United States
Environmental Protection
1=1 m m Agency
EPA/690/R-09/020F
Final
9-10-2009
Provisional Peer-Reviewed Toxicity Values for
N,N-Diphenyl-1,4-Benzenediamine
(CASRN 74-31-7)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

-------
COMMONLY USED ABBREVIATIONS
BMD
Benchmark Dose
IRIS
Integrated Risk Information System
IUR
inhalation unit risk
LOAEL
lowest-observed-adverse-effect level
LOAELadj
LOAEL adjusted to continuous exposure duration
LOAELhec
LOAEL adjusted for dosimetric differences across species to a human
NOAEL
no-ob served-adverse-effect level
NOAELadj
NOAEL adjusted to continuous exposure duration
NOAELhec
NOAEL adjusted for dosimetric differences across species to a human
NOEL
no-ob served-effect level
OSF
oral slope factor
p-IUR
provisional inhalation unit risk
p-OSF
provisional oral slope factor
p-RfC
provisional inhalation reference concentration
p-RfD
provisional oral reference dose
RfC
inhalation reference concentration
RfD
oral reference dose
UF
uncertainty factor
UFa
animal to human uncertainty factor
UFC
composite uncertainty factor
UFd
incomplete to complete database uncertainty factor
UFh
interhuman uncertainty factor
UFl
LOAEL to NOAEL uncertainty factor
UFS
subchronic to chronic uncertainty factor
1

-------
FINAL
9-10-2009
PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
N,N-DIPHENYL-1,4-BENZENEDIAMINE (CASRN 74-31-7)
Background
On December 5, 2003, the U.S. Environmental Protection Agency's (U.S. EPA) Office of
Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human
health toxicity values for Superfund risk assessments, establishing the following three tiers as the
new hierarchy:
1)	U.S. EPA's Integrated Risk Information System (IRIS).
2)	Provisional Peer-Reviewed Toxicity Values (PPRTVs) used in U.S. 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 U.S. EPA's IRIS. PPRTVs are developed according to a
Standard Operating Procedure (SOP) and are derived after a review of the relevant scientific
literature using the same methods, sources of data, and Agency guidance for value derivation
generally used by the U.S. EPA IRIS Program. All provisional toxicity values receive internal
review by two U.S. EPA scientists and external peer review by three independently selected
scientific experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the
multiprogram consensus review provided for IRIS values. This is because IRIS values are
generally intended to be used in all U.S. EPA programs, while PPRTVs are developed
specifically for the Superfund Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a 5-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV documents conclude that
a PPRTV cannot be derived based on inadequate data.
Disclaimers
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and Resource Conservation and Recovery Act (RCRA) program offices are advised to
carefully review the information provided in this document to ensure that the PPRTVs used are
appropriate for the types of exposures and circumstances at the Superfund site or RCRA facility
in question. PPRTVs are periodically updated; therefore, users should ensure that the values
contained in the PPRTV are current at the time of use.
It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
users are strongly encouraged to read the entire PPRTV document and understand the strengths
1

-------
FINAL
9-10-2009
and limitations of the derived provisional values. PPRTVs are developed by the U.S. EPA
Office of Research and Development's National Center for Environmental Assessment,
Superfund Health Risk Technical Support Center for OSRTI. Other U.S. 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 U.S. 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 U.S. EPA has not derived RfDs, RfCs, or estimates of carcinogenic potency for
N,N-diphenyl-l,4-benzenediamine (diphenyl-p-phenylene diamine or DPPD) (see Figure 1 for
chemical structure). No values are posted on IRIS (U.S. EPA, 2008a), the Drinking Water
Standards and Health Advisories list (U.S. EPA, 2006), or the Health Effects Assessment
Summary Tables (HEAST) (U.S. EPA, 1997). There are no entries for this chemical in the
Chemical Assessments and Related Activities (CARA) list (U.S. EPA, 1991, 1994). U.S. EPA
(2008b) has not derived Acute Exposure Guidelines (AEGLs) for DPPD for use in the event of a
sudden, unexpected release, and there are no occupational exposure guidelines for DPPD
(American Conference of Governmental Industrial Hygienists [ACGIH], 2007; National Institute
for Occupational Safety and Health [NIOSH], 2008; Occupational Safety and Health
Administration [OSHA], 2008).
Figure 1. Structure of N,N-Diphenyl-1,4-Benzenediamine
The National Toxicology Program (NTP, 2008) has not assessed the toxicity or
carcinogenicity of DPPD, and this compound is not included in the 11th Report on Carcinogens
(NTP, 2005). DPPD has not been the subject of a monograph by the International Agency for
Research on Cancer (IARC, 2008) or a toxicological profile by the Agency for Toxic Substances
and Disease Registry (ATSDR, 2008). CalEPA (2002, 2008a,b) has not derived exposure levels
for chronic toxicity or carcinogenic potency for DPPD.
2

-------
FINAL
9-10-2009
To identify toxicological information pertinent to the derivation of provisional toxicity
values for DPPD, literature searches were conducted on November 5, 2008, using the following
databases: MEDLINE, TOXLINE, BIOSIS (1999-November 5, 2008), Chemical Abstracts
(1999-November 2008), TSCATS1/2, CCRIS, DART, GENETOX, HSDB, RTECS, and Current
Contents (May 2007-November 2008). Except where noted, the literature searches are not
limited by date.
REVIEW OF PERTINENT DATA
Human Studies
Conde-Salazar et al. (2004) reported a case of contact dermatitis in association with
occupational exposure to a black rubber mixture that contained DPPD, N-cyclohexyl-N'-phenyl -
4-phenylenediamine, and N-isopropyl-N'-phenyl-4-phenylenediamine. The patient was an
18-year-old dental technician who developed dry hyperkeratotic lesions on the palm of one hand
that was in contact with a container used to prepare molds for dental prostheses. On patch
testing, the subject had a positive reaction to the black rubber mixture and its components. The
lesions went away when the subject stopped touching the container.
No other studies concerning the toxicity of DPPD in humans are identified in the existing
literature.
Animal Studies
Oral Exposure
There are no complete subchronic toxicity studies of DPPD, and there is only one chronic
oral study. The reproductive toxicity of DPPD has been studied extensively in older studies that
were conducted by the oral route of exposure.
Subchronic Studies—In a discussion of a reproductive toxicity study, Draper et
al. (1956) noted briefly that a group of weanling rats (sex and number, not specified) fed
0.5% DPPD (about 250 mg/kg-day based on measured body weight and food consumption) in a
vitamin E-deficient diet appeared healthy for the first 12 weeks on the diet, but then developed
"porphyrin" staining of the whiskers and coat in the area of the head and neck, rough "haircoat"
and moderate depression of weight gain. No significant changes in hemoglobin values or
prothrombin times were detected. According to the study authors, palatability of the food did not
appear to be a problem, based on food consumption and growth rate during the earlier portion of
the study. Effect levels could not be defined due to the limited information available.
Chronic Studies—In the only chronic oral study of DPPD, groups of 50 male and
50 female F344 rats were fed diets containing 0, 0.5, or 2% DPPD (0, 5000, or 20,000 ppm;
purity not reported, Tokyo Chemical Company, Inc.) dissolved in corn oil for 104 weeks
(Hasegawa et al., 1989). The study authors described the diet as "a powdered basal diet"
(Oriental M brand). Neither the constituents nor a dietary analysis was reported, but it appears
that the diet may have been a semipurified or purified diet based on a statement in the discussion
section of the report where the study authors attribute an observed endpoint, nephrocalcinosis, to
dietary insufficiency (discussed later). Surviving animals were killed 8 weeks after the end of
the treatment period. Daily intake of DPPD, based on food consumption and body-weight
3

-------
FINAL
9-10-2009
measurements taken throughout the study, was reported to be 0, 194, or 857 mg/kg-day in males
and 0, 259, or 1024 mg/kg-day in females. Urinalysis (pH, protein, glucose, ketone bodies,
bilirubin, occult blood, urobilinogen, specific gravity, and sediments) was performed 1 week
prior to the end of the treatment period. Hematology (erythrocyte, leukocyte and platelet counts;
hematocrit and hemoglobin), blood chemistry (aspartate aminotransferase [AST], alanine
aminotransferase [ALT]), alkaline phosphatase, total cholesterol, P-lipoprotein, total protein,
albumin/globulin ratio, urea nitrogen, glucose, sodium, potassium, chloride and calcium), organ
weights (brain, heart, liver, spleen, kidneys, adrenals, testes and ovaries) and comprehensive
histology were evaluated following sacrifice at the end of the posttreatment period (Study
Week 112). Necropsies and histological evaluations were also performed on the rats that died or
became moribund during the study.
Survival was not affected by DPPD; survival in the high-dose males was better than in
controls (statistics not reported) (Hasegawa et al., 1989). Body-weight gain was reduced in
treated males and females throughout the study. The decreases in weight gain were dose-related
and more pronounced in the females; final body weights in the low- and high-dose groups were
5.9 and 8.9% lower than controls in males, respectively, and 22.5 and 25.2% lower than controls
in females, respectively (statistical analysis not reported). Food consumption, however, was the
same in treated as in control groups. All treated groups of rats reportedly had statistically
significantly (p < 0.05) reduced absolute organ weights (heart, liver, and adrenal in both sexes;
kidney in females; testis in males) and relative organ weights (liver in both sexes) in comparison
with controls; data are not reported, so the magnitude of change is unknown. Hematological
effects included small—but statistically significant (p < 0.01)—increases (compared with
controls) in erythrocyte count (13—18%), hemoglobin (8-11%), and hematocrit (11—12%) in
females at >259 mg/kg-day and increased platelet count (15%) in males at 857 mg/kg-day, but
these changes were not considered toxicologically important by the study authors. Serum
calcium levels were statistically significantly reduced (7. 5%,p<0 .01) in males at
857 mg/kg-day. Histological examinations showed increased occurrence of renal calcification at
the corticomedullary junction (nephrocalcinosis) in treated males; incidences were reported as
2.0, 57.1, and 65.3% in the 0-, 194-, and 857-mg/kg-day groups, respectively. Incidences of
nephrocalcinosis were high (75—86%) in both control and treated female groups. The study
authors observed no other treatment-related nonneoplastic changes upon histological
examination of tissue samples. The results of this study indicate that the low dose, 5000 ppm
(194 mg/kg-day in males and 259 mg/kg-day in females), is a LOAEL associated with an
increased incidence of nephrocalcinosis in the males and biologically relevant decreased body
weight in the females (>20% below control values at termination).
A limitation of this study is that the long observation period (8 weeks after termination of
exposure) may have allowed for recovery of reversible lesions (Hasegawa et al., 1989). In
addition, nephrocalcinosis has been shown to occur in untreated rats fed "semipurified" or
"purified" diets (Phillips et al., 1986; NRC, 1995). The nature of the basal diet given to the rats
in the study by Hasegawa et al. (1989) is not reported. The study authors stated that it was not
clear whether the observed nephrocalcinosis was due to the feeding of DPPD or related to
nutritional imbalances in calcium or phosphorus, which might suggest that a semipurified or
purified diet was used. Feed intake rates in this study were lower than reference values and
consistent with values observed in purified diet studies (see discussion under "Studies using
vitamin E-deficient diets, with or without supplementation" below). In any event, the incidence
4

-------
FINAL
9-10-2009
of nephrocalcinosis in control male rats fed the same basal diet was only 2%, compared with
>57.1% in DPPD-treated male rats (Hasegawa et al., 1989), providing support that the
nephrocalcinosis in males was treatment related.
All rats that died (or were killed) after the appearance of the first tumor at 57 weeks were
included in the number of effective animals for statistical analysis of incidences of neoplastic
lesions. The incidences of neoplastic lesions were not significantly (p < 0.05) different in the
DPPD-treated groups as compared with controls. The incidence of squamous cell carcinoma of
the skin in females was lower (p < 0.05) in the treated groups than in the controls. The
incidences of testicular interstitial cell tumors were high in all groups of males (90, 89.8, and
87.8% in the control, low-, and high-dose groups, respectively). Thus, this study provides no
evidence of carcinogenicity in rats of either sex fed the chemical at dietary levels to produce
some toxicity (depression of body-weight gain, increased incidences of nephrocalcinosis in
males) without affecting survival.
Reproductive/Developmental Studies—A number of early studies examining the
effects of DPPD on reproduction were conducted decades ago. Some of these studies exposed
animals to DPPD in conjunction with vitamin E-deficient diets (referred to by the study authors
as "purified" or "semipurified" diets), in an effort to determine whether DPPD, an antioxidant,
could prevent the adverse effects of vitamin E deficiency on reproduction. The available studies
of reproductive effects of DPPD are discussed below; the studies are organized by whether stock
diet (presumed to contain sufficient vitamin E) or "purified/semipurified" diet (deficient in
vitamin E) was used in the study.
Studies using stock diets containing sufficient vitamin E
Bionetics Research Laboratories Inc. (1968) assessed the potential developmental toxicity
of DPPD in two strains of mice exposed via gavage. A group of 12 pregnant C57BL6 mice was
given 464 mg/kg of Agerite DPPD (no further characterization, purity not specified, dissolved in
a 50% honey and water solution) on Days 6-14 of gestation. An additional study was attempted
with AKR mice, but, due to the use of only one dam per dose group, no conclusions are possible.
Both untreated (two groups of 31 or 37 dams) and vehicle (32 dams) controls were included in
the study. All mice were fed a standard "baked diet" from a commercial supplier throughout the
study. Dams were sacrificed and weighed on Day 18 of gestation, and uterine contents were
examined. Maternal liver weights, but no other organ weights, were recorded. The study
authors reported no mortality or treatment-related clinical signs among the dams. There were no
statistically significant differences between DPPD-exposed dams and their vehicle controls with
respect to maternal weight gain. Relative liver weight of dams was reported to be significantly
(p < 0.05) increased compared with controls, but the difference was very small (6.37 ± 0.09% for
DPPD-exposed dams vs. 6.14 ± 0.08% for vehicle controls; p < 0.05).
There were no treatment-related effects on mean implantations per litter, mean number of
live fetuses per litter, or mean crown-rump length in C57BL6 mice (Bionetics Research
Laboratories Inc., 1968). The study authors reported significant (p < 0.05) decreases in fetal
mortality (7% mortality for DPPD-exposed; 14% for control litters); placental weight
(100 ± 4 mg for DPPD-exposed; 107 ± 2 mg for controls) and amniotic fluid per fetus
(174 ± 14 mg for DPPD-exposed; 202 ± 9 mg for controls). Mean fetal weight was significantly
(p < 0.05) increased in DPPD-exposed mice (1051 ± 28 mg) relative to vehicle controls of the
same strain (937 ± 24); the increase in fetal size may have contributed to the lower placental
5

-------
FINAL
9-10-2009
weight and amniotic fluid. According to the study authors, there were no statistically significant
differences between DPPD-exposed mice and their respective vehicle controls with regard to the
numbers or types of developmental anomalies observed. Based on results for C57BL6 mice, the
NOAEL for reproductive effects and changes in relative liver weights in this study is
464 mg/kg-day (the only dose tested). However, as shown in the reproductive toxicity studies
discussed below, the effects of DPPD on reproduction are usually manifested during delivery;
these effects include uterine hemorrhage, and maternal and fetal mortality. In this study, the
dams were sacrificed prior to delivery such that the effects of DPPD during delivery would not
have been observed.
B.F. Goodrich Company sponsored reproductive toxicity studies of DPPD (Ashe, 1956).
Several studies of rats and mice were conducted with both chemically pure DPPD and several
commercially available DPPD mixtures that contained small amounts of contaminants. Similar
studies were conducted for rats and mice, and the results were similar for both species.
Reporting of the mouse study was incomplete, while reporting for the rat study was relatively
complete; so only the rat study is reported in detail in this review. Groups of approximately
20 female Wistar rats were fed diets of Ralston-Purina Laboratory chow alone (controls) or with
chemically pure DPPD (99.5% pure) at concentrations of 300 or 1000 ppm (Ashe, 1956). Based
on default body weight and food consumption values1, these dietary concentrations are
equivalent to approximate doses of 31 and 103 mg/kg-day, respectively. Additional groups of
10 female Wistar rats were fed the control diet plus a commercial DPPD mixture (purity not
specified; contained small amounts of diphenylamine [DPA], hydroxydiphenylamine [HDA] and
blue tar; 300 or 1000 ppm), or the control diet plus various contaminants such as blue tar
(characterized as "mostly oxidized DPPD", 100 ppm), pure DPA (100 ppm), pure HDA
(100 ppm), or various combinations of DPPD (1000 ppm) with the aforementioned contaminants
(0.5% DPA, 3% HDA and/or 2% blue tar).
Ashe (1956) did not clearly indicate the length of time that these females were fed the
various diets before they were paired with males to initiate the reproductive phase of the study.
However, the report stated that all animals were observed for a week to 10 days before starting
the study. Test diets were fed to the females throughout pregnancy and lactation. Males were
fed the control diet except when they were paired with females, at which time they received
whatever diet the females were exposed to. Females were given up to five chances to become
pregnant (five 5-day pairings with males with 10-12-day intervals between the 5-day pairings).
The study authors acknowledged that they could not determine with accuracy the timing of
conception and gestation2. Females that did not become pregnant after the fifth pairing were
sacrificed and necropsied.
Ashe (1956) did not report maternal food consumption or body weights. Because the
timing of conception was not accurately determined for all test animals, the gestation times for
individual animals were reported as approximate ranges. In addition, the numbers of live and
dead births were not determined due to instances of undetected deliveries and subsequent
cannibalism (frequency of occurrence not reported). Individual animal data reported by the
1 Body weight of 0.156 kg and food consumption rate of 0.016 kg/day for female Wistar rats exposed for subchronic
duration (U.S. EPA, 1988).
2Ashe (1956) stated that "Careful examination of the vagina and the presence of a so-called 'copulation plug' was
not a reliable index of pregnancy in our hands."
6

-------
FINAL
9-10-2009
study authors are minimum, maximum, and midpoint of estimated gestation times; number of
live and dead offspring; number of pups raised to 21 days; and weight of pups on Day 21. A
summary table compared the results of each test group, reporting % fertility rate, % maternal
mortality, mean live births, mean dead births, mean gestation time, number of litters weaned per
number pregnant, and mean weanling weight on Postnatal Day 21. Table 1 summarizes these
data. No statistical analyses are reported, and neither are the variance data that would be needed
to conduct an independent statistical analysis.
Table 1. Reproductive Data from Rats Fed Diets Containing DPPD and its Contaminants11
Group
Fertility
Rate (%)
Maternal
Mortality
Rate (%)
Mean
Live
Birthsb
Mean
Dead
Birthsb
Mean
Gestation
Time (days)
Percent of
Litters
Weaned0
Mean
Weanling
Weight"1
(grams)
Control
85
0
9.0
0.4
20.9
80
46.1
Pure DPPD
300 ppm
75
5
4.6
4.1
22.6
38
47.0
1000 ppm
81
10e
1.9
5.4
22.6
7.7
36.0
Commercial DPPD
300 ppm
60
5
6.1
4.9
22.5
25
ND
1000 ppm
63
0
3.4
5.0
22.7
25
40.5
1000 ppm DPPD + Contaminants
HDA (3%)
70
0
1.1
7.5
24.1
14
38.8
DP A (0.5%)
80
20
6.0
2.6
25.1
12
37.0
Blue tar (2%)
70
0
3.0
4.1
22.8
14
35.6
DP A (0.5%) + HDA
(3%)
70
20
0.9
6.0
24.1
0
NA
HDA (3%) + Blue
tar (2%)
66
33
5.7
4.0
23.9
16
36.9
DP A (0.5%) + Blue
Tar (2%)
80
20
1.1
6.5
23.3
0
NA
Contaminants Alone
100-ppm HDA
100
0
4.9
2.3
22.2
20
40.6
100-ppm DPA
80
0
7.8
0.9
23.9
62
35.7
100-ppm Blue Tar
90
0
8.7
0.3
21.7
78
39.1
aAshe (1956), Table XVI, page 22 of report. Please note that slightly different numbers are reported in a second
version of this table shown on page 53 that appears to be a draft for review. No information other than mean
values is reported.
bAshe (1956) reports that these values may not be absolutely accurate due to unquantifiable cannibalism, but they
are still useful for comparing across groups
°Number litters weaned divided by the number dams pregnant x 100; determined on Postnatal Day 21.
dRecorded on Postnatal Day 21.
"Table XVI, page 22 of the report shows a maternal mortality of 15% for this group; an examination of the
individual animal data, however, indicates that 2/20 dams in this group died, for a mortality rate of 10%.
ND = Not Determined; NA = Not Applicable
7

-------
FINAL
9-10-2009
The data from Ashe (1956) suggest the following effects due to pure DPPD: (1) possible
treatment-related maternal mortality (0, 1/20, and 2/20 in control, 300-ppm, and 1000-ppm pure
DPPD groups, respectively; all maternal deaths occurred during labor); (2) possible slight
decrease in fertility (85%, 75%, and 81%, in control, 300 ppm and 1000 ppm pure DPPD groups,
respectively); (3) dose-related offspring mortality (increase in dead births and decreases in live
births and ratio of litters weaned to number of pregnant dams at 300 and 1000 ppm; offspring
mortality in DPPD-treated groups occurred primarily at birth), although there is some uncertainty
in these data due to problems determining the timing of conception; (4) possible increased
gestation time, although these data are highly uncertain due to problems determining the timing
of conception; and (5) an apparent decrease in pup weight on PND 21 at 1000 ppm DPPD.
Increased maternal and offspring mortality and reduced fertility were also observed in dams fed
commercial DPPD or DPPD preparations containing the various contaminants. Among the
contaminants alone, HDA appears to have effects on offspring viability as well. There is no
strong indication of adverse effects associated with the contaminants DPA or blue tar alone.
Ashe (1956) reported that all of the DPPD-treated dams—but none of the controls—
hemorrhaged abnormally during delivery; dams that survived were reported to be severely
anemic for many weeks (data not shown). The observed uterine hemorrhage is likely to have
caused the recorded maternal deaths, although this is not explicitly stated by the study author.
The study author reports that affected offspring were deeply cyanotic and concludes that fetal
deaths were due to anoxia resulting from partial or complete placental separation at term with
inadequate uterine contraction.
Ashe (1956) conducted gross and microscopic examinations of rats that failed to become
pregnant and those that died in labor. Data for these findings are not clearly summarized, and
the narrative mixes results for mice and rats. Based on the available information, a total of
26 rats were examined, of which three were controls that did not become pregnant, and 23 were
fed diets containing DPPD or DPPD contaminants (no further specification of what specific diets
these rats received). Of the 23 experimental (i.e., exposed to DPPD- or DPPD contaminants) rats
that were examined, 12 died during labor and the remainder were killed in moribund condition
before or after delivery of their litters. Histological examinations revealed no compound-related
effects on the heart, stomach or spleen (data not shown). Tubular degeneration of the kidneys
with casts in the upper and lower nephrons was observed in 11 of the 23 dams that died or were
killed; hyaline necrosis of the liver was also observed in five of these. The histology data are of
limited use given that the study authors do not clearly indicate the test material (pure DPPD,
commercial DPPD, or one of several contaminants) to which the 23 rats were exposed. The low
dose in this study (300 ppm, or 31 mg/kg-day) is a FEL for maternal and fetal mortality during
parturition.
In another reproductive study, groups of 10 female rats (strain not reported) were fed
diets containing 0, 0.025, 0.10, 0.40, or 1.60% (0, 250, 1000, 4000, or 16,000 ppm) commercial
grade DPPD (purity >95%) for 2 weeks prior to mating to untreated males, and subsequently
throughout gestation, parturition and lactation (Oser and Oser, 1956). The female rats were
selected from an established breeding colony; each female had previously produced and weaned
a normal litter. The basal diet was a stock-type diet consisting of a mixture of grains (whole
wheat, corn, alfalfa meal), nonfat dry milk, meat meal, liver, hydrogenated cottonseed oil,
brewer's yeast, vitamin supplements (B complex, A, D, E, and K in cottonseed oil providing
linoleic acid), and sodium chloride and manganese sulfate. Based on default values for body
8

-------
FINAL
9-10-2009
"3
weight and food consumption , estimated DPPD intakes were 0, 22, 88, 350, or 1400 mg/kg-day,
respectively. The endpoints evaluated in the study are duration of pregnancy; numbers of
resorptions, live and dead pups, and complete and partial litters; pup body weight; and maternal
(during parturition and postpartum) and pup (Days 4 and 21) survival.
The duration of gestation was prolonged in the DPPD-treated groups compared with
controls (Oser and Oser, 1956). The incidence of maternal mortality during parturition increased
with dose, as did pup mortality at birth, which was significantly increased at all dose levels.
There were postpartum maternal deaths in controls, 350-, and 1400-mg/kg-day groups, but the
incidence in treated groups was not statistically distinguishable from controls. Table 2 shows the
details of these findings. The pups born dead or found in the uterus of females that died at
parturition were reportedly 10-20% heavier than controls, although there was no clear indication
of abnormal weight in pups born alive (insufficient data were shown to conduct statistical
analyses). The study authors reported that the pups appeared large but, otherwise, normal. This
finding is consistent with the longer duration of gestation. To determine whether damage to the
posterior pituitary might be involved in the delay of parturition, histopathological examinations
of the posterior pituitary were performed in five of the females that died in parturition after
gestations of 24 or 25 days' duration, and in control rats killed on the 22nd day of gestation. The
study authors found no histological differences. The lowest dose of DPPD tested, 22 mg/kg-day,
is a FEL based on maternal and fetal mortality during parturition.
Table 2. Selected Reproductive Data from Rats Fed DPPD in Stock Diets"
Effect
DPPD Dose (mg/kg-day)
0
22
88
350
1400
Mean duration of
gestation (± SEM)
(days)
22.1 ±0.23
22.9 ± 0.23b
24.1 ± 0.30b
25.2 ± 0.68b
24.7 ± 0.54b
Maternal mortality
during parturition
0/10
1/10
3/10
3/10
5/10°
Pup mortality at birth
18/107
(17%)
42/79°
(53%)
21/35°
(60%)
18/20°
(90%)
20/24°
(83%)
Maternal postpartum
mortality
2/10
0/9
0/7
1/7
2/5
aSource: Oser and Oser (1956).
Statistically significant difference from controls, Student's t-test, p-valuc not specified.
Statistically significant difference from controls, (p < 0.05) by Fisher exact test conducted for this review.
Groups of 10-17 female rats of unspecified strain and body weight were fed 0, 0.0125,
0.0625, 0.313, or 1.55% (0, 125, 625, 3130, or 15,500 ppm) of DPPD ("feed grade", purity and
supplier not reported) in the diet starting 10 days prior to mating and continuing through
parturition and lactation (Ames et al., 1956). The diet was a "stock" diet consisting of corn,
wheat, dry skim milk, casein, "meat-bone scraps," alfalfa meal, liver, yeast, linseed meal,
3Food consumption of 0.022 kg/day and body weight of 0.25 kg for mature female rats (U.S. EPA, 1988). Values
for mature animals were used because the rats were from an established breeding colony, and, as such, were older
and weighed more than the assumed body weight for a young rat in a subchronic duration study.
9

-------
FINAL
9-10-2009
partially hydrogenated vegetable oil, calcium carbonate, and iodized salt (Ames et al., 1952,
1956). Based on default values for body weight and food consumption4, estimated average
DPPD intakes were 0, 11, 55, 275, and 1360 mg/kg-day. The study endpoints include a fertility
index (number of females pregnant/number mated), the litter efficiency (% pregnant animals
with at least 1 viable fetus), a mortality index (number of dams dying at parturition/number
pregnant), the duration of pregnancy, the litter size, a viability index (number of pups alive at
Neonatal Day 3/number born), and a lactation index (number of young weaned/number alive at
3 days).
Like Oser and Oser (1956), Ames et al. (1956) also observed prolonged gestation
associated with DPPD exposure as well as increased maternal mortality (at doses
>55 mg/kg-day) and markedly increased pup mortality on or before PND 3 (95-100% at all
doses, compared with 32% in controls). Table 3 shows the details. Deaths of the dams and pups
occurred mainly during parturition, and signs of difficult parturition (vaginal bleeding and
prolapse of the uterus) were occasionally observed. The study authors suggested that the
prolongation of gestation may have resulted in unusually large fetuses, such that the birth process
was difficult and prolonged. Pup weights are not reported. The lowest dose tested
(11 mg/kg-day) is a FEL based on markedly increased pup mortality.
Table 3. Selected Reproductive Data from Rats Fed DPPD in Stock Diets"
Effect
DPPD Dose (mg/kg-day)
0
11
55
275
1360
Mean duration of
gestation (days)
23
24
25
25
25
Mean litter size
10.6
7.9
4.9
5.3
4.7
Maternal mortality
1/17
0/12
5/17
5/10b
7/13b
Pup mortality (Postnatal
Day 3)
33/104
(32%)
75/79b
(95%)
49/49b
(100%)
16/16b
(100%)
14/14b
(100%)
"Source: Ames et al. (1956); no statistical analyses are reported.
Statistically significantly different from control (p < 0.05) by Fisher exact test conducted for this review.
In a study of the effects of antioxidants on fetal resorptions, a total dose of 0.5 g (500 mg)
of DPPD (purity not specified) was administered in the diet to mated female 200 g Walter
Reed-Carworth Farms rats (group size was not reported), starting after mating and continuing
through necropsy on Day 22 of gestation (Telford et al., 1962). The type of diet was not
specified, and so was assumed for this review to be a stock diet. The daily dose was estimated to
be 114 mg/kg-day (500 mg/ [0.2 kg x 22 days]). The young were delivered by caesarian section
after the dams were sacrificed and the uterus inspected for resorption sites. In comparison with
the control group (126 litters), the DPPD-treated group (23 litters) appeared to have an increased
rate of resorption (litters with resorptions = 40.8% in controls vs. 60.8% in DPPD-treated;
resorptions as % of total number of implantations = 10.6% control vs. 15.3% DPPD). The study
4Food consumption of 0.022 kg/day and body weight of 0.25 kg for mature female rats (U.S. EPA, 1988).
10

-------
FINAL
9-10-2009
authors considered this result to indicate a substantial increase in resorptions, but no additional
details were provided and statistical analyses were not reported. As such, effect levels were not
determined for this study.
Studies using vitamin E-deficient diets. with or without supplementation
A number of studies conducted in the 1950s and early 1960s were aimed at assessing
whether administration of DPPD, an antioxidant, could prevent the adverse effects of vitamin E
deficiency on reproduction (sterility). In these studies, "purified" or "semipurified" diets
(deficient in vitamin E) were used both with or without vitamin E supplementation. The doses of
vitamin E that were used in supplementation varied from 0.7 to 30 mg/week. The available
studies do not clearly discuss what level of dietary vitamin E is considered to be "sufficient" for
laboratory rodents. However, Ames (1974) reported that 0.7 mg DL-a-tocopherol acetate, given
6 times per week, was required for normal reproduction in older female rats. This translates to a
weekly requirement for 4.5 mg/week.
A study (Draper et al., 1956) that measured intake of a purified diet reported lower
consumption rates than for stock diets. Food consumption was measured for female
Sprague-Dawley rats fed DPPD in a purified diet from weaning through mating, gestation, and
parturition. Rats weighed 0.188 kg and had an estimated food consumption of 0.009 kg/day5.
This intake is about half of the reference value for food consumption predicted by the allometric
equations in U.S. EPA (1988) for a rat of that weight (0.018 kg/day). One possible explanation
for the reduced intake is that the purified diet was more concentrated (no fiber source was
included). Due to the possibility that intake of purified diets was lower than for stock diets,
default values for food consumption were not used to estimate doses of DPPD administered in
purified or semipurified diets. Instead, a food factor of 0.05 kg diet/kg bw-day (0.009 kg diet per
day divided by body weight of 0.188 kg) based on empirical data from Draper et al. (1956) was
used to estimate doses for studies where DPPD was administered in vitamin-E deficient diets.
Draper et al. (1956) fed weanling female Sprague-Dawley rats 0.005, 0.025, or 0.1% (50,
250, or 1000 ppm) of DPPD in a purified, vitamin E-deficient diet. An additional group of
10 rats was given 0.385 mg/week of DPPD orally, divided into three equal doses, to simulate the
level of intake that would result from 0.0006% (6 ppm) DPPD in the diet. Controls received just
the purified diet (25 females), or the purified diet supplemented with vitamin E at 30 mg/week
(19 females). The vitamin E supplementation at 30 mg/week appears to be adequate to support
successful reproduction in this study based on the requirement of 4.5 mg/week reported by Ames
(1974). The basal purified diet contained 64.6% cerelose (glucose), 20% casein, 10%
tocopherol-low (distilled) lard, salts (unspecified), and vitamins (including B, A, D, and K).
Using the food factor of 0.05 kg diet/kg bw-day derived above, concentrations of 6, 50, 250, and
1000 ppm DPPD in the purified diets are estimated to correspond to DPPD doses of 0.3, 2.5,
12.5, and 50 mg/kg-day. The rats were fed the DPPD-containing and control diets starting at
weaning and continuing through mating and parturition. The females, weighing 175-200 g
(188-g median) were then mated to healthy male rats that had been maintained on a stock diet.
5 A dose of 0.385 mg/week was estimated by the authors to correspond to a dietary level of 0.0006% (6 ppm) DPPD
(Draper et al., 1956). Dividing 0.385 mg/week by 7 days and by the median weight of the female rats at mating
(0.188 kg) gives an estimated DPPD dose of 0.3 mg/kg-day. A daily food ingestion value (kg-diet/day) can be
estimated using these values, as follows: (0.3 mg/kg bw/day x 0.188 kg bw) ^ 6 mg DPPD/kg diet =
0.009 kg diet/day.
11

-------
FINAL
9-10-2009
Endpoints include clinical observations, serum hemoglobin, prothrombin time, white blood cell
counts, number of conceptions (number pregnant), gestation length, number of pups born, and
live births.
Draper et al. (1956) reported that during the first 6 weeks on the diets (the growth
period), all the rats gained weight normally and appeared healthy, with no signs of DPPD
toxicity or vitamin deficiency. None of the rats in the control group that did not receive vitamin
E became pregnant. The conception rate for the other groups was not different from
vitamin E-supplemented controls. During gestation, signs of toxicity (anorexia, rough haircoat,
and anemia) were observed at the highest exposure level (1000 ppm DPPD). Table 4 shows the
other pertinent findings of the study. The average duration of gestation was increased among
dams fed the two highest concentrations of DPPD, and the incidence of stillbirths was increased
in all but the lowest dose group. The incidence of stillbirths at the low dose (6 ppm) was low
(12%) and less than the vitamin E-supplemented control group (43%); however, at the higher
doses, the incidence of stillbirth was >81%. At parturition, vaginal hemorrhage was observed in
the 1000-ppm dams; 3/10 animals in this group died and the rest were semimoribund. The three
deaths were attributed to hemorrhaging (only seven of these animals were pregnant, however,
and it is not clear whether hemorrhages were seen in the nonpregnant rats). The hemorrhagic
rats were acutely anemic, but their prothrombin times were normal. Hematological parameters
in the other groups were not affected. The total number of pups born per pregnant rat was
comparable to vitamin E-supplemented controls at the lower levels of DPPD, but it was very low
in the 1000-ppm group. Whether this was due to failure to deliver some of the pups (because the
dams died or were semi-moribund) is not discussed. During gestation and at parturition, no
adverse effects on any endpoint were seen in the 0.3 mg/kg-day (6 ppm) group; this dose is a
NOAEL. The next higher dose (2.5 mg/kg-day or 50 ppm) is considered to be a FEL based on
the marked increase in stillborn pups.
Table 4. Selected Reproductive Data from Rats Fed DPPD in Vitamin E-Deficient Diets3
Effect
DPPD Dose (mg/kg-day)
0
(no vitamin E)
0
(+ 30 mg vitamin
E/wk)
0.3
2.5
12.5
50
Mean duration of
gestation (days)
No pregnancies
22.8
Not reported
23.8
25.4
Not
reported
Vaginal
hemorrhage,
death
Not applicable
None reported
None
reported
None
reported
None
reported
3/10
Stillborn pups
Not applicable
67/155 (43%)
8/69
(12%)
93/108b
(86%)
66/7 lb
(93%)
17/2 lb
(81%)
aSource: Draper et al. (1956); no statistical analyses are reported.
Statistically significantly different from vitamin E-supplemented control (p < 0.05) by Fisher exact test conducted
for this review.
12

-------
FINAL
9-10-2009
Draper et al. (1958) conducted a series of four studies to determine whether vitamin E
deficiency could be reversed by supplementation with vitamin E or other antioxidants such as
DPPD. In the first study, groups of 10 weanling Sprague-Dawley rats were fed a purified,
vitamin E-deficient diet supplemented with (1) no supplement; (2) 0.7 mg D-a-tocopheryl
acetate; (3) 0.7 mg DL-a-tocopheryl acetate; (4) 0.4 mg DPPD/week given once weekly in
triacetin and estimated to be equivalent to 0.0006% DPPD (6 ppm) in the diet; or (5) 0.1%
(1000 ppm) DPPD in the diet, reduced in the second and third reproductive cycles to 0.025%
(250 ppm) and 0.0025% (25 ppm), respectively. Using the food factor of 0.05 kg diet/kg bw-day
for purified diets developed from data in the Draper et al. (1956) study, DPPD dietary levels of
6 ppm (0.4 mg/week) and 1000 ppm (followed by 250 and 25 ppm) correspond to estimated
doses of 0.3 and 50 mg/kg-day (followed by 12.5 and 1.3 mg/kg-day). The amount of vitamin E
that was added to the diets of groups 2 and 3 (0.7 mg, once per week) is well below the
4.5 mg/week reported to be required to support female reproduction in rats (Ames, 1974). The
diets were fed starting at weaning and continuing for 8 weeks, at which time the rats were mated
to normal males fed a stock diet; the female rats were continued on their respective diets as
above through two to four reproductive cycles (not specifically defined by the study authors;
however, a reproductive cycle appears to include mating and the production of a litter) over three
generations. There were five weanling females (Fl) from the first litters of the lowest-dose
DPPD group that were fed the same diet for two reproductive cycles, and five of the weanling
female pups (F2) from their first litters were then maintained on the diet through mating and
parturition. Criteria for reproductive performance included number of pregnancies and of
resorbed litters, number of litters, number of pups born (total and per pregnancy), and percent
born alive. Table 5 summarizes the results for this study. The reproductive performance of the
females receiving DPPD at an oral dose equivalent to 6 ppm in the diet is comparable to that of
the groups receiving vitamin E supplements (D-a- or DL-a-tocopheryl acetate) through two
reproductive cycles for each of two generations (Draper et al., 1958). Reproductive failure
(resorption) occurred in the third generation (F2 females); it was similar to that seen in the first
generation control group that received no vitamin E supplementation (controls were studied only
for one generation). The reproductive failure seen in the F2 females was of the type
characteristic of vitamin E deficiency (resorptions), and, thus, appears to reflect an inability of
DPPD to substitute totally for vitamin E during prolonged vitamin E deficiency rather than any
toxicity of DPPD itself (as the latter would typically be manifest as maternal or pup mortality at
delivery). Accordingly, this study provides support for a NOAEL 0.3 mg/kg-day (equivalent to
6 ppm) for reproductive effects of DPPD in a vitamin E-deficient purified diet.
The group that started on 1000 ppm (50 mg/kg-day) DPPD at weaning through the first
reproductive cycle, with decreased dietary levels (250 and 25 ppm, corresponding to 12.5 and
1.3 mg/kg-day) for the second and third cycles, experienced a high rate of stillbirth (calculated
from data regarding total pups born and the percent born alive) in the first two cycles, and a low
pregnancy rate in the third (Draper et al., 1958). Additional effects in the dams were the
prolongation of gestation time by 2-3 days, vaginal hemorrhages, and anemia. It was not
specified whether the prolongation of gestation, hemorrhages, and anemia occurred only in the
first cycle or in subsequent cycles as well. The number of dams dropped from 10 for the first
cycle to 8 for the second and third; whether this attrition was due to mortality or morbidity also
was not specified, but it appears likely from the description of effects in the dams.
13

-------
FINAL
9-10-2009
Table 5. Reproductive Performance of Female Rats Fed DPPD in Vitamin E-Deficient Diets3
Cycle No.b
Group
No.
Treatment
No.
Females
No. Females
with
Implantations
No. Females
with
Resorptions
No. of Pups Born
Percent Born Alive
Total
Per Implantation
First Generation
1
1
None
10
7
7
0
0
0
1
2
0.7 mg D-a-tocopheryl acetate/wk
10
4
0
30
7.5
100
1
3
0.7 mg DL-a-tocopheryl acetate/wk
10
7
0
64
9.1
100
1
4
0.3 mg DPPD/kg-day
10
5
0
35
7.0
100
1
5
50 mg DPPD/kg-day
10
10
0
21
2.1
15
2
1
None
10
10
7
15
1.5
100
2
2
0.7 mg D-a-tocopheryl acetate/wk
10
10
1
69
6.9
99
2
3
0.7 mg DL-a-tocopheryl acetate/wk
10
10
0
92
9.2
97
2
4
0.3 mg DPPD/kg-day
9
9
1
61
6.8
87
2
5
12.5 mg DPPD/kg-day
8
7
0
50
7.1
50
3
5
1.3 mg DPPD/kg-day
8
2
1
5
2.5
100
Second Generation
1
4
0.3 mg DPPD/kg-day
5
4
0
36
9.0
100
2
4
0.3 mg DPPD/kg-day
5
3
0
18
6.0
100
Third Generation
1
4
0.3 mg DPPD/kg-day
5
2
2
0
0
0
aSource: Draper et al. (1958), Table 1; no statistical analyses are reported.
bIncludes mating and gestation period/litter.
14

-------
FINAL
9-10-2009
The second experiment tested the ability of DPPD at dietary levels of 0.005% or 0.025%
(50 or 250 ppm, corresponding to 2.5 or 12.5 mg/kg-day estimated as above) to maintain
reproductive performance in female rats fed vitamin E-deficient purified diets through four
complete reproductive cycles (Draper et al., 1958). Diets were similar to those reported above
for other experiments in the same study; controls included a vitamin E-deficient group and a
group that received an oral supplement of vitamin E (30 mg D-a-tocopheryl acetate/week;
sufficient to support female reproduction). From weaning through the end of the second
reproductive cycle, the only fat source in the diet was cod-liver oil, which was known to
accelerate the appearance of vitamin E deficiency. These diets produced signs of essential fatty
acid deficiency (tails became scaly and developed cracks) in all groups by the time the animals
were mated, so supplementation with methyl linoleate was instituted. Nevertheless, the
percentage of dams that produced litters during the first cycle was low in all groups (<44%). For
cycles three and four, lard, rather than cod liver oil, was used as the source of dietary fat.
Table 6 shows the results from the second experiment reported by Draper et al. (1958).
No litters were born to the vitamin E-deficient controls. In all four reproductive cycles, the
percentage of stillbirths, calculated from data regarding total pups born and the percent born
alive, was higher in the 50-ppm (2.5 mg/kg-day) DPPD group than in the vitamin-E-
supplemented controls. The percentage of stillbirths was even higher in the 250-pm
(12.5 mg/kg-day) DPPD group—reaching 100% in the fourth cycle—and the number of pups
born in this cycle to this group was low relative to the vitamin E controls. The number of dams
decreased somewhat in all groups over the four cycles, but the decrease was more marked in the
250-pm group; whether the decrease was due to treatment-related mortality was not discussed
but in any event, was not statistically significant (Fisher exact test conducted for this review).
The study authors noted that chronic respiratory infection affected an unspecified number of rats
in each group as age advanced.
Additional studies in which one generation of female rats fed Vitamin E deficient diets
were allowed to mate, but were unable to reproduce. Their diet was supplemented with nothing,
with vitamin E (30 mg D-a-tocopherol acetate/week; adequate to support female reproduction)
or with 0.005%) DPPD (50 ppm or 2.5 mg/kg-day) during the second reproductive cycle of
mating and litter production. The results showed that DPPD restored fertility in terms of
supporting the ability to carry litters to term (Draper et al., 1958). The percentage of stillbirths,
however, was elevated in the DPPD group (37%>) relative to vitamin E-supplemented controls
(14%).
The Draper et al. (1956, 1958) series of studies defines a FEL of 2.5 mg/kg-day (50 ppm)
based on markedly increased incidence of stillbirths (see Table 6). The NOAEL for this series of
studies is 0.3 mg/kg-day (6 ppm). An important observation from the studies by
Draper et al. (1958) is that the effects of vitamin E deficiency on reproduction appear earlier in
gestation than the effects of DPPD. Specifically, vitamin E deficiency results in marked
increases in the rate of resorptions. In contrast, DPPD toxicity is manifested as stillbirths or pup
mortality at birth (see data in Tables 5 and 6).
15

-------
FINAL
9-10-2009
Table 6. Reproductive Performance of Female Rats fed DPPD in Vitamin E-Deficient Diets"





No. of Pups Born
Percent Born
Cycle No.b
Group No.
Treatment Group
No. Females
No. of Litters
Total
Per Litter
Alive
1
1
Control
25
0
0
0
0
1
2
30 mg D-a-tocopheryl acetate/wk
25
9
78
8.7
75
1
3
2.5 mg DPPD/kg-day
25
11
78
7.1
25
1
4
12.5 mg DPPD/kg-day
25
4
13
3.2
15
2
1
None
25
0
0
0
0
2
2
30 mg D-a-tocopheryl acetate/wk
23
17
155
9.1
57
2
3
2.5 mg DPPD/kg-day
22
16
108
6.7
14
2
4
12.5 mg DPPD/kg-day
21
10
71
7.1
7
3
1
None
21
0
0
0
0
3
2
30 mg D-a-tocopheryl acetate/wk
21
4
33
8.2
55
3
3
2.5 mg DPPD/kg-day
15
7
44
6.3
27
3
4
12.5 mg DPPD/kg-day
14
4
14
3.5
4
4
1
None
17
0
0
0
0
4
2
30 mg D-a-tocopheryl acetate/wk
17
6
39
6.5
67
4
3
2.5 mg DPPD/kg-day
14
7
43
6.1
35
4
4
12.5 mg DPPD/kg-day
11
6
11
1.8
0
aSource: Draper et al. (1958), Table 1; no statistical analyses are reported.
bIncludes mating and gestation period/litter.
16

-------
FINAL
9-10-2009
In a preliminary study of DPPD's ability to substitute for vitamin E, Ames et al. (1956)
fed nine vitamin E-depleted female rats DPPD at 0.2% (2000 ppm, or 100 mg/kg-day, estimated
as above for purified diets) in a purified vitamin E-deficient diet starting 7 days before mating
and continuing through gestation, parturition and lactation. A vitamin E-supplemented control
group of nine vitamin E-depleted female rats received 0.002% D-a-tocopheryl acetate in the diet
starting 7 days before mating, and a negative control group (three females) received only the
vitamin E-deficient diet. Although the negative controls became pregnant, the fetuses were
resorbed such that there were no viable fetuses at term. In the DPPD group, 3/9 dams had a least
one viable fetus versus 9/9 for the vitamin E controls. Gestation was prolonged to 25 days in the
DPPD group (as compared with a normal duration of 22 days), and 2/9 dams died (versus 0/9 in
the vitamin E controls and 0/3 in the negative controls). The viability index (number pups alive
at 3 days/number born) was 0/7 for the DPPD group and 63/70 for the vitamin E controls. The
only dose tested in this study (100 mg/kg-day) is a FEL for maternal and pup mortality.
In another study by these study authors, groups of 10-17 female rats of unspecified strain
and body weight were fed DPPD atO, 0.0125, 0.0625, 0.313, or 1.55% (0, 125, 625, 3130, or
15,500 ppm) of DPPD ("feed grade", purity and supplier not reported) in a purified diet to which
0.001%) vitamin E was added, starting on the day of mating and continuing through parturition
and lactation (Ames et al., 1956). It is not clear whether the amount of vitamin E added to this
diet could be considered "sufficient" because it is half the amount of vitamin E used in the
previously reported preliminary study in the positive control group. In that study,
0.002%) vitamin E added to the basal vitamin E-depleted diet was sufficient to prevent the
adverse effects on reproduction that were observed among dams fed the vitamin E-depleted diet.
The purified diet used in this and the preliminary study consisted of 60%> cerelose (glucose),
24%o casein, 12%> distilled lard, supplemental vitamins (B, K, A, and D), and a salt mixture
(content not specified) (Ames et al., 1956; Harris and Ludwig, 1949). Using the food factor of
0.05 kg diet/kg bw-day from the study of Draper et al., (1956), concentrations of 0-, 125-, 625-,
3130-, and 15,500-ppm DPPD in the purified diets of Ames et al. (1956) are estimated to
correspond to doses of 0, 6.3, 31, 157, and 775 mg/kg-day. Study endpoints included fertility
index (number of females pregnant/number mated), litter efficiency (%> pregnant animals with at
least 1 viable fetus), mortality index (number of dams dying at parturition/number pregnant),
duration of pregnancy, litter size, viability index (number of pups alive at Neonatal Day
3/number born), and lactation index (number of young weaned/number alive at 3 days).
Results from the latter experiment using purified diet were similar to those from the
experiment using stock diet and longer-term DPPD exposure (see description under "Studies
using stock diets containing sufficient vitamin E"), but most of the effects were less severe at any
given DPPD dietary level (Ames et al., 1956). Table 7 presents pertinent findings. As the table
shows, DPPD exposure resulted in a dose-related increase in gestation duration and pup
mortality. Maternal mortality (1-2 dams/dose) was observed in the 6.3-, 157-, and
775-mg/kg-day groups—but not in the 31 mg/kg-day group or in controls. Pup mortality was
42% at the lowest dose (6.3 mg/kg-day), compared with 14% in controls. Higher doses were
associated with 85-100%) pup mortality. The lowest dose in this study (6.3 mg/kg-day) is a FEL
for markedly increased pup mortality.
17

-------
FINAL
9-10-2009
Table 7. Selected Reproductive Data from Rats fed DPPD in Vitamin E-Deficient Diets
Partially Supplemented with Vitamin E (0.001 %)a
Effect
DPPD Dose (mg/kg-day)
0
6.3
31
157
775
Mean duration of
gestation (days)
22
23
24
25
26
Mean litter size
7.7
6.2
5.1
6.0
7.3
Maternal mortality
0/17
1/11
0/11
2/11
1/11
Pup mortality (Postnatal
Day 3)
12/85
(14%)
13/3 lb
(42%)
43/5 lb
(85%)
54/54b (100%)
29/29b
(100%)
"Source: Ames et al. (1956); no statistical analyses are reported.
Statistically significantly different from control (p < 0.05) by Fisher exact test conducted for this review.
Draper et al. (1964) maintained female Sprague-Dawley rats on a vitamin E-deficient
purified diet from weaning through mating and gestation so that sterility could be demonstrated;
they were then remated and given 0.75 mg DPPD/day until Day 21 of gestation, at which time
they were killed and the uteri examined for live fetuses and resorptions. Reproductive
performance of the DPPD-treated group was better than that of control groups supplemented
with vitamin E (5 or 10 mg/day of DL-a-tocopheryl acetate) in the same manner. However,
because dams were not allowed to go through the birthing process, the parturition-related
maternal and pup mortality observed in other studies of DPPD-exposure are not able to be
evaluated in this study.
King (1964) fed three dietary levels of DPPD (0.025, 0.050, and 0.075% [250, 500, and
750 ppm]) in purified, vitamin E-deficient diets to weanling female Holtzman rats through
mating and 21 days of gestation. These concentrations correspond to 12.5, 25, and
37.5 mg/kg-day calculated using the food factor of 0.05 kg diet/kg bw-day for purified diets.
Each concentration of DPPD was tested at three different levels of vitamin E supplementation:
none, 2 mg/day given on the first five days of gestation, and 2 mg given on Day 10 of gestation.
As with the previous study, the dams were not allowed to give birth. Results are variable, but
they did not reveal a pattern of significant differences in terms of percentages of live normal or
abnormal fetuses or dead or resorbed fetuses (per total number of implantation sites) among the
three dietary levels of DPPD tested, or within each dietary level under different levels of vitamin
E supplementation. The examination of fetuses for abnormalities was conducted under a
dissecting binocular microscope, and, thus, was not a rigorous examination for skeletal and soft
tissue abnormalities. Although the study included control groups receiving the three different
levels of vitamin E and no DPPD, the DPPD groups were not compared statistically with the
control groups, and the data are not presented in enough detail to support independent statistical
analysis. In addition, the author's statement that the experiments lasted for more than 2-years
raises the question of whether the various DPPD dietary levels and the controls were tested
concurrently. As such, effect levels cannot be determined from this study. The use of this study
is also limited because the critical event affected by DPPD exposure (i.e., parturition) was not
allowed to take place.
18

-------
FINAL
9-10-2009
Inhalation Exposure
No inhalation studies of DPPD were located in the available literature.
Other Studies
Although DPPD has been tested for antioxidant properties in a number of feeding studies
with animals, these studies were focused narrowly to address endpoints such as the effects of
DPPD on the prevention of atherosclerosis (e.g., Sparrow et al., 1992; Tangirala et al.,1995) or
on the distribution of mercury in body tissues (Welsh, 1979). As such, the usual toxicological
endpoints necessary for a reliable assessment of dose-response and the definition of "adverse"
effect levels (such as clinical chemistry, urinalysis, and gross and microscopic examination of
major organs) are not presented in these studies. A mouse study (Tangirala et al., 1995)
suggested some mortality and reduced body weights at a high dose, but the study used mice that
were genetically modified to develop atherosclerosis, and. as such, the results from the study are
of questionable value with respect to the general population.
Mechanistic Studies
Orally administered DPPD was found to inhibit processes indicative of inflammatory
response, including paw edema in rats, adjuvant arthritis in rats, and serum sickness in rabbits
(Levy and Kerley, 1974). The doses used in the studies ranged from 50 to 200 mg/kg with
durations of exposure ranging from 30 minutes to 21 days, depending on the study. The protocol
for administration of DPPD in these studies is not always specific and there are no toxicological
evaluations independent of the narrow focus on inflammatory response (i.e., no monitoring of
body weight, food consumption, or clinical signs and gross or microscopic pathology outside of
the endpoints relevant to mitigation of an artificially induced inflammatory condition). Levy and
Kerley (1974) hypothesized that the inhibitory effects of DPPD on inflammatory response are
due to an inhibition of prostaglandin synthesis or fatty acid peroxide formation that could also
explain the adverse effects of DPPD on the uterus during the birth process.
The effects of DPPD on estrone-induced uterine growth were investigated in young mice
in an attempt to explain previously observed effects of DPPD on gestation and pregnancy in
laboratory rodents (Sonnen et al., 1962). Groups of young 28-day-old Swiss-Webster mice (8 to
23 mice/group) were injected subcutaneously once each day for 3 consecutive days with
(1) estrone alone at doses ranging from 0.00321 to 3.21 |ig per mouse; (2) DPPD alone by
gavage at doses ranging from 0.03 to 9.6 mg/mouse; or (3) estrone injections (0.321 |ig per
mouse) in combination with orally administered DPPD at doses ranging from 0.0005 mg to
9.6 mg/mouse. A group of control mice received only injections of the carrier solvent (sesame
oil) for 3 days. All mice were sacrificed on the day after the last treatment. Body and uterine
weights were recorded. Estrone injections promoted an increase (greater than double the control
value) in uterine weight. DPPD alone had no effect on either body or uterine weight in
comparison with controls. However, DPPD antagonized estrone-induced increases in uterine
weight in a dose-related manner at doses of 0.004 mg/mouse (35.5% mean inhibition) up to the
highest dose tested of 9.6 mg/mouse (83.4%). Sonnen et al. (1962) suggested that because
DPPD was devoid of progestational6, antiprogestational, androgenic, antiandrogenic, and
estrogenic activity in other studies conducted in their laboratory (not discussed further; no data
shown), the observed results were likely due to a "unique pharmacologic property, independent
of hormonal activity."
^'progestational" and "antiprogestational" refer to progesterone agonism and antagonism, respectively.
19

-------
FINAL
9-10-2009
Genotoxicity
DPPD gave positive results for mutagenicity in Salmonella typhimurium TA98 and
TA100 with hamster liver S9—but not with rat liver S9 or in the absence of an activating system
(Zeiger et al., 1992). In another study, positive results were reported in Salmonella typhimurium
TA98 and TA1538 in the presence—but not in the absence—of rat liver S9 (Rannug et al.,
1984). DPPD produced positive results in an assay for point mutations in cultured Chinese
hamster V79 cells in the absence, but not in the presence, of an unspecified S9 preparation
(Donner et al., 1983). Similarly, DPPD was mutagenic in the L5178Y TK+/- lymphoma assay
only when tested without rat liver S9 and not in the presence of this activating system
(Microbiological Associates, Inc., 1987).
Results obtained in chromosomal aberration assays in cultured Chinese hamster ovary
cells are not consistent from study to study: negative results are reported with and without rat
liver S9 by Sofuni et al. (1990), there are equivocal positive results in the presence or absence of
rat liver S9 by Microbiological Associates, Inc. (1988), and there are positive results in the
absence of S9 by Sofuni et al. (1991). Use of longer treatment times appear to produce positive
results (Microbiological Associates, Inc., 1988; Sofuni et al., 1991). Positive results were
obtained for chromosomal aberrations in cultured Chinese hamster lung cells only without S9
(Sofuni et al., 1990, 1991).
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC ORAL RfD
VALUES FOR N,N-1,4-DIPHENYL-1,4-BENZENEDIAMINE (DPPD)
The available data clearly indicated high maternal mortality and marked increase in
stillborn pups at a dose (2.5 mg/kg-day) that is about 8 times higher than the NOAEL
(0.3 mg/kg-day) reported in a series of studies conducted by Draper et al. (1956, 1958).
Therefore, no provisional RfDs for either subchronic or chronic durations are developed.
However, the Appendix of this document contains a screening value that may be useful in certain
instances. Please see the attached Appendix for details.
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC INHALATION RfC
VALUES FOR N,N-1,4-DIPHENYL-1,4-BENZENEDIAMINE (DPPD)
A p-RfC cannot be derived for DPPD because inhalation toxicity data for humans and
animals are lacking. Furthermore, without pharmacokinetic data and information to rule out
portal-of-entry effects, there is no basis to support a route-to-route extrapolation from the oral
RfD.
20

-------
FINAL
9-10-2009
PROVISIONAL CARCINOGENICITY ASSESSMENT
FOR N,N-1,4-DIPHENYL-1,4-BENZENEDIAMINE (DPPD)
Weight-of-Evidence Descriptor
Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005), the
available evidence data provide "Inadequate Information to Assess \the\ Carcinogenic Potential'
of DPPD. No data regarding the potential carcinogenicity of DPPD in humans are available. In
the only available chronic animal study (Hasegawa et al., 1989), DPPD in the diet was not
carcinogenic to rats. DPPD gave mixed results in a small number of genotoxicity studies. DPPD
was not mutagenic in Salmonella typhimurium without activation, but it was mutagenic with
microsomal activation in some studies. DPPD increased point mutations in cultured mammalian
cells without activation, but it gave negative results in two out of three tests with activation.
Assays for chromosomal aberrations in cultured mammalian cell systems resulted in inconsistent
findings both with and without activation; positive findings were more common with longer
treatment times.
Quantitative Estimates of Carcinogenic Risk
Lack of data precludes derivation of quantitative estimates of cancer risk (i.e., p-OSF and
p-IUR) for DPPD.
21

-------
FINAL
9-10-2009
REFERENCES
ACGIH (American Conference of Governmental Industrial Hygienists). 2007. TLVs® and
BEIs®: Threshold Limit Values for Chemical Substances and Physical Agents, Biological
Exposure Indices. ACGIH, Cincinnati, OH.
Ames, S.R. 1974. Age, parity and vitamin A supplementation on the vitamin E requirement of
female rats. Am. J. Clin. Nutr. 27:1017-1025.
Ames, S.R., M.I. Ludwig, W.J. Swanson et al. 1952. Biochemical studies on vitamin A. X. A
nutritional investigation of synthetic Vitamin A in margarine. J. Amer. Oil Chem. Soc.
April: 151-153.
Ames, S.R., M.I. Ludwig, W.J. Swanson et al. 1956. Effect of DPPD, methylene blue, BHT,
and hydroquinone on reproductive process in the rat. Proc. Soc. Exp. Biol. Med. 93:39-42.
Ashe, W.F. 1956. Initial submission: Reproduction studies on DPPD and its impurities as a
food additive to the diets of white rats with cover letter dated 11/12/93. Submitted by B.F.
Goodrich TSCA 8E. Fiche #: OTS0556010. Doc#: 88-940000041.
ATSDR (Agency for Toxic Substances and Disease Registry). 2008. Toxicological Profile
Information Sheet. U.S. Department of Health and Human Services, Public Health Service,
Atlanta, GA. Online, http://www.atsdr.cdc. goy/toxprofites/index.asp.
Bionetics Research Laboratories Inc. 1968. Evaluation of carcinogenic, teratogenic, and
mutagenic activities of selected pesticides and industrial chemicals. Vol. II. Teratogenic study
in mice and rats. Prepared by Bionetics Research Laboratories Inc., for National Cancer
Institute, Bethesda, MD. 145 pgs.
CalEPA (California Environmental Protection Agency). 2002. Hot Spots Unit Risk and Cancer
Potency Values. Online, http://www.oehha.ca.gov/air/hot spots/pdf/TSDlookup2002.pdf.
CalEPA (California Environmental Protection Agency). 2008a. OEHHA/ARB Approved
Chronic Reference Exposure Levels and Target Organs. Online, http://www.arb.ca.eov/toxics/
healthy at/chroni c. pdf.
CalEPA (California Environmental Protection Agency). 2008b. Air Chronic Reference
Exposure Levels Adopted by OEHHA as of February 2005.
Conde-Salazar, L., R. Valks, C.G. Acebes et al. 2004. Occupational allergic contact dermatitis
from antioxidant amines in a dental technician. Dermatitis. 15(4): 197-200.
Donner, M., K. Husgafvel-Pursiainen, A. Jenssen et al. 1983. Mutagenicity of rubber additives
and curing fumes: Results from five short-term bioassays. Scand. J. Work Environ. Health.
9(Suppl.2):27-37.
Draper, H.H., S. Goodyear, K.D. Barbee et al. 1956. Tolerance of the rat for
N,N'-diphenyl-/?-phenylenediamine. Proc. Soc. Exp. Biol. 93:186-189.
22

-------
FINAL
9-10-2009
Draper, H.H., S. Goodyear, K.D. Barbee et al. 1958. A study of the nutritional role of anti-
oxidants in the diet of the rat. Brit. J. Nutr. 12:89-97.
Draper, H.H., J.G. Bergan, M. Chiu et al. 1964. A further study of the specificity of the
vitamin E requirement for reproduction. J. Nutr. 84:395-400.
Harris, P.L. and M.I. Ludwig. 1949. Relative vitamin E potency of natural and of synthetic a-
tocopherol. J. Biol. Chem. 179:1111-1115.
Hasegawa, R., S. Fukushima, A. Hagiwara et al. 1989. Long-term feeding study of
N,N'-diphenyl-p-phenylenediamine in F344 rats. Toxicology. 54(l):69-78.
IARC (International Agency for Research on Cancer). 2008. Search IARC Monographs.
Online, http://monographs.iarc.fr/.
King, D.W. 1964. Comparative effects of certain antioxidants on gestational performance and
teratogeny in vitamin E-deficient rats. J. Nutr. 83:123-132.
Levy, L; Kerley, TL. 1974. The use of DPPD (N,N'-diphenyl-P-phenylenediamine) as an anti-
inflammatory agent. Life Sci 14(10): 1917-1925.
Microbiological Associates, Inc. 1987. Test for chemical induction of mutation in mammalian
cells in culture—The L5178y TK+/- mouse lymphoma assay. Final report. TSCA 8(e)
submission. Fiche # OTS0545453.
Microbiological Associates, Inc. 1988. Cytogenicity study—Chinese hamster ovary (CHO) cells
in vitro. TSCA 8(e) submission. Fiche # OTS0545526.
NIOSH (National Institute for Occupational Safety and Health). 2008. NIOSH Pocket Guide to
Chemical Hazards. Index by CASRN.
NRC (National Research Council). 1995. Nutrient Requirements of Laboratory Animals.
Fourth Revised Edition. National Academy Press, Washington, DC. pp 36-38.
NTP (National Toxicology Program). 2005. 11th Report on Carcinogens. U.S. Department of
Health and Human Services, Public Health Service, National Institutes of Health, Research
Triangle Park, NC. Online, http://ntp-server.niehs.nih.gov/.
NTP. 2008. Testing Status of Agents at NTP. Online, http://ntp.mehs.nih.gov:8080/
index.html?col=010stat.
OSHA (Occupational Safety and Health Administration). 2008. OSHA Standard 1915.1000 for
Air Contaminants. Part Z, Toxic and Hazardous Substances.
Oser, B.L. and M. Oser. 1956. Inhibitory effect of feed grade diphenyl-/^-phenylenediamine
(DPPD) on parturition in rats. Agricult. Food Chem. 4(9):796-797.
23

-------
FINAL
9-10-2009
Phillips, J.C., C. Bex, D. Mendis et al. 1986. Studies on the mechanism of diet-induced
nephrocalcinosis: Calcium and phosphorus metabolism in the female rat. Food Chem. Toxic.
24(4):283-288.
Rannug, A., U. Rannug and C. Ramel. 1984. Genotoxic effects of additives in synthetic
elastomers with special consideration to the mechanism of action of thiurames and
dithiocarbamates. In: Industrial Hazards of Plastics and Synthetic Elastomers. J. Jarvisalo, P.
Pfaffli and H. Vainio, Ed. Alan R. Liss, Inc., New York. p. 407-419.
Sofuni, T., A. Matsuoka, M. Sawada et al. 1990. A comparison of chromosome aberration
induction by 25 compounds tested by two Chinese hamster cell (CHL and CHO) systems in
culture. Mutat. Res. 241(2):175-214.
Sofuni, T., N. Yamazake, A. Matsuoka et al. 1991. Effect of experimental protocols on
detection of chromosomal aberrations in two Chinese hamster cell lines (CHL and CHO). Mutat.
Res. 253(3):276-277.
Sonnen, N., R. Goldhammer and S. Carson. 1962. Anti-uterotropic effect of N^'-diphenyl-
phenylenediamine on immature mice. Endocrinology. 71:779-781.
Sparrow, C.P., T.W. Doebber, J. Olszewski et al. 1992. Low density lipoprotein is protected
from oxidation and the progression of atherosclerosis is slowed in cholesterol-fed rabbits by the
antioxidant n,n-diphenyl-phenylenediamine. J. Clin. Invest. 89:1885-1891.
Tangirala, R.K., F. Casanada, E. Miller et al. 1995. Effect of the antioxidant N,N" -diphenyl
1,4-phenylene diamine (DPPD) on atherosclerosis in ApoE-deficient mice. Arteroscl. Thromb.
Vase. Biol. 15:1625-1630.
Telford, I.R., C.S. Woodruff and R.H. Linford. 1962. Fetal resorption in the rat as influenced by
certain antioxidants. Am. J. Anat. 110:29-36.
U.S. EPA. 1988. Recommendations for and Documentation of Biological Values for Risk
Assessment. Prepared by the Office of Health and Environmental Assessment, Environmental
Criteria and Assessment Office, Cincinnati, OH. EPA/600/6-87/008.
U.S. EPA. 1991. Chemical Assessments and Related Activities (CARA). Office of Health and
Environmental Assessment, Washington, DC. April.
U.S. EPA. 1994. Chemical Assessments and Related Activities (CARA). Office of Health and
Environmental Assessment, Washington, DC. December.
U.S. EPA. 1997. Health Effects Assessment Summary Tables (HEAST). FY-1997 Update.
Prepared by the Office of Research and Development, National Center for Environmental
Assessment, Cincinnati, OH, for the Office of Emergency and Remedial Response, Washington,
DC. July. EPA/540/R-97/036. NTIS PB 97-921199.
U.S. EPA. 2005. Guidelines for Carcinogen Risk Assessment. Risk Assessment Forum,
National Center for Environmental Assessment, Washington, DC. EPA/630/P-03/001F. Online.
http ://www. epa. gov/cancerguidelines/.
24

-------
FINAL
9-10-2009
U.S. EPA. 2006. Drinking Water Standards and Health Advisories. Office of Water,
Washington, DC. Summer 2006. Online, http://www.epa.gov/waterscience/criteria/drinkine/
dwstandards.pdf.
U.S. EPA. 2008a. Integrated Risk Information System (IRIS). Online. Office of Research and
Development, National Center for Environmental Assessment, Washington, DC.
http ://www. epa. gov/iri s/.
U.S. EPA. 2008b. Acute Exposure Guideline Levels (AEGL). Office of Pollution, Prevention
and Toxics. Online, http://www.epa.gov/oppt/aegl/pubs/chemlist.htm.
Welsh, S.O. 1979. The protective effect of vitamin E and N,N -diphenyl-p-phenylenediamine
(DPPD) against methyl mercury toxicity in the rat. J. Nutr. 109:1673-1681.
Zeiger, E., B. Anderson, S. Haworth et al. 1992. Salmonella mutagenicity tests: V. Results
from the testing of 311 chemicals. Environ. Mol. Mutagen. 19(Suppl. 21): 2-141.
25

-------
FINAL
9-10-2009
APPENDIX A. DERIVATION OF SCREENING SUBCHRONIC AND CHRONIC ORAL
RfD VALUES FOR N,N-1,4-DIPHENYL-l,4-BENZENEDIAMINE (CASRN 74-31-7)
For reasons noted in the main PPRTV document, it is inappropriate to derive provisional
toxicity values for N,N-l,4-diphenyl-l,4-benzenediamine. However, information is available for
this chemical which, although insufficient to support derivation of a provisional toxicity value,
under current guidelines, may be of limited use to risk assessors. In such cases, the Superfund
Health Risk Technical Support Center summarizes available information in an Appendix and
develops a "screening value." Appendices receive the same level of internal and external
scientific peer review as the PPRTV documents to ensure their appropriateness within the
limitations detailed in the document. Users of screening toxicity values in an appendix to a
PPRTV assessment should understand that there is considerably more uncertainty associated
with the derivation of an appendix screening toxicity value than for a value presented in the body
of the assessment. Questions or concerns about the appropriate use of screening values should
be directed to the Superfund Health Risk Technical Support Center.
The toxicological database for DPPD is very limited. Apart from one comprehensive
chronic toxicity study (Hasegawa et al., 1989), there are a number of limited reproductive
toxicity studies conducted during the 1950s and 1960s. Table A-l summarizes the studies of
DPPD that provided information to define effect levels. The chronic study identifies a LOAEL
of 194-259 mg/kg-day for nephrocalcinosis in male rats and decreased body weight in female
rats. The reproductive toxicity studies identify effects at lower doses. However, most of these
studies examined very few endpoints, and all are limited by incomplete reporting. Many of the
studies were designed to determine whether DPPD administration could counteract the adverse
effects of vitamin E deficiency on reproduction. Several studies used vitamin E-deficient diets,
confounding the reproductive toxicity findings of DPPD exposure. In addition, there is evidence
from one study (i.e., Draper et al., 1956) that consumption of vitamin E deficient diets by
laboratory animals may have been lower than other diets, making the dose estimates for studies
administering DPPD in these diets uncertain.
Despite their limitations, these studies do provide consistent evidence, regardless of diet,
that DPPD exposure prior to and through gestation results in prolonged gestation and increased
incidences of uterine hemorrhage, maternal mortality during parturition, and stillbirths. In
addition, the series of studies conducted by Draper et al. (1958)—and shown in Tables 5 and 6—
demonstrate that the reproductive effects of vitamin E deficiency (increased resorptions) differ
from those observed with DPPD exposure. One possible mechanism, proposed by
Ames et al. (1956), for the effects of DPPD on survival of dams and pups during delivery is that
prolonged gestation leads to larger fetuses that cause difficulties during parturition. Some
studies (Bionetics Research Laboratories Inc., 1968; Oser and Oser, 1956) have reported
increased fetal weight or visibly larger fetuses after DPPD exposure.
26

-------
FINAL
9-10-2009
Table A-l. Oral Dose-Response Data for DPPD
Species, Strain and Route
(n/sex/group)
Exposure
NOAEL
(mg/kg-day)
Effect Level
(mg/kg-day)
Responses at the LOAEL
Reference
Rat, Chronic, Dietary,
possibly purified/semipurified, vitamin E
status unknown
(50/sex/group)
0, 0.5, or 2% DPPD (0, 194
or 857 mg/kg-day in males
and 0, 259, and 1024
mg/kg-day in females) for
104 weeks followed by 8
weeks observation
None
194 (M)
(LOAEL)
259 (F)
(LOAEL)
Increased incidence of
nephrocalcinosis in males and
decreased body weight in females.
Hasegawa et al. (1989)
Reproduction Studies Conducted with Stock Diets Containing Sufficient Vitamin E
Mouse, C57BL6 Strain, Gavage
(12 F/group)
464 mg/kg in 50% honey
and water on Days 6-15 of
gestation
464
None
Dams sacrificed prior to delivery
(when DPPD effects typically
observed).
Bionetics Research
Laboratories Inc.
(1968)
Rat, Wistar Strain, Diet
(20 F/group)
300 or 1000 ppm (31 or
103 mg/kg-d through
mating, pregnancy and
lactation; males were
exposed during mating
only.
None
31 (FEL)
Maternal and fetal mortality during
parturition
Ashe etal. (1956)
Rat, Strain not reported, Diet
(lOF/group)
0, 250, 1000, 4000, or
16000 ppm (0, 22, 88,350,
or 1400 mg/kg-d for 2
weeks prior to mating (to
untreated males), and
through gestation,
parturition and lactation
None
22 (FEL)
Maternal and fetal mortality during
parturition
Oser and Oser (1956)
Rat, Strain not reported, Diet
(10-17 F/group)
0, 125, 625, 3130, and
15,500 ppm (0, 11,55,275,
and 1360 mg/kg-day) 10
days prior to mating,
through mating, gestation
and early lactation
None
11 (FEL)
Markedly increased pup mortality
(>95%)
Ames et al. (1956)
27

-------
FINAL
9-10-2009
Table A-l. Oral Dose-Response Data for DPPD
Species, Strain and Route
(n/sex/group)
Exposure
NOAEL
(mg/kg-day)
Effect Level
(mg/kg-day)
Responses at the LOAEL
Reference
Reproduction Studies Conducted Using Vitamin E-Deficient Diets with or without Supplementation
Rat, SD, Diet
Multiple experiments
(5-25 F/group)
0, 6, 50, 250, or 1000 ppm
(0,0.3,2.5, 12.5, or 50
mg/kg-day) from weaning
through mating and
lactation, for multiple
reproductive cycles and
generations
0.3
2.5 (FEL)
Markedly increased stillbirths
(>81%)
Draper etal. (1956;
1958)
Rat, Strain not reported, Diet
(9F)
2000 ppm (100 mg/kg-day)
7 days before mating,
through gestation and
lactation.
None
100 (FEL)
Maternal and pup mortality
Ames et al. (1956)
Rat, Strain not reported, Diet
(10-17F/group)
0, 125, 625, 3130, and
15,500 ppm (0,6.3,31,
157, and 775 mg/kg-day)
from the day of
insemination through
gestation and lactation.
None
6.3 (FEL)
Markedly increased pup mortality
(>42%)
Ames et al. (1956)
28

-------
FINAL
9-10-2009
The reproductive studies observed frank effects (consisting of maternal mortality during
parturition or pronounced pup mortality and/or stillbirths) at the lowest dose tested in most of the
studies (as low as 2.5 mg/kg-day) (Table A-l). A NOAEL is identified in one study (i.e.,
Draper et al., 1956) and supported in a second study by the same study authors giving the same
NOAEL (Draper et al., 1958). Because frank effects were observed at the lowest doses
producing effects in the reproductive toxicity studies, benchmark dose modeling of the critical
effects would not be appropriate. Furthermore, BMD analysis was also performed using data
from Ames et al. (1956) and Oser and Oser (1956). The analysis did not yield BMDLio values
lower than the NOAEL, thus it was not used. The NOAEL from the reproductive/developmental
study of 0.3 mg/kg-day identified in the Draper et al. (1956, 1958) studies was used to calculate
a screening subchronic and chronic p-RfD for N,N-diphenyl-l,4-benzenediamine as follows:
Screening Subchronic and Chronic p-RfD =	NOAEL UF
=	0.3 mg/kg-day 1000
=	0.0003 or 3 x 10"4 mg/kg-day
The composite UF of 1000 is composed of the following:
•	UFh: A factor of 10 is applied for extrapolation to a potentially susceptible human
subpopulation because data for evaluating susceptible human response are
incomplete.
•	UFa: A factor of 10 is applied for animal-to-human extrapolation because data for
evaluating relative interspecies sensitivity are incomplete.
•	UFd: A factor of 10 is applied for database limitations. The toxicological database
for DPPD includes one incompletely reported chronic toxicity study in rats and a
number of limited reproductive toxicity studies in rats and one in mice. The available
reproductive toxicity studies are limited by inadequate reporting, limited evaluations,
use of vitamin E-deficient diets (in some cases), and failure to identify a LOAEL that
did not produce frank effects. The database lacks a comprehensive multigeneration
reproductive toxicity study (including exposure and evaluation of males) and studies
evaluating potential teratogenicity.
•	UFS: A factor of 1 is applied to derive the chronic RfD because further adjustments
for duration of exposure is not warranted when reproductive toxicity data are used.
Confidence in the principal studies (Draper et al., 1956, 1958) are low. While these
studies employed a long exposure period (from weaning through parturition) and, in some
experiments, over two to four reproductive cycles (mating, gestation and lactation) in more than
one generation, they suffer from a number of limitations. These include lack of detail in the
reporting of methods and results, limited evaluations of effects, failure to identify a LOAEL that
did not produce frank effects, and use of a specialized diet (purified, vitamin E-deficient) that
may have confounded the reproductive toxicity findings. However, the findings in these studies
are corroborated at higher doses by results in studies using stock diets that provided adequate
vitamin E. Despite the corroborating studies, confidence in the database is low because of the
lack of a subchronic or chronic study in a second species, the lack of comprehensive
multigeneration reproductive toxicity and developmental toxicity studies, and because of the
failure of the available studies to identify a LOAEL that did not produce frank effects.
Accordingly, confidence in the subchronic and chronic screening p-RfD is low.
29

-------