United States
Environmental Protection
1=1 m m Agency
EPA/690/R-15/01 OF
Final
9-25-2015
Provisional Peer-Reviewed Toxicity Values for
Picric Acid (2,4,6-Trinitrophenol)
(CASRN 88-89-1)
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 MANAGERS
Q. Jay Zhao, MPH, PhD, DABT
National Center for Environmental Assessment, Cincinnati, OH
Lucina E. Lizarraga, PhD
ORISE Postdoctoral Research Participant
CONTRIBUTORS
Dan D. Petersen, PhD, DABT
National Center for Environmental Assessment, Cincinnati, OH
Zhongyu (June) Yan, PhD
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
National Center for Environmental Assessment, Cincinnati, OH
PRIMARY INTERNAL REVIEWERS
Jason Lambert, PhD, DABT
National Center for Environmental Assessment, Cincinnati, OH
Jeff Swartout
National Center for Environmental Assessment, Cincinnati, OH
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 AND ACRONYMS iv
BACKGROUND 1
DISCLAIMERS 1
QUESTIONS REGARDING PPRTVs 1
INTRODUCTION 2
REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) 4
HUMAN STUDIES 7
Oral Exposures 7
Inhalation Exposures 7
ANIMAL STUDIES 7
Oral Exposures 7
Inhalation Exposures 9
OTHER DATA 9
DERIVATION 01 PROVISIONAL VALUES 14
DERIVATION OF ORAL REFERENCE DOSES 15
Derivation of a Subchronic Provisional RfD (Subchronic p-RfD) 15
Derivation of Chronic Provisional RfD (Chronic p-RfD) 17
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 17
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 17
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES 18
APPENDIX A. SCREENING PROVISIONAL VALUES 19
APPENDIX B. DATA TABLES 32
APPENDIX C. BENCHMARK DOSE MODELING RESULTS 38
APPENDIX D. REFERENCES 50
in
Picric Acid
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COMMONLY USED ABBREVIATIONS AND ACRONYMS
a2u-g
alpha 2u-globulin
MN
micronuclei
ACGIH
American Conference of Governmental
MNPCE
micronucleated polychromatic
Industrial Hygienists
erythrocyte
AIC
Akaike's information criterion
MOA
mode of action
ALD
approximate lethal dosage
MTD
maximum tolerated dose
ALT
alanine aminotransferase
NAG
N-acetyl-P-D-glucosaminidase
AST
aspartate aminotransferase
NCEA
National Center for Environmental
atm
atmosphere
Assessment
ATSDR
Agency for Toxic Substances and
NCI
National Cancer Institute
Disease Registry
NOAEL
no-observed-adverse-effect level
BMD
benchmark dose
NTP
National Toxicology Program
BMDL
benchmark dose lower confidence limit
NZW
New Zealand White (rabbit breed)
BMDS
Benchmark Dose Software
OCT
ornithine carbamoyl transferase
BMR
benchmark response
ORD
Office of Research and Development
BUN
blood urea nitrogen
PBPK
physiologically based pharmacokinetic
BW
body weight
PCNA
proliferating cell nuclear antigen
CA
chromosomal aberration
PND
postnatal day
CAS
Chemical Abstracts Service
POD
point of departure
CASRN
Chemical Abstracts Service Registry
PODadj
duration-adjusted POD
Number
QSAR
quantitative structure-activity
CBI
covalent binding index
relationship
CHO
Chinese hamster ovary (cell line cells)
RBC
red blood cell
CL
confidence limit
RDS
replicative DNA synthesis
CNS
central nervous system
RfC
inhalation reference concentration
CPN
chronic progressive nephropathy
RfD
oral reference dose
CYP450
cytochrome P450
RGDR
regional gas dose ratio
DAF
dosimetric adjustment factor
RNA
ribonucleic acid
DEN
diethylnitrosamine
SAR
structure activity relationship
DMSO
dimethylsulfoxide
SCE
sister chromatid exchange
DNA
deoxyribonucleic acid
SD
standard deviation
EPA
Environmental Protection Agency
SDH
sorbitol dehydrogenase
FDA
Food and Drug Administration
SE
standard error
FEVi
forced expiratory volume of 1 second
SGOT
glutamic oxaloacetic transaminase, also
GD
gestation day
known as AST
GDH
glutamate dehydrogenase
SGPT
glutamic pyruvic transaminase, also
GGT
y-glutamyl transferase
known as ALT
GSH
glutathione
SSD
systemic scleroderma
GST
glutathione-S-transferase
TCA
trichloroacetic acid
Hb/g-A
animal blood-gas partition coefficient
TCE
trichloroethylene
Hb/g-H
human blood-gas partition coefficient
TWA
time-weighted average
HEC
human equivalent concentration
UF
uncertainty factor
HED
human equivalent dose
UFa
interspecies uncertainty factor
i.p.
intraperitoneal
UFh
intraspecies uncertainty factor
IRIS
Integrated Risk Information System
UFS
subchronic-to-chronic uncertainty factor
IVF
in vitro fertilization
UFd
database uncertainty factor
LC50
median lethal concentration
U.S.
United States of America
LD50
median lethal dose
WBC
white blood cell
LOAEL
lowest-observed-adverse-effect level
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
PICRIC ACID (CASRN 88-89-1)
BACKGROUND
A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value
derived for use in the Superfund Program. PPRTVs are derived after a review of the relevant
scientific literature using established Agency guidance on human health toxicity value
derivations. All PPRTV assessments receive internal review by a standing panel of National
Center for Environment Assessment (NCEA) scientists and an independent external peer review
by three scientific experts.
The purpose of this document is to provide support for the hazard and dose-response
assessment pertaining to chronic and subchronic exposures to substances of concern, to present
the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to
characterize the overall confidence in these conclusions and toxicity values. It is not intended to
be a comprehensive treatise on the chemical or toxicological nature of this substance.
The PPRTV review process provides needed toxicity values in a quick turnaround
timeframe while maintaining scientific quality. PPRTV assessments are updated approximately
on a 5-year cycle for new data or methodologies that might impact the toxicity values or
characterization of potential for adverse human health effects and are revised as appropriate. It is
important to utilize the PPRTV database flittp://hhpprtv.ornl.gov) to obtain the current
information available. When a final Integrated Risk Information System (IRIS) assessment is
made publicly available on the Internet (http://www.epa.eov/iris). the respective PPRTVs are
removed from the database.
DISCLAIMERS
The PPRTV document provides toxicity values and information about the adverse effects
of the chemical and the evidence on which the value is based, including the strengths and
limitations of the data. All users are advised to review the information provided in this
document to ensure that the PPRTV used is appropriate for the types of exposures and
circumstances at the site in question and the risk management decision that would be supported
by the risk assessment.
Other U.S. Environmental Protection Agency (EPA) programs or external parties who
may choose to use PPRTVs are advised that Superfund resources will not generally be used to
respond to challenges, if any, of PPRTVs used in a context outside of the Superfund program.
This document has been reviewed in accordance with U.S. EPA policy and approved for
publication. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
QUESTIONS REGARDING PPRTVs
Questions regarding the contents and appropriate use of this PPRTV assessment should
be directed to the EPA Office of Research and Development's National Center for
Environmental Assessment, Superfund Health Risk Technical Support Center (513-569-7300).
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INTRODUCTION
Picric acid, CASRN 88-89-1, also known as 2,4,6-trinitrophenol, is a yellow, odorless
crystalline solid used in the manufacture of explosives, batteries, matches, and dyes for textiles.
Picric acid also has medical uses as an antiseptic and astringent. The chemical formula of picric
acid is C6H3N3O7 and its chemical structure is presented in Figure 1. A table of physicochemical
properties for picric acid is provided below (see Table 1).
o
N:
0
N +
O
Figure 1. Picric Acid Structure (CASRN 88-89-1)
Table 1. Physicochemical Properties of Picric Acid (CASRN 88-89-1)
Property (unit)
Value
Boiling point (°C)
300a
Melting point (°C)
122.5b
Density at 20°C (g/inL)
1.0a
Log P (unitless)
1.33b
Vapor pressure (lmnHg at 25°C)
7.5 x 10-?b
pH (unitless)
NV
Solubility in water (mg/L at 35°C)
1.27 x 104b
Relative vapor density (air = 1)
7.9a
Molecular weight (g/mol)
229.lb
aChemicalBook (2015).
bChemIDplus (2015).
NV = not available.
A summary of available toxicity values for picric acid from U.S. EPA and other
agencies/organizations is provided in Table 2.
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Table 2. Summary of Available Toxicity Values for Picric Acid (CASRN 88-89-l)a
Source/Parametera'b
Value (applicability)
Notes
Reference
Noncancer
ACGIH (TLV-TWA)
TLV-TWA: 0.1 mg/m3
TLV basis: skin
sensitization,
dermatitis, and eye
irritation
ACGIH (2015)
ATSDR
NV
NA
ATSDR (2015)
Cal/EPA
NV
NA
Cal/EPA (2014); Cal/EPA
(2015a): Cal/EPA (2015b)
NIOSH (REL, TWA)
REL = 0.1 mg/m3
TWA for up to a 10-h
workday
NIOSH (2015)
OSHA (PEL-TWA)
8-h PEL-TWA = 0.1 mg/m3
For skin
OSHA (2011): OSHA
(2006)
IRIS
NV
NA
U.S. EPA (2015)
DWSHA
NV
NA
U.S. EPA (2012)
HEAST
NV
NA
U.S. EPA (2011a)
CARA HEEP
NV
NA
U.S. EPA (1994)
WHO
NV
NA
WHO (2015)
Cancer
IRIS
NV
NA
U.S. EPA (2015)
HEAST
NV
NA
U.S. EPA (2011a)
IARC
NV
NA
IARC (2015)
NTP
NV
NA
NTP (2014)
Cal/EPA
NV
NA
Cal/EPA (2015a): Cal/EPA
(2011): Cal/EPA (2015b)
aSources: ACGIH = American Conference of Governmental Industrial Hygienists; ATSDR = Agency for
Toxic Substances and Disease Registry; Cal/EPA = California Environmental Protection Agency;
CARA = Chemical Assessments and Related Activities; DWSHA = Drinking Water Standards and Health
Advisories; HEAST = Health Effects Assessment Summary Tables; HEEP = Health and Environmental
Effects Profile; IARC = International Agency for Research on Cancer; IRIS = Integrated Risk Information
System; NIOSH = National Institute for Occupational Safety and Health; NTP = National Toxicology
Program; OSHA = Occupational Safety and Health Administration; WHO = World Health Organization.
Parameters: PEL-TWA = permissible exposure limit-time weighted average; REL = recommended
exposure limit; TLV-TWA = threshold limit value-time weighted average.
NV = not available; NA = not applicable.
Literature searches were conducted on sources published from 1900 through August 2015
for studies relevant to the derivation of provisional toxicity values for picric acid. The following
databases were searched by chemical name, synonyms, or CASRN: ACGIH, ANEUPL, ATSDR,
BIOSIS, Cal/EPA, CCRIS, CDAT, ChemlDplus, CIS, CRISP, DART, EMIC, EPIDEM,
ETICBACK, FEDRIP, GENE-TOX, HAPAB, HERO, HMTC, HSDB, IARC, INCHEM IPCS,
IP A, ITER, IUCLID, LactMed, NIOSH, NTIS, NTP, OSHA, OPP/RED, PESTAB, PPBIB,
PPRTV, PubMed (toxicology subset), RISKLINE, RTECS, TOXLINE, TRI, U.S. EPA IRIS,
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U.S. EPA HEAST, U.S. EPA HEEP, U.S. EPA OW, and U.S. EPA TSCATS/TSCATS2. The
following databases were searched for toxicity values or exposure limits: ACGIH, ATSDR,
Cal/EPA, U.S. EPA IRIS, U.S. EPA HEAST, U.S. EPA HEEP, U.S. EPA OW, U.S. EPA
TSCATS/TSCATS2, NIOSH, NTP, OSHA, and RTECS.
REVIEW OF POTENTIALLY RELEVANT DATA
(NONCANCER AND CANCER)
Tables 3A and 3B provide an overview of the relevant database for picric acid and
include all potentially relevant repeated-dose, short-term-, subchronic-, and chronic-duration
studies. Principal studies are identified in bold. The phrase "statistical significance," used
throughout the document, indicates ap-walue < 0.05, unless otherwise noted.
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Table 3A. Summary of Potentially Relevant Noncancer Data for Picric Acid (CASRN 88-89-1)
Category
Number of Male/Female,
Strain, Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAELa
BMDLb
LOAELa
Reference
(comments)
Notes0
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
Animal
1. Oral (mg/kg-d)
Short-termd
6 M/6 F, S-D rat, picric acid
administered by gavage,
newborn study, 18 d
ADD: 0,
4.1, 16.3, or
65.1
Increased relative and absolute liver weight in
males and females
16.3
41.2
Relative
liver weight
in males
65.1
Takahashi et
al. (2004)
PR
6 M/6 F, S-D rat, picric acid
administered by gavage,
young rat study, 28 d
ADD: 0,4,
20, or 100
Increased liver weights, hematological and
related splenic effects (increased spleen weights
and hematopoiesis) in males and females and
testicular effects (testicular atrophy, and
decreased sperm in the epididymis) in males
20
17.3
Absolute
spleen
weight in
males
100
Takahashi
PR,
PS
et al. (2004)
Subchronic
ND
Chronic
ND
Developmental
ND
Reproductive
ND
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Table 3A. Summary of Potentially Relevant Noncancer Data for Picric Acid (CASRN 88-89-1)
Category
Number of Male/Female,
Strain, Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAELa
BMDLb
LOAELa
Reference
(comments)
Notes0
2. Inhalation (mg/m3)a
ND
dosimetry: values were reported by the study authors as adjusted daily doses (ADD, in mg picric acid/kg-day).
bBenchmark dose (BMD) analyses were conducted using the U.S. EPA's Benchmark Dose Software (BMDS Version 2.4); doses are in units of mg picric acid/kg-day.
°Notes: PS = principal study; PR = peer reviewed.
•'Short-term = repeated exposure for >24 hours <30 days (U.S. EPA. 20021.
ND = no data; S-D = Sprague-Dawley.
Table 3B. Summary of Potentially Relevant Cancer Data for Picric Acid (CASRN 88-89-1)
Category
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry
Critical Effects
NOAEL
BMDL/
BMCL
LOAEL
Reference
(comments)
Notes
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
Animal
1. Oral (mg/kg-d)
ND
ND
2. Inhalation (mg/m3)
ND = no data.
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HUMAN STUDIES
Oral Exposures
The following effects have been reported after acute oral exposure to >28 mg/kg of picric
acid: headache, vertigo, nausea, vomiting, diarrhea, myalgia, yellow coloration of the skin,
hematuria, albuminuria, and at high doses, destruction of erythrocytes, hemorrhagic nephritis,
and hepatitis (ACGUI, 2015; NIOSH, 2015). No quantitative data have been found on the
toxicity of picric acid to humans following chronic- or sub chronic-duration oral exposure.
Inhalation Exposures
Acute inhalation of high concentrations of picric acid dust has caused temporary coma
followed by weakness, myalgia, anuria, and later polyuria in one worker (NIOSH. 2015). No
relevant data have been found on the toxicity of picric acid to humans following chronic- or
subchronic-duration inhalation exposure.
ANIMAL STUDIES
Oral Exposures
The effects of oral exposure of animals to picric acid were evaluated in two short-term
toxicity studies (Takahashi et aL 2004).
Short-Term-Duration Studies
Takahashi et al. (2004)
In a peer-reviewed, short-term-duration, toxicity study performed by Takahashi et al.
(2004) picric acid was suspended in a 0.5% carboxymethyl cellulose sodium salt aqueous
solution with 0.1% Tween-80 (purity: 81.4%) and given to 6 pup Sprague-Dawley (S-D)
rats/sex/dose daily via gavage. Test sample impurities included: 18.5% (w/w) water and 0.008%
(w/w) sulfuric acid (based on personal communication with the study corresponding author).
The study authors reported administered doses of 0, 4.1, 16.3, or 65.1 mg (as picric acid)/kg-day
to pups from Postnatal Day (PND) 4 to PND 21 (18 days). Pups in the main study were
sacrificed on PND 22. Another 6 pups/sex/dose in the maintenance-recovery groups were given
the same dosages for 18 days, then maintained for 9 weeks without chemical treatment and
sacrificed on PND 85. Twelve foster mothers were used to suckle the pups up to PND 22.
Animals were allowed free access to a sterilized basal diet (MF, Oriental Yeast, Tokyo, Japan)
after weaning. Animals were maintained in an environmentally controlled room at 24 ± 2°C
with a relative humidity of 55 ± 10% and a 12:12 hour light/dark cycle. The study authors
reported using good laboratory practice (GLP) principles.
General condition was observed twice daily for pups and foster mothers during the
dosing period and daily for pups during the recovery-maintenance period. All pups were
examined for developmental landmarks such as pinna detachment (PND 4), piliation (PND 8),
incisor eruption (PND 10), gait and eye opening (PND 15), testes descent (PND 21), and
preputial separation and/or vaginal opening (PND 42). Body weights were recorded and food
consumption was determined at least twice per week. Body weights were also measured on the
day of testes descent and preputial separation and/or vaginal opening. Blood was collected from
the abdominal vein on the day of sacrifice, and the following hematological parameters were
evaluated: erythrocyte count (RBC), hematocrit (Hct), hemoglobin (Hb), mean corpuscular
hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular
volume (MCV), total leukocyte count (WBC), differential leukocyte count, platelet count
(PLAT), mean platelet volume (MPV), cell morphology, prothrombin time (PT), and activated
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partial thromboplastin time (APTT). The following clinical chemistry parameters were also
examined: total protein (TP), triglycerides (TRI), albumin (A), globulin (G), albumin/globulin
ratio (A/G), glucose (GLU), cholesterol (CHOL), total bilirubin (TBILI), blood urea nitrogen
(BUN), creatinine (CREAT), alanine aminotransferase (ALT), aspartate aminotransferase (AST),
alkaline phosphatase (ALK), calcium (Ca), phosphorus (PHOS), sodium (Na), potassium (K),
and chloride (CI). After gross examination, the liver, kidney, spleen, thymus, pituitary gland,
adrenals, lungs, gonads, heart, and brain were weighed. Tissue samples from these organs were
also fixed, sectioned, and histologically examined.
No treatment-related effects were noted on food consumption, mortality, or behavior in
the main study. Yellowish fur was observed in all picric acid-treated rats but not in controls.
The study authors reported a statistically significant decrease in body weight on Days 4 and 8 of
the dosing period (max. 7% decrease) for males in the 65.1-mg/kg-day group (data not presented
in original publication). However, terminal body weights for treated groups in the main study
were not statistically different from controls (see Table B-l). No dose-dependent effects on
body weight or food consumption were observed during the maintenance-recovery period. As
shown in Table B-l, males and females in the 65.1-mg/kg-day dose groups showed statistically
significant increases (13 and 12%, respectively) in relative liver weights (liver-to-body weight
ratio) compared to controls. Although not statistically significant, absolute liver weights were
also increased in males and females in the 65.1-mg/kg-day dose groups (10% and 12%,
respectively). No other treatment-related organ weight effects were observed. Developmental
landmarks and sexual maturation were similar in treated and control groups. No
treatment-related changes in hematological parameters, urinalysis, clinical chemistry
measurements, or histopathological findings were reported in males or females. Based on
increased absolute and relative liver weights, the high dose of 65.1 mg/kg-day is considered the
lowest-observed-adverse-effect level (LOAEL) and the mid dose of 16.3 mg/kg-day is identified
as the corresponding no-observed-adverse-effect level (NOAEL) for both male and female rats.
In a separate study by Takahashi et al. (2004). picric acid was suspended in a 0.5%
carboxymethyl cellulose sodium salt aqueous solution with 0.1% Tween-80 (purity: 81.4%) and
given to young (5-week-old) S-D rats (6/sex/dose) daily via gavage. Test sample impurities
include: 18.5% (w/w) water and 0.008% (w/w) sulfuric acid (based on personal communication
with the study corresponding author). The study authors reported administering doses of 0, 4,
20, or 100 mg (as picric acid)/kg-day to rats in the main study for 28 days. Animals were
sacrificed the next day following an overnight fast. Another 6 rats/sex/dose in the
maintenance-recovery groups were given 0 or 100 mg/kg-day picric acid starting on Week 5 for
a total of 28 days, then maintained for 2 weeks without chemical treatment and sacrificed on
Week 11. Animals were allowed free access to a sterilized basal diet (MF, Oriental Yeast,
Tokyo, Japan) after weaning. Animals were examined for general condition, body weight, organ
weight, food consumption, urinalysis, hematology, blood biochemistry, necropsy, and
histopathological findings as described for the newborn study.
There were no treatment-related effects on mortality, food consumption or body weight
during the dosing or maintenance-recovery periods. Yellowish fur was observed in all picric
acid-treated rats but not in controls. As shown in Table B-2, there were statistically significantly
higher WBC and reticulocyte (Ret) counts and lower RBC and Hb levels in males at
100 mg/kg-day. In females exposed to the highest dose, there were statistically significant
increases in WBC, Ret, MCV, and lower RBC, Hb, and MCHC.
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Statistically significant changes in relative liver weight (12% increased), absolute spleen
weight (44% increased), relative spleen weight (45% increased), absolute epididymis weight
(23%) decreased), and relative epididymis weight (23% decreased) were observed at the end of
the dosing period in males at 100 mg/kg-day only (see Table B-3). In contrast, the only
statistically significant changes at the end of the maintenance-recovery period were in absolute
epididymis weight (25% decreased) and relative epididymis weight (17% decreased) in males at
the 100-mg/kg-day dose. In females, there were statistically significant increases in relative liver
weight (23%>), absolute spleen weight (92%), and relative spleen weight (100%) at the end of the
28-day dosing period at the highest dose only (see Table B-3). No statistically significant
changes in organ weight were observed in females at the end of the maintenance-recovery
period. Although statistically significant changes in absolute liver weight were not observed in
exposed male and female rats, biologically significant (>10%) increases occurred in the
high-dose group for both sexes. No other organ weight changes were reported. Statistically
significant histopathological changes occurred in males at the highest dose at the end of the
dosing period and included development of germinal centers and extramedullary hematopoiesis
in the spleen, testicular atrophy, and decreased sperm in the epididymis (see Table B-4). In
females at 100 mg/kg-day there was development of germinal centers, extramedullary
hematopoiesis, and hemosiderin deposition in the spleen at the end of the dosing period
(see Table B-5). At the end of the maintenance-recovery period, only hemosiderin deposition in
the spleen of both males and females and testicular atrophy in males were observed at
100 mg/kg-day (data not shown). No other changes were reported. Based on hematological and
related splenic effects, increased liver weights and testicular effects, the high dose of
100 mg/kg-day is identified as the LOAEL and the mid dose of 20 mg/kg-day is the
corresponding NOAEL.
Subchronic-Duration Studies
No studies have been identified.
Chronic-Duration Studies
No studies have been identified.
Reproductive Studies
No studies have been identified.
Developmental Studies
No studies have been identified.
Inhalation Exposures
No inhalation studies have been identified on the sub chronic-duration, chronic-duration,
developmental, or reproductive toxicity or on the carcinogenicity of picric acid in animals.
OTHER DATA
Other studies that utilized picric acid are described here. These studies are not adequate
for the determination of a provisional reference dose (p-RfD), provisional reference
concentration (p-RfC), provisional oral slope factor (p-OSF), or provisional inhalation unit risk
(p-IUR) values but provide supportive data supplementing a weight-of-evidence approach.
Table 4A provides an overview of genotoxicity studies while Table 4B provides an overview of
other supporting studies on picric acid, including mechanistic and toxicokinetic studies.
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Table 4A. Summary of Picric Acid (CASRN 88-89-1) Genotoxicity
Resultsb
Endpoint
Test System
Dose/Concentration3
Without
Activation
With
Activation
Comments
References
Genotoxicity studies in prokaryotic organisms
Mutation
Salmonella typhimurium
strains TA98, TA100,
TA1535, TA1537
(Activation using male S-
D rat liver S9 induced
with Aroclor 1254)
0-100 |-ig/platc
(")
TA98, TA100,
TA1535,
TA1537
(")
TA1535
(±)
TA100
(+)
TA98,
TA1537
Haworth et al.
(1983)
Mutation
Salmonella typhimurium
strains TA98, TA100,
TA1535, TA1537
(Activation using male
Syrian hamster liver S9
induced with
Aroclor 1254)
0-100 |-ig/platc
(")
TA98, TA100,
TA1535,
TA1537
(")
TA1535,
TA100
(+)
TA98,
TA1537
Haworth et al.
(1983)
Genotoxicity studies in nonmammalian eukaryotic organisms
Mutation
ND
Recombination induction
ND
CA
ND
Chromosomal
malsegregation
ND
Mitotic gene conversion
ND
Mitotic arrest
ND
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Table 4A. Summary of Picric Acid (CASRN 88-89-1) Genotoxicity
Resultsb
Endpoint
Test System
Dose/Concentration3
Without
Activation
With
Activation
Comments
References
Genotoxicity studies in mammalian cells—in vitro
CAs
Chinese hamster ovary
cells
0, 600, 800, l,000c (-S9)
0, 1,740, 2,485, 3,500, 5,000c
(+S9)
NTP (1985)
SCE
Chinese hamster ovary
cells
0, 50, 167, 500, 1,700° (-S9)
0, 167, 500, 1,670, 5,000c (+S9)
+
NTP (1985)
MN induction
ND
DNA damage (Comet
assay)
ND
DNA adducts
ND
Genotoxicity studies—in vivo
Mutagenicity (eye
wlw + assay)
ND
Mutagenicity (Wing spot
test)
ND
Mouse bone marrow
micronucleus test
ND
CAs
ND
SCE
ND
DNA damage
ND
DNA adducts
ND
Mouse biochemical or
visible specific locus test
ND
Dominant lethal
ND
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Table 4A. Summary of Picric Acid (CASRN 88-89-1) Genotoxicity
Resultsb
Endpoint
Test System
Dose/Concentration3
Without With
Activation Activation
Comments
References
Sex-linked recessive lethal
assay
Drosophila melanogaster
0, 450d (feeding)
0, 400d (injection)
0, 300, 500, 1,000, l,500d
(feeding)
0, 1,000, l,500d (injection)
0, l,250d (feeding)
0, l,500d (injection)
(feeding; injection)
(feeding; injection)
- (feeding)/+ (injection)
Data represent results from
three different laboratories.
All laboratories obtained
negative results from feeding
studies; however, exposure
after injection yielded
positive results in one
laboratory. The study
authors also noted that when
all experimental data are
combined, the findings
compared to controls are
significant (p = 0.02).
Woodruff et al.
(1985)
Reciprocal translocation
Drosophila melanogaster
0, l,500d (injection)
-
Woodruff et al.
(1985)
Genotoxicity studies in subcellular systems
DNA binding
ND
"Lowest effective dose for positive results, highest dose tested for negative results.
b+ = positive, (+) = weak positive, - = negative, ± = equivocal, NA = not applicable, ND = no data; NR = not reported.
"Picric acid concentrations expressed as |ig/mL.
dPicric acid concentrations expressed as parts per million.
ND = no data.
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Table 4B. Mechanistic and Other Studies of Picric Acid (CASRN 88-89-1) Exposure
Test
Materials and Methods
Results
Conclusions
References
Human studies
No studies were located regarding the toxicity or carcinogenicity of picric acid in humans.
Animal toxicity studies
Immunotoxicity
ND
Neurotoxicity
ND
Studies of absorption, distribution, metabolism, or elimination (ADME)
ADME
Blood and urine samples were
collected from F344 rats treated
via gavage with a single dose of
[14C] picric acid (100 mg/kg).
The following metabolites were isolated from urine: Y-acetylisopicramic acid (14.8%),
picramic acid (18.5%), \ -acctylpicramic acid (4.7%), and unidentified components
(2.4%). Most of the parent compound (60%) was excreted unchanged. The plasma
half-life for picric acid was 13.4 h with a gut absorption coefficient (ka) of 0.069 h
24 h postadministration of [14C] picric acid, the primary depots of radioactivity (per
gram tissue basis) were blood, spleen, kidney, liver, lung, and testes.
Wvmanet al. (1992)
Studies of mode of action/mechanism/therapeutic action
Mode of
action/mechanistic
ND
ND = no data.
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DERIVATION OF PROVISIONAL VALUES
Tables 5 and 6 present summaries of noncancer and cancer reference values, respectively.
Table 5. Summary of Noncancer Reference Values for Picric Acid (CASRN 88-89-1)
Toxicity Type (units)
Species/Sex
Critical Effect
p-Reference
Value
POD Method
PODiin)1
UFc
Principal Study
Subchronic p-RfD (mg/kg-d)
Rat/M
Increased absolute spleen
weight
1 X 10-2
BMDLi sd
4.2
300
Takaliaslii et al.
(2004)
Screening Chronic p-RfD (mg/kg-d)
Rat/M
Increased MetHb
9 x 1(T4
BMDLi sd
0.276 (based on
surrogate PODhed)
300
Reddv et al. (2001a):
Reddv et al. (1997)
Subchronic p-RfC (mg/m3)
NDr
Chronic p-RfC (mg/m3)
NDr
aHED expressed in mg/kg-d.
NDr = not determined.
Table 6. Summary of Cancer Values for Picric Acid (CASRN 88-89-1)
Toxicity Type
Species/Sex
Tumor Type
Cancer Value
Principal Study
Provisional Oral Slope Factor (p-OSF) (mg/kg-d) 1
NDr
Provisional Inhalation Unit Risk (p-IUR) (mg/m3)-1
NDr
NDr = not determined.
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DERIVATION OF ORAL REFERENCE DOSES
The database of oral toxicity studies for picric acid includes two short-term-duration
toxicity studies in rats, both of which were conducted by Takahashi et al. (2004). Both of these
studies were peer-reviewed and employed GLP guidelines. In the 18-day newborn-rat study, a
NOAEL of 16.3 mg/kg-day and a LOAEL of 65.1 mg/kg-day were identified for both males and
females based on increased absolute and relative liver weight. No treatment-related
histopathological findings were reported in the liver or any other organ examined. In the 28-day
young-rat study, a NOAEL of 20 mg/kg-day and a LOAEL of 100 mg/kg-day were identified for
males and females based on splenic, hematological, testis and liver effects.
Benchmark dose (BMD) analyses were conducted on the liver weight data from the
newborn rat study using the U.S. EPA's Benchmark Dose Software (BMDS Version 2.4).
Results of BMD modeling are summarized in Appendix C. The lowest benchmark dose lower
confidence limit (BMDL) identified from the newborn rat study is 31.8 mg/kg-day based on
increased absolute liver weight in males (see Table C-l).
Benchmark dose analyses were also conducted on the statistically significant blood and
organ weight data from the young rat study. Although statistically significant, histopathological
data from the young rat study are not amenable to BMD modeling because no clear
dose-response trend is observed with these data. Splenic lesions only occurred in males and
females at the highest treatment dose (100 mg/kg-day) and testicular lesions in males were also
reported at this dose. The lowest BMDL identified from the young rat study is 14.0 mg/kg-day
based on increased WBC count in males; however, the biological significance of the
corresponding benchmark response (1 standard deviation [SD]) for this endpoint is not clear
(see Table C-2). The next lowest BMDL from the young rat study is 17.3 mg/kg-day based on
increased absolute spleen weight in males. While spleen weights in male and female rats were
most prominently increased at the highest dose (100 mg/kg-day), slight elevations also occurred
at lower doses. Furthermore, trend test analyses revealed that treatment-related increments in
absolute spleen weight in males were highly significant (ANOVA contrast with equally spaced
coefficients; trend p = 9.6 x 10~5). Consistent findings of decreases in RBC and Hb levels in
both male and female rats, increases in absolute and relative spleen weights, and multiple
histopathological findings on the spleen suggest a treatment-induced hematological response and
point to the spleen as the major target organ. Thus, the BMDL of 17.3 mg/kg-day based on
increased absolute spleen weight in males from the young rat study is selected as the point of
departure (POD) for derivation of the subchronic provisional reference dose (p-RfD).
Derivation of a Subchronic Provisional RfD (Subchronic p-RfD)
EPA endorses a hierarchy of approaches to derive human equivalent oral exposures from
data from laboratory animal species, with the preferred approach being physiologically based
toxicokinetic modeling. Another approach may include using chemical-specific information,
including what is known about the toxicokinetics and toxicodynamics of the chemical, to derive
chemical-specific adjustments. In lieu of chemical-specific information to derive human
equivalent oral exposures, EPA endorses body-weight scaling to the 3/4 power (i.e., BW3/4) as a
default to extrapolate toxicologically equivalent doses of orally administered agents from all
laboratory animals to humans for the purpose of deriving an RfD under certain exposure
conditions (U.S. EPA. 2011b). More specifically, the use of BW3 4 scaling for deriving an RfD
is recommended when the observed effects are associated with the parent compound or a stable
metabolite but not for portal-of-entry effects. Because the selected critical effect is increased
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absolute spleen weight in male rats, the use of BW3/4 scaling to obtain a human equivalent dose
(HED) is considered appropriate in this case.
Following EPA guidance, the POD for the rat 28-day study (Takahashi et al.. 2004) is
converted to an HED through an application of a dosimetric adjustment factor (DAF) derived as
follows:
DAF = (BWa1/4 - BWh1/4)
where:
DAF = dosimetric adjustment factor
BWa = animal body weight
BWh = human body weight
Using a BWa of 0.25 kg for rats and a standard BWh of 70 kg for humans the resulting
DAF is 0.24. Applying this DAF to the BMDLisd obtained from modeling the absolute spleen
weight data from the 28-day young rat study yields a BMDLisdhed as follows:
BMDLisdhed for picric acid = BMDLisd (mg/kg-day) x DAF
= 17.3 (mg/kg-day) x 0.24
= 4.2 mg/kg-day
The subchronic p-RfD for picric acid, based on the BMDLisdhed of 4.2 mg/kg-day for
increased absolute spleen weight in male rats, is derived as follows:
Subchronic p-RfD for picric acid = BMDLisdhed ^ UFc
= 4.2 mg/kg-day -^300
= 1 x 10"2 mg/kg-day
Table 7 summarizes the uncertainty factors for the subchronic p-RfDs for picric acid.
Table 7. Uncertainty Factors for the Subchronic p-RfD for Picric Acid (CASRN 88-89-1)
UF
Value
Justification
UFa
3
A UFa of 3 (100 5) is applied to account for remaining uncertainty (e.g., the toxicodynamic
differences between rats and humans) following oral picric acid exposure. The toxicokinetic
uncertainty has been accounted for by calculation of a human equivalent dose (HED) through
application of a dosimetric adjustment factor (DAF) as outlined in the EPA's Recommended Use of
Bodv Weisht3/4 as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA. 2011b).
UFd
10
A UFd of 10 is applied because there are no acceptable developmental or two-generation
reproductive toxicity studies although there is limited examination of reproductive parameters in the
new born rat study. In addition, the database lacks repeated-dose studies beyond 28-d exposure.
UFh
10
A UFh of 10 is applied to account for human-to-human variability in susceptibility in the absence of
quantitative information to assess the toxicokinetics and toxicodynamics of picric acid in humans.
UFl
1
A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation because the POD is a BMDL.
UFS
1
A UFS of 1 is applied because a 28-day rat study was selected as the principal study.
UFC
300
Composite Uncertainty Factor = UFA x UFD x UFH x UFL x UFS
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The confidence in the subchronic p-RfD for picric acid is low as explained in Table 8
below.
Table 8. Confidence Descriptors for the Subchronic p-RfD for Picric acid
(CASRN 88-89-1)
Confidence Categories
Designation3
Discussion
Confidence in study
M
Confidence in the kev studv is medium. The Takaliaslii et al.
(2004) studv had a duration of onlv 28 d and it used a small
number of animals. However, this study is appropriate in the
number of endpoints analyzed; it is peer-reviewed, and
experiments were performed according to GLP guidelines.
Confidence in database
L
There are no acceptable developmental or two-generation
reproductive toxicity studies and no repeated-dose studies
beyond 28-d exposure.
Confidence in subchronic p-RfD
L
The overall confidence in the subchronic p-RfD is low.
aL = low; M = medium.
Derivation of Chronic Provisional RfD (Chronic p-RfD)
There are no chronic-duration studies available for picric acid. Furthermore, the longest
available study is 28 days in duration, which is not suitable for the derivation of a chronic p-RfD
due to increased uncertainty. However, Appendix A of this document contains a screening value
(screening chronic p-RfDs) using a surrogate (e.g., structural, metabolic, and toxicity-like)
approach, which may be of use under certain circumstances. Please see Appendix A for details
regarding the screening value.
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
Human and animal data are inadequate to derive subchronic or chronic p-RfCs for picric
acid.
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR
Table 9 identifies the cancer weight-of-evidence (WOE) descriptor for picric acid.
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Table 9. Cancer WOE Descriptor for Picric Acid (CASRN 88-89-1)
Possible WOE
Descriptor
Designation
Route of Entry (oral,
inhalation, or both)
Comments
"Carcinogenic to
Humans "
NS
NA
There are no human carcinogenicity data
identified to support this descriptor.
"Likely to Be
Carcinogenic to Humans "
NS
NA
There are no animal carcinogenicity studies
identified to support this descriptor.
"Suggestive Evidence of
Carcinogenic Potential"
NS
NA
There are no animal carcinogenicity studies
identified to support this descriptor.
"Inadequate Information
to Assess Carcinogenic
Potential"
Selected
Both
This descriptor is selected due to the lack of
any information on carcinogenicity of picric
acid.
"Not Likely to Be
Carcinogenic to Humans "
NS
NA
No evidence of noncarcinogenicity is available.
NA = not applicable; NS = not selected.
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
Because no cancer data are available, the cancer WOE descriptor for picric acid is
"Inadequate Information to Assess the Carcinogenic Potential' (for both oral and inhalation
routes of exposure; see Table 9). Genotoxicity assays of picric acid (see Table 4A) have yielded
mixed results. Under the proposed U.S. EPA (2005) cancer guidelines, the available data are
inadequate for an assessment of human carcinogenic potential.
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APPENDIX A. SCREENING PROVISIONAL VALUES
For reasons noted in the main provisional peer-reviewed toxicity value (PPRTV)
document, it is inappropriate to derive provisional toxicity values for picric acid. 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.
APPLICATION OF AN ALTERNATIVE SURROGATE APPROACH
The surrogate approach allows for the use of data from related compounds to calculate
screening values when data for the compound of interest are limited or unavailable. Details
regarding searches and methods for surrogate analysis are presented in Wane et al. (2012).
Three types of potential surrogates (structural, metabolic, and toxicity-like) are identified to
facilitate the final surrogate chemical selection. The surrogate approach may or may not be
route-specific or applicable to multiple routes of exposure. In this document, it is limited to the
oral noncancer effects only, based on the available toxicity data. All information was considered
together as part of the final weight-of-evidence (WOE) approach to select the most suitable
surrogate both toxicologically and chemically.
Structural Surrogates (Structural Analogs)
An initial surrogate search focused on the identification of structurally similar chemicals
with toxicity values from the Integrated Risk Information System (IRIS), Provisional Peer-
Reviewed Toxicity Value Reports (PPRTVs), and Health Effects Assessment Summary Tables
(HEAST) databases to take advantage of the well-characterized chemical-class information.
This was accomplished by searching U.S. EPA's DSSTox database (DSSTox. 2012) at similarity
levels >60% and the National Library of Medicine's ChemlDplus database (ChemlDplus. 2015)
at similarity levels >80%. Six structure analogs to picric acid were identified to have oral
toxicity values listed on IRIS or a PPRTV: 2-methyl-4,6-dinitrophenol (U.S. EPA, 2010);
2,4,6-trinitrotoluene (U.S. EPA. 1993); 2,4-dinitrophenol (U.S. EPA. 1991);
2-( 1 -methylpropyl)-4,6-dinitrophenol (U.S. EPA. 1989); 1,3,5-trinitrobenzene (U.S. EPA. 1997);
and 1.3-dinitrobenzene (U.S. EPA. 1988a). Table A-l summarizes their physicochemical
properties and similarity scores.
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Table A-l. Physicochemical Properties of Picric Acid (CASRN 88-89-1) and Candidate Structural Analogs
Chemical
2,4,6-Trinitrophenol
(picric acid)
2-Methyl-
4,6-dinitrophenol
(DNOC)
2,4,6-
Trinitro toluene
2,4-Dinitrophenol
(2,4 DNP)
2-(l-Methylpropyl)-
4,6-dinitrophenol
(Dinoseb)
1,3,5-Trinitrobenzene
1,3-Dinitrobenzene
Structure
o
\N_o
/Jr°'
o
OH 0"
H3C\ /L
^-0
O ^O
O
\\_o
/J*"0'
o
OH o"
0 ^-o
ch3
0 OH ^
, N* -I JL
-
0
0sN+°~
o-li+tXNJp
o d-
0-
N+:0
CASRN
88-89-1
534-52-1
118-96-7
51-28-5
88-85-7
99-35-4
99-65-0
Molecular weight3
229.10
198.133
227.132
184.11
240.214
213.105
168.108
DSSTox similarity
score (%)
100
78
58.3
99
60.6
73.6
73.6
ChemID Plus
similarity score (%)a
100
83.86
83.51
80.26
80.17
75.03
57.25
Melting point (°C)a
122.5
86.6
80.1
115.5
40
121.5
90
Boiling point (°C)a
300b
378
NV
NV
332
315
291
Vapor pressure
(mmHg [at °C])a
7.50 x 10-7(at25°C)
1.06 x 10-4 (at25°C)
8.02 x 10"« (at25°C)
3.90 x 10-4(at20°C)
NV
NV
NV
Henry's law constant
(atm-m3/mole
[at °C])a
1.70 x 10-n(at25°C)
1.4 x 10"6 (at25°C)
2.08 x 10-8 (at25°C)
8.60 x 10-8 (at 20°C)
4.56 x 10-7 (at25°C)
3.31 x 10-10
4.90 x 10-8
Water solubility
(mg/L [at C])a
1.27 x 104(at25°C)
198 (at 20°C)
130 (at25°C)
2,790 (at25°C)
52 (at25°C)
278 (at 15°C)
533 (at25°C)
Log Kowa
1.33
2.12
1.6
1.67
3.56
1.18
1.49
pKaa
0.38 (at25°C)
4.31 (at21°C)
NV
4.09 (at25°C)
4.62
NV
NV
"CliemlDplus (2015).
bChemicalBook (2015)
NV = not available.
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Metabolic Surrogates
Picric acid is eliminated from the body primarily as the parent compound, although
dinitrophenol derivatives were also identified in the urine of rats treated with picric acid (Wvman
et al.. 1992). Three of the six potential surrogates for picric acid identified by a structural
similarity search (2,4-dinitrophenol, 2-[l-methylpropyl]-4,6-dinitrophenol, and
1,3-dinitrobenzene) appear to have some commonalities with picric acid with regards to
metabolites (see Table A-2); however, a metabolic surrogate could not be identified because no
detailed information is available regarding the experimental design or results of these metabolic
studies. Due to limited information, an attempt to select metabolic surrogates is inconclusive.
Therefore, none of the six chemicals could be excluded based on metabolism analysis due to the
following: (1) 60% of absorbed picric acid is excreted as the parent compound, (2) picric acid
toxicity appears to be due to the parent compound, and (3) the parent compound is more than
50% structurally similar to all six potential surrogates.
Table A-2. Summary of Metabolites for Picric Acid (CASRN 88-89-1)
and Potential Surrogates
Chemical
Route
Species
Parent Compound and Metabolites Excreted
Reference
Picric acid
(2,4,6-trinitrophenol)
Oral
Rat
Parent compound (60%), Y-acctylisopicramic acid
(14.8%), picramic acid (18.5%), jV-acetylpicramic acid
(4.7%), and unidentified components (2.4%) in urine.
Wvman et al.
(1992)
2-Methyl-4,6-
dinitrophenol
(DNOC)
Oral
Rat
3,5 -Dinitro-2-hydroxybenzenemethanol, and
3,5 -diacetamido -2-hydroxytoluene.
Leeewater and
van der Greef
(1983)
2,4,6-Trinitrotoluene
Oral
Rat
4,6-Diamine, 2,6-diamine, and monoamines of
2,4,6-trinitrotoluene were the predominant metabolites
detected in the urine. Smaller quantities of 2- and
4-hydroxylamines, and azoxytoluene were present.
ATSDR (1995b)
2,4-Dinitrophenol
(2,4-DNP)
Oral
Rat
Nitrophenols, and 2-amino-4-nitrophenol in urine.
ATSDR (1995c)
2-(l -Methylpropyl)
-4,6-dinitrophenol
(Dinoseb)
Oral
Rat
2-(2-Hydro xy-l-methylpropyl)-4,6-dinitrophenol;
2-methyl-2-(2-hydroxy-3, 5 -dinitrophenyl)
propionic acid;
2-amino-6-(l-methylpropyl)-4-nitrophenol, and the
glucuronide in urine.
Hathwav (1970)
1,3,5-
Trinitrobenzene
Oral
Rat
1,3-Dinitro, 5-aniline, l,3-diamino-5-nitrobenzene, and
1,3,5-triaminobenzene in urine.
U.S. EPA (1997)
1,3 -Dinitrobenzene
Oral
Rat
3-Aminoacetanilide (22%), 4-acetamidophenylsulfate
(6%), 1,3-diacetamidobenzene (7%), and
3 - n i t ro a n i 1 i nc - A -glue u ro n i dc (4%).
ATSDR (1995a)
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Toxicity-Like Surrogates
Table A-3 summarizes available acute lethality and repeated-dose toxicity data for picric
acid and the six structurally similar analogs identified as potential surrogates. Lethality data
indicate that picric acid and related structural analogs share similarities in target organ of acute
toxicity, inducing adverse effects primarily in the central nervous system (CNS). Comparison of
oral acute toxicity studies in rats reveal that 1,3,5-trinitrobenzene has comparable median lethal
dose (LD50) values to picric acid. Other candidate analogs are either slightly less potent
(2,4,6-trinitotoluene) or more potent (2-methyl-4,6-dinitrophenol; 2,4-dinitrophenol;
2-[l-methylpropyl]-4,6-dinitrophenol; 1,3-dinitrobenzene) than picric acid.
As presented in the main PPRTV document, after 28-day administration, picric acid
exposure has been shown to result in liver, male reproductive, hematological, and splenic effects.
Increased absolute spleen weight was identified as the critical effect. The increased spleen
weight is considered a pathological consequence associated with hematological effects
(increased reticulocyte [Ret], decreased red blood cell [RBC] and hemoglobin [Hb]) which is
supported by extramedullary hematopoiesis observed in the spleen. Therefore, similar
hematological and associated splenic effects were anticipated from the potential surrogates,
preferably from rat toxicity studies, the animal species tested for picric acid.
Out of the six potential surrogates, 2-methyl-4,6-dinitrophenol and 2,4-dinitrophenol
resulted in significantly decreased body weight in rats starting at doses of 17.3 mg/kg-day and
46 mg/kg-day (subchronic-duration studies) with no hematological effects at dose levels up to
44.9 mg/kg-day and 182 mg/kg-day, respectively. These observations were in contrast to the
decreased Hb and RBC in rats treated with picric acid at a dose of 100 mg/kg-day, with no
significant effect in body weight (see Table A-3). Therefore, these two chemicals were not
considered toxicity-like surrogates. Based on the available toxicity information from chronic
studies, the critical effect of 2-(l-methylpropyl)-4,6-dinitrophenol is decreased fetal weight with
a free-standing lowest-observed-adverse-effect level (LOAEL) of 1 mg/kg-day from a
three-generation reproductive study in rats (U.S. EPA. 1989). In a 2-year feeding study in mice
(Dow Chemical Co, 1981), cystic endometrial hyperplasia and testicular atrophy/degeneration
with hypospermatogenesis were observed at all doses (1, 3, and 10 mg/kg-day); lenticular
opacities were observed at 3 and 10 mg/kg-day (low-dose animals not examined) (U.S. EPA.
1989). It is unclear if hematological effects were evaluated in this study. Further, no systemic
toxicity studies were conducted in rats and no toxicity information is available with respect to
hematological and splenic effects at doses greater than 10 mg/kg-day in mice. Therefore, due to
limited toxicity information for comparison purposes, 2-(l-methylpropyl)-4,6-dinitrophenol was
not considered as surrogate of picric acid (see Table A-3).
IRIS assessments for 1,3,5-trinitrobenzene and 1,3-dinitrobenzene have identified
hematological and splenic effects in rats (U.S. EPA. 1997. 1988a) (see Table A-3). Therefore,
1,3,5-trinitrobenzene and 1,3-dinitrobenzene were considered toxicity-like surrogates.
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Table A-3. Comparison of Available Repeated-Dose Toxicity Data for Picric Acid (CASRN 88-89-1) and Candidate Analogs
Chemical
2,4,6-Trinitrophenol
(Picric Acid)
2-Methyl-4,6-
dinitrophenol
(DNOC)
2,4,6-
Trinitrotoluene
2,4-Dinitrophenol
(2,4 DNP)
2-(l-Methylpropyl)-
4,6-dinitrophenol
(Dinoseb)
1,3,5-
T rinitrobenzene
1,3-Dinitrobenzene
Structure
0" OH G"
, nt X m*
0 ^0
OH O"
h,c . Js. ,r-it
1J "
0 ^0
q" ch3 g"
V
0 xo
OH 0"
C^°
0 ^0
cHj
CHJ
-
0"^ 0
°v°-
0-1i+^1n+i:i
0 0-
0-
N+:Q
CASRN
88-89-1
534-52-1
118-96-7
51-28-5
88-85-7
99-35-4
99-65-0
Acute lethality studies3
Rat Oral LD5o
(mg/kg)
200
7
607
30
25
275
59.5
Effect
Tremor, convulsions,
or effect on seisure
treshold and
chromodacyrorrea
NV
Respitory
stimulation,
changes in urine
composition,
infammation, and
necrosis of the
bladder
NV
Depressed behavioral
activity, convulsions or
effect on seisure
threshold, and
respitory stimulation
Dyspnea, rigidity, and
depressed behavioral
activity
Dysnea, depressed
behavioral activity,
and effect on skin
and appendages
Short-term- or subchronic-duration treatment (oral)
Subchronic RfD
(mg/kg-d)
1 X 10-2
8 x 10-4
NV
2 x 10-2
NV
NV
NV
Critical effects
Increased absolute
spleen weight
Reduced body
weight, excessive
perspiration and
fatigue, elevated
BMR and body
temperature, as
well as ocular
effects (based on
human study)
Increased liver
weight, change of
liver enzymes and
liver lesions
(26-wk study in
dogs)
Cataract formation
(human study)
NV
NV
Increased spleen
weight (16-wk
study in rats)
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Table A-3. Comparison of Available Repeated-Dose Toxicity Data for Picric Acid (CASRN 88-89-1) and Candidate Analogs
2-Methyl-4,6-
2-(l-Methylpropyl)-
2,4,6-Trinitrophenol
dinitrophenol
2,4,6-
2,4-Dinitrophenol
4,6-dinitrophenol
1,3,5-
Chemical
(Picric Acid)
(DNOC)
Trinitrotoluene
(2,4 DNP)
(Dinoseb)
T rinitrobenzene
1,3-Dinitrobenzene
Other effects
Hematological and
(1) Decreased body
Comprehensive
(1) No effects were
NV
Methemoglobinemia
Decreased body
related splenic effects
weight; no
hematological
observed at doses up
and spleen-erythroid
weight gain,
(hematopoiesis),
hematological
parameters were
to 10 mg/kg-d
cell hyperplasia;
decreased Hb,
increased liver
effects were
evaluated, but it is
(free-standing
increased relative
testicular atrophy,
weight, and testicular
specified at doses
unclear if those
NOAEL;
spleen and liver
and splenic
effects
up to 44.9 mg/kg-d
effects were
hematological
weight; and
hemosiderin
(evaluated
observed at a dose
endpoints were
decreased testes
hematological
up to 32 mg/kg-d
examined; 27-wk
weight (90- and
parameters
(26-wk study in
study in dogs).
180-d interim
included RBC,
dogs).
sacrifice in a 2-yr
WBC, and Hb;
(2) Decreased body
chronic-duration
182-d oral study in
No information
weight (less than
study in rats)
rats).
with respect to
10%), slight liver,
(2) Decreased
hematological
kidney, spleen
blood pyruvate, T3
effects in rats was
(congestion and
and T4 levels; no
available in IRIS
hemosiderosis), and
hematological
risk assessment.
testicular atrophy at a
toxicity was
dose of 46-mg/kg-d.
specified at doses
(However, toxic
No hematological
up to 41.0 mg/kg-d
effects on
effects were observed
(examined
hematologic
at doses up to
hematological
parameters and
182 mg/kg-d.
endpoints included
related splenic
(Hematological
RBC, Hb, MCH,
effects were
examination,
MCV, and WBC;
observed in other
including RBC and
90-d oral study in
subchronic-
Hb; 6-mo study in
rats). (3) Increased
duration studies in
rats).
percentages of
rats, mice, and
abnormal sperm
dogs at doses
(3) In a similar study
(reproductive study
higher than those
to picric acid study by
in male rats).
causing liver
Koizumi et al. (2001).
effects as
young rats were tested
described in
for behavior,
24
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Table A-3. Comparison of Available Repeated-Dose Toxicity Data for Picric Acid (CASRN 88-89-1) and Candidate Analogs
Chemical
2,4,6-Trinitrophenol
(Picric Acid)
2-Methyl-4,6-
dinitrophenol
(DNOC)
2,4,6-
Trinitrotoluene
2,4-Dinitrophenol
(2,4 DNP)
2-(l-Methylpropyl)-
4,6-dinitrophenol
(Dinoseb)
1,3,5-
T rinitrobenzene
1,3-Dinitrobenzene
Continued
Continued
Continued
AT SDR f1995b)
risk assessment
(p. 46/208).
hematological,
urinalysis,
biochemistry, organ
weight, and
histopathology.
Decreased locomotor
activity and
salivation were
observed at dose of
80 mg/kg-d. No
hematological, liver,
spleen, or testicular
effects were observed
(28-d study in rats).
Continued
Continued
Continued
POD
(mg/kg-d)
BMDLisd of 17.3
LOAEL of 0.8
NV
NV
NV
NV
NV
UFc
300
1,000
NV
NV
NV
NV
NV
Source
Subchronic RfC in
this assessment
U.S. EPA (2010)
U.S. EPA ("19931
U.S. EPA ("19911:
U.S. EPA (20071
NV
U.S. EPA (19971
U.S. EPA (1988a1
Chronic-duration treatment (oral)
Chronic RfD
(mg/kg-d)
NA
8 x 10-5
5 x 10-4
2 x 10~3
1 x 10~3
3 x 10-2
1 x 10-4
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Table A-3. Comparison of Available Repeated-Dose Toxicity Data for Picric Acid (CASRN 88-89-1) and Candidate Analogs
Chemical
2,4,6-Trinitrophenol
(Picric Acid)
2-Methyl-4,6-
dinitrophenol
(DNOC)
2,4,6-
Trinitrotoluene
2,4-Dinitrophenol
(2,4 DNP)
2-(l-Methylpropyl)-
4,6-dinitrophenol
(Dinoseb)
1,3,5-
T rinitrobenzene
1,3-Dinitrobenzene
Critical effects
NV
NV
IRIS summary
does not specify
toxic effects at
doses greater than
NV
Decreased fetal weight
(3-generation
reproductive study in
rats)
Methemoglobinemia
and spleen-erythroid
cell hyperplasia (2-yr
study in rats)
NV
Other effects
(oral)
NV
NV
0.4 mg/kg-d
(DOD. 1984) (2-vr
study in rats)
Decreases in body
weight at doses
greater than
47 mg/kg-d. No
treatment-related
effects in
histopathology at
doses up to
187 mg/kg-d.
It is unclear whether
hematology
parameters were
evaluated and what
tissues/organs were
evaluated
pathologically (2-yr
study in rats).
Cystic endometrial
hyperplasia and
testicular atrophy with
hypospermatogenesis
at doses >1 mg/kg-d
and lenticular
opacities at doses of 3
and 10 mg/kg-d. It is
unclear if
hematological effects
were evaluated.
It is unclear if Dinoseb
causes hematological,
splenic, or testicular
effects.
NV
POD (mg/kg-d)
LOAEL: 0.8
LOAEL: 0.5
LOAEL: 2
LOAEL: 1
NOAEL: 2.68
LOAEL: 0.4
UFC
10,000
1,000
1,000
1,000
100
3,000
Source
U.S. EPA (2010)
U.S. EPA (1993)
U.S. EPA (1991):
U.S. EPA (2007)
U.S. EPA (1989)
U.S. EPA (1997)
U.S. EPA (1988a)
;'C he ml Dolus (2015)
BMDL = lower confidence limit (95%) on the benchmark dose; BMR = base metabolism rate; Hb = hemoglobin; LOAEL = lowest-observed-adverse-effect level; NA = not
applicable; NOAEL = no-observed-adverse-effect level; NV = not available; RBC = red blood cell; WBC = white blood cell.
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For 2,4,6-trinitotoluene, liver effects (increased liver weight, alterations in liver enzyme
levels and liver lesions) were identified as critical effects with a LOAEL of 0.5 mg/kg-day, based
on a 26-week study in dogs (U.S. EPA. 1993). According to the IRIS assessment,
comprehensive endpoints including clinical chemistry, hematological evaluation, urinalyses,
periodic electrocardiography (ECG), and ophthalmic examinations were evaluated in this study,
but it is unclear whether hematological and splenic effects were observed at this dose or higher.
No information on 2,4,6-trinitotoluene with respect to hematological effects in rats was available
in the IRIS risk assessment (U.S. EPA. 1993). However, the effects of 2,4,6-trinitotoluene in
the hematological and splenic compartments were observed in other sub chronic-duration studies
in rats, mice, and dogs at doses higher than the dose which caused liver effects as described in
the ATS PR (1995b). Thus, 2,4,6-trinitotoluene is also considered a toxicity-like surrogate.
In conclusion, an attempt was made to identify a suitable surrogate to derive chronic
toxicity values for picric acid. Comparison of the potential surrogates
(2-methyl-4,6-dinitrophenol; 2,4-dinitrophenol; 2,4,6-trinitrotoluene;
2-[l-methylpropyl]-4,6-dinitrophenol; 1,3,5-trinitrobenzene; and 1,3-dinitrobenzene) was made
based on their profiles of structural similarity, metabolic profile, and tissue-specific toxicity, and
2,4,6-trinitrotoluene; 1,3,5-trinitrobenzene; and 1,3-dinitrobenzene were kept for the final
selection.
Weight-of-Evidence Approach
To select the best surrogate chemical, the following considerations were used in a WOE
approach: (1) lines of evidence from U.S. EPA assessments are preferred; (2) chemicals that
have chronic toxicity information are preferred; (3) if there are no clear indications as to the best
surrogate chemical based on the first two considerations, then the candidate surrogate with the
highest structural similarity may be preferred.
Overall, based on the WOE of all the information presented above, 1,3,5-trinitrobenzene
appears to be the most appropriate surrogate for picric acid because of the following factors:
1) U.S. EPA IRIS identified that the critical effect of 1,3,5-trinitrobenzene is
"Methemoglobinemia and spleen-erythroid cell hyperplasia," which are consistent
with the hematological and associated splenic effects observed in rats treated with
picric acid.
2) The critical effect for 1,3,5-trinitrobenzene is based on a 2-year chronic-duration
study in rats (compared to point of departures [PODs] based on subchronic-duration
studies for 2,4,6-trinitrotoluene and 1,3-dinitrobenzene IRIS assessments).
3) High structural similarity scores of 75.03 and 73.6% were found using the National
Library of Medicine's ChemlDplus database (ChemlDplus. 2015) and the EPA
DSSTox database, respectively.
4) Lethality studies in rats suggest that 1,3,5-trinitrobenzene and picric acid have similar
potencies (oral LDsos), and their acute toxic effects primarily target the CNS.
The 1,3,5-trinitrobenzene IRIS summary (U.S. EPA, 1997) cited (Reddv et at., 2001a;
Reddv et at.. 1997: Reddv et at., 1996) as the principal studies for the reference dose (RfD):
"Chronic toxic effects of 1,3,5-TNB in male andfemale Fisher 344 rats were
evaluated by feeding powdered certified laboratory chow diet supplemented with
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varied concentrations of TNB for 2 years. Based on food consumption, the
average TNB intake was calculatedfor both males andfemales.
The study was conducted in accordance with the U.S. EPA guidelines for chronic
toxicity studies as required by the GLP standards. One of the unique features of
this study is that 10 animals/sex were sacrificed at the end of 90 days, 6 months
and 1 year, and 25 or more rats were sacrificed at 2 years; complete
toxicological evaluations were performed during these periods.
High-dose animals showed decreased body weight gains associated with
decreasedfood consumption. Relative organ weight changes for the brain
(increase), spleen (increase), liver (increase) and testes (decrease in 90- and
180-day periods) were reported for all treated animals dosed with TNB at levels
higher than 3 mg/kg/day; adverse hematological findings (decreased hematocrit
and hemoglobin) and increased methemoglobulin) were consistently reported in
all animals treated at these levels. Histopathological findings in the 1-year study
revealed extramedullary hematopoiesis in rats treated with TNB at doses of
3 mg/kg-day or higher. In the 2-year study, these effects were seen only in rats
dosed with TNB at the high dosage level (13.23 mg/kg/day). The adverse effects,
such as increased methemoglobin, erythroid cell hyperplasia, and increased
relative organ weights, observed during interim sacrifices in rats receiving
60 ppm TNB did not persist and were not detected in rats fed 60 ppm TNB for
2 years, suggesting that an adaptive mechanism has taken place in order to
compensate adverse effects observed during interim sacrifices.
Results of this study exhibited clear evidence of toxicity of the hematopoietic
system as has been reportedfor other nitroaroniatics such as, dinitrobenzene and
trinitrotoluene. The NOAEL for this study is 2.68 mg/kg/day and the LOAEL for
hematological effects is 13.31 mg/kg/day. "
ORAL TOXICITY VALUES
Derivation of Screening Chronic Provisional Reference Dose (Screening Chronic p-RfD)
Based on the overall surrogate approach presented in this PPRTV assessment, IRIS
critical effects of methemoglobinemia, spleen-erythroid cell hyperplasia, and related effects for
1,3,5-trinitrobenzene established in female F344 rats from a 2-year study (Reddv et al.. 2001a;
Reddv et aL 1997) are identified as the potential surrogate critical effects for picric acid.
Benchmark dose (BMD) modeling was performed for all the related endpoints observed in male
and female rats. Among these endpoints, only the male methemoglobin (MetHb) data was
adequately fit with the available continuous models (see Appendix C for details).
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Table A-4. Summary of BMD Modeling of Data from Rats Treated with
Trinitrobenzene in Diet for 2 Years
Endpoint
Sex
NOAEL
(mg/kg-d)
LOAEL
(mg/kg-d)
BMR
BMDisd
(mg/kg-d)
BMDLisd
(mg/kg-d)
POD
(mg/kg-d)
Relative spleen
weight
M
2.64
13.44
NA
NA
2.64
MetHb
M
2.64
13.44
1 SD
2.14
1.15
1.15
Relative spleen
weight
F
2.68
13.31
NA
NA
2.68
MetHb
F
2.68
13.31
NA
NA
2.68
"Reddv et al. (2001a)
NA = not applicable; SD = standard deviation.
A benchmark dose lower confidence limit (BMDLisd) of 1.15 mg/kg-day based on
methemoglobinemia in male rats was identified as the most sensitive endpoint from the study
(see Table A-4). Although supporting evidence for the induction of MetHb with picric acid is
lacking, it should be emphasized that MetHb levels were not examined in the available
repeated-dose toxicity studies (Takahashi et al.. 2004). Indeed, increased MetHb levels are
associated with exposure to nitroaromatic compounds (Beard and Noe. 1981). including two of
the structural analogs (1,3,5-trinitrobenzene and 1,3-dinitrobenzene) and other nitrophenols (U.S.
EPA, 1997; ATSDR, 1992; U.S. EPA, 1988b). Furthermore, picric acid induced adverse effects
on the hematological system, spleen, and testes that were similar to those observed with
1,3,5-trinitrobenzene treatment; these effects included decreased RBC and Hb levels, increased
spleen weight, extramedullary hematopoiesis, and seminiferous tubular degeneration. Thus,
1,3,5-trinitrobenzene is considered an appropriate chemical surrogate for picric acid based on
similarities in structure and major target organs of toxicity. The BMDLisd of 1.15 mg/kg-day
identified for methemoglobinemia in male rats exposed to 1,3,5-trinitrobenzene is selected as a
POD for the derivation of the chronic p-RfD.
As described in the EPA's Recommended Use of Body Weight4 as the Default Method in
Derivation of the Oral Reference Dose (U.S. EPA, 201 lb), the POD of 1.15 mg/kg-day is
converted to a human equivalent dose (HED) through an application of a dosimetric adjustment
factor (DAF) derived as follows:
DAF = (BWa1/4 - BWh1/4)
where:
DAF = dosimetric adjustment factor
BWa = animal body weight
BWh = human body weight
Using a BW„ of 0.25 kg for rats and a default BWh of 70 kg for humans (U.S. EPA,
1988b), the resulting DAF is 0.24. Applying this DAF to the BMDLisd identified in the rat
study yields a surrogate PODhed as follows:
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Surrogate PODhed = BMDLisd (mg/kg-day) x DAF
= BMDLisd (mg/kg-day) x 0.24
= 1.15 mg/kg-day x 0.24
= 0.276 mg/kg-day
Wane et al. (2012) indicated that the uncertainty factors (UFs) typically applied to the
chemical of concern are the same as those applied to the surrogate unless additional information
is available. However, UFa for picric acid has been reduced from 10 to 3 due to the conversion
of the POD from animal dose to HED [the IRIS assessment for the 1,3,5-trinitrobenzene was
performed prior to the recommended use of BW3 4 scaling for noncancer effects (U.S. EPA.
201 lb)l. Further, the UFd of 10 was applied to account for limited information with regards to
reproductive toxicity and no information with regard to developmental toxicity for picric acid,
and systemic toxicity appears to be more sensitive than developmental and reproductive effects
for the surrogate chemical. To derive a screening chronic p-RfD for picric acid, a UFc of 300
has been applied to the surrogate PODhed (see Table A-5). A comparison of UF applications
between picric acid and 1,3,5-trinitrobenzene chronic RfDs is also presented in Table A-6. The
screening chronic p-RfD for picric acid is derived as follows:
Screening Chronic p-RfD = Surrogate PODhed ^ UFc
= 0.276 mg/kg-day -^300
= 9 x 10"4 mg/kg-day
Table A-5 summarizes the uncertainty factors for the screening chronic p-RfD for picric
acid, and Table A-6 compares uncertainty factor values for picric acid and the selected surrogate
chemical.
Table A-5. Uncertainty Factors for the Screening Chronic p-RfD for
Picric Acid (CASRN 88-89-1)
UF
Value
Justification
UFa
3
A UFa of 3 (10°5) has been applied to account for residual uncertainty, including toxicodynamic
differences between rats and humans following oral picric acid exposure. The toxicokinetic
uncertainty has been accounted for by calculation of a HED through application of a DAF as outlined
in the EPA's Recommended Use of Body Weight4 as the Default Method in Derivation of the Oral
Reference Dose ('U.S. EPA, 2011b).
UFd
10
A UFd of 10 has been applied based on unknown and unaccountable database deficiencies of picric
acid. For the surrogate chemical, systemic toxicity appears to be more sensitive than developmental
and reproductive effects.
UFh
10
A UFh of 10 is applied to account for human-to-human variability in susceptibility in the absence of
quantitative information to assess the toxicokinetics and toxicodynamics of picric acid in humans.
UFl
1
A UFl of 1 has been applied for LOAEL-to-NOAEL extrapolation because the POD is a BMDLi Sd.
UFS
1
A UFS of 1 has been applied because a chronic-duration study was selected as the principal study.
UFC
300
Composite Uncertainty Factor = UFA x UFD x UFH x UFL x UFS
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Table A-6. Comparison of Uncertainty Factors for Picric Acid and 1,3,5-Trinitrobenzene
for the Chronic p-RfD
Picric Acid
1,3,5-
T rinitrobenzene
Comments
UFa
3
10
The UFa for picric acid has been reduced from 10 to 3 based on the
calculation of a HED through the application of a default DAF. The
U.S. EPA (1997) assessment for 1.3.5-trinitrobenzene was performed
prior to the EPA's Recommended Use of Body Weight3'4 as the Default
Method in Derivation of the Oral Reference Dose (U.S. EPA. 201 lb),
therefore, a UFA of 10 was applied to account for inter-species
extrapolation.
UFd
10
1
A UFd of 10 for picric acid reflects unknown and unaccountable
database deficiencies, including the lack of information on potential
developmental and reproductive effects. The UFd for
1,3,5-trinitrobenzene was reduced from 10 to 1 due to the available
information from systemic, developmental and reproductive studies
that support hematological toxicity as the most sensitive effect.
UFh
10
10
NA
UFl
1
1
NA
UFS
1
1
NA
UFC
300
100
NA
NA = not applicable.
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APPENDIX B. DATA TABLES
Table B-l. Body and Organ Weight for Newborn Sprague-Dawley Rats Exposed to
Picric Acid (CASRN 88-89-1) for 18 Days (PNDs 4-21)ab
Dose (mg/kg-d)
0
4.1
16.3
65.1
Males
No. animals
6
6
6
6
Body weight (g)
63.4 ±4.9
63.0 ± 2.8 (-1%)
63.7 ±5.7 (0%)
61.8 ±4.8 (3%)
Absolute liver weight
(g)
2.69 ±0.22
2.74 ±0.14 (2%)
2.79 ± 0.24 (4%)
2.97 ± 0.38 (10%)c
Relative liver weight
(g/lOOgBW)
4.25 ±0.16
4.35 ±0.12 (2%)
4.38 ± 0.08 (3%)
(4.79 ±0.28)**
(13%)
Absolute spleen
weight (g)
0.34 ±0.07
0.35 ±0.06
0.38 ±0.04
0.37 ±0.06
Relative spleen weight
(g/lOOgBW)
0.54 ±0.07
0.56 ±0.08
0.60 ±0.05
0.60 ±0.05
Absolute kidney
weight (g)
0.74 ±0.12
0.73 ± 0.08 (-1%)
0.77 ± 0.03 (4%)
0.73 ±0.12 (-1%)
Relative kidney weight
(g/lOOgBW)
1.16 ± 0.12
1.16 ±0.09 (0%)
1.21 ±0.10 (4%)
1.18 ±0.12 (2%)
Absolute epididymis
weight (mg)
57.6 ±4.6
55.4 ±6.0
57.6 ±7.3
50.3 ±3.7
Relative epididymis
weight
(mg/100 g BW)
91.1 ± 6.9
87.9 ±7.2
91.3 ± 16.4
81.9 ±7.9
Absolute testes weight
(mg)
326 ± 47
302 ± 27
319 ±22
295 ± 20
Relative testes weight
(mg/100 g BW)
513 ±54
479 ± 26
504 ± 44
478 ± 27
Females
No. animals
6
6
6
6
Body weight (g)
59.0 ±3.3
59.6 ± 2.3 (1%)
57.0 ± 4.6 (-3%)
58.8 ± 5.3 (-2%)
Absolute liver weight
(g)
2.46 ± 0.22
2.44 ± 0.24 (-1%)
2.33 ± 0.25 (-5%)
2.75 ± 0.28 (12%)°
Relative live weight
(g/100 g BW)
4.18 ±0.35
4.09 ± 0.29 (-2%)
4.09 ±0.19 (-2%)
4.67 ±0.19* (12%)
Absolute spleen
weight (g)
0.32 ±0.04
0.33 ±0.04
0.29 ±0.05
0.37 ±0.05
Relative spleen weight
(g/100 g BW)
0.54 ±0.05
0.55 ±0.07
0.51 ±0.08
0.62 ±0.03
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Table B-l. Body and Organ Weight for Newborn Sprague-Dawley Rats Exposed to
Picric Acid (CASRN 88-89-1) for 18 Days (PNDs 4-21 )ab
Dose (mg/kg-d)
0
4.1
16.3
65.1
Absolute kidney
weight (g)
0.69 ±0.05
0.69 ± 0.06 (0%)
0.66 ± 0.06 (-4%)
0.70 ± 0.05 (1%)
Relative kidney weight
(g/100 g BW)
1.17 ±0.09
1.16 ±0.08 (-1%)
1.16 ± 0.10 (-1%)
1.20 ±0.06 (3%)
aTakahasM et al. (2004).
bValues are mean ± SD. (percent change compared with control); percent change control = [(treatment
mean - control mean) control mean] x 100.
°Not statistically significant but biologically relevant (>10% increase).
*Significant difference from control atp< 0.05.
**Significant difference from control atp< 0.01, as calculated by study authors.
BW = body weight; PND = postnatal day.
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Table B-2. Hematological Parameters for Young Sprague-Dawley Rats Exposed to
Picric Acid (CASRN 88-89-1) for 28 Daysa b
Dose (mg/kg-d)
0
4
20
100
Males
No. animals
6
6
6
6
WBC (x 102/mL)
93 ± 14
98 ± 14
112 ±22
146 ±38**
RBC (x 104/mL)
720 ± 32
720 ± 13
739 ±34
661 ±52*
Hb (g/dL)
14.3 ±0.3
14.6 ±0.5
14.8 ±0.7
13.4 ±0.7*
Ht (%)
40.9 ± 1.0
41.5 ± 1.8
42.6 ± 1.4
39.1 ±2.2
MCV (fL)
56.8 ± 1.6
57.7 ±2.3
57.8 ±2.3
59.3 ±2.7
MCHC (%)
35.0 ±0.7
35.2 ±0.6
34.8 ±0.6
34.1 ±0.5
Ret (%o)
31.4 ± 1.4
29.8 ±4.1
31.6 ± 3.8
54.7 ±7.6**
Females
No. animals
6
6
6
6
WBC (x 102/mL)
67 ± 18
79 ±27
73 ± 15
123 ±33**
RBC (x 104/mL)
706 ± 30
711 ± 47
713 ±41
608±19**
Hb (g/dL)
14.2 ±0.5
14.3 ±0.5
14.3 ±0.6
12.6 ±0.3**
Ht (%)
39.3 ± 1.2
40.3 ± 1.9
40.3 ± 1.8
37.3 ±0.9
MCV (fL)
55.8 ±0.9
56.9 ±3.4
56.6 ± 1.7
61.4 ±2.4**
MCHC (%)
36.2 ±0.9
35.6 ±0.6
35.6 ±0.7
33.9 ±0.3**
Ret (%o)
25.5 ±4.6
25.2 ± 1.0
24.1 ±3.3
65.5 ±5.9*
"TakahasM et al. (2004).
bValues are mean± SD.
*Significant difference from control atp< 0.05, as calculated by study authors.
**Significant difference from control atp< 0.01, as calculated by study authors.
WBC = total leukocyte count; RBC = erythrocyte count; Hb = hemoglobin levels; Ht = hematocrit levels;
MCV = mean corpuscular volume; MCHC = mean corpuscular hemoglobin concentration; Ret = reticulocyte
count.
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Table B-3. Body and Organ Weight for Young Sprague-Dawley Rats Exposed to
Picric Acid (CASRN 88-89-1) for 28 Daysa b
Dose (mg/kg-d)
0
4
20
100
Males
No. animals
6
6
6
6
Body weight (g)
374 ± 12
380 ±31 (2%)
384 ± 35 (3%)
367 ± 27 (-2%)
Absolute liver weight (g)
14.2 ± 1.3
14.0 ± 0.9 (-1%)
14.4 ± 1.8(1%)
15.6 ± 1.1 (10%)
Relative liver weight
(g/100 g BW)
3.79 ±0.31
3.69 ±0.19
(-3%)
3.73 ±0.23
(-2%)
4.24 ±0.24*
(12%)
Absolute spleen (g)°
0.82 ±0.08
0.76 ± 0.08 (-7%)
0.89 ±0.19 (9%)
1.18 ±0.16** (44%)
Relative spleen weight
(g/100 g BW)°
0.22 ±0.02
0.20 ±0.02
(-9%)
0.23 ±0.03
(5%)
0.32 ±0.03**
(45%)
Absolute kidney weight (g)
2.62 ±0.13
2.57 ±0.13 (-2%)
2.81 ±0.33 (7%)
2.72 ±0.13 (4%)
Relative kidney weight
(g/100 g BW)
0.70 ±0.03
0.68 ±0.05
(-3%)
0.73 ± 0.06
(4%)
0.74 ±0.03
(6%)
Absolute testes weight (g)
3.08 ±0.32
3.09 ±0.19
3.13 ±0.25
3.29 ±0.35
Relative testes weight
(g/100 g BW)
0.82 ±0.09
0.82 ±0.06
0.82 ±0.05
0.90 ±0.05
Absolute epididymis weight
(g)
0.82 ±0.06
0.78 ±0.06
(-5%)
0.78 ±0.07
(-5%)
0.63 ±0.10**
(-23%)
Relative epididymis weight
(g/100 g BW)
0.22 ± 0.02
0.21 ±0.02
(-5%)
0.20 ±0.01
(-9%)
0.17 ±0.03**
(-23%)
Females
No. animals
6
6
6
6
Body weight (g)
242 ± 19
241 ± 17 (0%)
237 ± 29 (-2%)
233 ± 14 (-4%)
Absolute liver weight (g)
8.2 ±0.7
8.0 ± 0.8 (-2%)
8.2 ± 1.5 (0%)
9.7 ± 1.2(18%)
Relative liver weight
(g/100 g BW)
3.38 ± 0.11
3.32 ±0.15
(-2%)
3.45 ±0.19
(2%)
4.16 ±0.27**
(23%)
Absolute spleen weight (g)°
0.51 ±0.08
0.58 ± 0.05 (14%)
0.54 ± 0.08 (6%)
0.98 ±0.12** (92%)
Relative spleen weight
(g/100 g BW)°
0.21 ±0.04
0.24 ±0.02
(14%)
0.23 ±0.20
(10%)
0.42 ±0.05**
(100%)
Absolute kidney weight (g)
1.77 ±0.16
1.73 ±0.20
(-2%)
1.67 ±0.20
(-6%)
1.86 ±0.17
(5%)
Relative kidney weight
(g/100 g BW)
0.74 ± 0.07
0.71 ±0.04
(-4%)
0.71 ±0.05
(-4%)
0.80 ±0.06
(8%)
"Takahashi et al. (2004).
bValues are mean ± SD (percent change compared with control); percent change control = [(treatment
mean - control mean)/control mean] x 100.
Statistically significant as calculated for this review (ANOVA contrast with equally spaced coefficients); trend
p < 0.01.
*Significant difference from control atp< 0.05, as calculated by study authors.
**Significant difference from control atp< 0.01, as calculated by study authors.
BW = body weight.
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Table B-4. Histopathological Parameters for Young Male Sprague-Dawley Rats
Exposed to Picric Acid (CASRN 88-89-1) for 28 Daysa'b
Dose (mg/kg-d)
0
4
20
100
Males
No. animals examined
6
6
6
6
Spleen
Development, germinal center
+
0
0
0
5*
Extramedullary hematopoiesis, erythrocyte
+
0
0
0
Hemosiderin deposition
Total
0
0
0
4
+
0
0
0
3
++
0
0
0
1
Cecum
Ulcer
Total
0
0
0
4
+
0
0
0
1
++
0
0
0
2
+++
0
0
0
1
Liver
Hypertrophy, hepatocytes, centrilobular
+
0
0
0
4
Testis
Atrophy, seminiferous tubules, diffuse
Total
0
0
0
+
0
0
0
Epididymis
Cell debris, lumen
Total
0
0
0
4
+
0
0
0
3
++
0
0
0
1
Decrease in sperm
Total
0
0
0
6*
+
0
0
0
5*
++
0
0
0
1
aTakahasM et al. (2004).
bGrade sign: +, mild; ++, moderate; +++, marked.
*Significant difference from control atp< 0.05, as calculated by study authors.
**Significant difference from control atp< 0.01, as calculated by study authors.
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Table B-5. Histopathological Parameters for Young Female Sprague-Dawley Rats
Exposed to Picric Acid (CASRN 88-89-1) for 28 Daysa'b
Dose (mg/kg-d)
0
4
20
100
Females
No. animals examined
6
6
6
6
Spleen
Development, germinal center
+
0
0
0
5*
Extramedullary hematopoiesis, erythrocyte
+
0
0
0
Hemosiderin deposition
Total
0
0
0
+
0
0
0
3
++
0
0
0
3
Cecum
Ulcer
++
0
0
0
3
Liver
Hypertrophy, hepatocytes, centrilobular
+
0
0
0
3
aTakafaaste et al. (2004).
bGrade sign: +, mild; ++, moderate; +++, marked.
*Significant difference from control atp< 0.05.
**Significant difference from control atp< 0.01.
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APPENDIX C. BENCHMARK DOSE MODELING RESULTS
MODELING PROCEDURE FOR CONTINUOUS DATA
The benchmark dose (BMD) modeling of continuous data was conducted with EPA's
Benchmark Dose Software (BMDS) (Version 2.4). For these data, all continuous models
available within the software were fit using a default benchmark response (BMR) of 1 standard
deviation (SD) relative risk. For liver weight changes, a BMR of 10% relative risk was also
used. An adequate fit was judged based on the %2 goodness-of-fitp-value (p> 0.1), magnitude
of the scaled residuals in the vicinity of the BMR, and visual inspection of the model fit. In
addition to these three criteria forjudging adequacy of model fit, a determination was made as to
whether the variance across dose groups was homogeneous. If a homogeneous variance model
was deemed appropriate based on the statistical test provided in BMDS (i.e., Test 2), the final
BMD results were estimated from a homogeneous variance model. If the test for homogeneity of
variance was rejected (p < 0.1), the model was run again while modeling the variance as a power
function of the mean to account for this nonhomogeneous variance. If this nonhomogeneous
variance model did not adequately fit the data (i.e., Test 3; p-v alue < 0.1), the data set was
considered unsuitable for BMD modeling. Among all models providing adequate fit, the lowest
benchmark dose lower confidence limit (BMDL) was selected if the BMDLs estimated from
different models varied greater than threefold; otherwise, the BMDL from the model with the
lowest Akaike's Information Criteria (AIC) was selected as a potential point of departure (POD)
from which to derive the provisional reference dose (p-RfD).
BMD Modeling of Data from the Newborn Rat Study (Takahashi et al.. 2004)
Table C-l. Summary of BMD Modeling of Data from Newborn Sprague-Dawley Rats
Treated with Picric Acid (CASRN 88-89-1) via Gavage for 18 Days
Endpoint
Sex
Model
/>-Valuc"
AIC for
Fitted Model
Scaled
Residual
BMD io
(mg/kg-d)
BMDLio
(mg/kg-d)
Increased absolute
liver wt
M
Power
0.89
-41.23
-0.09
64.5
31.8
Increased absolute
liver wt
F
Polynomial
0.55
-38.17
0.02
58.1
34.0
Increased relative
liver wt
M
Exponential (M2)
0.56
-59.92
0.04
55.1
41.2
Increased relative
liver wt
F
Polynomial
0.74
-37.69
0.01
59.0
39.8
aValues <0.10 fail to meet conventional goodness-of-fit criteria.
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose; wt = weight.
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BMD Modeling of Data from the Young Rat Study ( I akahashi et al.. 2004)
Table C-2. Summary of BMD Modeling of Data from Young Sprague-Dawley Rats
Treated with Picric Acid (CASRN 88-89-1) via Gavage for 28 Days
Endpoint
Sex
Model
/>-Value"
AIC for
Fitted
Model
Scaled
Residual
BMD
(mg/kg-d)
BMDL
(mg/kg-d)
Increased WBC
M
Power
0.52
173.13
0
25.0
14.0
Increased WBC
F
Exponential (M2)
0.58
179.91
-0.55
50.1
38.0
Decreased RBC
M
No lit
Decreased RBC
F
Polynomial
0.92
197.65
-0.002
68.5
62.0
Decreased Hb
M
Polynomial
0.26
1.72
-0.008
78.0
46.5
Decreased Hb
F
Polynomial
0.89
-8.65
-0.002
64.4
26.4
Increased MCV
F
No lit
Decreased MCHC
F
Linear
0.29
5.56
0.14
32.3
25.1
Increased Ret
M
No lit
Increased Ret
F
No lit
Increased absolute liver wt
M
Polynomial
0.99
40.75
-0.002
100
84.6
Increased absolute liver wt
F
Linear
0.87
31.87
-0.37
49.1
29.5
Increased relative liver wt
M
Polynomial
0.74
-39.05
0.0008
90.5
54.0
Increased relative liver wt
F
Linear
0.47
-54.39
-0.58
41.0
32.8
Increased absolute spleen wt
M
Linear
0.22
-67.86
0.42
27.5
17.3
Increased absolute spleen wt
F
Polynomial
0.28
-89.53
-0.45
43.3
19.6
Increased relative spleen wt
M
Exponential (M2)
0.23
-147.53
0.21
26.3
20.7
Increased relative spleen wt
F
No lit
Decreased absolute
epididymis wt
M
Linear
0.67
-98.32
0.29
39.1
27.9
Decreased relative epididymis
wt
M
Exponential (M2)
0.68
-158.47
-0.59
31.9
18.4
aValues <0.10 fail to meet conventional goodness-of-fit criteria.
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose; Hb = hemoglobin levels; MCHC = mean corpuscular hemoglobin concentration; MCV = mean
corpuscular volume; RBC = erythrocyte count; Ret = reticulocyte count; WBC = white blood cell; wt = weight.
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For increased absolute spleen weight in young male Sprague-Dawley (S-D) rats, with
nonconstant variance model applied, all models except the Exponential Model 5 and Hill Model
provided an adequate fit to the variance and the means. Compared to the other adequately fitted
models, the Exponential Model under estimates control data point, therefore, this model is
excluded for further consideration. BMDLs for rest of models providing adequate fit were
sufficiently close (differed by less than two- to three-fold), so the model with the lowest AIC was
selected (Linear Model).
Table C-3. Modeling Results for Increased Absolute Spleen Weight in Young Male
Sprague-Dawley Rats Treated with Picric Acid via Gavage for 28 Days"
Model
Variance
/>-Valucb
Means
/>-Valucb
Scaled
Residuals0
AIC
BMDisd
(mg/kg-d)
BMDLisd
(mg/kg-d)
Constant variance
Exponential (Model 2)d
0.1329
0.1909
0.5802
-67.56478
32.8705
22.1982
Exponential (Model 3)d
0.1329
0.1909
0.5802
-67.56478
32.8705
22.1982
Exponential (Model 4)d
0.1329
0.1054
-0.08294
-66.25554
17.5529
6.61834
Exponential (Model 5)d
0.1329
NA
-0.5917
-65.63357
19.2349
8.5157
Hilld
0.1329
NA
-0.592
-65.633572
19.0606
9.56034
Linear®
0.1329
0.2212
0.422
-67.859042
27.5402
17.3281
Polynomial (2-degree)6
0.1329
0.2212
0.422
-67.859042
27.5402
17.3281
Polynomial (3-degree)6
0.1329
0.2212
0.422
-67.859042
27.5402
17.3281
Power"1
0.1329
0.2212
0.422
-67.859042
27.5402
17.3281
aTakahasM et ai. 2004
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals for dose group near the BMD.
dPower restricted to >1.
"Coefficients restricted to be negative.
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose; NA = not applicable.
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BMD Output of Linear Model for Increased Absolute Spleen Weight in Young Male
Sprague-Dawley Rats Treated with Picric Acid via Gavage for 28 Days
Linear Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
1.4
1.3
a)
to
c
o
Q.
to
a)
a:
c
ro
a)
1.2
1.1
0.9
0.8
0.7
Linear
BMDL
09:52 07/09 2014
BMDS Model Run
dose
The form of the response function is:
Y[dose] = beta 0 + beta l*dose + beta 2*dose/s2 + ...
Dependent variable = Mean
Independent variable = Dose
Signs of the polynomial coefficients are not restricted
The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)
Total number of dose groups = 4
Total number of records with missing values = 0
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
lalpha = -3.98325
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rho =
beta_0 =
beta 1 =
0.791321
0.00390901
Asymptotic Correlation Matrix of Parameter Estimates
lalpha
rho
beta_0
beta 1
lalpha
1
0. 61
0.006
-0.0025
rho
0.61
1
0. 018
-0.017
beta_0
0.006
0.018
1
-0.51
beta_l
-0.0025
-0.017
-0.51
1
Parameter Estimates
Interval
Variable
Limit
lalpha
0.363161
rho
6.25068
beta_0
0.847097
beta_l
0.00544589
Estimate
-3.9346
-4.64638
2.14829
0.790409
0.00394387
Std. Err.
-3.22282
2.0931
0.0289231
0.00076635
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
-1.9541
0.733721
0.00244185
Table of Data and Estimated Values of Interest
Dose
Obs Mean
Est Mean Obs Std Dev Est Std Dev Scaled Res.
0
4
20
100
0.82
0.76
0.89
1.18
0.79
0.806
0.869
1.18
0.08
0.08
0.19
0.16
0.109
0.111
0.12
0.168
0. 667
-1.02
0. 422
-0.07
Model Descriptions for likelihoods calculated
Model A1: Yij = Mu(i) + e(ij)
Var{e(ij)0 = Sigma^2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)0 = Sigma(i)^2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)0 = exp(lalpha + rho*ln(Mu(i)))
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)0 = Sigma^2
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Likelihoods of Interest
Model Log(likelihood) # Param's AIC
A1 37.986865 5 -65.973730
A2 41.456478 8 -66.912957
A3 39.438338 6 -66.876676
fitted 37.929521 4 -67.859042
R 26.187755 2 -48.375509
Explanation of Tests
Test 1:
Test
Test
Test
Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Are Variances Homogeneous? (A1 vs A2)
Are variances adeguately modeled? (A2 vs. A3)
Does the Model for the Mean Fit? (A3 vs. fitted)
(Note: When rho=0 the results of Test 3 and Test 2 will be the same.)
Tests of Interest
Test
-2*log(Likelihood Ratio) Test df
p-value
Test
Test
Test
Test
30.5374
6. 93923
4.03628
3.01763
<.0001
0.07386
0.1329
0.2212
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is less than .1. A non-homogeneous variance
model appears to be appropriate
The p-value for Test 3 is greater than .1. The modeled variance appears
to be appropriate here
The p-value for Test 4 is greater than .1. The model chosen seems
to adeguately describe the data
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
Confidence level = 0.95
BMD = 27.5402
BMDL = 17.3281
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BMD Modeling of Data from Two-Year Trinitrobenzene Rat Study (Reddv et al.. 2001a;
Reddv et al.. 199?)
Table C-4. Spleen Weight and Percent Methemoglobin (MetHb) Levels in Rats
Treated with Trinitrobenzene in Diet for 2 Yearsa'b
Males
N
0
0.22
2.64
13.44
Relative spleen weight
10
0.84 ±0.22
1.00 ±0.25
0.44 ±0.08
0.30 ± 0.02°
MetHb
10
0.66 ±0.30
0.57 ±0.41
1.10 ±0.44
1.92 ± 0.55°
Females
N
0
0.23
2.68
13.31
Relative spleen weight
10
0.71 ±0.25
0.94 ±0.20
1.02 ±0.31
0.41 ± 0.06°
MetHb
10
1.0 ±0.63
0.87 ±0.29
1.16 ±0.28
2.49 ± 0.65°
"Reddv et al. (700lb): U.S. EPA fl.997)
bMean ± Standard Deviation.
Significantly different for controls (p = 0.05) by Dunnett's test.
MetHb = methemoglobin; N = number of rats.
BMD modeling was performed on all the data listed in Table C-4, and only male MetHb
was adequately fitted by available continuous models. With constant variance model applied, all
models except Exponential Model 5 and the Hill Model provided an adequate fit to the variance
and the means. Visual inspection of the adequately fitted models indicated that the Exponential
Model 4 provided best fit to the data set at the low dose range which is supported by a low scaled
residual at the a response level close to BMR. Therefore, this model was selected.
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Table C-5. Modeling Results for Percent MetHb Levels in Male Rats Treated with
Trinitrobenzene in Diet for 2 Years"
Model
Variance
/>-Valucb
Means
/>-Valucb
Scaled
Residuals0
AIC
BMDisd
(mg/kg-d)
BMDLisd
(mg/kg-d)
Constant variance
Exponential (Model 2)d
0.3141
0.1052
1.639
-20.44595
6.41983
5.40249
Exponential (Model 3)d
0.3141
0.1052
1.639
-20.44595
6.41983
5.40249
Exponential (Model 4)d
0.3141
0.449
0.07419
-22.37688
2.13524
1.14655
Exponential (Model 5)d
0.3141
NA
-1.42 x 10~7
-20.71215
2.56145
1.18721
Hilld
0.3141
NA
1.63 x 10-6
-20.712148
2.55283
1.08078
Linear6
0.3141
0.259
1.29
-22.2483
4.48567
3.49636
Polynomial (2-degree)6
0.3141
0.259
1.29
-22.2483
4.48567
3.49636
Polynomial (3-degree)6
0.3141
0.259
1.29
-22.2483
4.48567
3.49636
Power"1
0.3141
0.259
1.29
-22.2483
4.48567
3.49636
aReddv et al. (200lb): U.S. EPA fl997)
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals for dose group near the BMD.
dPower restricted to >1.
"Coefficients restricted to be negative.
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose; NA = not applicable.
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Exponential Model 4, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Level for BMD
2.5
Exponential
2
.5
1
0.5
BMDL
BMD
0 2 4 6 8 10 12 14
dose
10:01 07/22 2014
Exponential Model. (Version: 1.9; Date: 01/29/2013)
Input Data File:
C:/Users/j zhao/Documents/BMDS25 0/Data/exp_malesMetHb_Exp-ConstantVariance-BMRlStd-Up. (
d)
Gnuplot Plotting File:
Wed Aug 27 15:09:11 2014
BMDS Model Run
The form of the response function by Model:
Model 2
Model 3
Model 4
Model 5
Y[dose] = a * exp{sign * b * dose0
Y[dose] = a * exp{sign * (b * dose)Ad0
Y[dose] = a * [c-(c-l) * exp{-b * dose0]
Y[dose] = a * [c-(c-l) * exp{-(b * dose)^d0]
Note: Y[dose] is the median response for exposure
sign = +1 for increasing trend in data;
sign = -1 for decreasing trend.
dose;
Model 2 is nested within Models 3 and 4.
Model 3 is nested within Model 5.
Model 4 is nested within Model 5.
Dependent variable = Mean
Independent variable = Dose
Data are assumed to be distributed: normally
Variance Model: exp(lnalpha +rho *ln(Y[dose]))
rho is set to 0.
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A constant variance model is fit.
Total number of dose groups = 4
Total number of records with missing values = 0
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
MLE solution provided: Exact
Initial Parameter Values
Variable
lnalpha
rho(S)
a
b
c
d
Model 4
-1.77375
0
0.5415
0.202372
3.72299
1
(S)
Specified
Parameter Estimates
Variable Model 4
lnalpha -1.75942
rho 0
a 0.594907
b 0.147119
c 3.58708
d 1
Table of Stats From Input Data
Dose N Obs Mean Obs Std Dev
0 10 0.66 0.3
0.22 10 0.57 0.41
2.64 10 1.1 0.44
13.44 10 1.92 0.55
Estimated Values of Interest
Dose Est Mean Est Std Scaled Residual
0 0.5949 0.4149 0.4961
0.22 0.6439 0.4149 -0.5634
2.64 1.09 0.4149 0.07419
13.44 1.921 0.4149 -0.006889
Other models for which likelihoods are calculated:
Model A1: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
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Model A2 : Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)^2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = exp(lalpha + log(mean(i)) * rho)
Model R: Yij = Mu + e(i)
Var{e(ij)} = Sigma^2
Likelihoods of Interest
Model Log(likelihood) DF AIC
A1 15.47505 5 -20.9501
A2 17.2511 8 -18.50219
A3 15.47505 5 -20.9501
R -4.251447 2 12.50289
4 15.18844 4 -22.37688
Additive constant for all log-likelihoods = -36.76. This constant added to the
above values gives the log-likelihood including the term that does not
depend on the model parameters.
Explanation of Tests
Does response and/or variances differ among Dose levels? (A2 vs. R)
Are Variances Homogeneous? (A2 vs. Al)
Are variances adeguately modeled? (A2 vs. A3)
Test 6a: Does Model 4 fit the data? (A3 vs 4)
Test
1:
Test
2 :
Test
3:
Test
Test 1
Test 2
Test 3
Test 6a
Tests of Interest
-2*log(Likelihood Ratio)
43. 01
3.552
3.552
0.5732
D. F.
6
3
3
1
p-value
< 0.0001
0.3141
0.3141
0.449
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose
levels, it seems appropriate to model the data.
The p-value for Test 2 is greater than .1. A homogeneous
variance model appears to be appropriate here.
The p-value for Test 3 is greater than .1. The modeled
variance appears to be appropriate here.
The p-value for Test 6a is greater than .1. Model 4 seems
to adeguately describe the data.
Benchmark Dose Computations:
Specified Effect = 1.000000
Risk Type = Estimated standard deviations from control
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Confidence Level
BMD
BMDL
0.950000
2.13524
1.14655
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