EPA/690/R-20/008F | September 2020 | FINAL
United States
Environmental Protection
Agency
xvEPA
Provisional Peer-Reviewed Toxicity Values for
Picric Acid (2,4,6-Trinitrophenol)
(CASRN 88-89-1)
arid
Ammonium Picrate
(CASRN 131-74-8)
U.S. EPA Office of Research and Development
Center for Public Health and Environmental Assessment
-------
JBI 1#%, United Slates
Environmental Protection
ImI M * Agency
EPA/690/R-20/008F
September 2020
https://www.epa.gov/pprtv
Provisional Peer-Reviewed Toxicity Values for
Picric Acid (2,4,6-Trinitrophenol)
(CASRN 88-89-1)
and
Ammonium Picrate
(CASRN 131-74-8)
Center for Public Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
ii
Picric acid and ammonium picrate
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Q. Jay Zhao, PhD, MPH, DABT
Center for Public Health and Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
SRC, Inc.
7502 Round Pond Road
North Syracuse, NY 13212
CONTRIBUTORS
Daniel D. Petersen, MS, PhD, DABT, ATS, ERT
Center for Public Health and Environmental Assessment, Cincinnati, OH
Zhongyu (June) Yan, PhD
Center for Public Health and Environmental Assessment, Cincinnati, OH
Lucina E. Lizarraga, PhD
Center for Public Health and Environmental Assessment, Cincinnati, OH
PRIMARY INTERNAL REVIEWERS
Roman Mezencev, PhD
Center for Public Health and Environmental Assessment, Cincinnati, OH
Margaret Pratt, PhD
Center for Public Health and 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 content of this PPRTV assessment should be directed to the U.S. EPA
Office of Research and Development (ORD) CPHEA website at
https://www.epa.gov/pprtv/forms/contact-us-about-pprtvs.
in
Picric acid and ammonium picrate
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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS AND ACRONYMS v
BACKGROUND 1
QUALITY ASSURANCE 1
DISCLAIMERS 2
QUESTIONS REGARDING PPRTVs 2
INTRODUCTION 3
REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) 7
HUMAN STUDIES 11
Oral Exposures 11
Inhalation Exposures 11
ANIMAL STUDIES 12
Oral Exposures 12
Inhalation Exposures 16
OTHER DAT A 16
Genotoxicity 27
Supporting Human Studies 27
Supporting Animal Toxicity Studies 27
Metabolism/Toxicokinetic Studies 28
DERIVATION 01 PROVISIONAL VALUES 29
DERIVATION 01 ORAL REFERENCE DOSES 29
Derivation of a Subchronic Provisional Reference Dose 29
Derivation of a Chronic Provisional Reference Dose 33
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 33
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 34
DERIVATION OF PROVISIONAL CANCER RISK ESTIMATES 34
APPENDIX A. SCREENING PROVISIONAL VALUES 35
APPENDIX B. DATA TABLES 52
APPENDIX C. BENCHMARK DOSE MODELING RESULTS 62
APPENDIX D. REFERENCES 71
iv Picric acid and ammonium picrate
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COMMONLY USED ABBREVIATIONS AND ACRONYMS
a2u-g
alpha 2u-globulin
IVF
in vitro fertilization
ACGIH
American Conference of Governmental
LC50
median lethal concentration
Industrial Hygienists
LD50
median lethal dose
AIC
Akaike's information criterion
LOAEL
lowest-observed-adverse-effect level
ALD
approximate lethal dosage
MN
micronuclei
ALT
alanine aminotransferase
MNPCE
micronucleated polychromatic
AR
androgen receptor
erythrocyte
AST
aspartate aminotransferase
MOA
mode of action
atm
atmosphere
MTD
maximum tolerated dose
ATSDR
Agency for Toxic Substances and
NAG
7V-acetyl-P-D-glucosaminidase
Disease Registry
NCI
National Cancer Institute
BMC
benchmark concentration
NOAEL
no-observed-adverse-effect level
BMCL
benchmark concentration lower
NTP
National Toxicology Program
confidence limit
NZW
New Zealand White (rabbit breed)
BMD
benchmark dose
OCT
ornithine carbamoyl transferase
BMDL
benchmark dose lower confidence limit
ORD
Office of Research and Development
BMDS
Benchmark Dose Software
PBPK
physiologically based pharmacokinetic
BMR
benchmark response
PCNA
proliferating cell nuclear antigen
BUN
blood urea nitrogen
PND
postnatal day
BW
body weight
POD
point of departure
CA
chromosomal aberration
PODadj
duration-adjusted POD
CAS
Chemical Abstracts Service
QSAR
quantitative structure-activity
CASRN
Chemical Abstracts Service registry
relationship
number
RBC
red blood cell
CBI
covalent binding index
RDS
replicative DNA synthesis
CHO
Chinese hamster ovary (cell line cells)
RfC
inhalation reference concentration
CL
confidence limit
RfD
oral reference dose
CNS
central nervous system
RGDR
regional gas dose ratio
CPHEA
Center for Public Health and
RNA
ribonucleic acid
Environmental Assessment
SAR
structure activity relationship
CPN
chronic progressive nephropathy
SCE
sister chromatid exchange
CYP450
cytochrome P450
SD
standard deviation
DAF
dosimetric adjustment factor
SDH
sorbitol dehydrogenase
DEN
diethylnitrosamine
SE
standard error
DMSO
dimethylsulfoxide
SGOT
serum glutamic oxaloacetic
DNA
deoxyribonucleic acid
transaminase, also known as AST
EPA
Environmental Protection Agency
SGPT
serum glutamic pyruvic transaminase,
ER
estrogen receptor
also known as ALT
FDA
Food and Drug Administration
SSD
systemic scleroderma
FEVi
forced expiratory volume of 1 second
TCA
trichloroacetic acid
GD
gestation day
TCE
trichloroethylene
GDH
glutamate dehydrogenase
TWA
time-weighted average
GGT
y-glutamyl transferase
UF
uncertainty factor
GSH
glutathione
UFa
interspecies uncertainty factor
GST
glutathione-S-transferase
UFC
composite uncertainty factor
Hb/g-A
animal blood-gas partition coefficient
UFd
database uncertainty factor
Hb/g-H
human blood-gas partition coefficient
UFh
intraspecies uncertainty factor
HEC
human equivalent concentration
UFl
LOAEL-to-NOAEL uncertainty factor
HED
human equivalent dose
UFS
subchronic-to-chronic uncertainty factor
i.p.
intraperitoneal
U.S.
United States of America
IRIS
Integrated Risk Information System
WBC
white blood cell
Abbreviations and acronyms not listed on this page are defined upon first use in the
PPRTV document.
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
PICRIC ACID (CASRN 88-89-1) AND AMMONIUM PICRATE (CASRN 131-74-8)
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.
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.
Currently available PPRTV assessments can be accessed on the U.S. Environmental
Protection Agency's (EPA's) PPRTV website at https://www.epa.gov/pprtv. PPRTV
assessments are eligible to be updated on a 5-year cycle and revised as appropriate to incorporate
new data or methodologies that might impact the toxicity values or affect the characterization of
the chemical's potential for causing adverse human-health effects. Questions regarding
nomination of chemicals for update can be sent to the appropriate U.S. EPA Superfund and
Technology Liaison (https://www.epa.gov/research/fact-sheets-regional-science).
QUALITY ASSURANCE
This work was conducted under the U.S. EPA Quality Assurance (QA) program to ensure
data are of known and acceptable quality to support their intended use. Surveillance of the work
by the assessment managers and programmatic scientific leads ensured adherence to QA
processes and criteria, as well as quick and effective resolution of any problems. The QA
manager, assessment managers, and programmatic scientific leads have determined under the
QA program that this work meets all U.S. EPA quality requirements. This PPRTV was written
with guidance from the CPHEA Program Quality Assurance Project Plan (PQAPP), the QAPP
titled Program Quality Assurance Project Plan (PQAPP) for the Provisional Peer-Reviewed
Toxicity Values (PPRTVs) and Related Assessments/Documents (L-CPAD-0032718-QP), and the
PPRTV development contractor QAPP titled Quality Assurance Project Plan—Preparation of
Provisional Toxicity Value (PTV) Documents (L-CPAD-0031971-QP). As part of the QA
system, a quality product review is done prior to management clearance. A Technical Systems
Audit may be performed at the discretion of the QA staff.
All PPRTV assessments receive internal peer review by at least two Center for Public
Health and Environmental Assessment (CPHEA) scientists and an independent external peer
review by at least three scientific experts. The reviews focus on whether all studies have been
correctly selected, interpreted, and adequately described for the purposes of deriving a
provisional reference value. The reviews also cover quantitative and qualitative aspects of the
provisional value development and address whether uncertainties associated with the assessment
have been adequately characterized.
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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. 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 content of this PPRTV assessment should be directed to the
U.S. EPA Office of Research and Development (ORD) CPHEA website at
https://www.epa.gov/pprtv/forms/contact-us-about-pprtvs.
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INTRODUCTION
Picric acid, CASRN 88-89-1, also known as 2,4,6-trinitrophenol, is a pale yellow,
odorless crystalline solid used in the manufacture of explosives, batteries, matches, and dyes for
textiles (O'Neil et al.. 2013). The chemical formula of picric acid is C6H3N3O7 and its chemical
structure is presented in Figure 1.
N'""
Figure 1. Picric Acid (CASRN 88-89-1) Structure
Ammonium picrate, CASRN 131-74-8, is the ammonium salt of picric acid. Ammonium
pi crate occurs as bright yellow scales or orthorhombic crystals (O'Neil et al.. 2013). It is used in
explosives, fireworks, and rocket propel 1 ants (O'Neil et al.. 2013). The empirical formula for
ammonium picrate is C6H6N4O7 (see Figure 2).
0 o
Figure 2. Ammonium Picrate (CASRN 131-74-8) Structure
A table of physicochemical properties for picric acid and ammonium picrate is provided
below (see Table 1). Both picric acid and ammonium picrate are water soluble. The low pKa of
0.38 for picric acid corresponds to a high pKb of 13.62 for its conjugate base, the picrate anion.
The high pKb indicates that in aqueous solution at neutral pH, the picrate ion will be mostly
dissociated from any spectator cation that is present (e.g., hydrogen ion for picric acid,
ammonium ion for ammonium picrate). As a result, both picric acid and ammonium picrate
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rapidly dissociate to form the pi crate anion when dissolved in water (Thorne and Jenkins. 1997).
Even in the acid environment of the stomach (pH = 3-4 in rats, pH = 1.5-3.5 in humans), both
ammonium picrate and picric acid will occur primarily as dissociated picrate anions, and will do
so as well in other parts of the body with more neutral pH levels. Therefore, ammonium picrate
is expected to behave like picric acid in the body, and the systemic toxicities of the two
chemicals are expected to be very similar. Although available absorption, distribution,
metabolism, and excretion (ADME) studies are limited, they have shown the picrate anion to be
present in the blood and urine of rabbits after dosing (intraperitoneal [i.p.] or dermal) with both
picric acid and ammonium picrate (Weeks et al.. 1983). Distribution of the picrate anion was
widespread in rabbits and guinea pigs following inhalation exposure to ammonium picrate.
Additionally, although toxicity data for ammonium picrate are limited, lethal doses by i.p.
injection in rats were similar for both compounds (Weeks et al.. 1983). Further support for the
similarities of ammonium picrate and picric acid is provided in the analogue approach presented
in Appendix A. In the review that follows, data from both picric acid and ammonium picrate
exposures are considered together in developing assessments of picrate anion toxicity that are
applicable to both picrate source compounds.
Table 1. Physicochemical Properties of Picric Acid (CASRN 88-89-1)
and Ammonium Picrate (CASRN 131-74-8)
Property (unit)
Picric Acid Value
Ammonium Picrate Value
Boiling point (°C)
300 (explodes)3
423 (explodes)3
Melting point (°C)
122.5b
Decomposes3
Density (g/mL at 20°C)
1.76a
1.723
Vapor pressure (mm Hg at 25°C)
7.5 x ur7b
3.4 x 10 11 (estimated)13
Henry's law constant (atm-m3/mole
at 25°C)
1.70 x 10 11 (estimated)13
2.9 x 10 22 (estimated)13
pH (unitless)
0.2 (colorless) to 1 (yellow)0
NV
Solubility in water (mg/L at 25°C)
1.27 x 104b
1.6 x io5 (estimated)13
Relative vapor density (air = 1)
7.9a
NV
Log Kow
1.33b
-1.40 (estimated)13
pKa (at 25°C)
0.38b
NA
pKb
NA
13.62d
Molecular weight (g/mol)
229. lb
246. lb
•'Lewis (21)12).
bChemIDplus (2018).
cChemieal6ook (2017).
Calculated using the equation pKb = pKw - pKa (conjugate acid) = 14 - 0.38 = 13.62, where Kw is the ionic
equilibrium constant for water (10 '
NA = not applicable; NV = not available.
A summary of available toxicity values for picric acid and ammonium picrate 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 88-89-1)
and Ammonium Picrate (CASRN 131-74-8)
Source
(parameter)3'b
Value
(applicability)
Notes
Reference0
Noncancer
IRIS
NV
NA
U.S. EPA (2018b)
HEAST
NV
NA
U.S. EPA (2011a)
DWSHA
NV
NA
U.S. EPA (2018a)
ATSDR
NV
NA
ATSDR (2019)
IPCS
NV
NA
IPCS (2018)
CalEPA
NV
NA
CalEPA (2018a):
CalEPA (2018b)
OSHA (PEL-TWA)
0.1 mg/m3 (picric acid)
Skin designation; 8-hr TWA
for general industry,
construction, and shipyard
employment
OSHA (2018a):
OSHA (2018b):
OSHA (2020)
NIOSH (REL-TWA)
0.1 mg/m3 (picric acid)
Skin designation; TWA for up
to a 10-hr workday
NIOSH (2016)
NIOSH (STEL-TWA)
0.3 mg/m3 (picric acid)
15-min TWA exposure that
should not be exceeded at any
time during a workday
NIOSH (2016)
NIOSH (IDLH)
75 mg/m3 (picric acid)
Based on acute oral toxicity
data in humans and animals
NIOSH (1994)
ACGIH (TLV-TWA)
0.1 mg/m3 (picric acid)
Based on skin sensitization,
dermatitis, and eye irritation
ACGIH (2001):
ACGIH (2018)
DOE (PAC)
PAC-1: 0.3 mg/m3 (picric acid)
PAC-2: 17 mg/m3
PAC-3: 100 mg/m3
Based on TEELs
DOE (2016)
DOE (PAC)
PAC-1: 30 mg/m3 (ammonium
picrate)
PAC-2: 330 mg/m3
PAC-3: 2,000 mg/m3
Based on TEELs
DOE (2016)
USAPHC (air-MEG)
1-hr critical: 75 mg/m3 (picric acid)
1-hr marginal: 15 mg/m3
1-hr negligible: 0.30 mg/m3
8-hr negligible: 0.10 mg/m3
14-d negligible: 0.034 mg/m3
1-yr negligible: 0.034 mg/m3
1-hr values based on TEELs;
other values based on TLV for
skin sensitization, dermatitis,
and eye irritation
U.S. APHC (2013)
USAPHC (air-MEG)
1-hr critical: 250 mg/m3 (ammonium
picrate)
1-hr marginal: 50 mg/m3
1-hr negligible: 30 mg/m3
Based on TEELs
U.S. APHC (2013)
Cancer
IRIS
NV
NA
U.S. EPA (2018b)
HEAST
NV
NA
U.S. EPA (2011a)
DWSHA
NV
NA
U.S. EPA (2018a)
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Table 2. Summary of Available Toxicity Values for Picric Acid (CASRN 88-89-1)
and Ammonium Picrate (CASRN 131-74-8)
Source
(parameter)3'b
Value
(applicability)
Notes
Reference0
CalFPA
NV
NA
CalEPA (2018a):
CalEPA (2018b)
NTP
NV
NA
NTP (2016)
IARC
NV
NA
IARC (2018)
ACGIH
NV
NA
ACGIH (2018)
aSources: ACGIH = American Conference of Governmental Industrial Hygienists; ATSDR = Agency for Toxic
Substances and Disease Registry; CalEPA = California Environmental Protection Agency; DOE = Department of
Energy; DWSHA = Drinking Water Standards and Health Advisories; HEAST = Health Effects Assessment
Summary Tables; IARC = International Agency for Research on Cancer; IPCS = International Programme on
Chemical Safety; IRIS = Integrated Risk Information System; NIOSH = National Institute for Occupational Safety
and Health; NTP = National Toxicology Program; OSHA = Occupational Safety and Health Administration;
USAPHC = U.S. Army Public Health Command.
Parameters: IDLH = immediately dangerous to life or health; MEG = military exposure guideline;
PAC = protective action criteria; PEL = permissible exposure limit; REL = recommended exposure limit;
STEL = short-term exposure limit; TEEL = temporary emergency exposure limit; TLV = threshold limit value;
TWA = time-weighted average.
°Reference date is the publication date for the database and not the date the source was accessed.
NA = not applicable; NV = not available.
A non-date-limited literature search was last updated in April 2020 for picric acid
(CASRN 88-89-1), and non-date-limited searches were conducted in April 2020 for studies
relevant to the derivation of provisional toxicity values for ammonium picrate
(CASRN 131-74-8). The database searches for PubMed, TOXLINE (including TSCATS1), and
Web of Science were conducted by an information specialist and records stored in the
U.S. EPA's Health and Environmental Research Online (HERO) database. The following
additional databases were searched for health-related data: American Conference of
Governmental Industrial Hygienists (ACGIH), Agency for Toxic Substances and Disease
Registry (ATSDR), California Environmental Protection Agency (CalEPA), Defense Technical
Information Center (DTIC), European Centre for Ecotoxicology and Toxicology of Chemicals
(ECETOC), European Chemicals Agency (ECHA), U.S. EPA Chemical Data Access Tool
(CDAT), U.S. EPA ChemView, U.S. EPA Health Effects Assessment Summary Tables
(HEAST), U.S. EPA Integrated Risk Information System (IRIS), U.S. EPA Office of Water
(OW), International Agency for Research on Cancer (IARC), Japan Existing Chemical Data
Base (JECDB), National Institute for Occupational Safety and Health (NIOSH), National
Toxicology Program (NTP), Organisation for Economic Co-operation and Development (OECD)
Existing Chemicals Database, OECD Screening Information Dataset (SIDS) High Production
Volume Chemicals via International Programme on Chemical Safety (IPCS) INCHEM,
Occupational Safety and Health Administration (OSHA), U.S. Army Public Health Command
(APHC), and World Health Organization (WHO).
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REVIEW OF POTENTIALLY RELEVANT DATA
(NONCANCER AND CANCER)
Tables 3A and 3B provide an overview of the relevant data for picric acid and
ammonium picrate and include all potentially relevant repeated-dose, short-term, subchronic, and
chronic studies. Principal studies used in the PPRTV assessment for derivation of provisional
toxicity values are identified in bold. The phrase "statistical significance" or the term
"significant," used throughout the document, indicates ap-value < 0.05 unless otherwise noted.
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Table 3A. Summary of Potentially Relevant Noncancer Data for Picric Acid (CASRN 88-89-1) and
Ammonium Picrate (CASRN 131-74-8)
Category"
Number of Male/Female,
Strain, Species, Study Type,
Reported Doses, Study
Duration
Dosimetryb
Critical Effects
NOAEL
LOAEL
Reference
(comments)
Notes0
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
Animal
1. Oral (mg/kg-d)
Short term
4 M/4 F, S-D rat, picric acid
administered by gavage,
newborn rat dose-finding
study, daily for 14 d
(PND 4-17)
Reported doses: 0, 16.3, 81.4,
or 407 mg/kg-d
0, 16.3,
81.4, or 407
Decreased body weight in males and females,
decreased relative and absolute kidney weight in
males, and increased relative liver weight in
males and females. Mortality was observed in
males and females.
16.3
81.4 (FEL)
TakahasM et al.
(2004)
PR
Short term
6 M/6 F, S-D rat, picric acid
administered by gavage,
0,4.1, 16.3,
or65.1
Increased relative and absolute liver weight in
males and females, decreased absolute
16.3
65.1
TakahasM et al.
(2004)
PR
newborn rat main study, daily
for 18 d (PND 4-21)
epididymis weight in males and increased relative
spleen weight in females.
Reported doses: 0, 4.1, 16.3,
or 65.1 mg/kg-d
Short term
3 M/3 F, S-D rat, picric acid
administered by gavage,
0, 20, 100,
or 500
Hematological effects in females, increased liver
weight in males and females, increased relative
20
100
TakahasM et al.
(2004)
PR
young rat dose-finding study
daily for 14 d
spleen weight in males.
Reported doses: 0, 20, 100, or
500 mg/kg-d
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Table 3A. Summary of Potentially Relevant Noncancer Data for Picric Acid (CASRN 88-89-1) and
Ammonium Picrate (CASRN 131-74-8)
Category"
Number of Male/Female,
Strain, Species, Study Type,
Reported Doses, Study
Duration
Dosimetryb
Critical Effects
NOAEL
LOAEL
Reference
(comments)
Notes0
Short term
6 M/6 F, S-D rat, picric acid
administered by gavage,
young rat main study, daily
for 28 d
Reported doses: 0,4,20, or
100 mg/kg-d
0,4,20, or
100
Increased absolute liver weights,
hematological and related splenic effects
(increased absolute and relative spleen weights
and hematopoiesis) in males and females, and
testicular effects (testicular atrophy, decreased
sperm in the epididymis) in males
20
100
Takahashiet
PR,
PS
al. (2004)
2. Inhalation (mg/m3)
ND
aDuration categories are defined as follows: Acute = exposure for <24 hours; short term = repeated exposure for 24 hours to <30 days; long term (subchronic) = repeated
exposure for >30 days <10% lifespan for humans (>30 days up to approximately 90 days in typically used laboratory animal species); and chronic = repeated exposure
for >10% lifespan for humans (>~90 days to 2 years in typically used laboratory animal species) (U.S. EPA. 2002).
bDosimetry: Doses are presented as ADDs (mg/kg-day) for oral noncancer effects and as TWA concentrations (mg/m3) for inhalation noncancer effects.
°Notes: PS = principal study; PR = peer reviewed.
ADD = adjusted daily dose; F = females; FEL = frank effect level; LOAEL = lowest-observed-adverse-effect level; M = males; ND = no data;
NOAEL = no-observed-adverse-effect level; PND = postnatal day; S-D = Sprague-Dawley; TWA = time-weighted average.
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Table 3B. Summary of Potentially Relevant Cancer Data for Picric Acid (CASRN 88-89-1) and
Ammonium Picrate (CASRN 131-74-8)
Category
Number of Male/Female,
Strain, Species, Study
Type, Reported Doses,
Study Duration
Dosimetry
Critical Effects
Reference (comments)
Notes
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
Animal
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
ND = no data.
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HUMAN STUDIES
Oral Exposures
ACGIH (2001) reports that ingestion of 1-2 g picric acid (approximately 14-28 mg/kg)
can cause severe poisoning in humans. Effects attributed to picric acid poisoning include
headache, vertigo, nausea, vomiting, abdominal cramps, diarrhea, yellow coloration of the skin
and conjunctiva, myalgia, hematuria, albuminuria, and at high doses, destruction of erythrocytes,
gastroenteritis, hemorrhagic nephritis, acute hepatitis, stupor, convulsions, and death (ACGIH.
2018; NIOSH. 2016; II.(). 2011; Weeks et al.. 1983). No data were located on the toxicity of
ammonium picrate to humans following repeated oral exposure.
Inhalation Exposures
Exposure to picric acid dust in the air has been reported to produce skin and eye irritation
and sensitization in exposed persons (ACGIH. 2001). No data on systemic or respiratory effects
of inhaled picric acid were found. One published study evaluated potential effects of
occupational inhalation exposure to ammonium picrate dust for 2-24 months (Sunderman et al..
1945).
Sunderman et al. (1945)
Sunderman et al. (1945) evaluated the potential health effects in 71 individuals who
worked predominately with ammonium picrate for 2-24 months. Other chemicals that the
workers may have been exposed to included potassium nitrate and chlorinated diphenyl. The
employees worked in 10 different types of positions with varying exposure to ammonium picrate
dust. Those working in "milling" and "preforming" positions (n= 18) were exposed to the most
dust, with atmospheric concentrations at these sites ranging from 0.0088 to 0.1942 mg/m3, based
on 20-minute samples collected every 2 hours over several working days. Respirators from two
milling workers collected 52 and 156 mg of ammonium picrate during a 6-hour operation.
Atmospheric dust concentrations were not reported for other working areas (pressing, examining,
coating, firing, pack house, laundry, or experimental). Physical examinations were conducted
over a 1-15-month period in all workers, including urinalysis, complete blood counts, and skin
evaluation. Upon report of epistaxis (acute hemorrhage from the nostril, nasal cavity, or
nasopharynx) by two workers, examination of the nasal cavity, pharynx, ear canal, drum
membrane, epipharynx, larynx, hearing ability with 4,090 double vibrations (d.v.) frequency
tuning fork, and inspection and palpation of the neck for enlarged lymph nodes was conducted
on 18 randomly selected workers.
Results of the urinalysis and blood testing were not reported. Physical examination of the
workers found swelling and excoriation (skin lesions due to chronic skin-picking) of the nasal
mucous membranes and nasal mucosa that bled at slight manipulation in several individuals.
Yellowish skin coloration (particularly around the hairline, nape of the neck, and palms) and
dermatitis were also observed in several workers, but this was attributed to direct skin exposure
to dust rather than inhalation exposure. Dermatitis was observed in 7/71 workers on exposed
parts of the body (hands/forearms). All seven workers were in "low exposure" areas (coating,
firing, laundry, experimental). Lesions, characterized as erythematous patches containing
papules and vesicles, cleared when individuals were removed from contact with ammonium
picrate. Two of the individuals developed eczematous lesions and could not continue to work
with ammonium picrate; the other five individuals were able to return to work with additional
protection (i.e., gloves). In the detailed evaluation of the upper respiratory tract, yellow
discoloration of the nasal vestibules and yellow stained mucus in the anterior turbinate were
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observed. The investigators found that blowing ammonium picrate dust into the nose caused a
slight reflexive spasm in the palatal pharyngeal area, which was associated with a "tight feeling,
sharp taste, odor" described by most of the workers. No alterations in the other examined
endpoints were attributable to inhalation of ammonium picrate dust.
The investigators concluded that inhalation of ammonium picrate dust does not cause
prominent pathological changes in humans. However, available data are inadequate to establish
no-observed-adverse-effect level/lowest-observed-adverse-effect level (NOAEL/LOAEL) values
because of the lack of a control group; relatively small study size; limitations in characterization
of exposure and evaluation of health endpoints, and reporting deficiencies in the publication.
ANIMAL STUDIES
Oral Exposures
The effects of oral exposure of animals to picric acid were evaluated in two short-term
toxicity studies and two corresponding dose-finding studies (Takahashi et al.. 2004).
Short-Term Studies
Takahashi et al. (2004): Newborn Rat Dose-Finding Study
Sprague-Dawley (S-D) rat pups (4/sex/dose) were administered picric acid (81.4%
purity) by daily gavage at doses of 0, 16.3, 81.4, or 407 mg (as picric acid)/kg-day from
Postnatal Days (PNDs) 4-17 (14 days total). For the gavage doses, the picric acid was
suspended in a 0.5% carboxymethyl cellulose sodium salt aqueous solution with 0.1%
Tween-80. One foster mother nursed four male and four female pups. The animals were
allowed free access to a sterilized basal diet (manufacturer [MF]: Oriental Yeast, Tokyo, Japan)
and were maintained in an environmentally controlled room at 22 ± 2°C with a relative humidity
of 55 ± 10%) and a 12:12-hour light/dark cycle. All pups were sacrificed on PND 18 and
necropsies conducted. General condition, body weight, hematology, blood biochemistry, and
organ weights were examined.
In the high-dose group, all pups died by Day 4 of the dosing period. In the mid-dose
group, one male pup died on Day 3, a female pup died on Day 6, and a second female pup died
on Day 7 of the dosing period. Prior to their deaths, these pups showed hypoactivity, bradypnea,
and hypothermia. In surviving pups in the mid-dose group, hypoactivity was observed on
Days 3, 5, or 8 of the dosing period. Yellowish fur was observed in all picric acid-treated rats
but not in the controls; the study authors stated that this "did not seem to be an adverse effect"
because the pups' "hair roots and skin showed no anomalies." The mid-dose treatment resulted
in statistically significant decreases in body weight in the male pups (13%> lower than controls).
Decreased body weights were also observed in the female pups (15%> lower than controls), but
the decreases were not statistically significant (see Table B-l). The study authors reported that
no treatment-related effects were observed on food consumption or behavior. They also reported
that treatment at the mid dose resulted in statistically significant increases in relative liver
weights (13%o, liver-to-body-weight ratio) in male pups and statistically nonsignificant increases
in relative liver weights in female pups (22%>; see Table B-l). The mid-dose animals also
showed statistically significant decreases in absolute (26%) and relative (14%>) kidney weight in
male pups and statistically nonsignificant decreases in absolute (22%) and relative (8.2%>) kidney
weight in female pups. No other significant body-weight and organ-weight changes were
reported. The study authors also stated that there were no other consistent changes related to the
administration of picric acid in hematological results, blood biochemical parameters, or necropsy
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findings at any dose. A frank effect level (FEL) of 81.4 mg/kg-day is identified for this study
based on mortality observed in male and female rat pups; a corresponding NOAEL of
16.3 mg/kg-day is determined. Significant (statistically and/or biologically) changes in body,
kidney, and liver weight in male and/or female rats were also observed at 81.4 mg/kg-day.
Takahashi et al. (2004): 18-Day Newborn Rat Main Study
In a peer-re vie wed, short-term, 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 (81.4% purity) and given to six pup 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 PNDs 4-21
(18 days). The pups in the main study were sacrificed on PND 22. Another six 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. The animals were allowed free access to a sterilized basal
diet (MF: Oriental Yeast, Tokyo, Japan) after weaning and 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), testis 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 testis 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 or red blood cell (RBC) count, hematocrit (Hct), hemoglobin (Hb), mean
corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean
corpuscular volume (MCV), total leukocyte or white blood cell (WBC) count, differential
leukocyte count, platelet count (PLAT), mean platelet volume (MPV), cell morphology,
prothrombin time (PT), and activated 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 (P), 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 the controls.
The study authors reported a statistically significant decrease in body weight on Days 4 and 8 of
the dosing period (maximum 7% decrease) for males in the 65.1-mg/kg-day group (data not
presented in publication). However, terminal body weights for treated groups in the main study
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were not statistically different from controls (see Table B-2). No dose-dependent effects on
body weight or food consumption were observed during the maintenance-recovery period. As
shown in Table B-2, 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 with controls. Absolute liver weights were also increased in males and females
in the 65.1-mg/kg-day dose groups (10 and 12%, respectively), although these increases did not
reach statistical significance. The males in the same dose group had statistically significant
decreases in absolute (but not relative) epididymis weight, and the females had statistically
significant increases in relative (but not absolute) spleen weight. No other treatment-related,
organ-weight effects were observed. Developmental landmarks and sexual maturation were
similar in the 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 in both
sexes, decreased absolute epididymis weight in males and increased relative spleen weight in
females, the high dose of 65.1 mg/kg-day is considered the LOAEL and the mid dose of
16.3 mg/kg-day is identified as the corresponding NOAEL for both male and female rats.
Takahashi et al. (2004): Young Rat Dose-Finding Study
Five-week-old S-D rats (3/sex/dose) were administered picric acid (81.4% purity) by
daily gavage at doses of 0, 20, 100, or 500 mg (as picric acid)/kg-day for 14 days. Picric acid
was suspended in a 0.5% carboxymethyl cellulose sodium salt aqueous solution with 0.1%
Tween-80. The animals were sacrificed on the day following the last dose after overnight fasting
and necropsies were conducted. General condition, body weight and food consumption,
hematology results, and organ weights were examined.
In the high-dose group, all male rats and one female rat died by Day 2 of the dosing
period. No deaths were observed in the control, low- and mid-dose groups. Yellowish fur was
observed in all picric acid-treated rats but not in controls; the study authors stated that this "did
not seem to be adverse" because the rats' "hair roots and skin showed no anomalies." They also
reported that the body weights of males and females in the low- and mid-dose groups did not
significantly differ from those of controls during the dosing period. No treatment-related effects
were reported on food consumption or behavior. Both males and females in the mid-dose group
exhibited significantly increased absolute liver weights (13% increase in males and 25% in
females) and relative liver weights (9.7% and not statistically significant in males and 18% in
females; see Table B-3). Mid-dose males exhibited a statistically significant increase in relative
spleen weight (14% increase). No other statistically significant organ-weight changes were
reported. Mid-dose females exhibited significantly lower Hb and Hct values and a higher
reticulocyte (Ret) ratio relative to controls (see Table B-4). A LOAEL of 100 mg/kg-day with a
corresponding NOAEL of 20 mg/kg-day is identified for this study based on biologically
significantly (>10%) increased liver weights in male (absolute) and female (absolute and
relative) rats and increased relative spleen weight in male rats.
Takahashi et al. (2004):28-Day Young Rat Main Study
In a separate study by Takahashi et al. (2004). picric acid was given to young
(5-week-old) S-D rats (six/sex/dose) daily via gavage. This study used the same vehicle with
identical measured impurities as the newborn rat study. 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. The animals were sacrificed the next day following an overnight fast. Another
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six 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. The animals were allowed free access to a sterilized basal
diet (MF: Oriental Yeast, Tokyo, Japan) after weaning. They 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 the controls. As shown in Table B-5, there were statistically
significantly higher WBC and 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.
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-6). 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-6). 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-7).
Females at 100 mg/kg-day showed the development of germinal centers, extramedullary
hematopoiesis, and hemosiderin deposition in the spleen at the end of the dosing period
(see Table B-8). 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 Studies
No studies have been identified.
Chronic Studies
No studies have been identified.
Reproductive Studies
No studies have been identified.
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Developmental Studies
No studies have been identified.
Inhalation Exposures
No adequate inhalation studies have been identified on the subchronic, chronic,
developmental, or reproductive toxicity or on the carcinogenicity of picric acid or ammonium
picrate in animals.
OTHER DATA
Table 4 A provides an overview of genotoxicity studies of picric acid and ammonium
picrate, and Table 4B provides and overview of other supporting studies on picric acid and
ammonium picrate.
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Table 4A. Summary of Picric Acid (CASRN 88-89-1) and Ammonium Picrate (CASRN 131-74-8) Genotoxicity
Endpoint
Test System
Dose/
Concentration3
Results
without
Activationb
Results with
Activationb
Comments
References
Genotoxicity studies in prokaryotic organisms
Mutagenicity
(picric acid)
Salmonella typhimurium
strains TA98, TA100,
TA1535, and TA1537
0-100 |ig picric acid/plate
(")
TA98, TA100,
TA1535,
TA1537
(")
TA1535
(±)
TA100
(+)
TA98, TA1537
Activation using male
S-D rat liver S9 induced with
Aroclor 1254
Haworth et al.
(1983)
Mutagenicity
(picric acid)
S. typhimurium strains
TA98, TA100, TA1535,
and TA1537
0-100 |ig picric acid/plate
(")
TA98, TA100,
TA1535,
TA1537
(")
TA1535,
TA100
(+)
TA98, TA1537
Activation using male Syrian
hamster liver S9 induced
with Aroclor 1254
Haworth et al.
(1983)
Mutagenicity
(ammonium picrate)
S. typhimurium strains
TA98, TA100, TA1535,
TA1537, and TA1538
0, 0.5, 1.0, 10, 100, 500,
1,000 ng ammonium
picrate/plate
Plate incorporation
Litton
Bionetics
(1979)
Genotoxicity studies in nonmammalian eukaryotic organisms
Mutagenicity
(ammonium picrate)
Saccharomyces cerevisiae
strain D4
0, 0.5, 1.0, 10, 100, 500,
1,000 ng ammonium
picrate/plate
Plate incorporation
Litton
Bionetics
(1979)
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Table 4A. Summary of Picric Acid (CASRN 88-89-1) and Ammonium Picrate (CASRN 131-74-8) Genotoxicity
Endpoint
Test System
Dose/
Concentration3
Results
without
Activationb
Results with
Activationb
Comments
References
Genotoxicity studies in mammalian cells—in vitro
Mutagenicity
(ammonium picrate)
L5178Y/TK+/- mouse
lymphoma cells
0, 500-3,000 ng ammonium
picrate/mL (-S9)
0, 31.3-1,000 ng ammonium
picrate/mL (+S9)
Cytotoxicity (>50%) was
observed at >500 ng/mL
without activation and
>400 |ig/mL with activation.
In the assay with activation,
only 220 cells were scored at
1,000 |ig/mL due to high
toxicity.
Precipitation was noted at
concentrations
>1,000 ng/mL; these
concentrations were obtained
by mixing the weighed
compound directly into
growth medium.
Litton
Bionetics
(1979)
Clastogenicity [CA]
(picric acid)
CHO cells
0, 600, 800, 1,000 ng picric
acid/mL (-S9)
0, 1,740, 2,485, 3,500, 5,000 ng
picric acid/mL (+S9)
NA
NTP (1985)
Clastogenicity [SCE]
(picric acid)
CHO cells
0, 50, 167, 500, 1,700 ng picric
acid/mL (-S9)
0, 167, 500, 1,670, 5,000 ng
picric acid/mL (+S9)
+
NA
NTP (1985)
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Table 4A. Summary of Picric Acid (CASRN 88-89-1) and Ammonium Picrate (CASRN 131-74-8) Genotoxicity
Endpoint
Test System
Dose/
Concentration3
Results
without
Activationb
Results with
Activationb
Comments
References
Clastogenicity [SCE]
(ammonium picrate)
L5178Y/TK+/- mouse
lymphoma cells
0, 15.6,31.3,62.5, 125, 250 ng
ammonium picrate/mL (-S9);
0, 1.00,2.00,3.90,7.80, 15.6,
31.3 |ig ammonium picrate/mL
(+S9)
+
Significantly increased SCE
frequency was observed at
concentrations >15.6 ng/mL
(compared with solvent
control) and at 250 ng/mL
(compared with negative
control).
Cytotoxicity was observed at
concentrations >31.3 ng/mL
in the presence of S9
activation: cultures at these
concentrations did not
contain scoreable cells.
Litton
Bionetics
(1979)
Genotoxicity studies—in vivo
Mutagenicity [dominant
lethal]
(ammonium picrate)
Male CD-I mice
(10/group); ammonium
picrate administered in
deionized water orally
once/d for 5 d and mated
weekly to untreated virgin
females over 7 wk;
females were sacrificed
after 14 d
0, 2.23, 7.43, 22.3 mg
ammonium picrate/kg
No significant difference
from concurrent and/or
historical controls for the
parameters measured.
Litton
Bionetics
(1979)
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Table 4A. Summary of Picric Acid (CASRN 88-89-1) and Ammonium Picrate (CASRN 131-74-8) Genotoxicity
Endpoint
Test System
Dose/
Concentration3
Results
without
Activationb
Results with
Activationb
Comments
References
Mutagenicity [sex-linked
recessive lethal]
(picric acid)
Drosophila melanogaster
0, 450 ppm picric acid
(feeding);
0, 400 ppm picric acid
(injection)
0, 300, 500, 1,000, 1,500 ppm
picric acid (feeding);
0, 1,000, 1,500 ppm picric acid
(injection)
0, 1,250 ppm picric acid
(feeding);
0, 1,500 ppm picric acid
(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)
Clastogenicity [mouse
bone marrow CA]
(ammonium picrate)
Male Ha/ICR mice
(32/group); ammonium
picrate administered in
deionized water orally for
1 (single dose) or 5 d;
sacrifice at 6, 24, or 48 hr
following single exposure;
or 6 hr following final
exposure of 5 d.
0,2.23, 7.43, or 22.3 mg
ammonium picrate/kg
NA
Litton
Bionetics
(1979)
Clastogenicity [reciprocal
translocation]
(picric acid)
D. melanogaster
0, 1,500 ppm picric acid
(injection)
NA
Woodruff et al.
(1985)
aLowest effective dose for positive results, highest dose tested for negative results.
b+ = positive, (+) = weak positive, - = negative, ± = equivocal.
CA = chromosomal aberration; CHO = Chinese hamster ovary; NA = not applicable; S-D = Sprague-Dawley; SCE = sister chromatid exchange.
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Supporting evidence—human studies
Dermal [patch test]
(ammonium picrate)
Patch-testing of 23 volunteers,
(11 picrate workers,
12 never-exposed individuals).
Patches of acetone saturated with
ammonium picrate were placed
on the inside of the arm for 5 d.
Ten days after removal of the
patch, the patch was reapplied for
2 d. Skin was evaluated for
irritation.
No skin irritation was observed in any
subject after the initial exposure. Two
individuals reacted after reapplication
of patches to the same area (one picrate
worker and one nonpicrate worker).
However, the nonpicrate worker was a
guard who had previously reported
itchy, burning, reddened eyelids upon
making rounds in the building
containing ammonium picrate.
Ammonium picrate is not a primary irritant
but may cause sensitization in some
individuals.
Sunderman et al.
(1945)
Dermal [case study]
(picric acid)
A 61-yr-old woman presented
with severe acute eczema on
dorsum of right hand after using
Queratil® burn cream for 3 d.
The cream contained 10%
alcoholic solution of picric acid
along with several other
compounds. After the burn
healed and eczema cleared, patch
testing was conducted.
Patch testing was positive for a
reaction to picric acid. Patch testing
was negative to other cream
components.
Observed allergic reaction was due to
sensitization to picric acid.
Aguirre et al. (1993)
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Supporting evidence—animal studies
Acute [oral]
(picric acid)
The LD5o was determined in
groups of rats (at least
3/sex/group) exposed to picric
acid at doses of 0, 50, 100, 200,
600, 400, or 800 mg/kg via
gavage. Rats were observed for
14 d.
Separate groups of males
(2-3/group) were exposed to 0,
100, 200, 300, or 400 mg/kg.
Arterial blood acid-base
parameters were measured
35-40 min after exposure.
Mortality in males was 0% at
<200 mg/kg, 70-80% at
300-400 mg/kg, and 100% at
800 mg/kg. In females, death was 0%
at <100 mg/kg, -25% at 200 mg/kg,
and 100% at >300 mg/kg. Death
occurred within 60 min at lethal doses.
Clinical signs at lethal doses included
tremors leading to tonic/clonic
convulsions and discharge from the
eyes. Cause of death was attributed to
acidosis, with significant fall in blood
pH at 400 mg/kg.
Rat LD50:
Male: 290 ± 57.5 mg/kg
Female: 200 ± 42.9 mg/kg
Wvtnan et al. (1992)
Acute [oral]
(ammonium picrate;
picric acid)
The LD50 was determined in
groups of male and female rats
exposed to ammonium picrate or
picric acid in polyethylene glycol
via gavage. Rats were observed
for 14 d.
Clinical signs at lethal doses
(compound not specified) included
ataxia, convulsions, and red discharge
from eyes.
Rat LD50 (CI = 95%):
Ammonium picrate
Male: 1,690 (1,590-1,830) mg/kg
Female: 720 (240-2,120) mg/kg
Picric acid
Male: 629 (536-737) mg/kg
Female: 520 (430-620) mg/kg
Weeks etal. (1983)
Acute [oral]
(picric acid)
The LDlo for oral picric acid was
determined in groups of cats and
rabbits. No further details were
provided.
NA
Cat LDlo = 250 mg/kg
Rabbit LDlo =120 mg/kg
Weeks etal. (1983)
Acute [i.p.]
(ammonium picrate;
picric acid)
The ALD was determined in
groups of male and female rats
exposed to ammonium picrate or
picric acid in polyethylene glycol
via i.p. injection. Rats were
observed for 14 d.
Clinical signs at lethal doses
(compound not specified) included
ataxia, convulsions, and red discharge
from eyes.
Rat ALD:
Ammonium picrate
Male: 168 mg/kg
Female: 168 mg/kg
Picric acid
Male: 378 mg/kg
Female: 168 mg/kg
Weeks etal. (1983)
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Chronic [inhalation]
(ammonium picrate)
Four rabbits and 8 guinea pigs
were housed in cages in the
milling and preforming areas of a
factory with reported ammonium
picrate dust concentrations of
0.0088-0.1942 mg/m3. No
control animals were included.
Sacrifices were scheduled at 6 wk
(2 rabbits) and 12 mo (remaining
animals). At death or sacrifice,
"tissues" were examined for
histopathological changes.
Three guinea pigs died during exposure
(Wk 1, Wk 3, and 9 mo).
Histopathological examination of the
guinea pig that died after 3 wk showed
congestion, inflammation, and erosion
of the nasal mucosa and turbinates,
lung congestion, and hyaline
degeneration of the heart.
Inflammation of the trachea and
periductal fibrosis and glycogen
infiltration of the liver was observed in
rabbits sacrificed at 6 wk. After
12 mo, histopathological findings
included minor inflammatory lesions in
the lungs and yellow picrate deposits in
several organs, most notably the lungs
and liver but also the heart, thyroid,
and kidney.
Very few conclusions can be drawn from this
study because of the lack of controlled
exposure, limited exposure level data, and
lack of a control group. However, the study
suggests that ammonium picrate may damage
the respiratory tract.
Sunderman et al.
(1945)
Acute [dermal]
(ammonium picrate;
picric acid)
Six rabbits were exposed once to
0.5 g ammonium picrate or picric
acid under occlusion on intact or
abraded skin. The skin was
evaluated 24 hr, 72 hr, and 7 d
after a single application.
No skin irritation was observed.
Neither ammonium picrate nor picric acid are
primary skin irritants.
Weeks etal. (1983)
Acute [dermal]
(ammonium picrate)
Guinea pigs (10/group) were
injected with 0 or 1% solution of
ammonium picrate in
polyethylene glycol for 3 wk.
After a 2-wk rest, guinea pigs
were challenged with single
challenge dose.
2/10 guinea pigs showed sensitization
reactions (compared with
0/10 controls).
Ammonium picrate is a mild skin sensitizer.
Weeks etal. (1983)
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Supporting evidence—studies of ADME
ADME [oral]
(picric acid)
Blood and urine samples were
collected from F344 rats treated
via gavage with a single dose of
[14C] picric acid (100 mg/kg).
[14C]-label absorption from the gut
increased continuously for 1 hr, then
was eliminated in a biphasic manner.
A large majority remained in the gut
(absorption coefficient was 0.069 hr1).
24 hr after exposure, the primary
depots of radioactivity (per gram tissue
basis) were blood (plasma protein
binding), spleen, kidney, liver, lung,
and testes. 50.55% of radioactivity
was found in the urine, 22.75% was in
the gut contents, and 6.19% was in
feces.
The following metabolites were
isolated from urine:
\ -acctylisopicramic acid (14.8%),
picramic acid (18.5%),
\ -acctylpicramic acid (4.7%), and
unidentified components (2.4%). Most
of the radioactivity was in the parent
compound (60%) that was excreted
unchanged.
Absorption from the gut is limited.
Absorption and elimination are biphasic with
an initial rapid phase. The slow phase is
attributed to plasma protein binding.
Distribution is widespread.
Primary metabolic pathways are reduction of
the ortho or para nitro group on the aromatic
ring and acetylation of the amine.
Primary urinary excretion product is the
parent compound (which exists as dissociated
picrate anion under physiological
conditions).
Wvtnan et al. (1992)
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
ADME [i.v.]
(picric acid)
Blood and urine samples were
collected from F344 rats
following i.v. injection of [14C]
picric acid (50 mg/kg).
Blood distribution period of 2 hr.
Elimination followed first-order
kinetics, with a plasma half-life of
13.4 hr (rapid phase) followed by a
slower elimination phase attributed to
plasma protein binding (demonstrated
in vitro).
24-hr post-injection, 81.5% of i.v. dose
was cleared from the blood, with
58.9% excreted in urine and 12.2%
eliminated in feces. Low levels still
found in urine and feces 14 d after i.v.
dose.
Elimination is biphasic, with initial (rapid)
half-life of 13.4 hr. Low levels are retained
in the body for long periods of time
(attributed to plasma protein binding). The
primary excretion path is urinary.
Wvtnan et al. (1992)
ADME [i.p.]
(ammonium picrate;
picric acid)
Blood and urine samples were
collected from rabbits following
i.p. injection of ammonium
picrate or picric acid (60 mg/kg).
Ammonium picrate: Blood
concentrations of picrate ion were
135.7, 87.8, 12.1, and 0 ng/mL at 1 hr,
6 hr, 24 hr, and 7 d, respectively.
Urine concentrations of picrate ion
were 2.6, 0.7, 0, and 0 ng/mL at 24 hr,
48 hr, 72 hr, and 7 d, respectively.
Picric acid: Blood concentrations of
picrate ion were 93.5, 7.7, and
0 iig/mL at 6 hr, 24 hr, and 7 d,
respectively.
Urine concentrations of picrate ion
were 4.7, 1.5, 1.2, and 0 ng/mL at
24 hr, 48 hr, 72 hr, and 7 d,
respectively.
Data suggest biphasic elimination, with peak
blood concentrations at <24 hr, but detectable
picrate ion in urine at 3 d.
Weeks etal. (1983)
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
ADME [dermal]
(ammonium picrate;
picric acid)
Blood and urine samples were
collected from rabbits following
dermal exposure to ammonium
picrate or picric acid for 24 hr
under occluded conditions
(600 mg/kg).
Ammonium picrate: Blood
concentrations of picrate ion were 8.8,
31.3, and 7.9 ng/mL at 6 hr, 24 hr, and
7 d, respectively.
Urine concentrations of picrate ion
were 11.6, 14.4, 21.8, and 2 ng/mL at
24 hr, 48 hr, 72 hr, and 7 d,
respectively.
Picric acid: Blood concentrations of
picrate ion were 84.7, 69.7, and
0 iig/mL at 6 hr, 24 hr, and 7 d,
respectively.
Urine concentrations of picrate ion
were 112.5, 18.0, 10.3, and 1.3 ng/mL
at 24 hr, 48 hr, 72 hr, and 7 d,
respectively.
Data suggest biphasic elimination, with peak
blood concentrations at <24 hr, but detectable
picrate ion in urine at 7 d.
Weeks etal. (1983)
ADME [inhalation]
(ammonium picrate)
Tissue samples were examined
for picrate deposits in rabbits and
guinea pigs housed in cages in
the milling and preforming areas
of a factory for 12 mo. Reported
ammonium picrate dust
concentrations of
0.0088-0.1942 mg/m3.
Picrate deposits as demonstrated as
guanidine (as a fixing agent) picrate
granules were found in various organs,
with highest concentrations in lung and
liver, followed by moderate
concentrations in heart, thyroid, and
kidneys. Small amounts were seen in
the chondrocytes of the bronchial
cartilages and adrenal cells.
Distribution is widespread following
inhalation exposure.
Stmderman et al.
(1945)
ADME = absorption, distribution, metabolism, excretion; ALD = approximate lethal dose; CI = confidence interval; i.p. = intraperitoneal; i.v. = intravenous;
LD5o = median lethal dose (dose at which 50% mortality occurs); LDLo = lowest observed lethal dose; NA = not applicable.
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Genotoxicity
Evidence for genotoxic activity of picric acid and ammonium picrate is limited. Neither
picric acid nor ammonium picrate was mutagenic to Salmonella typhimurium without metabolic
activation (1 la worth et al.. 1983; Litton Bionetics. 1979). In the presence of metabolic
activation, however, picric acid was mutagenic to S. typhimurium strains TA98 and TA1537
(Haworth et al.. 1983). while ammonium picrate was not (Litton Bionetics. 1979). Ammonium
picrate was also not mutagenic to Saccharomyces cerevisiae strain D4 or mouse L5178Y/TK+/-
lymphoma cells with or without metabolic activation (Litton Bionetics. 1979). Results were
negative in in vivo assays for dominant lethal mutations in mice treated orally with ammonium
picrate (Litton Bionetics. 1979) and in sex-linked recessive lethal mutations in Drosophila
melanogaster treated with picric acid orally or by injection (one injection study was positive, but
this result was not confirmed in two additional studies) (Woodruff et al.. 1985).
Studies evaluating clastogenicity found that both picric acid and ammonium picrate
induced sister chromatid exchanges (SCE) in mammalian cells without, but not with, metabolic
activation (N I P. 1985; Litton Bionetics. 1979). Picric acid did not increase chromosomal
aberrations (CAs) in Chinese hamster ovary (CHO) cells with or without metabolic activation in
vitro (N I P. 1985). and ammonium picrate was negative in an assay for CAs in mouse bone
marrow cells in vivo (Litton Bionetics. 1979). Picric acid was also negative in a test for
reciprocal translocations in I), melanogaster in vivo (Woodruff et al.. 1985).
Supporting Human Studies
As already discussed, dermatitis observed in several workers exposed to ammonium
picrate dust for 2-24 years was suggestive of skin sensitization (Sunderman et al.. 1945).
Follow-up patch testing showed no evidence of primary irritation but did show positive
sensitization reactions in 2/23 volunteers (Sunderman et al.. 1945). A case of allergic contact
dermatitis due to picric acid in a burn cream has also been reported (Aguirre et al.. 1993).
Supporting Animal Toxicity Studies
Oral LDso values in rats ranged from 200-629 mg/kg for picric acid and from
720-1,690 mg/kg for ammonium picrate (Wyman et al.. 1992; Weeks et al.. 1983). Minimum
lethal oral doses for picric acid were reported as 250 mg/kg for cats and 120 mg/kg for rabbits
(Weeks et al.. 1983). Approximate lethal doses in rats treated by intraperitoneal (i.p.) injection
were 168 mg/kg for ammonium picrate and 168-378 mg/kg for picric acid (Weeks et al.. 1983).
Clinical signs at lethal doses in these studies included ataxia, tremors, convulsions, and discharge
from the eyes (Wvman et al.. 1992; Weeks et al.. 1983).
Sunderman et al. (1945) evaluated potential effects of industrial exposure to ammonium
picrate dust in four rabbits and eight guinea pigs. The animals were housed in cages in the
milling and preforming areas of a factory with reported dust concentrations of
0.0088-0.1942 mg/m3 for up to 12 months. Three guinea pigs died during exposure (Week 1,
Week 3, and 9 months). Histopathological examinations of the animals that died and those
sacrificed after 12 months indicate that exposure may be associated with damage to the
respiratory tract. However, no conclusions can be drawn from this study because of the lack of a
control group; the small group sizes; limitations in characterization of exposure and evaluation of
health endpoints, and reporting deficiencies.
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Neither ammonium picrate nor picric acid produced primary skin irritation in rabbits
exposed topically to 0.5 g under occlusion on intact or abraded skin (Weeks et al.. 1983).
Ammonium picrate tested positive for dermal sensitization in 2/10 guinea pigs (Weeks et al..
1983).
Metabolism/Toxicokinetic Studies
Wvman et al. (1992) evaluated the toxicokinetics of picric acid in rats following oral or
intravenous exposure. This study indicated that gastrointestinal absorption is approximately
60-80%. Distribution of the absorbed compound was widespread, with the largest disposition in
the blood (due to plasma protein binding), followed by the spleen, kidney, liver, lung, and testes.
Elimination is biphasic, with an initial rapid plasma half-life of 13.4 hours, followed by evidence
of slower elimination (detectable levels in urine 14 days postexposure). Excretion is primarily
via urine, and the majority of the excretion product (60%) is the parent compound, which exists
as dissociated picrate anion under physiological conditions. Metabolites identified in the urine
(A-acetylisopicramic acid, picramic acid, A-acetyl pi cram ic acid) indicate that the primary
metabolic pathways are reduction of the ortho or para nitro group on the aromatic ring and
acetylation of the amine. Studies evaluating picrate deposits following dermal or i.p. exposure to
picric acid or ammonium picrate support biphasic elimination, with peak blood concentrations at
<24 hours but low levels still detectable 3-7 days after exposure (Weeks et al.. 1983).
Sunderman et al. (1945) reported widespread deposition of picrate in rabbits and guinea
pigs housed in cages in a factory with ammonium picrate dust concentrations of
0.0088-0.1942 mg/m3 for up to 12 months. The highest concentrations were in the lungs and
liver, with lower concentrations detected in the heart, thyroid, and kidneys.
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DERIVATION OF PROVISIONAL VALUES
DERIVATION OF ORAL REFERENCE DOSES
The database of the oral toxicity studies for picric acid includes two short-term toxicity
studies in rats and two dose-finding studies in rats, all of which were conducted by Takahashi et
al. (2004). The dose-finding studies were not considered for deriving the p-RfDs because the
follow-up primary studies reported in the same publication were more comprehensive and
because the dose-finding studies had some limitations, such as smaller sample sizes. No
repeated-dose oral toxicity studies have been identified for ammonium picrate.
Derivation of a Subchronic Provisional Reference Dose
The studies by Takahashi et al. (2004) were peer reviewed and employed GLP principles.
In the 18-day newborn-rat study, a NOAEL of 16.3 mg/kg-day and a LOAEL of 65.1 mg/kg-day
are identified for both males and females based on increased absolute and relative liver weight in
males and females, decreased absolute epididymis weight in males, and increased relative spleen
weight in females. 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 are identified for males and females based on splenic, hematological,
testicular, and liver effects.
In U.S. EPA's Recommended Use of Body Weight3/4 as the Default Method in Derivation
of the Oral Reference Dose (U.S. EPA. 201 lb), the Agency 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. Other approaches may
include using some chemical-specific information without a complete physiologically based
toxicokinetic model. In lieu of chemical-specific models or data to inform the derivation of
human equivalent oral exposures, U.S. EPA endorses body-weight scaling to the 3/4 power
(i.e., BW3/4) to extrapolate toxicologically equivalent doses of orally administered agents from
all laboratory animals to humans for deriving an oral reference dose (RfD) under certain
exposure conditions. 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. A validated human physiologically based
toxicokinetic model for picric acid is not available for use in extrapolating doses from animals to
humans. Furthermore, the most sensitive endpoints being considered are not portal-of-entry
effects. The BW3'4 scaling factor was not applied to effects in newborn rats because empirical
data are currently lacking on whether BW3'4 scaling is appropriate for extrapolating from
neonates or juveniles across species. However, scaling by BW3'4 is considered relevant for
deriving HEDs for effects observed in the young rats that were 5 weeks old when treatment
began.
Following U.S. EPA (2011b) guidance, the doses administered resulting in the most
sensitive endpoints are converted to HEDs through application of a dosimetric adjustment factor
(DAF) derived as follows:
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DAF = (BWa1 4 - BWh1 4)
where
DAF = dosimetric adjustment factor
BWa = animal body weight
BWh = human body weight
Study-specific body weight (i.e., terminal body weight in Table B-6) is used to calculate
the DAF for each dose group (U.S. EPA. 2011b). Calculated HEDs for the young-rat main
experiment in the Takahashi et al. (2004) study can be found in Tables B-3 through B-6.
Benchmark dose (BMD) analyses were conducted on the liver weight, absolute
epididymis weight, and relative spleen weight data from the newborn rat study using the
U.S. EPA's Benchmark Dose Software (BMDS; Version 2.7). Animal doses reported by the
study authors were used in the BMD modeling for effects in newborn rats as discussed above.
All the data were adequately fitted with the BMD model suite and results of BMD modeling are
summarized in Appendix C. The lowest benchmark dose lower confidence limit (BMDL)
identified from the newborn rat study is 34 mg/kg-day (BMDLio) based on increased absolute
liver weight in females (see Table C-l).
BMD analyses were also conducted on the statistically or biologically significant blood,
organ-weight, and epididymis-weight data from the young rat study. Although statistically
significant, histopathological data from the young rat study are not amenable to BMD modeling
because changes only occurred at the highest dose and no changes occurred at low and
mid-doses. Specifically, 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 only at this
dose. Before BMD modeling, all experimental doses were converted to a human equivalent dose
(HED) based on animal terminal body weight reported in the study. The lowest BMDL
identified from the young rat study is 1.2 mg/kg-day (HED) based on increased WBC count in
males; however, the level of response for this effect to be considered biologically significant is
unclear (see Table C-2). The next lowest BMDL from the young rat study is 1.8 mg/kg-day
(HED) based on increased absolute spleen weight in males. Although spleen weights in male
and female rats were most prominently increased at the highest dose (26.9 mg/kg-day [HED]),
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 (analysis
of variance [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 BMDLisd (HED) of 1.8 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 deriving the
subchronic provisional reference dose (p-RfD). The POD of 1.8 mg/kg-day (HED) based on the
young rat study is lower than the most sensitive POD of 34 mg/kg-day (ADD) based on
increased absolute liver weight in newborn females; therefore, it will be protective for effects
observed in newborn animals exposed to picric acid.
The POD (HED) based on picric acid data reflects toxicity of the picrate anion, and for
reasons described in the "Introduction" section, is considered to be applicable to both picric acid
and ammonium picrate, which are equivalent picrate sources in the environment and in the body.
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Because the molecular weight of ammonium picrate (formula weight [FW] = 246.1) is slightly
higher than picric acid (FW = 229.1), the same POD (FLED) of 1.8 mg/kg-day for picric acid is
used, for practical reasons, for ammonium picrate in deriving the subchronic p-RfD without
formula weight adjustment.
The subchronic p-RfD for picric acid and ammonium picrate, based on the BMDLisd
(FLED) of 1.8 mg/kg-day for increased absolute spleen weight in male rats exposed to picric acid,
is derived as follows:
Subchronic p-RfD = BMDLisd (FLED) UFc
= 1.8 mg/kg-day -^300
= 6 x 10"3 mg/kg-day
Table 5 summarizes the uncertainty factors for the subchronic p-RfD for picric acid and
ammonium picrate.
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Table 5. Uncertainty Factors for the Subchronic p-RfD for Picric Acid (CASRN 88-89-1)
and Ammonium Picrate (CASRN 131-74-8)
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 picrate exposure. The toxicokinetic uncertainty has been
accounted for by calculation of an HED through application of a DAF as outlined in the U.S. EPA's
Recommended Use of Body Weight4 as the Default Method in Derivation of the Oral Reference Dose
OJ.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 newborn rat
study. In addition, the database lacks repeated-dose oral studies beyond 28-d exposure for picric acid
and repeated-dose oral studies of any duration for ammonium picrate.
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 or ammonium
picrate 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-d rat study was selected as the principal study.
UFC
300
Composite UF = UFA x UFD x UFH x UFL x UFS.
BMDL = benchmark dose lower confidence limit; DAF = dosimetric adjustment factor; HED = human equivalent
dose; LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level; POD = point of
departure; p-RfD = provisional reference dose; UF = uncertainty factor; UFA = interspecies uncertainty factor;
UFC = composite uncertainty factor; UFD = database uncertainty factor; UFH = intraspecies variability uncertainty
factor; UFL = LOAEL-to-NOAEL uncertainty factor; UFS = subchronic-to-chronic uncertainty factor.
Confidence in the subchronic p-RfD for picric acid and ammonium picrate is low, as
explained in Table 6 below.
Table 6. Confidence Descriptors for the Subchronic p-RfD for Picric Acid
(CASRN 88-89-1) and Ammonium Picrate (CASRN 131-74-8)
Confidence Categories
Designation3
Discussion
Confidence in study
M
Confidence in the kev studv is medium. The TakahasM 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 the
experiments were performed according to GLP principles.
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.
aThe overall confidence cannot be greater than lowest entry in table (low).
GLP = Good Laboratory Practice; L = low; M = medium; p-RfD = provisional reference dose.
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Derivation of a Chronic Provisional Reference Dose
There are no chronic oral studies available for picric acid or ammonium picrate.
Furthermore, the longest available study is 28 days in duration, which is not suitable for deriving
a chronic p-RfD because of increased uncertainty in extrapolating to a chronic-duration time
frame. However, Appendix A of this document contains a screening value (screening chronic
p-RfD) using an analogue (e.g., structural, metabolic, and toxicity-like) approach, which may be
of use under certain circumstances. Based on the overall analogue approach presented in
Appendix A, 1,3,5-trinitrobenzene was selected as the most appropriate analogue for picric acid
and ammonium picrate for deriving a screening chronic p-RfD.
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
Human and animal data are inadequate to derive subchronic or chronic p-RfCs for picric
acid and ammonium picrate. The only available repeated-exposure study is an occupational
study by Sunderman et al. (1945) that reported a lack of prominent pathological changes in
humans exposed to ammonium picrate dust for 2-24 months; however, this study has major
limitations (including lack of adequate exposure monitoring) that preclude identification of a
NOAEL or LOAEL value. Sunderman et al. (1945) also evaluated potential adverse effects in
laboratory animals by housing rabbits and guinea pigs in the factory near areas of high exposure.
This study suggested potential damage to the respiratory tract but was not adequate to draw any
conclusions.
Table 7 summarizes noncancer oral and inhalation reference values derived.
Table 7. Summary of Noncancer Reference Values for Picric Acid (CASRN 88-89-1)
and Ammonium Picrate (CASRN 131-74-8)
Toxicity Type
(units)
Species/
Sex
Critical
Effect
p-Reference
Value
POD
Method
POD
(HED)
UFc
Principal
Study
Subchronic
p-RfD
(mg/kg-d)
Rat/M
Increased absolute
spleen weight
6 x 1(T3
BMDLisd
1.8
(based on
picric acid)
300
Takahashi et
al. (2004)
Screening
chronic p-RfD
(mg/kg-d)
Rat/M
Increased MetHb
and spleen-erythroid
cell hyperplasia
2 x 1(T3
NOAEL
0.643
(based on the
selection of
1,3,5-trinitrobenzene
as the analogue)
300
Reddv et al.
(2001);
Reddv et al.
(1997)
Subchronic
p-RfC (mg/m3)
NDr
Chronic p-RfC
(mg/m3)
NDr
BMDL = benchmark dose lower confidence limit; HED = human equivalent dose; M = male;
MetHb = methemoglobin; NDr = not determined; NOAEL = no-observed-adverse-effect level; POD = point of
departure; p-RfC = provisional reference concentration; p-RfD = provisional reference dose; SD = standard
deviation; UFC = composite uncertainty factor.
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CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR
Table 8 identifies the cancer weight-of-evidence (WOE) descriptor for picric acid and
ammonium picrate. No cancer data are available for either chemical. Genotoxicity assays of
both picric acid and ammonium picrate (see Table 4A) have yielded mixed results. Under the
U.S. EPA (2005) cancer guidelines, the available data are inadequate to assess human
carcinogenic potential, and the cancer WOE descriptor for picric acid and ammonium picrate is
"Inadequate Information to Assess the Carcinogenic Potential" (for both oral and inhalation
routes of exposure).
Table 8. Cancer WOE Descriptor for Picric Acid (CASRN 88-89-1)
and Ammonium Picrate (CASRN 131-74-8)
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 or ammonium picrate.
"Not Likely to Be
Carcinogenic to Humans"
NS
NA
No evidence of noncarcinogenicity is available.
NA = not applicable; NS = not selected; WOE = weight of evidence.
DERIVATION OF PROVISIONAL CANCER RISK ESTIMATES
Due to lack of carcinogenicity data for picric acid and ammonium picrate, derivation of
cancer risk estimates is precluded (see Table 9).
Table 9. Summary of Cancer Risk Estimates for Picric Acid (CASRN 88-89-1)
and Ammonium Picrate (CASRN 131-74-8)
Toxicity Type (units)
Species/Sex
Tumor Type
Cancer Risk Estimate
Principal Study
p-OSF (mg/kg-d) 1
NDr
p-IUR (mg/m3)
NDr
NDr = not determined; p-IUR = provisional inhalation unit risk; p-OSF = provisional oral slope factor.
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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 chronic provisional toxicity values for picric acid and
ammonium picrate. However, information is available for these chemicals, which although
insufficient to support deriving a provisional toxicity value under current guidelines, may be of
use to risk assessors. In such cases, the Center for Public Health and Environmental Assessment
(CPHEA) 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
provisional reference values 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 could be 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 CPHEA.
APPLICATION OF AN ALTERNATIVE ANALOGUE APPROACH
The analogue 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 analogue analysis are presented in Wang et al. (2012). Three
types of potential analogues (structural, metabolic, and toxicity-like) are identified to facilitate
the final analogue chemical selection. The analogue approach may or may not be route specific
or applicable to multiple routes of exposure. All information is considered together as part of the
final WOE approach to select the most suitable analogue both toxicologically and chemically.
In this document, the analogue approach has been applied with the goal of deriving a
chronic p-RfD. No adequate data were available to support derivation of inhalation toxicity
values via the analogue approach. Furthermore, this document has been developed for picric
acid and ammonium picrate, which are both ready (water soluble and rapidly dissociated to form
picrate anion), and equivalent sources of picrate anion in the environment and in the body. In
application of the analogue approach below, only picric acid was explicitly considered, because
available metabolic and repeated-dose toxicological data were only available for this chemical.
Nevertheless, the results are considered applicable to both picrate source compounds.
Structural Analogues
An initial analogue search focused on identifying structurally similar chemicals with
toxicity values from the Integrated Risk Information System (IRIS), PPRTV, and Health Effects
Assessment Summary Tables (HEAST) databases to take advantage of the well-characterized
chemical-class information. The search was accomplished through U.S. EPA's DSSTox
database (DSSTox. 2012) at similarity levels >60% and the National Library of Medicine's
(NLM's) ChemlDplus database (ChemlDpius, 2018) at similarity levels >80%. Six structural
analogues 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, 1989b);
2,4-dinitrophenol (U.S. EPA, 1991); 2-( 1 -methylpropyl)-4,6-dinitrophenol (U.S. EPA, 1989a);
1,3,5-trinitrobenzene (U.S. EPA, 1997); and 1,3-dinitrobenzene (U.S. EPA, 1988a). Table A-1
summarizes their physicochemical properties and similarity scores.
35
Picric acid and ammonium picrate
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September 2020
Table A-l. Physicochemical Properties of Picric Acid (CASRN 88-89-1) and Candidate Structural Analogues
Type of Data
2,4,6-Trinitrophenol
(Picric Acid)
2-Methyl-4,6-dinitro
phenol (DNOC)
2,4,6-Trinitro-
toluene (TNT)
2,4-Dinitrophenol
(2,4-DNP)
2-(l -Me thy lp ropy 1)-
4,6-dinitrophenol
(Dinoseb)
1,3,5-Trinitrobenzene
1,3-Dinitrobenzene
Structure
Cf OH tf
0 ^0
OH O"
K.C Jv ,Nt
^1 0
0 ^0
O" CHj O"
^ n! .nX.
o ^f|
0 ^0
OH o'
0 ^0
ch?
0 OH f
0 """ 0'
N
H l-i+
1 1
0" 0"
o „ ,o"
- N«
N*
II
O
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
ChemlDplus
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
(mm Hg [at °C])a
7.50 x 10-7
(at25°C)
1.06 x 10-4
(at25°C)
8.02 x 10-6
(at25°C)
3.90 x 10-4
(at 20°C)
NV
NV
NV
Henry's law constant
(atm-m3/mole
[at °C])a
1.70 x 10-11
(at25°C)
1.4 x 10-6
(at 25°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
(at20°C)
130
(at25°C)
2,790
(at25°C)
52
(at25°C)
278
(at 15°C)
533
(at 25°C)
Log Kowa
1.33
2.12
1.6
1.67
3.56
1.18
1.49
pKaa
0.38 (at 25°C)
4.31 (at21°C)
NV
4.09 (at25°C)
4.62
NV
NV
aChemIDplus (2018).
bChemical6ook (2017).
NV = not available.
36
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September 2020
Metabolic Analogues
Table A-2 summarizes the available toxicokinetic data for picric acid and the structurally
similar compounds identified as candidate analogues. Experimental data indicate that picric acid
and all six potential analogues are absorbed following oral exposure, have widespread
distribution, and are primarily eliminated via the urine. While no identical metabolites have been
identified between picric acid and candidate analogues, nitro reduction is a common metabolic
pathway for all compounds (see Table A-2). Common subsequent or secondary pathways
included acetylation (picric acid, 2-methyl-4,6-dinitrophenol, 2,4,6-trinitrotoluene,
1,3-dintrobenzene), conjugation with sulfate or glucuronic acid (Dinoseb, 1,3-dintrobenzene),
ring hydroxylation (2,4,6-trinitrotoluene, 1,3-dintrobenzene), and oxidation of methyl group
(2-methyl-4,6-dinitrophenol, 2,4,6-trinitrotoluene, Dinoseb). Taken together, available data
indicate similar patterns of absorption, distribution, and excretion, as well as a common primary
metabolic pathway (nitro reduction) for picric acid and candidate analogues. Therefore, all
candidate analogues are considered potential metabolic analogues for picric acid.
37
Picric acid and ammonium picrate
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Table A-2. Comparison of Available ADME Data for Picric Acid (CASRN 88-89-1) and Candidate Analogues
2,4,6-Trinitrophenol
(Picric acid)
CASRN 88-89-1
2-Methyl-4,6-dinitrophenol
(DNOC)
CASRN 534-52-1
2,4,6-T rinitrotoluene
(TNT)
CASRN 118-96-7
2,4-Dinitrophenol
(2,4-DNP)
CASRN 51-28-5
2-(l-Methylpropyl)-
4,6-dinitrophenol
(Dinoseb)
CASRN 88-85-7
1,3,5-
Trinitrobenzene
CASRN 99-35-4
1,3-Dinitrobenzene
CASRN 99-65-0
o
//' "q
o
OH 0"
/L
O ^0
cr ch, o
1 | II
cr
OH O"
y °
0 ^0
XH,
o oh r
0^ 0"
o. cf
"N
°" + 1 ' + '°
"N 'N '
O O"
CK ,cr
1
a
Absorption
Oral absorption in rats
was -60-80% based
on radioactivity in
blood, urine, and
tissues.
Oral absorption in humans
was -40%, based on
radioactivity in urine and
blood.
Oral absorption in rats was
-60%, based on radioactivity
in the blood, urine, and
tissues.
Oral absorption in rats,
mice, and dogs was
>60%, based on
radioactivity in urine.
Oral absorption is
rapid in mice;
extent of absorption
was not reported.
Oral absorption in
rats and mice was
>80% based on
elimination via urine
and feces (see
below).
Oral absorption in rats
was >24-39% based
on urine and expired
air (see below); fecal
elimination was low,
and it is uncertain
whether the balance of
the dose was retained
in the body or if
overall recovery was
low.
Oral absorption in
rabbits was >80%
based on elimination
via urine (see below).
Distribution
Rats exDosed orallv:
Widespread, with
highest radioactivity in
the blood, followed by
the spleen, kidney,
liver, lungs, and testes.
Rats exposed orallv:
Widespread, with highest
radioactivity in the blood,
liver, kidney, and spleen.
Rats exposed orallv:
Highest radioactivity
in liver, skeletal
muscle, blood, and fat
(<0.1-5.4% of dose
24 hr postdosing).
Mice exposed
orallv:
Highest
radioactivity in
serum, followed by
the liver and
kidney.
Mice exposed orallv:
Widespread; highest
radioactivity in the
plasma, followed by
the liver and kidney.
Rats exposed orallv:
Highest radioactivity
in liver, kidney, skin,
and lungs
(0.02-0.03% of dose/g
tissue 96 hr
postdosing).
ND
38
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September 2020
Table A-2. Comparison of Available ADME Data for Picric Acid (CASRN 88-89-1) and Candidate Analogues
2-(l-Methylpropyl)-
2,4,6-Trinitrophenol
2-Methyl-4,6-dinitrophenol
2,4,6-T rinitrotoluene
2,4-Dinitrophenol
4,6-dinitrophenol
1,3,5-
(Picric acid)
(DNOC)
(TNT)
(2,4-DNP)
(Dinoseb)
Trinitrobenzene
1,3-Dinitrobenzene
CASRN 88-89-1
CASRN 534-52-1
CASRN 118-96-7
CASRN 51-28-5
CASRN 88-85-7
CASRN 99-35-4
CASRN 99-65-0
Metabolic pathway(s)
• Nitro reduction
• Nitro reduction
• Sequential nitro
• Nitro reduction
• Nitro reduction
• Nitro reduction
• Sequential nitro
• Acetylation
• Acetylation
reduction followed
• Oxidation of
reduction followed
• Oxidation of methyl group
by \-acctvlation or
methyl group
by \-acctylation or
• Conjugation of the
ring hydroxylation
• Conjugation of
ring hydroxylation
hydroxyl group represents a
• Formation of
some metabolites
• Conjugation of
minor pathway
hydroxy lamine
with glucuronic
some metabolites
intermediates
acid
with sulfate or
• Oxidation of methyl
glucuronic acid
group and benzene
ring represents a
minor pathway
Metabolites
Rats cxDoscd orallv
Rats exposed orallv
Humans
Humans exoosed
Rats exoosed orallv
Rats exoosed orallv
Rats exoosed orallv
Urinary:
Urinary:
Urinary:
orallv or via
Urinary:
Urinary:
Urinary:
• Parent compound
• Parent compound (5%)
• 2-Amino-4,6-dintro-
inhalation:
• 2-(2-Hydroxy-l-
• 3,5-Dinitroaniline
• 3-Aminoacetanilide
(60%)
• 4,6-Diacetamido-o-cresol
toluene
Urinary:
methylpropyl)-
• l,3-Diamino-5-nitro-
(22%)
• 2-Amino-4,6-dinitro-
(18%)
• 4-Amino-2,6-dinitro-
• Parent compound
4,6-dinitrophenol
benzene
• 4-Acetamido
phenol (picramic
• 4,6-Dinitro-2-
toluene
• 2-Amino-4-nitro-
• 2-Methyl-2-(2-
• 1,3,5-Triamino-
phenyl sulfate (6%)
acid) (18.5%)
hydroxymethyl-phenol
• 2,4-Diamino-6-nitro-
phenol
hydroxy-3,5-
benzene
• 1,4-Diacetamido
• Y-acctvlisopicramic
(4-5%)
toluene
• 4-Amino-2-
dinitro-phenyl)
benzene (7%)
acid (14.8%)
• 6-Acetamido-4-nitro-o-
• 4-Hydroxyl
nitrophenol
propionic acid
Fecal:
• 3-Nitroanilinc-Y-
• Y-acctvlpicramic
cresol (2-3%)
amino-2,6-dintro-
• 2,4-Diamino-
• 2-Amino-6-(l-
• l,3-Diamino-5-nitro-
glucuronide (4%)
acid (4.7%)
toluene
phenol
methylpropyl)-
benzene
• Unidentified
• 4-Amino-2,6-dinitro-
4-nitrophenol
• 1,3,5-Triamino-
components (2.4%)
m-cresol
• Glucuronide-
benzene
conjugated
metabolites
39
Picric acid and ammonium picrate
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Table A-2. Comparison of Available ADME Data for Picric Acid (CASRN 88-89-1) and Candidate Analogues
2-(l-Methylpropyl)-
2,4,6-Trinitrophenol
2-Methyl-4,6-dinitrophenol
2,4,6-T rinitrotoluene
2,4-Dinitrophenol
4,6-dinitrophenol
1,3,5-
(Picric acid)
(DNOC)
(TNT)
(2,4-DNP)
(Dinoseb)
Trinitrobenzene
1,3-Dinitrobenzene
CASRN 88-89-1
CASRN 534-52-1
CASRN 118-96-7
CASRN 51-28-5
CASRN 88-85-7
CASRN 99-35-4
CASRN 99-65-0
Continued:
Continued:
Continued:
Continued:
Continued:
Continued:
Continued:
• 4-Acetamido-6-nitro-o-
Similar metabolites
Rats cxooscd orallv
Rabbits cxooscd
cresol (1-2%)
identified in rat,
Urinary:
orallv
• 6-Amino-4-nitro-o-cresol
mouse, rabbit, and dog
• Nitrophenols
Urinary:
(1-2%)
urine.
• 2 - Amino -4 -nitro -
• 3-Nitroaniline and
•Unidentified metabolites
phenol
1,3-benzene
and conjugates
diamine (35%)
Rabbits cxooscd
• 2,4-Diaminophenol
Similar metabolites in rabbit
orallv
(31%)
Urinary:
• 2-Amino-4-nitro-
• 2,4-Diamino-
phenol (14%)
phenol
• 4-Amino-2-nitro-
phenol (2%)
Mice cxooscd
• 30% of the
orallv
metabolites were
Plasma:
conjugated with
• 2-Amino-4-
glucuronic acid and
nitrophenol
6% with sulfate
• 4-Amino-2-
nitrophenol
40
Picric acid and ammonium picrate
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September 2020
Table A-2. Comparison of Available ADME Data for Picric Acid (CASRN 88-89-1) and Candidate Analogues
2,4,6-Trinitrophenol
(Picric acid)
CASRN 88-89-1
2-Methyl-4,6-dinitrophenol
(DNOC)
CASRN 534-52-1
2,4,6-T rinitrotoluene
(TNT)
CASRN 118-96-7
2,4-Dinitrophenol
(2,4-DNP)
CASRN 51-28-5
2-(l-Methylpropyl)-
4,6-dinitrophenol
(Dinoseb)
CASRN 88-85-7
1,3,5-
Trinitrobenzene
CASRN 99-35-4
1,3-Dinitrobenzene
CASRN 99-65-0
Excretory pattern
Rats cxDoscd orallv
Rat cx nosed orallv (% dose):
Rats. mice. doss, and
Urine is the primary
elimination
pathway in humans
and animals.
Rats cx nosed orallv
Rats cxnoscd orallv
Rabbits cxnoscd
(% dose at 24 hr):
• Urine: 50.55 ± 17.97
• Feces: 6.19 ± 4.18
• Expired air:
0.14 ±0.09
• Urine: 29-41
• Feces: 10-23
Rabbits exposed orallv
(% dose):
• Urine: 25-38
rabbits exposed orallv
(% dose at 24 hr):
• Urine: 59-74
(% dose in 72 hr):
• Urine :60-65%
• Feces: 25-30%
Mice cxnoscd orallv
(% dose in 72 hr):
• Urine:35-40%
• Feces: 35-40%
(% dose):
• Urine: 21-36 (in 4 d)
• Feces: 4 (in 4 d)
• Expired air: 3-5 (in
2d)
orallv (% dose in
2d):
• Urine: 81
• Feces: 0.3-5.2
• Expired air: ND
Wvtnan et al. (1992)
ATSDR (2018)
ATSDR (1995b)
ATSDR (1995c)
Gibson and Rao
(1973): Bandal and
Casida (1972):
U.S. EPA (1997):
ATSDR (1995a):
U.S. EPA (2006):
ATSDR (1995a):
Cossum and Rickert
Hathwav (1970)
(1985)
ADME = absorption, distribution, metabolism, and excretion; ND = no data.
41
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Toxicity-Like Analogues
Table A-3 summarizes available acute lethality and repeated-dose toxicity data for picric
acid and the six structurally similar chemicals identified as potential analogues. Comparison of
oral acute toxicity studies in rats suggests that 1,3,5-trinitrobenzene and 2,4,6-trinitrotoluene
have comparable median lethal dose (LD50) values to picric acid. The other candidate analogues
seem to be more potent than picric acid. Clinical signs were suggestive of central nervous
system (CNS) effects for picric acid and the candidate analogues with data.
As presented in the main PPRTV document, after a 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 Ret, decreased RBC and Hb), which is supported by extramedullary hematopoiesis
observed in the spleen. Therefore, similar hematological and associated splenic effects were
expected from the potential analogues, preferably from rat toxicity studies, the animal species
tested for picric acid.
Out of the six potential analogues, 2-methyl-4,6-dinitrophenol and 2,4-dinitrophenol
resulted in significantly decreased body weight in rats starting at doses of 17.3 and 46 mg/kg-day
(subchronic studies), with no hematological effects at doses up to 44.9 and 182 mg/kg-day,
respectively. These observations contrasted with 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 analogues. 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 LOAEL of 1 mg/kg-day
from a three-generation reproductive study in rats (U.S. EPA. 1989a). In a 2-year feeding study
in mice [Dow Chemical Co. (1989) as cited in U.S. EPA (1989a)1. 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. HP A. 1989a). It is unclear from U.S. EPA (1989a)
whether hematological effects were evaluated in this study, and the original report is not readily
available. 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 a toxicity-like analogue 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. HP A. 1997. 1988a) (see Table A-3). Therefore,
1,3,5-trinitrobenzene and 1,3-dinitrobenzene were considered toxicity-like analogues.
42
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Table A-3. Comparison of Available Repeated-Dose Toxicity Data for Picric Acid (CASRN 88-89-1) and Candidate Analogues
Type of Data
2,4,6-Trinitrophenol
(Picric Acid)
2-Methyl-4,6-
dinitrophenol
(DNOC)
2,4,6-Trinitro-
toluene (TNT)
2,4-Dinitrophenol
(2,4-DNP)
2-(l-Methylpropyl)-
4,6-dinitrophenol
(Dinoseb)
1,3,5-Trinitro-
benzene
1,3-Dinitrobenzene
Structure
0 OH 0
X
0 CH. 0
O. „0"
- N
CASRN
3-89-1
534-52-1
118-96-7
51-28-5
3-85-7
99-35-4
99-65-0
Acute lethality studies3
Rat oral LD5o
(mg/kg)
200-629
607
30
25
275
59.5
Effect
Ataxia, tremors,
convulsions,
circumorbital
discharge
(chromodactorea) of
the eyes
NV
Respiratory
stimulation,
changes in urine
composition,
infammation, and
necrosis of the
bladder
NV
Depressed behavioral
activity, convulsions
or effect on seisure
threshold, and
respiratory
stimulation
Dyspnea, rigidity,
and depressed
behavioral activity
Dyspnea, depressed
behavioral activity,
and effect on skin
and appendages
Short-term or subchronic treatment (oral)
Subchronic RfD
(mg/kg-d)
1 x 10-2
10"
NV
2 x 10-2
NV
NV
NV
Critical effects
Increased absolute
spleen weight
Reduced body
weight, excessive
perspiration and
fatigue, elevated
basal metabolic
rate 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)
43
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Table A-3. Comparison of Available Repeated-Dose Toxicity Data for Picric Acid (CASRN 88-89-1) and Candidate Analogues
2-Methyl-4,6-
2-(l-Methylpropyl)-
2,4,6-Trinitrophenol
dinitrophenol
2,4,6-Trinitro-
2,4-Dinitrophenol
4,6-dinitrophenol
1,3,5-Trinitro-
Type of Data
(Picric Acid)
(DNOC)
toluene (TNT)
(2,4-DNP)
(Dinoseb)
benzene
1,3-Dinitrobenzene
Other effects
Hematological and
(1) Decreased
Comprehensive
(1) No effects were
NV
Methemoglobi-
Decreased
related splenic effects
body weight; no
hematological
observed at doses up
nemia and
body-weight gain,
(hematopoiesis),
hematological
parameters were
to 10 mg/kg-d
spleen-erythroid
decreased Hb,
increased liver
effects were
evaluated, but it is
(free-standing
cell hyperplasia;
testicular atrophy,
weight, and testicular
specified at doses
unclear whether
NOAEL;
increased relative
and splenic
effects
up to
those effects were
hematological
spleen and liver
hemosiderin
44.9 mg/kg-d
observed at doses
endpoints were
weight; and
(evaluated
up to 32 mg/kg-d
examined; 27-wk
decreased testis
hematological
(26-wk study in
study in dogs).
weight (90- and
parameters
dogs).
180-d interim
included RBC,
(2) Decreased body
sacrifice in a 2-yr
WBC, and Hb;
No information
weight (<10%),
chronic study in
182-d oral study
with respect to
slight liver, kidney,
rats)
in rats).
hematological
spleen (congestion
(2) Decreased
effects in rats was
and hemosiderosis),
blood pyruvate,
available in IRIS
and testicular
T3, and T4 levels;
risk assessment.
atrophy at a dose of
no hematological
46-mg/kg-d. No
toxicity was
However, toxic
hematological
specified at doses
effects on
effects were
up to
hematologic
observed at doses up
41.0 mg/kg-d
parameters and
to 182 mg/kg-d.
(examined
related splenic
(Hematological
hematological
effects were
examination,
endpoints
observed in other
including RBC and
included RBC,
subchronic studies
Hb; 6-mo study in
Hb, MCH, MCV,
in rats, mice, and
rats.)
and WBC; 90-d
dogs at doses
oral study in rats).
higher than those
(3) In a similar study
(3) Increased
causing liver
to the picric acid
percentages of
effects as
studv bv Koizumi et
abnormal sperm
described in
al. (2001). vouns
(reproductive
rats
44
Picric acid and ammonium picrate
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Table A-3. Comparison of Available Repeated-Dose Toxicity Data for Picric Acid (CASRN 88-89-1) and Candidate Analogues
Type of Data
2,4,6-Trinitrophenol
(Picric Acid)
2-Methyl-4,6-
dinitrophenol
(DNOC)
2,4,6-Trinitro-
toluene (TNT)
2,4-Dinitrophenol
(2,4-DNP)
2-(l-Methylpropyl)-
4,6-dinitrophenol
(Dinoseb)
1,3,5-Trinitro-
benzene
1,3-Dinitrobenzene
Continued:
Continued:
Continued:
study in male
rats).
Continued:
ATSDR (1995b)
risk assessment
(p. 46/208).
Continued:
were tested for
behavior,
hematological,
urinalysis,
biochemistry, organ
weight, and
histopathology.
Decreased
locomotor activity
and salivation were
observed at a 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 p-RfC in
this assessment
U.S. EPA (2010)
U.S. EPA f1989b)
U.S. EPA (1991):
U.S. EPA (2007)
NV
U.S. EPA (1997)
U.S. EPA (1988a)
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Table A-3. Comparison of Available Repeated-Dose Toxicity Data for Picric Acid (CASRN 88-89-1) and Candidate Analogues
Type of Data
2,4,6-Trinitrophenol
(Picric Acid)
2-Methyl-4,6-
dinitrophenol
(DNOC)
2,4,6-Trinitro-
toluene (TNT)
2,4-Dinitrophenol
(2,4-DNP)
2-(l-Methylpropyl)-
4,6-dinitrophenol
(Dinoseb)
1,3,5-Trinitro-
benzene
1,3-Dinitrobenzene
Chronic treatment (oral)
Chronic RfD
(mg/kg-d)
NA
8 x 1(T5
5 x 1(T4
2 x 10-3
1 X 10-3
3 x 10-2
1 x 1(T4
Critical effects
NV
NV
IRIS summary
does not specify
toxic effects at
doses
>0.4 mg/kg-d
(DOD. 1984s)
(2-yr study in
rats)
NV
Decreased fetal
weight (3-generation
reproductive study in
rats)
Methemoglobi-
nemia and
spleen-erythroid
cell hyperplasia
(2-yr study in rats)
NV
Other effects
(oral)
NV
NV
Decreases in body
weight at doses
>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
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Table A-3. Comparison of Available Repeated-Dose Toxicity Data for Picric Acid (CASRN 88-89-1) and Candidate Analogues
Type of Data
2,4,6-Trinitrophenol
(Picric Acid)
2-Methyl-4,6-
dinitrophenol
(DNOC)
2,4,6-Trinitro-
toluene (TNT)
2,4-Dinitrophenol
(2,4-DNP)
2-(l-Methylpropyl)-
4,6-dinitrophenol
(Dinoseb)
1,3,5-Trinitro-
benzene
1,3-Dinitrobenzene
POD (mg/kg-d)
NV
LOAEL: 0.8
LOAEL: 0.5
LOAEL: 2
LOAEL: 1
NOAEL: 2.68
LOAEL: 0.4
UFC
NV
10,000
1,000
1,000
1,000
100
3,000
Source
NV
U.S. EPA (2010)
U.S. EPA (1989b)
U.S. EPA (1991):
U.S. EPA (2007)
U.S. EPA (1989a)
U.S. EPA (1997)
U.S. EPA (1988a)
'Data for picric acid from Tabic 4B in main body of this report; data for candidate analogues from ChemlDplus (2018).
BMDL = benchmark dose lower confidence limit; Hb = hemoglobin; IRIS = Integrated Risk Information System; LD5o = median lethal dose;
LOAEL = lowest-observed-adverse-effect level; MCH = mean corpuscular hemoglobin; MCV = mean corpuscular volume; NA = not applicable;
NOAEL = no-observed-adverse-effect level; NV = not available; POD = point of departure; p-RfC = provisional reference concentration; RBC = red blood cell
(erythrocyte); RfD = oral reference dose; SD = standard deviation; T3 = triiodothyronine; T4 = thyroxine; UFC = composite uncertainty factor; WBC = white blood cell
(leukocyte).
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For 2,4,6-trinitrotoluene, 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. 1989b). 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-trinitrotoluene with respect to hematological effects in rats was
available in the IRIS risk assessment (U.S. HP A. 1989b). However, the effects of
2,4,6-trinitrotoluene in the hematological and splenic compartments were observed in other
subchronic studies in rats, mice, and dogs at doses higher than the dose that caused liver effects
as described in ATSDR (1995b). Thus, 2,4,6-trinitrotoluene is also considered a toxicity-like
analogue.
In conclusion, an attempt was made to identify a suitable analogue to derive chronic
toxicity values for picric acid. Comparison of the potential analogues (2-methyl-4,6-dinitro-
phenol; 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 analogue chemical for picric acid, 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; and (3) if there are no clear
indications as to the best analogue chemical based on the first two considerations, then the
candidate analogue with the highest structural similarity may be preferred.
Overall, based on the WOE of all the information presented above, 1,3,5-trinitrobenzene
seems to be the most appropriate analogue 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 is 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 study in rats
(compared to PODs based on subchronic studies for 2,4,6-trinitrotoluene and
1,3-dinitrobenzene IRIS assessments).
3) Relatively high structural similarity scores of 75.03 and 73.6% were found using the
NLM's ChemlDplus database (ChemlDpius, 2018) and the U.S. 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 Reddy et al. (2001),
Reddv et al. (1997), and Reddv et al. (1996) as the principal studies for the reference dose (RfD):
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"Chronic toxic effects of 1,3,5-TNB in male andfemale Fischer 344 rats were
evaluated by feeding powdered certified laboratory chow diet supplemented with
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 [sic] were consistently reported
in all animals treated at these levels. Histopathologicalfindings 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 [sic] 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
Based on the overall analogue approach presented in this PPRTV assessment, the critical
effects identified in the IRIS assessment (U.S. EPA, 1997) of methemoglobinemia and
spleen-erythroid cell hyperplasia for 1,3,5-trinitrobenzene established in F344 rats from a 2-year
study (Reddv et al.. 2001; Reddv et al.. 1997) are identified as the potential analogue critical
effects for picric acid. The NOAEL of 2.68 mg/kg-day identified in female rats exposed to
1,3,5-trinitrobenzene is selected as the POD for derivation of the chronic p-RfD for picric acid.
As described in the U.S. EPA's Recommended Use of Body Weight3/4 as the Default
Method in Derivation of the Oral Reference Dose (U.S. EPA. 201 lb), the POD of
2.68 mg/kg-day is converted to an HED through an application of a DAF derived as follows:
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DAF = (BWa1 4 - BWh1 4)
where
DAF = dosimetric adjustment factor
BWa = animal body weight
BWh = human body weight
Using a reference BWa of 0.229 kg for female F344 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 NOAEL
identified in the rat study yields an analogue POD (FLED) as follows:
Analogue POD (FLED) = NOAEL (mg/kg-day) x DAF
= NOAEL (mg/kg-day) x 0.24
= 2.68 mg/kg-day x 0.24
= 0.643 mg/kg-day
The POD based on 1,3,5-trinitrobenzene as an analogue of picric acid is considered
applicable to both picric acid and ammonium picrate for reasons described in the "Introduction"
section. Both are equivalent sources of picrate in the environment and in the body. Furthermore,
the analogue chemical analysis presented here shows that the spectrum of effects associated with
picric acid (primarily hematological, splenic, and testicular effects) is consistent with the
structurally related chemicals considered as analogues. These chemicals also all share a common
primary metabolic pathway (i.e., nitro reduction). The findings link the picrate anion to the
effects observed after dosing with picric acid. Because the same picrate anion is formed from
ammonium picrate, and in the same amounts, confidence is high that the conclusions made in the
PPRTV assessment for picric acid (based on the picrate anion) are applicable to ammonium
picrate as well.
To derive the screening chronic p-RfD for picric acid and ammonium picrate, a
composite uncertainty factor (UFc) of 300 (see Table A-4) was applied to the analogue POD
(HED). As described in Wang et al. (2012) the uncertainty factors typically applied to the
chemical of concern are the same as those applied to the analogue unless additional information
is available. In this case, the interspecies uncertainty factor (UFa) for picric acid was reduced
from 10 to 3 due to the conversion of the POD from an animal dose to an 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. HP A. 201 lb)1. Further, the database uncertainty factor (UFd)
for picric acid was raised from 1 to 10 to account for limited information with regard to
reproductive toxicity and no information with regard to developmental toxicity for picric acid
[reproductive and developmental toxicity data for 1,3,5-trinitrobenzene were available and
indicated that these were not sensitive endpoints for that chemical (U.S. HP A. 201 lb)1.
The screening chronic p-RfD for picric acid and ammonium picrate is derived as follows:
Screening Chronic p-RfD = Analogue POD (HED) UFc
= 0.643 mg/kg-day -^300
= 2 x 10"3 mg/kg-day
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Table A-4. Uncertainty Factors for the Screening Chronic p-RfD for
Picric Acid (CASRN 88-89-1) and Ammonium Picrate (CASRN 131-74-8)
UF
Value
Justification
UFa
3
A UFa of 3 (100 5) is applied to account for residual uncertainty, including toxicodynamic differences
between rats and humans following oral picrate exposure. The toxicokinetic uncertainty has been
accounted for by calculation of an HED by applying a DAF as outlined in the U.S. EPA's
Recommended Use of Body Weight4 as the Default Method in Derivation of the Oral Reference
Dose (U.S. EPA. 201 lbV
UFd
10
A UFd of 10 is applied based on unknown and unaccountable database deficiencies of picric acid and
ammonium picrate. For the analogue chemical, systemic toxicity seems 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 or ammonium
picrate in humans.
UFl
1
A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation because the POD is a NOAEL.
UFS
1
A UFS of 1 is applied because a chronic study was selected as the principal study.
UFC
300
Composite UF = UFA x UFD x UFH x UFL x UFS.
DAF = dosimetric adjustment factor; HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect
level; NOAEL = no-observed-adverse-effect level; POD = point of departure; p-RfD = provisional reference dose;
UF = uncertainty factor; UFA = interspecies uncertainty factor; UFC = composite uncertainty factor; UFD = database
uncertainty factor; UFH = intraspecies variability uncertainty factor; UFL = LOAEL-to-NOAEL uncertainty factor;
UFS = subchronic-to-chronic uncertainty factor.
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APPENDIX B. DATA TABLES
Table B-l. Body and Organ Weight for Newborn S-D Rats in Dose-Finding Study
Exposed to Picric Acid (CASRN 88-89-1) for 14 Days (PNDs 4-17)a'b
Dose (mg/kg-d)c
0
16.3
81.4
Male
Number of animals
4
4
3
Body weight (g)
48.9 ±3.7
47.7 ± 2.6 (-2.5%)
42.3 ± 2.0* (-13%) d
Absolute liver weight (g)
1.73 ±0.14
1.67 ±0.13 (-3.5%)
1.70 ±0.13 (-1.7%)
Relative liver weight (g/100 g BW)
3.55 ±0.10
3.49 ±0.12 (-1.7%)
4.01 ±0.13** (13%) d
Absolute spleen weight (g)
0.21 ±0.04
0.21 ± 0.02 (0%)
0.17 ±0.01 (-19%)
Relative spleen weight (g/100 g BW)
0.44 ± 0.07
0.45 ± 0.05 (2.3%)
0.40 ±0.03 (-9.1%)
Absolute kidney weight (g)
0.58 ±0.03
0.56 ±0.04 (-3.4%)
0.43 ± 0.05** (-26%)
Relative kidney weight (g/100 g BW)
1.18 ±0.04
1.17 ±0.05 (-0.8%)
1.02 ±0.08** (-14%)
Female
Number of animals
4
4
2
Body weight (g)
45.2 ±2.2
47.5 ±3.1 (5.1%)
38.6 (-15%) d
Absolute liver weight (g)
1.57 ±0.08
1.72 ±0.09 (9.6%)
1.64 (4.5%)
Relative liver weight (g/100 g BW)
3.48 ±0.25
3.62 ±0.10 (4.0%)
4.23 (22%) d
Absolute spleen weight (g)
0.20 ±0.03
0.20 ± 0.04 (0%)
0.17 (-15%)
Relative spleen weight (g/100 g BW)
0.43 ± 0.04
0.43 ± 0.06 (0%)
0.44 (2.3%)
Absolute kidney weight (g)
0.55 ±0.02
0.57 ±0.05 (3.6%)
0.43 (-22%)
Relative kidney weight (g/100 g BW)
1.22 ±0.06
1.20 ±0.06 (-1.6%)
1.12 (-8.2%)
aTakahashi et al. (2004).
bValues are mean ± SD (percent change compared with control); percent change control = [(treatment
mean - control mean) control mean] x 100.
°A11 animals in the 407-mg/kg-day dose group died by Day 4; therefore, data from this dose group are excluded
from this table.
dNot statistically significant but biologically relevant (>10% increase).
*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; PND = postnatal day; SD = standard deviation; S-D = Sprague-Dawley.
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Table B-2. Body and Organ Weight for Newborn S-D Rats in Main Study Exposed to
Picric Acid (CASRN 88-89-1) for 18 Days (PNDs 4-21)a b
Dose (mg/kg-d)
0
4.1
16.3
65.1
Male
Number of 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/100 g BW)
4.25 ±0.16
4.35 ±0.12 (2%)
4.38 ± 0.08 (3%)
4.79 ±0.28**
(13%)c
Absolute spleen weight
(g)
0.34 ±0.07
0.35 ± 0.06 (3%)
0.38 ± 0.04 (12%)
0.37 ± 0.06 (9%)
Relative spleen weight
(g/100 g BW)
0.54 ±0.07
0.56 ± 0.08 (4%)
0.60 ±0.05 (11%)
0.60 ±0.05 (11%)
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/100 g BW)
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 testis weight
(mg)
326 ± 47
302 ± 27
319 ±22
295 ± 20
Relative testis weight
(mg/100 gBW)
513 ±54
479 ± 26
504 ± 44
478 ± 27
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Table B-2. Body and Organ Weight for Newborn S-D Rats in Main Study Exposed to
Picric Acid (CASRN 88-89-1) for 18 Days (PNDs 4-21)a b
Dose (mg/kg-d)
0
4.1
16.3
65.1
Female
Number of 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%)c
Relative liver 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 (3%)
0.29 ± 0.05 (-9%)
0.37 ± 0.05 (16%)
Relative spleen weight
(g/100 g BW)
0.54 ±0.05
0.55 ± 0.07 (2%)
0.51 ±0.08 (-6%)
0.62 ±0.03** (15%)
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%)
aTakahashi 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; SD = standard deviation; S-D = Sprague-Dawley.
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Table B-3. Body and Organ Weight for Young S-D Rats in Dose-Finding Study Exposed
to Picric Acid (CASRN 88-89-1) for 14 Daysa'b
ADD (mg/kg-d)c
0
20
100
Male
HED (mg/kg-d)
0
4.9
25.1
Number of animals
3
3
3
Body weight (g)
267 ± 15
257 ± 7 (-3.7%)
276 ± 9 (3.4%)
Absolute liver weight (g)
10.8 ±0.4
10.9 ± 0.7 (0.9%)
12.2 ± 0.2* (13%) d
Relative liver weight (g/100 g BW)
4.04 ±0.12
4.26 ± 0.39 (5.4%)
4.43 ±0.17 (9.7%)
Absolute spleen weight (g)
0.77 ±0.10
0.75 ± 0.03 (-2.6%)
0.91 ±0.07 (18%)
Relative spleen weight (g/100 g BW)
0.29 ±0.03
0.29 ± 0.02 (0%)
0.33 ± 0.02* (14%)
Absolute kidney weight (g)
2.29 ±0.25
2.12 ±0.16 (-7.4%)
2.39 ±0.12 (4.4%)
Relative kidney weight (g/100 g BW)
0.86 ±0.06
0.83 ±0.04 (-3.5%)
0.87 ±0.02 (1.2%)
Female
HED (mg/kg-d)
0
4.5
22.4
Number of animals
3
3
3
Body weight (g)
165 ±9
172 ± 4 (4.2%)
175 ± 8 (6.1%)
Absolute liver weight (g)
6.4 ±0.8
6.7 ±0.1 (4.7%)
8.0 ± 0.6* (25%) d
Relative liver weight (g/100 g BW)
3.85 ±0.28
3.90 ±0.07 (1.3%)
4.54 ±0.23* (18%) d
Absolute spleen weight (g)
0.49 ±0.13
0.45 ±0.13 (-8.2%)
0.56 ± 0.05 (14%)
Relative spleen weight (g/100 g BW)
0.30 ±0.06
0.26 ± 0.08 (-13%)
0.32 ±0.01 (6.7%)
Absolute kidney weight (g)
1.40 ±0.03
1.45 ±0.13 (3.6%)
1.52 ±0.17 (8.6%)
Relative kidney weight (g/100 g BW)
0.85 ±0.03
0.84 ±0.07 (-1.2%)
0.87 ± 11 (2.4%)
aTakahashi et al. (2004).
bValues are mean ± SD (percent change compared with control); percent change control = [(treatment
mean - control mean) control mean] x 100.
°Most animals in the 500-mg/kg-day dose group died by Day 2; therefore, data from this dose group are excluded
from this table.
dNot statistically significant but biologically relevant (>10% increase).
*Significant difference from control atp< 0.05 as calculated by study authors.
**Significant difference from control atp< 0.01 as calculated by study authors.
ADD = adjusted daily dose; BW = body weight; HED = human equivalent dose; SD = standard deviation;
S-D = Sprague-Dawley.
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Table B-4. Hematological Parameters for Young S-D Rats in Dose-Finding Study Exposed
to Picric Acid (CASRN 88-89-1) for 14 Daysa'b
ADD (mg/kg-d)
0
20
100
Male
HED (mg/kg-d)
0
4.9
25.1
Number of animals
3
3
3
WBC (x 102/mL)
117 ±26
94 ±20
108 ±21
RBC (x 104/mL)
682 ± 13
651 ±24
646 ± 32
Hb (g/dL)
14.0 ±0.6
13.8 ±0.2
13.8 ±0.6
Hct (%)
40.9 ± 1.4
41.3 ± 1.5
40.9 ±2.7
MCV (fL)
60.0 ±3.1
63.4 ±1.1
63.3 ± 1.7
MCHC (%)
34.2 ±0.3
33.5 ± 1.0
33.7 ±0.9
Ret (%o)
59.8 ±5.6
61.1 ± 3.7
72.6 ±8.2
Female
HED (mg/kg-d)
0
4.5
22.4
Number of animals
3
3
3
WBC (x 102/mL)
82 ±7
70 ± 12
98 ±31
RBC (x 104/mL)
711 ± 6
690 ±31
639 ±47
Hb (g/dL)
14.6 ±0.1
14.5 ±0.3
13.5 ±0.7*
Hct (%)
42.4 ±0.3
41.4 ±0.6
38.5 ± 1.7**
MCV (fL)°
59.6 ±0.8
60.0 ±3.6
60.3 ± 1.8
MCHC (%)
34.5 ±0.4
35.2 ±0.3
35.0 ±0.3
Ret (%o)
37.6 ± 1.5
39.6 ±6.9
56.3 ±3.6**
aTakahashi et al. (2004).
bValues are mean ± SD.
°Femtoliters (10 | s L).
*Significant difference from control atp< 0.05 as calculated by study authors.
**Significant difference from control atp< 0.01 as calculated by study authors.
ADD = adjusted daily dose; Hb = hemoglobin levels; Hct = hematocrit levels; HED = human equivalent dose;
MCHC = mean corpuscular hemoglobin concentration; MCV = mean corpuscular volume; RBC = red blood cell
(erythrocyte); Ret = reticulocyte; SD = standard deviation; S-D = Sprague-Dawley; WBC = total white blood cell
(leukocyte).
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Table B-5. Hematological Parameters for Young S-D Rats in Main Study Exposed to
Picric Acid (CASRN 88-89-1) for 28 Days3'b
ADD (mg/kg-d)
0
4
20
100
Male
HED (mg/kg-d)
0
1.1
5.4
26.9
Number of 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*
Hct (%)
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**
Female
HED (mg/kg-d)
0
1.0
4.8
24.0
Number of 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**
Hct (%)
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*
aTakahashi et al. (2004).
bValues are mean ± SD.
°Femtoliters (10 | s L).
*Significant difference from control atp< 0.05 as calculated by study authors.
**Significant difference from control atp< 0.01 as calculated by study authors.
ADD = adjusted daily dose; Hb = hemoglobin levels; Hct = hematocrit levels; HED = human equivalent dose;
MCHC = mean corpuscular hemoglobin concentration; MCV = mean corpuscular volume; RBC = red blood cell
(erythrocyte); Ret = reticulocyte; SD = standard deviation; S-D = Sprague-Dawley; WBC = total white blood cell
(leukocyte).
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Table B-6. Body and Organ Weight for Young S-D Rats in Main Study Exposed to
Picric Acid (CASRN 88-89-1) for 28 Days3'b
ADD (mg/kg-d)
0
4
20
100
Male
HED (mg/kg-d)
0
1.1
5.4
26.9
Number of 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%) d
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%) d
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 testis weight (g)
3.08 ±0.32
3.09 ±0.19
3.13 ±0.25
3.29 ±0.35
Relative testis 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%)
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Table B-6. Body and Organ Weight for Young S-D Rats in Main Study Exposed to
Picric Acid (CASRN 88-89-1) for 28 Days3'b
ADD (mg/kg-d)
0
4
20
100
Female
HED (mg/kg-d)
0
1.0
4.8
24.0
Number of 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%)d
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%)d
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.
dNot statistically significant but biologically relevant (>10% increase).
*Significant difference from control atp< 0.05 as calculated by study authors.
**Significant difference from control atp< 0.01 as calculated by study authors.
ADD = adjusted daily dose; ANOVA = analysis of variance; BW = body weight; HED = human equivalent dose;
S-D = Sprague-Dawley; SD = standard deviation.
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Table B-7. Histopathological Parameters for Young Male S-D Rats in Main Study
Exposed to Picric Acid (CASRN 88-89-1) for 28 Daysa'b
ADD (HED), mg/kg-d
0
4 (1.1)
20 (5.4)
100 (26.9)
Number of animals examined
Grade
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
aTakahashi 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.
ADD = adjusted daily dose; HED = human equivalent dose; S-D = Sprague-Dawley.
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Table B-8. Histopathological Parameters for Young Female S-D Rats Exposed to
Picric Acid (CASRN 88-89-1) for 28 Days3'b
ADD (HED), mg/kg-d
0
4 (1.0)
20 (4.8)
100 (24.0)
Number of animals examined
Grade
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
aTakahashi et al. (2004).
bGrade sign: +, mild; ++, moderate; +++, marked.
*Significant difference from control atp< 0.05.
**Significant difference from control atp< 0.01.
ADD = adjusted daily dose; HED = human equivalent dose; S-D = Sprague-Dawley.
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APPENDIX C. BENCHMARK DOSE MODELING RESULTS
MODELING PROCEDURE FOR CONTINUOUS DATA
Benchmark dose (BMD) modeling of continuous data is conducted with U.S. EPA's
Benchmark Dose Software (BMDS; Version 2.7). All continuous models available within the
software are fit using a benchmark response (BMR) of 1 standard deviation (SD) relative risk or
10% extra risk when a biologically determined BMR is available (e.g., BMR 10% relative
deviation [RD] for liver weight based on a biologically significant increase of 10%), as outlined
in the Benchmark Dose Technical Guidance (U.S. EPA, 2012). An adequate fit was judged
based on the %2 goodness-of-fit p-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 for
judging 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 criterion
(AIC) was selected as a potential point of departure (POD) from which to derive the provisional
reference dose (p-RfD).
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Table C-l. Summary of BMD Modeling of Data from Newborn S-D Rats Treated with Picric Acid (CASRN 88-89-1) via Gavage for
18 Days3
Endpoint
Sex
Model
/>-Valucb
/7-Value
Test 2
/7-Value
Test 3
AIC for Fitted
Model
Scaled Residual
BMD (mg/kg-d)
BMDL (mg/kg-d)
Increased absolute liver weight
M
Linear
0.9606
0.1215
0.1215
-38.99
-0.0424
67
37
Increased absolute liver
weight0
F
Polynomial
0.5452
0.9475
0.9475
-40.00
0.0138
58
34
Increased relative liver weight
M
Exponential (M2)
0.5781
0.0203
0.1161
-59.99
0.0283
55
41
Increased relative liver weight
F
Polynomial
0.7372
0.336
0.336
-37.69
0.005
59
53
Decreased epididymis weight
M
Polynomial
0.6799
0.3803
0.3803
108.84
-0.006
60
51
Increased relative spleen weight
F
Polynomial
0.4153
0.1272
0.1272
-107.17
0.016
57
34
'BMD modeling of data from the newborn rat study (TakahasM et al.. 20041.
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
°Selected model.
AIC = Akaike's information criterion; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the benchmark dose; F = female(s); M = male(s);
S-D = Sprague-Dawley.
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Table C-2. Summary of BMD Modeling of Data from Young S-D Rats Treated with Picric Acid (CASRN 88-89-1) via Gavage
for 28 Days3
Endpoint
Sex
Model
/>-Valucb
/7-Value
Test 2
/7-Value
Test 3
AIC for Fitted
Model
Scaled
Residual
BMD (mg/kg-d)
BMDL
(mg/kg-d)
Increased WBC
M
Exponential (M4)
0.9489
0.0346
0.9449
173.82
0.059
3.2
1.2
Increased WBC
F
Exponential (M2)
0.5816
0.2119
0.2119
179.91
-0.552
12
9.1
Decreased RBC
M
No fit
Decreased RBC
F
Polynomial
0.9163
0.176
0.176
197.65
-0.002
16
6.6
Decreased Hb
M
Polynomial
0.2588
0.1937
0.1937
1.7196
-0.008
21
19
Decreased Hb
F
Polynomial
0.8906
0.4384
0.4384
-8.646
-0.002
15
14
Increased MCV
F
No fit
Decreased MCHC
F
Linear
0.4844
0.0987
0.4539
3.53
0.132
7.7
6.0
Increased Ret
M
No fit
Increased Ret
F
No fit
Increased absolute liver
weight
M
Exponential (M2)
0.9313
0.3651
0.3651
39.03
0.001
25
15
Increased absolute liver
weight
F
Polynomial
0.9379
0.2233
0.2233
30.23
-0.002
17
14
Increased relative liver
weight
M
Polynomial
0.7402
0.6843
0.6843
-41.03
0.0009
24
15
Increased relative liver
weight
F
Exponential (M2)
0.609
0.1685
0.1685
-53.23
-0.358
10
8.7
Increased absolute spleen
weight0
M
Exponential (M4)
0.1039
0.0739
0.1329
-66.23
-0.074
4.8
1.8
Increased absolute spleen
weight
F
Polynomial
0.2851
0.227
0.227
-89.53
-0.445
10
9.2
Increased relative spleen
weight
M
Exponential (M2)
0.2249
0.589
0.589
-147.5
0.2107
7.1
5.6
Increased relative spleen
weight
F
No fit
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Table C-2. Summary of BMD Modeling of Data from Young S-D Rats Treated with Picric Acid (CASRN 88-89-1) via Gavage
for 28 Days3
Endpoint
Sex
Model
/>-Valucb
/7-Value
Test 2
/7-Value
Test 3
AIC for Fitted
Model
Scaled
Residual
BMD (mg/kg-d)
BMDL
(mg/kg-d)
Decreased absolute
epididymis weight
M
Linear
0.6673
0.5124
0.5124
-98.33
0.289
11
7.5
Decreased relative
epididymis weight
M
Exponential (M2)
0.6783
0.0988
0.1138
-158.48
-0.5923
8.6
4.9
'BMD modeling of data from the young rat study (Takahashi et at. 20041.
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
°Selected model.
AIC = Akaike's information criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the benchmark dose; F = female(s); Hb = hemoglobin levels;
M = male(s); MCHC = mean corpuscular hemoglobin concentration; MCV = mean corpuscular volume; RBC = red blood cell (erythrocyte); Ret = reticulocyte;
S-D = Sprague-Dawley; WBC = white blood cell (leukocyte).
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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. Among all models providing adequate
fit, the BMDLs estimated from different models varied greater than threefold. Therefore,
BMDLisd (human equivalent dose [HED]) of 1.8 mg/kg-day from the Exponential 4 model was
selected.
Table C-3. Modeling Results for Increased Absolute Spleen Weight in Young Male S-D
Rats Treated with Picric Acid (CASRN 88-89-1) via Gavage for 28 Days3
Model
Variance
/>-Valucb
Goodness-of-
Fit />-Valucb
Scaled
Residuals0
AIC
BMDisd
(mg/kg-d)
BMDLisd
(mg/kg-d)
Constant variance
Exponential (Model 2)d
0.1329
0.1905
0.578
-67.56478
8.84
6.00
Exponential (Model 3)d
0.1329
0.1905
0.578
-67.56478
8.84
6.00
Exponential (Model 4)df
0.1329
0.1039
-0.07437
-66.25554
4.76
1.80
Exponential (Model 5)d
0.1329
NA
-0.5917
-65.63357
5.19
2.32
Hilld
0.1329
NA
-0.592
-65.633572
5.15
NA
Linear6
0.1329
0.2203
0.419
-67.859042
7.41
4.71
Polynomial (2-degree)6
0.1329
0.2203
0.419
-67.859042
7.41
4.71
Polynomial (3-degree)6
0.1329
0.2203
0.419
-67.859042
7.41
4.71
Power"1
0.1329
0.2203
0.419
-67.859042
7.41
4.71
aTakahashi et al. (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.
Selected model.
AIC = Akaike's information criterion; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose; NA = not applicable; S-D = Sprague-Dawley; SD = standard deviation.
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Exponential 4 Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
0 5 10 15 20 25
dose
20:51 08/17 2020
Figure C-l. BMD Output of Exponential (M4) Model for Increased Absolute Spleen
Weight in Young Male S-D Rats Treated with Picric Acid (CASRN 88-89-1) via Gavage for
28 Days (Takahashi et al., 2004)
BMD Output for Figure C-l:
Exponential Model. (Version: 1.11; Date: 03/14/2017)
Input Data File: C:/Users/jzhao/OneDrive - Environmental Protection Agency
(EPA)/Profile/Documents/new working files/PPRTV/2019/amitionium
picrate/clearance/Phi Hip
review/exp_abs_spleen_wt_male_Exp-ModelVariance-BMRlStd-Up.(d)
Gnuplot Plotting File:
Tue Aug 18 17:35:17 2020
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 * dose}
Y[dose] = a * exp{sign * (b * dose)Ad}
Y[dose] = a * [c-(c-l) * exp{-b * dose}]
Y[dose] = a * [c-(c-l) * exp{-(b * dose)Ad}]
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.
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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]))
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
MLE solution provided: Exact
Initial Parameter Values
Variable Model 4
lnalpha -3.92971
rho 3.23346
a 0.722
b 0.0803637
c 1.71607
d 1 Specified
Parameter Estimates
Variable Model 4
lnalpha
rho
a
b
c
-3.89679
2 . 65484
0.779191
0.0389032
1.77578
Std. Err.
0.393798
2.41999
0.0325225
0. 059696
0.714234
Table of Stats From Input Data
Dose
0
1.1
5.4
26.9
Obs Mean
0.82
0.76
0.89
1.18
Obs Std Dev
0. 08
0. 08
0.19
0.16
Dose
0
1.1
5.4
26.9
Estimated Values of Interest
Est Mean Est Std Scaled Residual
0.7792
0.8045
0.8937
1.171
0.1023
0.1068
0.1228
0.1758
0.9769
-1. 021
-0.07437
0.1198
Other models for which likelihoods are calculated:
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Model A1:
Model A2:
Model A3:
Model R:
Yij
= Mu(i) + e(i j )
Var{e(ij)}
= Sigma^2
Yij
= Mu(i) + e(i j )
Var{e(ij)}
= Sigma(i)^2
Yij
= Mu(i) + e(i j)
Var{e(ij)}
= exp(lalpha + log(mean(i)) * rho)
Yij
= Mu + e(i)
Var{e(ij)}
= Sigma^2
Model
A1
A2
A3
R
4
Likelihoods of Interest
Log (likelihood) DF
37.98686 5
41.45648 8
39.43834 6
26.18775 2
38.11594 5
AIC
-65.97373
-66.91296
-66.87668
-48 .37551
-66.23187
Additive constant for all log-likelihoods = -22.05. 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)
30.54
6.939
4.036
2 . 645
D. F.
6
3
2
1
p-value
< 0.0001
0.07386
0.1329
0.1039
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 6a is greater than .1. Model 4 seems
to adeguately describe the data.
Benchmark Dose Computations:
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Specified Effect = 1.000000
Risk Type = Estimated standard deviations from control
Confidence Level = 0.950000
BMD = 4.7672 8
BMDL = 1.79969
BMDU = 11.8 933
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