United States
Environmental Protection
1=1 m m Agency
EPA/690/R-07/017F
Final
9-25-2007
Provisional Peer Reviewed Toxicity Values for
2,4-Dinitrophenol
(CASRN 51-28-5)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

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

Act of 1980
CNS
central nervous system
cu.m
cubic meter
DWEL
Drinking Water Equivalent Level
FEL
frank-effect level
FIFRA
Federal Insecticide, Fungicide, and Rodenticide Act
g
grams
GI
gastrointestinal
HEC
human equivalent concentration
Hgb
hemoglobin
i.m.
intramuscular
i.p.
intraperitoneal
i.v.
intravenous
IRIS
Integrated Risk Information System
IUR
inhalation unit risk
kg
kilogram
L
liter
LEL
lowest-effect level
LOAEL
lowest-observed-adverse-effect level
LOAEL(ADJ)
LOAEL adjusted to continuous exposure duration
LOAEL(HEC)
LOAEL adjusted for dosimetric differences across species to a human
m
meter
MCL
maximum contaminant level
MCLG
maximum contaminant level goal
MF
modifying factor
mg
milligram
mg/kg
milligrams per kilogram
mg/L
milligrams per liter
MRL
minimal risk level
MTD
maximum tolerated dose
MTL
median threshold limit
NAAQS
National Ambient Air Quality Standards
NOAEL
no-ob served-adverse-effect level
NOAEL(ADJ)
NOAEL adjusted to continuous exposure duration
NOAEL(HEC)
NOAEL adjusted for dosimetric differences across species to a human
NOEL
no-ob served-effect level
OSF
oral slope factor
p-IUR
provisional inhalation unit risk
p-OSF
provisional oral slope factor
p-RfC
provisional inhalation reference concentration
p-RfD
provisional oral reference dose
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PBPK
physiologically based pharmacokinetic
ppb
parts per billion
ppm
parts per million
PPRTV
Provisional Peer Reviewed Toxicity Value
RBC
red blood cell(s)
RCRA
Resource Conservation and Recovery Act
RDDR
Regional deposited dose ratio (for the indicated lung region)
REL
relative exposure level
RfC
inhalation reference concentration
RfD
oral reference dose
RGDR
Regional gas dose ratio (for the indicated lung region)
s.c.
subcutaneous
SCE
sister chromatid exchange
SDWA
Safe Drinking Water Act
sq.cm.
square centimeters
TSCA
Toxic Substances Control Act
UF
uncertainty factor
Hg
microgram
|imol
micromoles
voc
volatile organic compound
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PROVISIONAL PEER REVIEWED TOXICITY VALUES FOR
2,4-DINITROPHENOL (CASRN 51-28-5)
Background
On December 5, 2003, the U.S. Environmental Protection Agency's (EPA's) Office of
Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human
health toxicity values for Superfund risk assessments, establishing the following three tiers as the
new hierarchy:
1.	EPA's Integrated Risk Information System (IRIS).
2.	Provisional Peer-Reviewed Toxicity Values (PPRTV) used in EPA's Superfund
Program.
3.	Other (peer-reviewed) toxicity values, including:
~	Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR),
~	California Environmental Protection Agency (CalEPA) values, and
~	EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in EPA's Integrated Risk Information System (IRIS). PPRTVs are
developed according to a Standard Operating Procedure (SOP) and are derived after a review of
the relevant scientific literature using the same methods, sources of data, and Agency guidance
for value derivation generally used by the EPA IRIS Program. All provisional toxicity values
receive internal review by two EPA scientists and external peer review by three independently
selected scientific experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the
multi-program consensus review provided for IRIS values. This is because IRIS values are
generally intended to be used in all EPA programs, while PPRTVs are developed specifically for
the Superfund Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a five-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV manuscripts conclude
that a PPRTV cannot be derived based on inadequate data.
Disclaimers
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and RCRA program offices are advised to carefully review the information provided
in this document to ensure that the PPRTVs used are appropriate for the types of exposures and
circumstances at the Superfund site or RCRA facility in question. PPRTVs are periodically
updated; therefore, users should ensure that the values contained in the PPRTV are current at the
time of use.
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It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
users are strongly encouraged to read the entire PPRTV manuscript and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center for OSRTI. Other EPA programs or external parties who may
choose of their own initiative to use these PPRTVs are advised that Superfund resources will not
generally be used to respond to challenges of PPRTVs used in a context outside of the Superfund
Program.
Questions Regarding PPRTVs
Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on
chemicals not covered, or whether chemicals have pending IRIS toxicity values) may be directed
to the EPA Office of Research and Development's National Center for Environmental
Assessment, Superfund Health Risk Technical Support Center (513-569-7300), or OSRTI.
INTRODUCTION
The HEAST (U.S. EPA, 1997) listed a chronic RfD of 2E-3 mg/kg-day for
2,4-dinitrophenol (DNP) based on the IRIS database. The assessments were based on a LOAEL
of 2 mg/kg-day for cataract in humans treated with 2,4-DNP as a weight reducing agent (Horner,
1942). The derivation for the chronic RfD included an uncertainty factor (UF) of 1000 (10 for
extrapolation from subchronic exposure to chronic exposure, 10 for protecting sensitive
individuals, and 10 for the use of a LOAEL). The HEAST also lists a subchronic RfD by
adopting the chronic oral RfD from IRIS. The chronic RfD for 2,4-DNP was developed by U.S.
EPA in 1991, and is listed on the IRIS database. However, it was not listed on the Drinking
Water Standards and Health Advisories list (U.S. EPA, 2006). The toxicity of 2,4-DNP has been
summarized in the Ambient Water Quality Criteria for Nitrophenols by U.S. EPA (1980) and the
Toxicological Profile for Dinitrophenol by ATSDR (1995). ATSDR proposed an acute-duration
Minimal Risk Level (MRL) of 0.01 mg/kg-day, but not for intermediate- or chronic-duration oral
exposure to 2,4-DNP.
An RfC for 2,4-DNP was not listed on the HEAST (U.S. EPA, 1997). The health effect
data for 2,4-DNP were reviewed by the U.S. EPA RfD/RfC Work Group in 1991 and were
determined to be inadequate for the derivation of an inhalation RfC. The American Conference
of Governmental Industrial Hygienists has not established a Threshold Limit Value (TLV)-
Time-Weighted Average (TWA) and the National Institute for Occupational Safety and Health
(NIOSH, 2005) has not established a REL-TWA for this chemical. The Occupational Safety and
Health Administration (OSHA) has not developed a Permissible Exposure Limit (PEL)-TWA for
2,4-DNP either.
The 1997 HEAST did not include a cancer assessment for 2,4-DNP. No cancer
assessment for this chemical has been developed by IRIS or IARC.
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The WHO (2007) has not reviewed the toxicology of 2,4-DNP. Literature searches were
conducted for the period from 1980 to August 2007 to identify data relevant for the derivation of
provisional RfD, RfC, and cancer assessments for 2,4-DNP. The following databases were
searched: TOXLINE, MEDLINE, CANCERLIT, TOXLIT/BIOSIS, Registry of Toxic Effects of
Chemical Substances (RTECS), HSDB, GENETOX, CCRIS, TSCATS, EMIC/EMICBACK,
and DART/ETICBACK.
This document has passed the Superfund Health Risk Technical Support Center (STSC)
quality review and peer review evaluation indicating that the quality is consistent with the SOPs
and standards of the STSC and is suitable for use by registered users of the PPRTV system.
REVIEW OF PERTINENT LITERATURE
Human Studies
Numerous occasions of human poisoning by 2,4-DNP have been reported in the
literature. The earliest cases of fatal 2,4-DNP intoxication related to its usage as a component of
explosives during World War I. Thirty-six cases of fatal occupational dinitrophenol poisoning
occurred among employees of the munitions industry in France between 1916 and 1918 (Perkins,
1919). A literature review by von Oettingen (1949) revealed 27 reported cases of fatal
occupational dinitrophenol poisoning in the United States for the years 1914 to 1916. In
addition, Gisclard and Woodward (1946) reported two fatal cases of dinitrophenol poisoning
during manufacture of picric acid where 2,4-DNP was produced as an intermediate. Swamy
(1953) also described a case of suicidal poisoning by 2,4-DNP.
Early in the 1930s, 2,4-DNP was widely recommended as a treatment for obesity, and it
resulted in both toxic side effects and fatalities. Horner (1942) reported a total of nine deaths
resulting from the use of dinitrophenol as a slimming agent. The toxic manifestations of
dinitrophenol exposure as reviewed by Horner (1942) included subacute symptoms such as
gastrointestinal disturbances (nausea, vomiting, colic, diarrhea, anorexia), profuse sweating,
weakness, dizziness, headache, and loss of weight. Acute poisoning has resulted in the sudden
onset of pallor, burning thirst, agitation, dyspnea, profuse sweating, and hyperpyrexia. Intense
and rapid onset of rigor mortis after death has also been described.
Perkins (1919) reported that postmortem examination of dinitrophenol victims
demonstrated no characteristic lesions. Acute edema of the lungs was mentioned but was
believed to be secondary to the toxic effects on the vasomotor system. Microscopic lesions of
the liver and kidney cells were inconstant and typical changes were lacking elsewhere.
The widespread use of 2,4-DNP as a weight reducing agent in humans during the 1930s
also provided some information regarding the chronic effects of this compound in humans.
Recommended therapeutic doses of 2,4-DNP for weight control on humans ranged from 2 to 5
mg/kg-day (Dunlop, 1934; Horner, 1942; Tainter et al., 1933). Tainter et al. (1933) administered
2,4-DNP (average daily dose of 0.3 g) to 113 obese patients for as long as four months without
demonstrating evidence of cumulative or toxic effects. Based on an assumption of body weight
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of 70 kg, the corresponding average daily dose was 4.3 mg/kg-day. The most important side
effect noted by the investigators was a skin rash observed in about 7% of the patients treated.
The rash was manifested usually after a one-day period of mild itching and consisted of a
maculopapular or urticarial type of rash. The itching was intense and in some cases there was
considerable swelling. Symptoms subsided in 2 to 5 days following withdrawal from the drug.
The next most important side effect noted by the authors was a loss of taste for salt and sweets
observed in 5.3% of the patients. This effect also subsided following withdrawal from 2,4-DNP.
The investigators failed to detect any effect of 2,4-DNP on liver or kidney function, pulse, blood
pressure, or oxygen capacity of the blood. No cases of anemia, agranulocytosis, or malignant
neutropenia appeared. Three cases of mild gastrointestinal upset were reported, however.
In a later publication, Horner (1942) reviewed the acute and chronic toxicity of use of
2,4-DNP (including cataract formation) resulting from therapeutic use of the compound.
Gastrointestinal symptoms consisting of nausea, vomiting, and loss of appetite were common as
a result of 2,4-DNP administration. Cutaneous lesions were the most frequent side effect with an
incidence of 8 to 23%. Although the majority of lesions were mild, others were severe. Bone
marrow effects of dinitrophenol have also been reported. Eight cases of agranulocytosis were
reported, with three fatalities. Thirty cases of neuritis including aberrations of taste and multiple
regional involvement, particularly affecting the feet and legs, were recorded. Symptoms
appeared after an average of ten weeks, followed ordinary therapeutic doses and persisted for
weeks or months. Electrocardiographic evidence of functional heart damage was offered by
several investigators and fragmentation of the heart muscle was reported at autopsy in one fatal
case. It was generally agreed that 2,4-DNP was rarely injurious to the liver and kidneys when
administered in therapeutic doses.
The development of cataracts following dinitrophenol therapy was first described by
Horner et al. (1936). Later, over 100 cases of cataract formation following dinitrophenol therapy
were reviewed by Horner (1942). Horner described the following characteristic features of 2,4-
DNP induced cataracts: (1) they occurred in young women who were physically normal except
suffering varying degrees of obesity and were in an age group in which senile cataracts do not
occur; (2) they were bilateral and appeared either during or after period of dinitrophenol
treatment; (3) an interval of months or years might elapse between the time the last dose was
taken and the onset of blurred vision; (4) lenticular changes were strikingly similar and could be
demonstrated with the biomicroscope at a time when vision for distance and reading was still
normal; (5) after gradual onset, the lenticular changes progressed with startling rapidity until the
vision was obscured; (6) treatment was without effect in staying their progress; and (7) surgical
removal of the lens was uniformly successful in restoring vision. Cataract formation appears to
be the primary reason 2,4-DNP was withdrawn from medical use.
The length of time that 2,4-DNP was taken and the amount of the drug consumed varied
widely among cataract victims. In 29 cases, the duration of treatment varied from 3 months to
24 months with an average of 11 months. Neither the length of treatment nor the total dose
seemed to have any bearing on the occurrence of cataracts. Individual susceptibility appeared to
be a more important factor. Horner (1942) estimated that the incidence of cataracts in patients
who had taken dinitrophenol exceed one percent.
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The available data do not allow the calculation of a minimum effect level for 2,4-DNP-
induced cataract formation in humans. Cataractogenic activity in humans has been observed in a
small proportion of patients receiving as little as 2 mg/kg-day. An assessment of the no-effect-
level for cataract formation awaits further investigation. Such an assessment is further
complicated by the fact that cataract formation in humans, following DNP administration, differs
significantly from the situation seen in experimental animal studies.
The existing review documents (U.S. EPA, 1980, 1984; ATSDR, 1995) and an updated
literature search did not identify relevant studies regarding the carcinogenicity of
2,4-dinitrophenol in humans following oral or inhalation exposure.
Animal Studies
Short Term Animal Studies
Attempts to find a suitable animal to study cataract development in humans exposed to
2,4-DNP have generally been unsuccessful. Normal mammalian animals have not developed
cataracts after oral exposure to 2,4-DNP, although cataracts could be induced in a special strain
of mouse (yellow adipose), in vitamin C-deficient guinea pigs, in ducks, and in chickens
(ATSDR 1995). Formation of cataracts by acute exposure to DNP was first demonstrated in
animals almost 10 years after the problem was known to exist in humans (Gehring and Buerge,
1969a; Ogino and Yasukura, 1957; Feldman et al., 1959, 1960; Bettman, 1946). Experimental
cataracts, first produced in ducks and chickens, differ from DNP-induced human cataracts in that
they can be formed in acute exposures and may appear in less than one hour. Furthermore, these
lesions will disappear spontaneously in animals within 25 hours (Howard et al., 1976). Hence,
the usefulness of data on the formation of cataracts in experimental animals following DNP
administration in assessing human hazard to dinitrophenol is questionable.
Langerspectz and Tarkkonen (1961) failed to detect histological changes in the adrenals
or the liver during 30 day treatment of Swiss albino male mice with twice daily doses of 10 mg
of 2,4-DNP/kg (20 mg/kg-day) via the subcutaneous injection.
Subchronic Animal Studies
Tainter and Cutting (1933) administered 2,4-DNP to dogs at intervals of three or more
days over a period of 2 to 3 months. Abnormal liver and kidney pathology were not detected but
an effect on spleen tissue was noted. Over large areas of the material containing "numerous
large faintly staining cells with vesicular polyhedral nuclei." This study is limited due to the lack
of dose information in the summary document (U.S. EPA, 1980).
Groups of three male dogs received daily oral dose of 0, 5 or 10 mg/kg 2,4-DNP in
capsules for 6 day/week for 27 weeks (Tainter et al., 1934). There were no important changes in
body weight as a result of the continuous administration of 2,4-DNP. Estimations at intervals of
three weeks of the amount of sugar, and albumin in the urine and of the hemoglobin and red,
white and differential blood cell counts, urea content, icteric index and oxygen capacity of the
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blood and fragility of the red cells showed no significant or consistent deviations from the
normal or control values. At the end of treatment, the dogs were killed for complete necropsy
and histological study of the tissues. No significant pathologic changes were noticed grossly or
microscopically. Thus, the highest dose of 10 mg/kg-day (equivalent to continuous dose of 8.6
mg/kg-day) is considered a free-standing NOAEL.
Spencer et al. (1948) studied the subchronic toxicity of 2.4-DNP in rats. Male rats
(10-20/dose) were fed diets containing 0, 0.01, 0.02, 0.05, 0.1, or 0.2 g of 2,4-DNP per lOOg of
food. Rats were maintained on diets containing 2,4-DNP for six months and both hematological
and pathological investigations on surviving animals were performed. Based on rat food intake
in a subchronic study (U.S. EPA, 1988), the average daily food intake factor for male rat of
unknown species is assumed to be 0.091 kg food/kg body weight/day. Thus, the estimated doses
were 0, 9.1, 18, 46, 91, and 182 mg/kg-day, respectively. Hematological examination included
erythrocyte counts, hemoglobin concentrations, leukocyte counts, differential counts, and bone
marrow counts at autopsy. Both gross and microscopic examination of liver, kidney, spleen,
lung, heart, adrenal, pancreas, and stomach tissues were also performed. Rats maintained on
diets containing 0.02% 2,4-DNP (18 mg/kg-day) grew at a normal rate and the investigators
failed to detect discernible ill effects of pathological changes at autopsy. Similarly, pathological
changes were not found upon microscopic examination of tissues from rats receiving diets
containing 0.05% 2,4-DNP (46 mg/kg-day) although growth of these rats fell 5 to 10% below
that of the controls throughout the six-month experimental period. At autopsy the only changes
observed in these animals were a very slight depletion of body fat and a very slight increase in
the average weight of the kidneys. More reduced growth was also seen in the rats treated with
0.1% of 2,4-DNP (91 mg/kg-day). At the highest dose of 2,4-DNP in their diets (182 mg/kg-
day) rats occasionally died and survivors lost weight rapidly. Examination of surviving animals
revealed marked emaciation, an empty gastrointestinal tract, a slightly enlarged and dark spleen,
and undersized testes. Microscopic examination showed slight congestion and cloudy swelling
of the liver, very slight parenchymatous degeneration of the epithelium of the renal tubules,
slight congestion and hemosiderosis of the spleen and testicular atrophy. No significant
pathological changes were observed in the lung, heart, adrenals, pancreas, or stomach of these
animals. Based on these observations, a NOAEL for 2,4-DNP in rats was 18 mg/kg-day.
Chronic Animal Studies
Groups of 5-6 white rats (sex unknown) received 2,4-DNP in the food beginning shortly
after weaning when they weighted about 30 g, and continuing until death (Tainter, 1938). The
treatment doses included 0, 0.001, 0.005, 0.01, 0.02, 0.04, 0.06, 0.08, 0.12 and 0.24% of
2,4-DNP in the diet. Based on rat food intake in a chronic study (U.S. EPA, 1988), the average
daily food intake factor for rat of unknown sex and speices is assumed to be 0.078 kg food/kg
body weight. Thus, the estimated doses were 0, 0.78, 3.9, 7.8, 16, 31, 47, 62, 94 and 187 mg/kg-
day, respectively. The food intakes, growth curves, final weights, and life spans were compared
with those of untreated controls. At the time of death, necropsies and histological studies of the
tissues were made. The rats were observed closely throughout the entire duration of the
experiment. The food intakes were similar for all the groups. Doses of 2,4-DNP in the diet
ranging from 0.78 to 31 mg/kg-day did not appreciably modify the growth curves or final
weights. Doses of 47 to 94 mg/kg-day decreased the rate of growth, and diminished the final
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average weight about 75 g. The average duration of life of about two years was not decreased by
doses of 2,4-DNP up to 62 mg/kg-day; a dose of 94 mg/kg-day decreased it by about one-half
and 187 mg/kg-day killed the rats in about one month. There was no evidence of any toxic
effects of 2,4-DNP on the eyes of these rats, as indicated by direct observations, and
ophthalmoscopic study or slit lamp microscopy. At necropsy, and histologically, the tissues of
the treated rats were indistinguishable from those of the untreated controls, there being no lesions
which could be ascribed to the action of the 2,4-DNP. The NOAEL in rats was identified to be
31 mg/kg-day based on significant decreases in body weight.
Reproductive and Developmental Animal Studies
Based on the available data it appears unlikely that the 2,4-DNP pose a teratogenic
hazard to humans. Gibson (1973) examined developmental toxicity of 2,4-DNP in mice.
Groups of pregnant mice (7-8 animals/dose) received intraperitoneal (7.7 or 13.6 mg/kg) or oral
(25.5 or 38.3 mg/kg) administration of 2,4-DNP during early organogenesis (gestation day 10-
12). Nine pregnant females received water served as control. Caesarean section was performed
on day 19 of gestation, and the number and position of live, dead and resorbed fetuses was
examined. Individual fetuses were weighted, and examined for external anomalies. Fetal crown-
rump distance was measured for each fetus. Each litter was divided into two sub-groups for
further examination for soft-tissue or skeletal anomalies. Very limited results were provided in
the original report. Among the all the endpoints (including number of implantations, resorptions,
fetal body weight and fetal crown-rump length) presented in a summary table (Table 8 in the
original paper), increased resorptions (mean response/litter), decreased fetal body weight, and
fetal crown-rump length occurred in almost all the treated groups (including both i.p. and oral
treatments); however, only the decreases in the fetal body weight and crown-rump length in the
high i.p. dose group (13.6mg/kg-day) was statistically significant. No other details were provided
in the original report. Gibson (1973) ambiguously concluded that dinitrophenol does not
produce morphological defects in the offspring, but embryo toxicity occurs at the higher dose
levels. The higher doses also produced overt toxic signs (hyperexcitability and hyperthermia) in
the dams, but were not lethal. However, it is not clear whether the author referred the higher
doses to i.p. high dose of 13.6 mg/kg or to doses >13.6 mg/kg-day including i.p. high dose and
two oral doses (25.5 and 38.3 mg/kg-day). Based on limited information available from the
original report, the low i.p. dose of 7.7 mg/kg-day was considered NOAEL.
The toxicity of 2,4-DNP was examined in newborn rats by Koizumi et al. (2001). Groups
of Sprague-Dawley rats (6/sex/dose) were administered 2,4-DNP at 0, 3, 10 or 20 mg/kg-day by
gastric intubation daily from days 4 to 21 after birth, and killed after overnight starvation
following the last treatment. Recovery-maintenance groups at the same dosages were
maintained for 9 weeks without chemical treatment and fully examined at 12 weeks old. General
behavior was observed daily, and body weight and food consumption were measured more than
once a week. At treatment day 17 or 18, papillary reflex, corneal reflex, surface righting, mid-air
righting and auricular reflexes were examined as parameters of reflex ontogeny. Furthermore,
fur appearance, incisor eruption and eye opening were noted in the lactating period as evidence
of physical development, and testes descent and vaginal opening during the early recovery-
maintenance period for assessment of sexual maturation. Color, pH, occult blood, protein,
glucose, ketone bodies, bilirubin, urobilinogen, urine sediment and volume of the urine were
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examined only at the end of the recovery-maintenance period. The blood samples were analyzed
for complete hematological parameters as well as blood biochemistry. All the organs were
weighted and examined for gross and histopathological changes at the end of treatment. No
clinical signs or deaths were encountered. The body weights at 20 mg/kg-day were significantly
below control values from dosing day 7 in the males and dosing day 10 in females in the
scheduled-sacrifice group. There was also statistically significant lowering of body weight in the
20 mg/kg-day males for the first quarter of the recovery-maintenance period, but not in females.
No definitive changes in abdominal fur appearance, incisor eruption, eye opening and testis
descent or vaginal opening as well as reflex ontogeny parameters were detected in any dose
groups. There were significant changes in absolute weights of testes at >10 mg/kg-day, and
changes in absolute and relative organ weights in several other organs at 20 mg/kg-day. No
chemical-related histopathological changes were noted in either scheduled-sacrifice or recovery-
maintenance groups. Significant increase in RBC was observed in females receiving 20 mg/kg-
day after the treatment but not after the recovery-maintenance period. Although increases in
serum glutamate oxaloacetate transaminase (GOT) in males and total bilirubin in females were
detected at 10 and 20 mg/kg-day after treatment, those were not considered to be chemical-
induced because they were very slight and there was no dose-relationship. The authors
considered the dose of 10 mg/kg-day as the NOAEL at which only the lowering of absolute testis
weight was observed.
The toxicity of 2,4-DNP was also examined in young rats by Koizumi et al. (2001).
Groups of 5 to 6-week old Sprague-Dawley rats (6/sex/dose) were administered 2,4-DNP at 0, 3,
10, 30 or 80 mg/kg-day by gastric intubation daily for 28 days, and killed after overnight
starvation following the last treatment. Recovery-maintenance groups at the 0, 30 or 80 mg/kg-
day were maintained for 2 weeks without chemical treatment and fully examined at 11-12 weeks
of age. Rats were examined for general behavior, body weight, food consumption, urinalysis,
hematology and blood biochemistry, necropsy finding, organ weights and histopathological
finding. Clear toxic signs, such as decrease in locomotor activity, prone position, ptosis, panting,
crawling position and salivation, were observed repeatedly during the dosing period at 80 mg/kg-
day in both sexes, and two males and six females died in the same dose group. However,
decrease in locomotor activity and salivation in the 30 mg/kg-day group were mostly observed
only after the first dosing. The relative liver weights were increased in both sexes of the 80
mg/kg-day scheduled-sacrifice group, and this persisted through the recovery period. Relative
organ weights for brain, kidneys and testes were increased only in 80 mg/kg-day males. On
histopathological examination, mineralization of the corticomedullary junction in kidneys was
observed in both sexes at 80 mg/kg-day in the scheduled-sacrifice and recovery groups, but the
change was only statistically significant in males of the scheduled-sacrifice group. On
hematological examination, increase in hemoglobin and hematocrit in the recovery period were
observed, limited to 80 mg/kg-day males. Although blood chlorine levels were slightly
decreased in 30 and 80 mg/kg-day males and total bilirubin was slightly increased in females
receiving 10 mg/kg and more, no changes in histopathology or organ weights were observed at
30 mg/kg-day or lower. Prior to the this experiment, a dose-finding study in the same age group
(4/sex/group) at doses of 0, 0.6, 2, 6, 20 or 60 mg/kg-day had been conducted. The study results
from these animals after treatment for 14days were consistent with the main study. Thus, the
authors considered 20 mg/kg-day from the dose-finding study as the NOAEL based on decrease
in locomotor activity and salivation at 30 mg/kg-day.
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Wulff et al. (1935) examined the effects of 2,4-DNP on the fertility, gestation, and fetal
life of rats in an one-generation study. A group of 20 female rats (unknown strain) received 20
mg/kg 2,4-DNP 8 days prior the introduction of males. Nine females received no treatment and
five females received 1% sodium bicarbonate solvent served as control. Dinitrophenol was
administered intragastrically twice daily throughout cohabitation, and gestation until the
respective litters were weaned. The daily average dose was estimated to be 40 mg/kg-day. The
average number born in each litter was not affected by dinitrophenol treatment, and the treatment
did not appreciably affect the body weight gains of mothers during pregnancy. Neonatal
malformations were not detected. Among 2,4-DNP treated rats, however, 25% of the total
number of pups were stillborn while only 6.8% of the pups were stillborn in the control group
(two groups combined). In addition, the mortality during the nursing period of viable pups born
to mothers administering 2,4-DNP was 30.9% as compared with 13.4% for young of control
mothers. Two possible explanations for this latter phenomenon were offered by the authors:
treated mothers neglected their pups while in a febrile state, and only the more vigorous of the
offspring manage to reach the mother for nursing; or, a toxic agent was passed to the young
through the milk. Data to distinguish between the two possibilities are not available. Based on
developmental toxicity (stillbirth and mortality during lactation), this study provided a free
standing LOAEL of 40 mg/kg-day.
Other Studies
Bowman (1967) has studied the effect of 2,4-DNP on the developing chick embryo in
vitro. At 2,4-DNP concentrations of 18 mg/L or 370 mg/L a syndrome of abnormalities resulted
consisting of degeneration and sometimes complete absence of neural tissue accompanied by a
reduction in the number of somites. The 2,4-DNP concentrations used in this study are
extremely high and the relevance of the experimental findings to the in vivo situation in
mammals is not clear.
Genotoxicity data for 2,4-DNP were reviewed by ATSDR (1995). Test results were
negative for 2,4-DNP in multiple assays for reverse mutation in Salmonella typhimurium, with or
without metabolic activation. However, the two major metabolites of 2,4-DNP are mutagenic in
S. typhimurium (and other systems), suggesting that the negative results for 2,4-DNP may
indicate failure of the S9 activating system used in these assays to metabolize this chemical.
Mixed results were reported for 2,4-DNP in studies of reverse mutation in Escherischia coli.
There is little evidence that 2,4-DNP produces DNA damage. Assays for phase induction in E.
coli, SOS response in S. typhimurium, unscheduled DNA synthesis in rat hepatocytes, and DNA
damage (alkali elution) in Chinese hamster V79 cells were negative. DNA damage (alkali
elution) was reported in mouse leukemia L1210 cells and human HeLa cells, but was associated
with depletion of ATP. Depletion of ATP was also observed in studies showing decreases in
DNA synthesis and mitotic index after exposure to 2,4-DNP. Therefore, positive findings in
these studies probably reflects cytotoxicity (decreased cellular metabolic rate), rather than
genotoxicity. In vivo, 2,4-DNP produced chromosomal aberrations in bone marrow cells of mice
treated by intraperitoneal injection.
In a study designed to measure tumor promoting activity, Boutwell and Bosch (1959)
examined the ability of 2,4-DNP to promote tumor formation following a single initiating dose
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of dimethylbenzanthracene. The 2,4-DNP failed to promote skin tumors in mice in this
experiment. In a similar experiment, Stenback and Garcia (1975) also examined the ability of
2,4-DNP to promote skin tumor formation in mice, and found no tumor promoting activity.
The existing review documents (U.S. EPA, 1984; ATSDR, 1995) and an updated
literature search did not identify relevant studies regarding the carcinogenicity of 2,4-
dinitrophenol in animals following oral exposure.
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC
ORAL RfD VALUES FOR 2,4-DINITROPHENOL
2,4-DNP is considered a classic uncoupler of oxidative phosphorylation and is widely
used by biochemists to determine whether a given biochemical process is energy dependent. The
toxic action of the dinitrophenol is generally attributed to their ability to uncouple oxidative
phosphorylation. It prevents the utilization of the energy provided by cellular respiration and
glycolysis by inhibiting the formation of high energy phosphate bonds. All energy dependent
biochemical processes are therefore affected by the action of the compounds. The large number
of clinical effects attributed to dinitrophenol toxicity result essentially from the shortcircuiting of
metabolism in cells which absorb sufficient dinitrophenol. At concentrations higher than those
necessary to uncouple oxidative phosphorylation, a number of inhibitory effects of the
dinitrophenol isomers on certain enzymatic reactions may occur. The dinitrophenol may also act
directly on the cell membrane, thus causing toxic effects on cells which do not depend on
oxidative phosphorylation for their energy requirements. More detailed information on the
mechanism of toxicity has been summarized by U.S. EPA (1980).
The database for 2,4-DNP toxicity is relatively comprehensive, and it covers human case
studies and results from experimental exposure. In addition, the database also includes animals
studies ranging from short-term studies, subchronic studies in dogs and rats, to a chronic study in
rats, accompanied by developmental studies in mice and young rats, and an one-generation study
in rats.
The toxicity of 2,4-DNP in humans ranges from mortality due to high dose exposure to
minor effects such as gastrointestinal symptoms and cutaneous lesions due to therapeutic use of
the compound as a weight reducing agent. The development of cataracts following the
dinitrophenol therapy was first described by Horner et al. (1936), and similar response was also
reported from treated animals. The length of time that 2,4-DNP was taken and the amount of the
drug consumed varied widely among cataract victims. In 29 cases, the duration of treatment
with the compound varied from 3 months to 24 months. Neither the length of treatment nor the
total dose seemed to have any bearing on the occurrence of cataracts. The available data do not
allow the calculation of a minimum effect level for 2,4-DNP-induced cataract formation in
humans. Since cataractogenic activity in humans has been observed in a small proportion of
patients receiving as little as 2 mg/kg-day dose of 2,4-DNP, this dose is considered as a LOAEL
for cataract in humans after subchronic exposure to the chemical.
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The formation of cataracts by acute exposure to 2,4-DNP has been reported in ducks and
chickens treated with the compound. However, the experimental cataracts in the animals differ
from DNP-induced human cataracts in that they can be formed in acute exposures and may
appear in less than one hour. Furthermore, these lesions will disappear spontaneously in animals
within 25 hours.
Subchronic studies in dogs and rats did not identify a specific target organ for 2,4-DNP,
and a free-standing NOAEL of 8.6 mg/kg-day in dogs and a NOAEL of 18 mg/kg-day in rats
based on decreased growth rate were identified (Spencer et al., 1948; Tainter et al., 1938).
Similar to the subchronic studies, the chronic study in rats (Tainter et al., 1938) identified a
NOAEL of 31 mg/kg-day based on significant decreases in body weight. There was no evidence
of any toxic effect on the eyes in the rats treated chronically with 2,4-DNP at dose levels up to
187 mg/kg-day. Since the subchronic rat study (Spencer et al., 1948) did not include a dose at 30
mg/kg-day range, the subchronic NOAEL of 18 mg/kg-day from that study is considered
consistent with the chronic NOAEL of 31 mg/kg-day (Tainter et al., 1938) because the latter
study included smaller dose spacing in the experiment which allowed identification of a NOAEL
higher than that from the subchronic study.
The toxicity of 2,4-DNP in developmental studies did not demonstrate more sensitive
responses than the systemic effects observed in the subchronic or chronic studies. 2,4-DNP does
not produce morphological defects in the offspring, but it could produce embryo toxicity at dose
level of > 13.6 mg/kg-day, and the same doses also produced overt toxic signs (hyperexcitability
and hyperthermia) in the dams (Gibson, 1973). The developmental study provided a NOAEL of
7.7 mg/kg-day based on i.p. treatment dose. Short-term treatment (7 days) with 2,4-DNP in
newborn rats resulted in decreased testis weight and body weight at the dose of 20 mg/kg-day,
and the next lower dose of 10 mg/kg-day was identified as the NOAEL (Koizumi et al., 2001).
2,4-DNP treatment (28 days) in young rats (5-6 weeks old) resulted in decreases in locomotor
activity and salivation at the dose level of > 30 mg/kg-day (Koizumi et al., 2001), and this study
identified a NOAEL of 20 mg/kg-day in young rats. Comparison of the effective doses in
newborn and young rats suggested a less sensitivity to 2,4-DNP in young rats than newborn rats.
The one-generation reproductive study (Wulff et al., 1935) showed that 2,4-DNP
treatment at dose level of 40 mg/kg-day resulted in increased stillbirth and mortality during
lactation. It is not clear whether the mortality during lactation was due to toxicity in dams or
fetuses exposed to the compound through milk. Therefore, this dose (40 mg/kg-day) is
considered a free-standing LOAEL.
Based on all the data available, the cataracts developed in humans after therapeutic use of
the compound as a weight reducing agent is considered the critical effect, and the estimated
minimal dose of 2 mg/kg-day causing this effect is considered as the point of departure in
deriving a provisional subchronic RfD. Using this point of departure is further supported by the
relative rich data from animal studies. A provisional subchronic RfD of 2xl0"2 mg/kg-day for
2,4-DNP is derived by applying a composite uncertainty factor of 100 (10 for the use of a
LOAEL, and 10 to protect sensitive individuals) to the point of departure of 2 mg/kg-day.
Because the database for 2,4-DNP included subchronic studies, chronic studies, developmental
studies and an one-generation reproductive study, it is unlikely to identify more sensitive
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responses from additional animal studies. Therefore, an uncertainty factor of 1 is used as the
database factor. Because the critical effect in humans was observed after subchronic treatment
of the compound, no extra uncertainty factor for exposure duration is needed for deriving a
provisional subchronic RfD.
subchronic p-RfD = POD / UF
= (subchronic LOAEL) / (100)
= 2 mg/kg-day / 100
= 0.02 or 2xl0"2 mg/kg-day
The current chronic RfD of 2E-3 mg/kg-day on IRIS (U.S. EPA, 1991) was based on the
same critical effect and point of departure with an extra uncertainty factor of 10 to cover the
extrapolation from subchronic to chronic duration. A chronic RfD of 2xl0"3 mg/kg-day for 2,4-
DNP was derived by applying to the human LOAEL of 2 mg/kg-day a composite uncertainty
factor of 1000 (10 for the use of a LOAEL, 10 for extrapolation from subchronic to chronic
duration, and 10 to protect sensitive individuals). The chronic RfD on IRIS is still valid.
Chronic RfD = POD / UF
= (subchronic LOAEL) / (1000)
= 2 mg/kg-day / 1000
= 0.002 or 2xl0"3 mg/kg-day
Confidence in the principal study is low. Higher study confidence is precluded because
the principal study only describes anecdotal data which provides limited formation in the dosing
and exposure duration, minimal data reporting, and the lack of reliable data on no effect levels
for the critical effect in critical study. Confidence in the database is high because the database
for 2,4-DNP toxicity not only includes experimental studies in humans, but also covers relative
comprehensive studies in animals including short-tem studies, subchronic studies in multiple
species, a chronic study, developmental studies in pregnant mice and young rats, as well as a
one-generation study. Therefore, it is unlikely to identify other more sensitive responses from
additional animal studies. Overall confidence in the subchronic p-RfD values is medium, as the
strengths in the database, particularly the supportive data from comprehensive animal studies,
somewhat outweigh the low confidence in the principal human study. A chronic p-RfD based on
the same point of departure with an additional uncertainty factor to cover the extrapolation from
subchronic to chronic duration. The overall confidence for the chronic p-RfD would be the same
as the one for subchronic p-RfD. The using of the subchronic data as the point of departure for
chronic p-RfD is appropriate because neither the length of treatment nor the total dose seems to
have any bearing on the occurrence of critical effect.
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DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION RfC VALUES FOR 2,4- DINITROPHENOL
No data were located for the subchronic or chronic inhalation toxicity of 2,4-DNP in
humans or animals. Due to the lack of data, no provisional RfC was derived for 2-4-DNP.
PROVISIONAL CARCINOGENICITY ASSESSMENT
FOR 2,4- DINITROPHENOL
Weight-of-Evidence Descriptor
No studies were located examining associations between cancer and exposure of humans
to 2,4-DNP. Thus, there were inadequate human data to assess the carcinogenicity of 2,4-DNP.
Data examining the potential for 2,4-DNP to produce cancer in animals were restricted to
several mouse skin tumor assays that found no DNP-induced increases in incidence of skin
tumors. U.S. EPA guidelines (2005) indicated that, in order to classify the compound as not
likely to be carcinogenic to humans, no increased incidence of neoplasms should be found in at
least two-well designed and well-conducted animal studies of adequate power and dose in
different species. Thus, the available animal data for 2,4-DNP were not sufficient to classify
them as providing no evidence of carcinogenicity. Additional well-conducted testing in other
animal species with long-term exposure, preferably via oral and inhalation exposure, is necessary
to provide reasonable assurance as to whether 2,4-DNP may or may not be carcinogenic in
animals or humans.
Mixed results in genotoxicity of 2,4-DNP were reported in several short-term
mutagenesis assays in bacteria, and in in vitro and in vivo mammalian systems. The majority of
the in vitro genotoxicity studies showed negative responses with some exceptions in DNA
damage in mouse leukemia cells and human HeLa cells, although the positive findings in these
studies probably reflects cytotoxicity rather than genotoxicity. An in vivo study produced
chromosomal aberrations in bone marrow cells.
Following U.S. EPA (2005) guidelines for compounds with inadequate human data and
inadequate animal data, 2,4-DNP was classified as having inadequate information to assess
carcinogenic potential.
Quantitative Estimates of Carcinogenic Risk for 2,4-DNP
Due to inadequate information to assess carcinogenic potential, a quantitative cancer risk
estimate for neither an oral slope factor nor an inhalation unit risk could be derived for 2,4-DNP.
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