2.0
2.2
Spleen
Congestion
0/10
10/10**
10/10**
10/10**
10/10**
10/10**
Hematopoietic cell proliferation
0/10
10/10**
9/10**
10/10**
10/10**
10/10**
Pigmentation
0/10
10/10**
10/10**
10/10**
10/10**
10/10**
Bone Marrow
Hyperplasia
0/10
3/10
5/10*
8/10**
6/10**
5/10*
Thyroid Gland
Follicular cell hypertrophy
0/10
0/10
10/10**
10/10**
10/10**
10/10**
Females
Liver
Hypertrophy
0/9
10/10**
10/10**
10/10**
10/10**
10/10**
Severity13
0
1.0
2.0
1.8
2.0
2.0
Kidney
Hyaline droplet accumulation
0/10
10/10**
10/10**
10/10**
10/10**
10/10**
Nephropathy
3/10
2/10
3/10
5/10
8/10*
10/10**
Spleen
Congestion
0/9
10/10**
10/10**
10/10**
10/10**
10/10**
Hematopoietic cell proliferation
0/9
10/10**
10/10**
10/10**
10/10**
10/10**
Pigmentation
0/9
10/10**
10/10**
10/10**
10/10**
10/10**
Bone Marrow
Hyperplasia
0/10
7/10**
7/10**
10/10**
9/10**
10/10*
Thyroid Gland
Follicular cell hypertrophy
0/10
0/10
10/10**
10/10**
10/10**
10/10**
Urinary Bladder
Inflammation
0/9
0/10
0/10
0/10
1/9
6/10**
Transitional epithelium hyperplasia
0/9
0/10
0/10
0/10
0/9
9/10**
aNTP (2005b)
bAverage severity grade: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked
°A11 the animals including all of the controls developed nephropathy
*p < 0.05 (Fischer's exact test)
**p < 0.01
35
9,10-Anthraquinone

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FINAL
2-17-2011
Table B.5. Selected Hematology Parameters in Male and Female B6C3Fi Mice
Exposed to Oral (in Feed) 9,10-Anthraquinone for 14-Weeksa
Males
Endpoint
Average Daily Dose (mg/kg-day)
0
250
500
1050
2150
4300
Number of animals
10
10
10
9
10
10
Hematocrit (%)
55.6 ± 1.2b
55.2 ±1.1
53.3 ±0.7
52.4 ±0.9
52.5 ± 1.3
49.4 ± 1.2**
Hemoglobin (g/dL)
17.8 ±0.2
17.9 ±0.3
17.3 ±0.2
17.1 ±0.2
17.4 ±0.3
16.7 ±0.3*
Erythrocytes
(106/|iL)
11.45 ±0.28
11.23 ±0.25
10.79 ±0.16
10.63 ±0.17
*
10.70 ±0.29
*
9.98 ±0.28**
Reticulocytes
(106/hL)
0.12 ±0.02
0.17 ±0.02
0.17 ±0.01*
0.16 ±0.01
0.20 ±0.02
**
0.20 ±0.03**
Mean cell volume
(Pg)
15.6 ±0.2
16.0 ±0.2
16.1 ±0.2
16.1 ±0.1
16.3 ±0.2*
16.8 ±0.2*
Mean cell
hemoglobin
concentration (g/dL)
32.0 ±0.3
32.5 ±0.4
32.5 ±0.2
32.7 ±0.3
33.3 ±0.2
**
33.8 ±0.3**
Platelets (10:V|iL)
844.1 ±34.4
888.8 ±35.6
951.5 ±53.6
896.1 ±34.0
1000.9 ±
53.8*
1005.5 ±26.5
**
Females
Endpoint
Average Daily Dose (mg/kg-day)
0
300
640
1260
2600
5300
Number of animals
10
10
10
10
10
10
Hematocrit (%)
49.8 ±0.3
48.3 ±0.3**
46.7 ±0.5**
47.8 ±0.5**
46.6 ±0.3**
45.6 ±0.6**
Hemoglobin (g/dL)
16.6 ±0.1
16.2 ±0.1*
15.8 ±0.1**
16.1 ±0.1**
15.9 ±0.2**
15.7 ±0.1**
Erythrocytes
(106/|iL)
10.32 ±0.05
9.77 ±0.05
**
9.46 ±
0.10**
9.64 ±
on**
9.44 ±0.06
**
9.09 ±0.09**
Reticulocytes
(106/hL)
0.10 ±0.01
0.16 ±0.02*
0.19 ±0.02
**
0.20 ± 0.02
**
0.19 ±0.02
**
0.26 ±0.02**
Mean cell volume
(fL)
48.2 ±0.1
49.3 ±0.2**
49.4 ±0.2**
49.7 ±0.2
**
49.3 ±0.2**
50.3 ±0.3**
Mean cell
hemoglobin (pg)
16.1 ±0.1
16.6 ±0.1**
16.7 ±0.1**
16.7 ±0.1
**
16.9 ±0.1**
17.3 ±0.1**
Mean cell
hemoglobin
concentration (g/dL)
33.3 ±0.2
33.7 ±0.1
33.8 ±0.1
33.7 ±0.2
34.1 ±0.2**
34.5 ±0.2**
Platelets (lO^L)
889.2 ±13.9
993.1 ± 16.5
**
971.9 ± 14.2
**
1012.1 ±31
**
1065.3 ±
18.5 **
1096.6 ± 18.3
**
aNTP (2005b)
bValues are presented as mean ± standard error
*p < 0.05 (Dunn's or Shirley's test)
**p < 0.01
36
9,10-Anthraquinone

-------
FINAL
2-17-2011
Table B.6. Selected Relative Organ Weights in Male and Female B6C3Fi Mice
Exposed to Oral (in Feed) 9,10-Anthraquinone for 14-Weeksa'

Males

Average Daily Dose (mg/kg-day)
Endpoint
0
250
500
1050
2150
4300
Number of
10
10
10
10
10
10
animals






Necropsy body
38.7 ±0.9
39.8 ±0.8
39.5 ± 1.0
39.6 ±0.7
37.0 ±0.6
37.8 ±0.6
weight






Right kidney0
7.563 ±
0.122
7.355 ±
0.154
7.901 ±0.336
7.548 ±0.174
7.943 ±0.174
8.518 ±
0.183**
Liver0
44.558 ±
49.669 ±
53.105 ±
59.312 ±
69.203 ±
80.206 ±

0.469
0.456**
0.546**
0.965**
0.947**
0.862**
Females

Average Daily Dose (mg/kg-day)
Endpoint
0
300
640
1260
2600
5300
Number of
10
10
10
10
10
10
animals






Necropsy body
29.8 ±0.6
32.3 ±0.8
31.3 ± 1.0
31.6 ±0.8
31.2 ± 0.5
30.3 ±0.8
weight






Liver0
40.110 ±
44.497 ±
49.332 ±
51.365 ±
60.412 ±
74.799 ±

1.029
0.753*
0.642**
0.729**
1.016**
2.121**
aNTP (2005b)
bValues are presented as mean ±	error
°Relative organ weights are presented as mg organ weight/g body weight
*p < 0.05 (Williams or Dunnett's test)
**p < 0.01
37
9,10-Anthraquinone

-------
FINAL
2-17-2011
Table B.7. Incidence of Selected Nonneoplastic Lesions in Male and Female
B6C3Fi Mice Exposed to Oral (in Feed) 9,10-Anthraquinone for 14-Weeksa
Males
Endpoint
Average Daily Dose (mg/kg-day)
0
250
500
1050
2150
4300
Liver
Centrilobular hypertrophy
0/10
1/10
9/10**
10/10**
10/10**
10/10**
Severity13
0
1.0
1.6
2.8
3.0
3.1
Urinary Bladder
Transitional epithelium, cytoplasmic
alteration
0/10
10/10**
10/10**
10/10**
10/10**
10/10**
Severity15
0
1.1
2.5
3.1
3.2
3.8
Spleen
Hematopoietic cell proliferation
0/10
6/10**
10/10**
10/10**
10/10**
9/10**
Pigmentation
0/10
10/10**
10/10**
10/10**
10/10**
9/10**
Females
Endpoint
Average Daily Dose (mg/kg-day)
0
300
640
1260
2600
5300
Liver
Centrilobular hypertrophy
0/10
2/10
5/10*
9/10**
7/10**
10/10**
Severity13
0
1.0
1.0
1.1
1.7
2.4
Urinary Bladder
Transitional epithelium, cytoplasmic
alteration
0/10
10/10**
10/10**
10/10**
10/10**
10/10**
Severity15
0
1.0
1.0
1.7
2.8
3.5
aNTP (2005b)
bAverage severity grade 1 = minimal, 2 = mild, 3 = moderate, 4 = marked
All the animals including all of the controls developed nephropathy
*p < 0.05 (Fischer's exact test)
**p < 0.01
38
9,10-Anthraquinone

-------
FINAL
2-17-2011
Table B.8. Body-Weight Gain and Incidence of Selected Nonneoplastic Lesions in Male
F344 Rats Exposed to Oral (in Feed) 9,10-Anthraquinone for 2 Years"
Endpoint
Average Daily Dose (mg/kg-day)
0
20
45
90
180
Body-weight gain
317b
293
286
301
289
Kidney
Hyaline droplet accumulation
3/50°
14/50**
10/50
16/50**
16/50**
Medulla mineralization
30/50
42/50**
46/50**
47/50**
49/50**
Transitional epithelium hyperplasia
28/50
45/50**
44/50**
48/50**
48/50**
Liver
Centrilobular hypertrophy
0/50
4/50
21/50**
13/50**
29/50**
Cystic degeneration
9/50
31/50**
36/50**
28/50**
29/50**
Inflammation
13/50
30/50**
28/50**
30/50**
27/50**
Eosinophilic focus
9/50
22/50**
30/50**
29/50**
20/50**
Mixed cell focus
4/50
12/50*
15/50**
13/50*
10/50
Cytoplasmic vacuolization
5/50
18/50**
23/50**
17/50**
23/50**
Spleen
Congestion
6/50
35/50**
37/50**
30/50**
31/50**
Pigmentation
12/50
36/50 **
38/50**
33/50**
28/50**
Bone Marrow
Hyperplasia
25/50
28/50
37/50*
36/50*
33/50
aNTP (2005b)
bCalculated from the weekly body-weight tables
°Number of animals with lesions/number of animals examined
*p < 0.05 (Fischer's exact test)
**p < 0.01
39
9,10-Anthraquinone

-------
FINAL
2-17-2011
Table B.9. Body-Weight Gain and Incidence of Selected Nonneoplastic Lesions in Female
F344 Rats Exposed to Oral (in Feed) 9,10-Anthraquinone for 2 Years
a

Average Daily Dose (mg/kg-day)
Endpoint
0
25
50
100
200
Body-weight gain
242b
204
188
184
170
Kidney
Hyaline droplet accumulation
33/50°
48/50**
45/50**
44/50**
44/50**
Nephropathy
39/50
49/50**
47/50*
49/50**
49/50**
Pigmentation
27/50
50/50**
48/50**
50/50**
47/49**
Medulla mineralization
17/50
25/50
27/50*
28/50*
20/49
Renal tubule hyperplasia
0/50
12/50**
13/50**
15/50**
11/49**
Transitional epithelium hyperplasia
0/50
5/50*
12/50**
3/50
10/50**
Liver
Centrilobular hypertrophy
0/50
18/50**
23/50**
19/50**
26/50**
Cystic degeneration
0/50
5/50*
10/50**
10/50**
6/49*
Inflammation
25/50
46/50**
44/50**
38/50*
46/50**
Eosinophilic focus
8/50
32/50**
34/50**
39/50**
34/50**
Mixed cell focus
3/50
30/50**
20/50**
23/50**
13/49**
Angiectasis
3/50
15/50**
18/50**
15/50**
21/49**
Spleen
Congestion
1/50
46/50**
42/50**
44/50**
45/50**
Pigmentation
33/50
45/50**
48/50**
48/50**
47/49**
Hematopoietic cell proliferation
39/50
50/50**
47/50*
47/50*
46/49*
Bone Marrow
Hyperplasia
19/50
31/50*
28/50
19/50
23/50
Atrophy
4/50
13/50*
13/50*
11/50
13/50*
aNTP (2005b)
bCalculated from the weekly body-weight tables
°Number of animals with lesions/number of animals examined
*p < 0.05 (Fischer's exact test)
**p < 0.01
40
9,10-Anthraquinone

-------
FINAL
2-17-2011
Table B.10. Incidence of Selected Neoplastic Lesions in Male F344 Rats

Exposed to Oral (in Feed) 9,10-Anthraquinone for 2 Years3



Human Equivalent Dose (mg/kg-day)b
Endpoint
0
5.4
12.2
24.4
48.9
Kidney
Renal tubule adenoma
1/50°
3/50
9/50*
5/50
3/50
Transitional epithelial papilloma
0/50
0/50
2/50
0/50
1/50
Urinary Bladder
Transitional epithelial papilloma
0/50
1/50
3/50
7/50
3/49
Liver
Hepatocellular adenoma or carcinoma
1/50
3/50
4/50
5/50
3/50
aNTP (2005b)
bHuman Equivalent Dose = Average daily dose x body-weight adjustment, e.g., (180 mg/kg-day) x
(0.38 kg 70 kg)(025)
°Number of animals with lesions/number of animals examined
*p <0.05 (Fischer's exact test)
**p < 0.01
Table B.ll. Incidence of Selected Neoplastic Lesions in Female F344 Rats
Exposed to Oral (in Feed) 9,10-Anthraquinone for 2-Yearsa


Human Equivalent Dose (mg/kg-day)b
Endpoint
0
6.0
12.0
23.9
47.8
Kidney
Renal tubule adenoma
0/50°
4/50
9/50**
7/50*
12/49**
Renal tubule adenoma or carcinoma
0/50
6/50*
9/50**
8/50**
14/49**
Liver
Hepatocellular adenoma
0/50
2/50
6/50*
4/50
3/50
Urinary bladder
Transitional epithelial papilloma or carcinoma
0/49
0/49
0/49
1/50
2/49
aNTP (2005b)
bHuman Equivalent Dose = Average daily dose x body-weight adjustment, e.g., (200 mg/kg-day) x
(0.229 kg - 70 kg)(0 25)
°Number of animals with lesions/number of animals examined
*p < 0.05 (Fischer's exact test)
**p < 0.01
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Table B.12. Body-weight Gain and Incidence of Selected Nonneoplastic Lesions in
Female B6C3Fi Mice Exposed to Oral (in Feed) 9,10-Anthraquinone for 2 Years"
Endpoint
Average daily dose (mg/kg-day)
0
80
235
745
Body-weight gain
37.8b
38.2
36.2
33.9
Liver
Centrilobular hypertrophy
1/49°
27/50**
22/50**
39/49**
Degeneration, fatty, focal
2/49
3/50
1/50
9/49*
Eosinophilic focus
6/49
15/50*
11/50
22/49**
Spleen
Hematopoietic cell proliferation
9/45
17/49
17/48
26/48**
Urinary Bladder
Intracytoplasmic inclusion body
0/44
40/48**
43/46**
46/48**
aNTP (2005b)
bCalculated from the weekly body-weight tables
°Number of animals with lesions/number of animals examined
*p < 0.05 (Fischer's exact test)
**p < 0.01
Table B.13. Body-Weight Gain and Incidence of Selected Nonneoplastic Lesions in
Male B6C3Fi Mice Exposed to Oral (in Feed) 9,10-Anthraquinone for 2-Yearsa
Endpoint
Average daily dose (mg/kg-day)
0
90
265
825
Body-weight gain
26.lb
24.5
24.2
16.2
Liver
Centrilobular hypertrophy
24/50°
34/50*
41/50**
33/50**
Degeneration, fatty, focal
0/50
7/50**
6/50*
0/50
Hepatocyte, erythrophagocytosis
1/50
9/50**
13/50**
6/49*
Eosinophilic focus
14/50
17/50
24/50*
20/49*
Focal necrosis
2/50
3/50
3/50
8/49*
Spleen
Hematopoietic cell proliferation
12/50
14/50
12/49
30/42
Urinary Bladder
Intracytoplasmic inclusion bodyd
0/50
46/49**
46/49**
42/45**
Thyroid Gland
Follicular cell hyperplasia
7/50
10/50
15/49
21/46**
aNTP (2005b)
bCalculated from the weekly body-weight tables
°Number of animals with lesions/number of animals examined
Statistical analysis was not reported in the study report. Fischer's exact test was conducted on the data.
*p < 0.05 (Fischer's exact test)
**p < 0.01
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Table B.14. Incidence of Selected Neoplastic Lesions in
Male B6C3Fi Mice Exposed to
Oral (in Feed) 9,10-Anthraquinone for 2 Years
a


Human Equivalent Dose (mg/kg-day)b
Endpoint
0
13.7
40.3
125.3
Liver
Hepatocellular adenoma
21/50°
32/50*
38/50**
41/50**
Hepatocellular carcinoma
8/50
13/50
17/50*
21/50**
Hepatoblastoma
1/50
6/50
11/50**
37/49**
Hepatocellular adenoma, carcinoma, or hepatoblastoma
26/50
35/50*
43/50**
48/49**
Thyroid Gland
Follicular cell adenoma
0/50
0/50
2/49
2/46
Total
Animals with malignant neoplasms
18/50
28/50*
33/50**
45/50**
aNTP (2005b)
bHuman Equivalent Dose = Average daily dose x body-weight adjustment, e.g.,
(825mg/kg-day) x (0.0373 kg - 70 kg)(025)
°Number of animals with lesions/number of animals examined
*p < 0.05 (Fischer's exact test)
**p < 0.01
Table B.15. Incidence of Selected Neoplastic Lesions in Female B6C3Fi Mice Exposed to
Oral (in Feed) 9,10-Anthraquinone for 2 Years"


Human Equivalent Dose (mg/kg-day)b
Endpoint
0
12.0
35.2
111.6
Liver
Hepatocellular adenoma
6/49°
28/50**
27/50**
40/49**
Hepatocellular carcinoma
2/49
3/50
8/50
8/49*
Hepatocellular adenoma or carcinoma
6/49
30/50**
30/50**
41/49**
Thyroid Gland
Follicular cell adenoma
1/45
1/48
2/48
2/48
Follicular cell carcinoma
0/45
0/48
0/48
2/48
Follicular cell adenoma or carcinoma
1/45
1/48
2/48
4/48
aNTP (2005b)
bHuman Equivalent Dose = Average daily dose x body-weight adjustment, e.g.,
(745 mg/kg-day) x (0.0353 kg - 70 kg)(0 25)
°Number of animals with lesions/number of animals examined
*p < 0.05 (Fischer's exact test)
**p < 0.01
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APPENDIX C. BMDS OUTPUTS
Table C.l. BMD Values for Cancer Data" from Dichotomous Multistage-Cancer Model
for 9,10-Anthraquinone for Derivation of the p-OSF
Tumor Type
Species/
Sex
BMDio
(mg/kg-day)
BMDL10
(mg/kg-day)
Goodness-of-Fit
p-Valueb
Conclusions
Hepatocellular
adenoma,
carcinoma, or
hepatoblastoma
Mouse/M
3.8
2.61
0.8591
Selected as POD
for p-OSF
Hepatocellular
adenoma or
carcinoma
Mouse/F
6.0
4.5
0.0005
Failed to meet
goodness of fit
All malignant
neoplasms
Mouse/M
7.0
5.1
0.6492
Maximum order
beta = 0
Hepatocellular
adenoma
Mouse/M
10.3
6.8
0.0835
Failed to meet
goodness of fit
Renal tubule
adenoma or
carcinoma
Rat/F
13.0
8.8
0.094
Failed to meet
goodness of fit
Hepatoblastoma
Mouse/M
18.1
11.2
0.5119

Hepatocellular
carcinoma
Mouse/M
37.5
21.0
0.4782

aNTP (2005b)
Values <0.10 fail to meet conventional goodness-of-fit criteria


BMD = benchmark dose; BMDL lower confidence limit (95%) on the BMD; BMDi0 and BMDL10 = BMD and
BMDL for a BMR of 10% extra risk
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Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
fSMDL
60
dose
21:37 06/01 2010
Figure C.l. Dichotomous Multistage-Cancer BMD Model for Hepatocellular Adenoma,
Carcinoma, or Hepatoblastoma in Male Mouse Data (NTP, 2005b)
Text Output for Dichotomous Multistage-Cancer BMD Model for Hepatocellular
Adenoma, Carcinoma, or Hepatoblastoma in Male Mouse Data (NTP, 2005b)
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: C:\27\NTP_2005_2yr_adcarchepato_mice_m_MultiCanc_l.(d)
Gnuplot Plotting File: C:\27\NTP_2005_2yr_adcarchepato_mice_m_MultiCanc_l.plt
Tue Jun 01 21:37:25 2010
Hepatocellular adenoma, carcinoma, or hepatoblastoma in Male B6C3F1 Mice at 2 yr
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl-beta2*dose/s2-beta3* dose^S)]
The parameter betas are restricted to be positive
45
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Dependent variable = DichPerc
Independent variable = Dose
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 4
Total number of specified parameters = 0
Degree of polynomial = 3
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background =	0.5 69271
Beta (1) = 0.0246892
Beta(2) =	0
Beta(3) =	0
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Beta(2) -Beta(3)
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Background	Beta(l)
Background	1	-0.5 6
Beta (1)	-0.56	1
Parameter Estimates
Interval
Variable
Limit
Background
Beta(1)
Beta(2)
Beta(3)
Estimate
0.535757
0.028002
0
0
Std. Err.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-90.2903
-90.4333
-108 .781
Param's
4
2
1
Deviance Test d.f.
0.286058
36.9812
P-value
0.8667
<.0001
AIC:
184.867
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Dose
Goodness of Fit
Est._Prob. Expected Observed	Size
Scaled
Residual
0.0000
13.7000
40.3000
125.3000
0.5358
0.6837
0.8498
0.9861
26.788
34.184
42.490
48.319
26.000
35.000
43.000
48.000
50
50
50
49
-0.223
0.248
0.202
-0.389
Chi^2 = 0.30
d.f. = 2
P-value = 0.8591
Benchmark Dose Computation
Specified effect =	0.1
Risk Type =	Extra risk
Confidence level =	0.95
BMD =	3.7 62 61
BMDL =	2.60971
BMDU =	8.06013
Taken together, (2.60971, 8.06013) is a 90	% two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor =	0.0383185
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