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04750
V-89
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hypertrophy or the presence of testlcular degeneration. In addition to the
tumors presented In Table V-11, myelomonocytlc leukemia, mammary flbro-
adenoma, clltoral gland carcinoma and uterine endometrlal stromal polyps
were observed In one or more rats, but their Incidences 1n treated animals
did not dffer significantly from those Found 1n controls.
The lumber of mice bearing hepatocellular carcinomas was significantly
Increasec In both male and female groups receiving the high dose (780 mg/kg
bw) of D:HP and In female mice receiving the low dose (390 mg/kg bw) (see
t
Table V-ll). Trend tests showed significant dose-related effects for both
sexes. Metastases of the hepatocellular tumors to the lungs were found In
12 male and 8 female DEHP treated mice bearing hepatocellular carcinomas.
Pulmonarj metastases were not found 1n any of the control mice with liver
tumors. Incidences of mice with hepatocellular adenomas did not differ
significantly from controls; however, the Incidences of mice with either
hepatocellular carcinoma or adenoma was significantly Increased at both dose
levels IT both sexes, and significant dose-related trends were present.
Lymphomas, hemanglomas, mammary gland adenocarclnomas and alveolar or
bronchlolar carcinomas or adenomas were also found In one or more treated
mice, but Incidences did not differ significantly from those observed 1n
controls. In conclusion, the DEHP feeding studies In rats and mice Indicate
that statistically significant Increases 1n hepatocellular carcinomas,
neoplastlc nodules and adenomas occurred. These tumors were found 1n both
species and both sexes. There were metastases of hepatocellular tumors to
the lungj of treated mice.
04750 V-90 07/03/91
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A summary of the Interpretation of the bloassay results by NTP 1s shown
In Table V-12. The only compound for which there was clear evidence of car-
diogenlclty was OEHP. In both cases, the effect observed was an Increased
Incidence of liver carcinomas In mice and rats (Huff and Kluwe, 1984).
Northrup et al. (1982) criticized the conclusions drawn from the NTP
DEHP study (Kluwe et al., 1982a,b; Huff and Kluwe, 1984) claiming that the
results could not be Interpreted as showing a carcinogenic effect. Northrup
cited as one problem that the designated maximum tolerated dose (MTO) had
been exceeded (based on differences 1n body weight gain) In both rats and
m1-:e at several of the treated groups. Another criticism of the study was
that there was a significantly lower Incidence of tumor bearing animals
among female mice used 1n the control groups. When all the control groups
(both rats and mice) were pooled, the Incidence of total primary tumors
associated with DEHP treatment was no different for all control groups
except among male rats, which showed a decrease 1n total number of tumors.
Finally, Northrup et al. (1982) claimed that critical data on food consump-
tion, nutritional status, clinical signs, clinical pathology and Intestinal
microorganisms were lacking. Northrup et al. (1982) also felt that the
Incidence of liver tumors could have been Influenced by the altered Intes-
tinal flora Induced by DEHP. The authors postulated that the effects of
DEHP were attributable to eplgenetlc mechanisms of cardnogen1c1ty such as
chronic tissue Injury, nutritional deficiency, hormonal Imbalance or promo-
tional activity, since evidence of direct genotoxlc effects were lacking.
Also, Northrup et al. (1982) felt that because DEHP 1s metabolized
differently 1n rats than In humans, effects In these rodents cannot be
extrapolated to Indicate human risk.
0^750
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TABLE V-12
Summary of the Carcinogenic Effects of OEHP on the NTP Bloassays
and Interpretation of These Findings*
Jest
Chemical
Species
Sex
Neoplasms
Interpretation15
DEHP
rats
Liver neoplastlc
nodules/carcinomas
Some evidence
rats
mice
F
M&F
Liver carcinomas
Liver carcinomas
Clear evidence
Clear evidence
aSource: Huff and Kluwe, 1984
''Evidence of CardnogenlcUy-- Five categories of Interpretative conclu-
sions h.ive been adopted for use 1n the NTP Technical Reports series to
specifically emphasize consistency and the concept of actual evidence of
carclnocenHHy. For each definitive study result (male rats, female rats,
male mice, female mice) one category 1s selected to describe the findings.
This caiegory refers to the strength of the experimental evidence and not
to either potency or mechanism (Huff and Kluwe, 1984).
047SO
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On the other hand, Kluwe et al. (1983) defended the conclusions reached
in the NTP study on DEHP (KTuwe et al., 1982a) by noting that the MTD was
estimated based on prechronlc oral studies, and that the MTD was not tech-
nically exceeded since survival of animals was not adversely affected. In
response to the other criticisms, H was noted that the Hver tumors were
Increased regardless of which set of historical control data were used; the
DEHP bloassay was conducted using state-of-the-art procedures for animal
cardnogenlclty testing, and that the results of the bloassay were approved
by Independent peer review panels. Kluwe et al. (1983) also noted that the
metabolic difference between rodents and humans, cited by Northrup et al.
(1982), would not be expected to affect the response to the hepatocarclno-
genlc effects of DEHP observed In rodents. The authors noted that the
International Agency for Research on Cancer (1ARC, 1982) working group
reviewed the study and concluded that there was "sufficient evidence for
cardnogenlclty of DEHP 1n mice and rats". There Is some evidence
suggesting that peroxlsome proliferation, which occurs In both mice and rats
at the dose levels used 1n the NTP bloassay. Is Involved 1n a secondary
mechanism of cancer Induction (Reddy et al., 1986). Peroxlsomal prolifera-
tion Is discussed In detail 1n Chapter VII, Mechanisms of Toxlclty.
Similar results were reported In cynomolgus monkeys (Short et al.,
1937). In this study no treatment-related evidence of hepatic peroxlsomal
proliferation was found In monkeys exposed to levels <500 mg/kg/day DEHP.
Exposure to similar levels (100, 1000, 6000, 12000 and 25000 ppm) of DEHP In
rats produced hepatic peroxlsomal proliferation. It Is difficult to compare
exposure levels since monkeys were administered bolus doses and rats were
administered feed. For further detail see the metabolism section of
Chapter III.
04750
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Ward et al. (1983) studied the patterns of promotion of hepatocellular
neoplasla by OEHP and phenobarbHol (PB) following Initiation by 1.p.
dlethylnltrosamlne (OEN). B6C3F1 mice were given a single l.p. Injection of
80 mg/kg of DEN at 4 weeks of age followed by oral administration of PB or
DEHP beginning 2 weeks after DEN Injection and continuing for <6 months.
DEHP was administered In the diet at concentrations of 3000, 6000 or 12.000
ppm, and PB was given In drinking water at 500 ppm. Few fod of hyperplasla
were fourd In the liver at 2, 4 or 6 months In animals exposed only to DEN,
PB or DEHP, while numerous fod and hepatocellular neoplasms were found 1n
/
mice tree ted with DEHP or PB after Initiation with DEN. The pattern of
response of DEHP differed from that of PB. In DEHP-exposed mice, the
numbers cf foci did not Increase between 4 and 6 months as they did for PB,
but the fod did Increase In mean diameter and volume as the study pro-
gressed. Fod and tumors appeared earlier 1n the higher dose group of DEHP
and, alUough the number of fod per unit volume of liver was similar for
all DEHP dose groups, the volumes of the foci were dose-related. The type
of hepatccytes found In the foci and neoplasms differed for PB and DFHP;
those fot PB were predominantly eoslnophlllc hepatocytes while those In
DEHP-trealed mice were predominantly basophlllc and were more malignant In
appearanc;. After 6 months exposure, the neoplasms 1n the high-dose DEHP
and DEN n.lce were significantly larger (p<0.02) than those for PB and DEN,
although hlstochemlstry revealed similarities In the lesions. DEHP did not
exhibit liltlatlng action when given once orally followed by PB for 6 months
In drinking water.
Ward et al. (1986) found that DEHP did not cause tumor promotion In
female F3U/NCr rat livers. Rats were Initially Injected with 282 mg/kg DEN
04750 V-94 07/03/91
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ami thtm fed diets containing 12,000 ppm DEHP or placed on drinking water
containing 500 ppm PB. Animals were sacrificed after 14 days of exposure.
DEHP failed to Increase the number or size of focal hepatocellular prollf-
eratlve lesions (FHPL). The FHPL were morphologically similar between DEN
and DEN-DEHP treated rats. Based on the above studies (Ward et al., 1983,
1986) the Investigators suggest that liver cell replication 1s not a
requirement for tumor promotion and that the hepatomegaly Induced by DEHP
appears to be a consequence of Increased size of parenchymal cells.
Garvey et al. (1987) found that a single oral dose of 10 g/kg or 12
weeks of feeding 1.2% DEHP did not serve to Initiate carclnogenesls In
female F344 rats. Promoting agents were 2-acetylamlnofluorene, for the
single dose, and carbon tetrachloMde and P8 for the 12-week study. In
addition Williams et al. (1987) demonstrated that DEHP had no Initiating or
enhancing effect on male rat carclnogenesls when DEHP was given alone for 24
weeks or for 7 weeks followed by the PB. The absence of enhanced
development of foci In DEHP-treated rats Is also Indicative of a lack of
prDmotlng activity (Williams et al.. 1987).
B6P. BBP was fed for ~2 years to both male and female rats and male
and female mice at concentrations of 0, 6000 and 12,000 ppm (0, 780 and 1S60
mg/kg/day, respectively) (Kluwe et al.. 1982b; NTP, 1982b). Body weight
gains were decreased In male and female mice and In female rats Ingesting
BBP; however, survival among these groups was not affected. Excessive
mortality occurred among male rats treated with 6000 and 12.000 ppm BBP due
to apparent Internal hemorrhaglng, after -3 months of exposure. Due to the
high mortality the study of male rats was terminated early, precluding
04750
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evaluatlcn of the animals for tumorlgenlc responses. Incidences of tumors
at specific anatomical sites In BBP-treated male or female mice did not
differ s gnlflcantly from controls. However, the Incidence of mononuclear
cell leukemia was greater among the high-dose female rats than among
controls. The Incidences of leukemia In female rats are presented In Table
V-13. Although the Increase In leukemia was statistically significant, the
biologic relevance of this finding was questioned due to considerable
variation 1n the background Incidence of mononuclear cell leukemia In Fisher
344 rats. The conclusions reached by the peer review group of this study
Indicated that BBP "was probably carcinogenic 1n F344 female rats". A
summary cf the Interpretive conclusions drawn from the NTP carclnogenesls
testing o: 68P Is shown 1n Table V-14. BBP was not carcinogenic in mice of
either sex. In reports of a 26-week subchronlc study, NTP (1985, 1986)
revealed :Ignlflcantly reduced total bone marrow cell counts at the 0.03 and
2.5% dose groups, but not at the 0.09, 0.28 or 0.83X dose groups when
compared vlth controls. This change was comprised primarily of decreases In
neutrophll metamyelocytes, bands, segmenters, lymphocytes, and basophlllc
rubrlcytes.
The N" P 1s currently repeating the rat portion of the cacner bloassay
for BBP. Testing began 1n Oune, 1989 (NTP, 1991). When Information from
this study becomes available, the welght-of-evldence for the carclnogenlclty
of BBP will be re-evaluated.
Using the results of the NTP cardnogenlsls bloassays, Kluwe (1986)
compared tie carcinogenic effects of OEHP and BBP and related compounds to
determine the structure-activity relationships. Among the PAEs shown to be
04750 V-96 08/08/91
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TABLE V-13
Incidences of Female Rats with Tumors of the HefKatopo1et1c System
1n the NTP Cardnogen1c1ty Bloasssay of BBPa>D
Hematopoletlc System Tumor
Myelomonocytlc leukemia
Lymphoma
Hyelomonocytlc leukemia or lymphoma
Control
7/49
0/49
7/49
Incidence
Low-Dose
7/49
0/49
7/49
High-Dose
18/50C
1/50
19/50C
aSource: Kluwe at al.. 1982b; NTP, 1982b
^Female Fischer 344 rats were fed diets containing 0 (control), 6000 (low-
dose), 12,000 ppm (high-dose) of BBP for -2 years. The ratios of female
rats bearing tumors of the hematopoletlc system to the total number of
female rats examined microscopically are depicted.
cS1gn1fIcantly greater than controls, p<0.05
04750
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TABLE V-14
Summary of the Carcinogenic Effects of BBP In the NTP Bloassays
and Interpretation of These Findings3
Test
Chemical
Species
Sex
Neoplasms
Interpretat1onb
BBP
rats
rats
mice
M
F
M&F
Leukemia
Inadequate study
Some evidence
No evidence
aSource: luff and Kluwe, 1984
^Evidence of Carclnogenlclty-- Five categories of Interpretative conclu-
sions have been adopted for use 1n the NTP Technical Reports series to
spedflcjlly emphasize consistency and the concept of actual evidence of
carclnogjnlcHy. For each definitive study result (male rats, female rats.
male mice, female mice) one category Is selected to describe the findings.
This category refers to the strength of the experimental evidence and not
to elthe- potency or mechanism (Huff and Kluwe, 1984).
04750
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potentially carcinogenic, the target sites of carcinogenic action varied.
For example, DEHP Induced hepatocellular carcinoma, while 8BP was associated
with effects of the hematopoletlc system. It was concluded, therefore, that
the carclnogenlclty of PAEs may not be due to the acH1v1ty of the phthalate
moiety but rather determined by the moiety attached to the phthalate, or to
a metabolic byproduct. Support for such an argument 1s given by studies of
compounds containing the 2-ethylhexyl moiety. Comparison of results
obtained for DEHP, DEHA and two other compounds [dt(2-ethylhexylJphosphate
and 2-ethylhexylsulfate] containing the 2-ethylhexyl moiety revealed that
all four compounds possessed some hepatocarclnogenlc activity In female
mice. The related compounds will not be discussed In this document. Thus,
these results may Indicate that compounds containing the 2-ethylhexyl group
may have a propensity for causing hepatocardnogenlclty 1n female mice.
DSP. Data regarding the carclnogenUHy of OBP could not be located
1n the available literature.
DE_P. Data regarding the carclnogenlclty of DEP could not be located
In the available literature.
PHP. Data regarding the carclnogenlclty of DMP could not be located
In the available literature.
Summary
The acute toxUHy of PAEs tends to be Inversely related to the molecu-
lar weight of the compound. Signs of long-term toxlclty Include decreased
body weight gain and Increased liver, and In some cases, kidney weights.
Target organs of PAEs, particularly DEHP, are the testes, liver and kidney.
04750
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The fepatotoxH effects of PAEs have been studied by numerous Investi-
gators 1n a variety of species. Seth (1982) reviewed the hepatic effects of
PAEs anc described both the morphologic and biochemical alterations
attrlbutatjle to PAE exposure. Host Investigators have used DEHP as the
representative PAE 1n testing. Generally, enlargement of the Hver has been
observed following oral or l.p. administration of PAEs. Examination of
tissue fi om enlarged mouse, rat, hamster and monkey livers has revealed
changes 1n morphology and biochemical constituents. Oral administration of
OEHP for 21 days was reported to cause dilation of smooth and rough endo-
plasmlc retlculum, mltochondrlal swelling and Increase.1n mlcrobodles In rat
Hver (La
-------
TesUcular Injury Induced by PAEs appears to be species specific to some
extent. The rat, mouse, guinea pig and ferret were susceptible to testlcu-
lar Injury from OEHP and DSP while the hamster appeared to be resistant to
the gonadal effects of these compounds and the corresponding monoesters at
the dose levels and durat'ons tested (GangolU, 1982}.
Studies on the embryotoxUHy of PAEs seem consistent with other data
obtained using different toxlcologlc endpolnts {Tyl, 1988; NTP 1984a,b,
1985; Mitchell et a!., 1985; Dostal et al., 1987a), that 1s, there 1s a
range of toxlcltles that varies as a function of the PAE being tested and,
1n -general, high concentrations of this chemical are required to produce a
teratogenlc response. Most studies used the mouse or the rat as the test
subject and 1n those situations wherein a teratogenlc response occurred, the
target was generally the skeletal system (Shlota and N1sh1mura, 1982; Tomlta
et al., 1982a; Singh et al., 1972). Based on the high doses used and the
differences 1n PAE metabolism between man and these test species, It 1s
difficult at this time to define clearly the risk for the human population.
PAEs are generally regarded as nonmutagenlc although mutagenlc responses
have been shown for some PAEs 1n some tests. DEHP 1s apparently metabolized
to a nongenotoxlc form 1n Intact animals but not by tissue preparations.
DEHP and BBP have been tested for cardnogenlclty In 2-year NCI/NTP car-
c1f>ogen1c1ty bloassays. DEHP was found to Induce hepatocellular carcinomas
1n both rats and mice. Increased mononuclear cell leukemia was observed in
female rats exposed to BBP. Data regarding the carclnogenlclty of DBP. DEP
and DMP could not be located 1n the available literature.
04750
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VI. HEALTH EFFECTS IN HUMANS
Introduction
Althojgh PAEs are considered to have a low order of toxUHy, much con-
cern has been generated by the discovery that PAEs, such as DEHP, may leach
from the plastic tubing and plastic bags used for blood storage (Jaeger and
Rubin, 1970, 1972, 1973; Peck et al., 1979). Studies on the effects of PAEs
In humans have largely provided Information about the pharmacoklnetlcs of
the compounds. Associations between exposure to PAEs and toxic effects In
humans han-e been limited by the Inability to discern doses and responses In
light of the ubiquity of phthalates In the environment. DEHP has been
detected in both transfused and nontransfused patients (Wallln et al., 1974;
Rubin and Nalr, 1973; Jaeger and Rubin, 1972). In addition children may be
exposed to DEHP In products such as pacifiers, teethers. squeeze toys,
plastic b.iby pants and vinyl fabrics covering playpen pads. A report by the
Consumer deducts Safety Commission estimated possible Increased cancer risk
to children exposed to the above products (CPSC, 1983). The widespread
presence of PAEs 1n air, water, food and stored blood Indicates that humans
are subject to environmental and Industrial exposures to PAEs.
Clinical
-------
Shaffer et al. (1945) also examined the effects of DEHP after dermal
exposures to the plastldzer. Undiluted DEHP was applied to the backs of 23
human subjects as patch tests. The compound was left 1n contact for 7 days
and then reappHed on the same spots after 10 days. These exposures did not
result In any type of erythema or other effects, suggesting the Irritating
and sensitizing potentials of DEHP are minimal.
Jacobson et al. (1974) examined the effects of DEHP on tissue cultures
of ligman dlplold flbroblasts established from skin biopsies. DEHP was
solublllzed 1n sera collected and stored 1n polyvlnyl chloride {PVC} blood
packs under standard blood bank conditions (4°C). Tissue culture medium
containing 15% of the plastic stored serum was used for Incubation of
cells. The degree of growth Inhibition of the human dlplold flbroblasts
Increased with DEHP concentration. At Incubation concentrations of 0.10 mM
and 0.18 mM OEHP, cell growth was Inhibited by 20% and 50%. respectively.
These DEHP levels were comparable with concentrations detected 1n whole
blood stored 1n PVC blood packs at 4°C for 14 and 26 days, respectively. A
70% Inhibition of cell growth was observed when 0.70 mM DEHP was used, which
was the concentration detected 1n platelet concentrations stored at 22°C for
48 hours.
Chromosomal effects of DEHP (Stenchever et al., 1976) were Investigated
on human leukocytes from the blood of two male and two female healthy donors
1n their early twenties and on fetal lung cells established from a 16-week
fetus delivered by hysterotomy. DEHP was solublllzed In Polysorbate 80
(Tween) (1:3, vol:vol) and dispersed 1n fetal calf serum by sonlcatlon.
AHquots were diluted to final concentrations of 0.06, 0.6, 6.0 and 60.0
jig OEHP/mt of blood for the leukocyte Incubations. Incubations with
04760
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07/28/88
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DEHP were for 4 hours at 37°C. Phytohemaglutlnln was then added for 30
minutes t<> Initiate cell division and cells were cultured for 72 hours.
Mitosis was Inhibited by addition of democoldne 2 hours prior to harvest-
Ing. Metaphase spreads were scored blindly for chromosome abnormalities on
Glemsa-stalned slides prepared from these cultures. Pooled data from the
four donors showed no statistical differences In chromosome breaks, gaps or
abnormal forms at any of the Incubation concentrations when compared with
control ciltures. Fetal lung cells were Incubated with 6.0 vq DEHP (In
Polysorbat? 80)/mi medium for 5 days. No significant difference 1n
aneuploldy between study and control cultures was seen.
Ishlka^a et al. (1983) determined that platelet function decreased as
DEHP concentrations Increased In PVC blood storage bags. Platelets demon-
strated a decrease In ADP-lnduced aggregation after at least 2 hours of
exposure 1o 100, 300 or 500 yg DEHP/ma. Maximum aggregation gradually
decreased with Incubation time, depending on the DEHP dose. Platelets
renewed wl :h fresh plasma showed a restoration of aggregablHty. The degree
of restora:1on was decreased with Increasing DEHP dose.
The ef:ects of DEHP on cultures of the human dlplold cell strain, WI-38,
were Investigated by Jones et al. (1975). Cultures treated with 51, 69 and
160 yM DEHP (soluble concentrations In Incubation media) showed a sta-
tistically significant decrease In cell protein and longer generation times
when compired with control cultures. As Indicated by figures these
decreases were dose dependent. Cells treated with 160 uM DEHP were no
longer viable at day 9 and exhibited decreased cell density on day 6 of
treatment. The dose, which caused 50% growth Inhibition (IDrg), was cal-
culated to be 70 iiM. These toxic effects were greater In replicating cell
04760
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08/15/88
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populations than In those treated after reaching confluency. Cells grown 1n
160 yM for 3 days and subsequently subcultured Into control medium showed
only 60% of control growth after 5 days 1n control medium.
BBP. Malette and von Haam (1952) Investigated the dermal effects of
various phthalates. A 100% solution of BBP had moderately Irritating and
slightly sensitizing effects when applied to white rabbits (methodology not
specified). Patch tests were performed on 15-30 human subjects and sensltl-
zatlon tests 2 weeks after primary Irritation tests. A 10% solution
(vehicle not defined), of BBP was applied to the human subjects. A light
reaction (not described) was seen In 12% of those tested. The Irritative
effect was classified as moderate and no sensitizing effect was reported.
DBF. Atmospheric exposures to DBP were studied by Hen'shlkova
(1971). A human olfactory threshold was found to range from 0.26-1.47
mg/m3. Abnormal encephalographlc responses were noted In three subjects
at atmospheric DBP levels of 0.12 and 0.15 mg/m3. At 0.093 mg DBP/m3,
conditioned reflexes were not observed. A maximum atmospheric concentration
of 0.1 mg DBP/m3 was recommended.
A single case of accidental 1ngest1on of DBP by a 23-year-old adult male
has been reported (Lefaux, 1968). The Individual mistakenly Ingested a
spoonful (-10 g) of DBP Instead of a laxative. The Individual was hospita-
lized the next day with complaints of nausea and vertigo. The subject
exhibited signs of keratltls and toxic nephritis (excess albumen and red and
04760
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-------
white corpuscles 1n the urine). Unspecified treatment Initiated Immediately
allowed Ue subject to leave the hospital after 2 weeks without any after-
effects.
Epldemlolcqlc Studies
MUkov et al. (1973) performed a cross-sectional Investigation of
workers e;:posed to phthalate plastldzers In the manufacture of artificial
leather and PVC-based films. The phthalates In use Included predominantly
DBP and higher alkyl phthalates (DAP-789), but periodically DEHP and BBP.
Some formulations contained small amounts of the sebacates [dlbutyl sebacate
(DBS) and dloctyl sebacate (DOS)] or adlpates [dlbutyl adlpate (DBA) and
dlocytl aclpate (DOA)]. TMcresyl phosphate (TCP) was a component of the
Incombustible materials produced 1n 10-20% of machines assigned to various
workers. The presence of these other agents without any attempt to account
for confounding Is a major criticism of this study.
The study population consisted of 147 persons, 87 women and 60 men. The
majority |75%) of the population was <40 years of age (mean and range not
given). Exposure duration was divided Into three categories: 0.5-5.0 years
for 54, 6-10 years for 28 and 10-19 years for 65 workers, respectively (mean
and range not specified). Job categories Included: 60 primers, 28 calender
and mill )perators, 35 mixing apparatus and paint millers, and 24 winders
and final product Inspectors. A control population was not Identified.
Ambient exposure levels to vapors or aerosols of the plastlclzers (mixed
esters) 11 the working zone of the primers ranged from 10-66 mg/m3.
04760 VI-5 08/15/88
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Similar results were reported for the work station of the mm and calender
operators. The plastldzer level in the mixture preparation section was
found to be 1.7-40 mg/m3. Other contaminants (vinyl chloride, carbon
monoxide and hydrochloric acid) around the calenders and rollers were either
below their maximum allowable concentrations or not detected.
"he test procedures Included algeslmetry, olfactometry, audlometry,
vibration sensitivity and vestlbular function by the caloric method with
cold water (60 ml at 19°C for 20 seconds). Clinical and biochemical blood
studies {sedimentation -rate and blllrubln level) were also performed.
The most frequently cited complaint was of pain In the upper and lower
extremities accompanied by numbness and spasms, reported 1n 51.7% of the
subjects with a length of service 6-10 years and In 81.6% In those with >10
years, PolyneurlUs was found In 47 persons, 32 with an autonomlc-sensory
and 15 with a mixed form. The Incidence of polyneurltls Increased with
length of service. In 3.4% of the cases, organic disease of a nonoccupa-
tlonal character was noted In the nervous system. An elevation 1n the
threshold for sensitivity to pain was noted In 66.7% of the subjects, and
sensitivity to vibration was lowered to some extent 1n 33.8%. A marked
depression In vibration sensitivity was seen only In those subjects also
manifesting a significant depression of pain sensitivity. Of 81 subjects
undergoing vestlbular receptor Investigations, 78% were found to have a
depression of the vestlbulosomatlc reactions (absence or lowering of exdt-
abl'Hy). This depression began with the first years of this occupational
contact, often In the absence of any health status complaints. The majority
of subjects showed an elevated threshold of excitability when tested by
047 (JO
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olfactometry, especially for thymol (82.1%) but also for camphor and tar
(50%) and less for rosemary (33.4%). This elevation Increased with duration
of servlct . Audlometry did not reveal any pathology 1n auditory sensitiv-
ity. Blocd studies revealed a tendency to slight lowering of the number of
platelets and leukocytes, hemoglobin level and blood color Index. A slight
retlculocytosls and a tendency to acceleration of the erythrocyte sedimenta-
tion rate among the female subjects (statistically significant, but p value
not stated) was also noted.
t
Thless et al. (1978a) performed a morbidity study on 101 workers (97
males, 4 females) employed 1n a DEHP production plant. The age range of the
workers w.is from 22-60 years (no mean was given but the majority were
between 35 and 55 years of age). Duration of exposure was between 4 months
and 35 years, with an average of 12 years. In 1966 the plant changed from a
batch process to a continuous process production so that exposure to workers
was reduced to only the processes of removing samples In control passages
and during decantatlon. The Investigators concluded that the negative
results reported may be attributed to lower exposures after the processing
change In 1966. Samples of current exposure concentrations In the work
areas In question ranged between Q.0006 and 0.01 ppm (detection limit not
stated). A clinical and occupational history was taken and the clinical
examlnatloi Included vital statistics, EKG, lung X-ray, and a complete
urinary status with uric add and creatlnlne clearance. The blood analyses
Included < differential count and sedimentation rate, and thymol, total
protein, >GOT, SGPT,
-------
All the results of the examinations were compared with those obtained
from two In-house control groups with possible exposure to styrene and
dlmethylcarbamlc acid chloride (DMCC). No significant differences between
the study group and control groups were found. Even when the study group
was divided by age and time of exposure (greater and less than 12 years to
presumably account for the change 1n production process), no significant
differences were seen. Workers with duration of exposure >20 years (n*6),
with an average exposure of 26.3 years, were given a neurologic examination
that Included: tests of the cerebral nerves; reflexes of the arms, legs and
abdominal skin; and sensitivity to depth, pain and vibration. No neurologic
disease or toxic nerve damage was Indicated. Analyses of absenteeism,
accident rate and of a questionnaire regarding premature births,
miscarriages and malformations were also negative. Although the study was
comprehensive In scope, 1t lacked exposure data prior to the conversion to a
continuous process production so that a definitive conclusion can not be
ascertained. As such, however. It represents only one of two epidemlologic
studies reported to date on subjects with DEHP or a specific phthalate
exposure.
Thless et al. (1978b) also reported a mortality study on these DEHP pro-
duction workers. The study was a prospective cohort survey of 221 workers
con-pared with the general population. The study considered data prior to
1916. The population was derived from 28 workers who had worked prior to
1955, 85 workers who had started between 1940-1965, 135 workers who started
after 1965 and 109 workers employed at the time of the study. Selection
criteria for Inclusion In the study population were not provided. The
average observation period was 11.5 years. Half of the expected deaths were
observed 1n the exposed population. Eight cases of death were due to
04760
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cancer. Tiless et al. (1978b) reported one case of bladder papllloma, which
was significantly different from that expected. However, this was
attributed to a single case and was not considered to represent an Increased
health risk. Analysis of natural death cases, after minimal observation of
5-10 years, on workers exposed to durations of 5, 10, 15 or >15 years, did
not reveal an Increase 1n mortality with exposure duration.
Thless and Flelg (1979) also performed chromosomal analysis on blood
lymphocyte* from a subset of this same study population. Lymphocytes were
cultured from 10 exposed production workers according -to a modified method
of Hoorhead et al. (1960). The workers' duration of exposure ranged from
10-34 years (mean = 22.1 years). Lymphocytes from 20 age-matched workers
served as controls. It was not mentioned whether these controls were also
exposed to styrene and DMCC as mentioned previously. One hundred metaphases
were scored for abnormalities on lymphocytes from each worker. The specific
structural abnormalities were not defined, but were categorized with and
without gaps. Neither category appeared to be different from the controls
although statistical analyses were not stated.
In addition to Individuals who are occupatlonally exposed, research
Indicates that persons who undergo blood transfusions or hemodlalysls may
receive extensive amounts of PAEs (over background Intakes) as a result of
the leaching of compounds, such as I1p1d soluble OEHP, from plastic
containers or catheter tubing (Marcel and Noel, 1970; Jaeger and Rubin,
1970. 1972, 1973).
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Hlllman et al. (1975) studied DEHP levels 1n neonatal heart and GI tis-
sues. The study tissues were obtained from three Infants who previously had
umbT.lcal catheters In place but had never received blood products and from
14 Infants who previously had umbilical catheters and varying quantities of
blood products. Control tissues were obtained from eight stillborn Infants,
two Hveborn Infants who had died without administration of any blood prod-
ucts or Insertion of catheters, and three older subjects who had not
received blood products. The maximal amount of DEHP that could have leached
Into the blood was determined to be 13.9+fl.l mg of DEHP/5 cm of catheter,
based upon the mean i'S.E. of the extraction of four'No. 5 French cath-
eters. The maximal amount contributed by blood products was estimated at 4
vg/mi, based upon a reference to Marcel (1973). The potential dosage
thus ranged from 0.04 mg In Infants receiving only 10 ml of blood to 1.4
mg in those receiving double exchange (460 ml). The minimum detection
limit for DEHP was -0.02 yg/g of tissue under the conditions used. No
correlation could be made between hours of catheterlzatlon and DEHP levels.
In general, the OEHP levels In heart tissue reflected the combined dosage of
the numbers of catheters and amount of blood products 1n Infants who died In
<24 hours. In Infants who lived longer, levels were generally lower and
less correlated with dosage, suggesting that some blotransformatlon and
clearance of DEHP was taking place. The mean levels of DEHP for heart
residue and pressed extract of the study tissues, 1.27*0.42 and 0.66+0.22
ng/g, respectively, were significantly higher than the corresponding
control levels, <0.07+.0.03 and <0.07*0.04 yg/g. Three Infants who died of
necrotlzlng enterocolHIs and who previously had arterial umbilical
catheters In place and removed, had gut residue levels of 0.47, 0.63 and
0.16 yg/g. These levels were significantly higher (p<0.05) than those 1n
GI tissues from Infants without this disease. These higher DEHP levels
04760
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may represent Increased uptake or decreased metabolism by a dying bowel. A
direct causative link could not be determined between exposure to DEHP and
the deve'opment of necrotlzlng enterocoHUs. However, the study demon-
strated that DEHP accumulated 1n the tissues of critically 111 Infants.
Component; of the catheters, Including DEHP, should be further Investigated
as poten .lal vascular or GI toxins, according to the authors of this
Investlga ;1on.
Another group that could be at high risk for the development of toxic
responses to exposure' to PAEs may be Individuals who undergo hemodlalysls.
Gibson et al. (1976) studied blood samples from nine patients requiring
maintenance hemodlalysls both before, during and after the hemodlalysls
process tj quantify the levels of DEHP received by these Individuals from
blood transfusion bags and/or plastic hemodlalysls tubing. Hemodlalysls was
performed using reclrculatlng single-pass machines and colls. Samples for
DEHP analyses were obtained at 15 and 30 minutes, 1, 2, 3, 4 and 5 hours,
and 1mmed ately after dialysis unless dialysis was terminated earlier. The
metabolic fate and toxldty of these DEHP levels were not determined. Esti-
mates of the total amount of DEHP delivered to a patient during hemodlaly-
sls rangeil from 1.5-150 mg for dlalyses that lasted from 15 minutes to 5
hours.
Neergaard et al. (1971) reported that exposures to DEHP may have been
assodatec with the development of abnormal liver function tests 1n three
patients (two men, aged 25 and 40 years, and one 25-year-old woman), follow-
ing the u: e of a new set of PVC blood tubings In hemodlalysls. IR-analyses
of DEHP In salt solution perfusates through this blood tubing set ranged
from 10-2) mg/l. UV-determ1nat1ons using the perfusate directly gave a
04760 VI-11 08/15/88
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somewhat higher range, 20-50 mg/l. DEHP could not be washed out by
perfuslon of three other commercially available blood tubing. Over a 5
month period dialysis machines with this tubing were used 93 times. Of
these dlalyses, 75 were performed upon the three patients who developed
symptoms. Symptoms presented by the three patients after 10-15 dlalyses
(estimated dose of DEHP not given) Included malaise, fever, abdominal pains,
nausea, abnormal serum enzyme levels (LDH and SGOT), Increased serum
blllrubln, and 1n one case, jaundice. Liver biopsies In one patient
revealed changes 1n accordance with so-called nonspecific reactive
hepatitis; and 1n another patient, a hlstologlc picture compatible with a
diagnosis of viral hepatitis. Upon removal from the new dialysis machines
to dialysis systems 1n which OEHP was not detected, the conditions of the
three patients Improved. A patient who was returned to the new dialysis
machine developed a more severe relapse of the symptomology until she was
removed to a different dialysis system. Evidence as to the exact etiology
of Illness associated with the use of the new dialysis machines could not be
determined.
In a recent study (Pollack et al., 1985b), circulating concentrations of
OtHP and Its desterlfled phthallc acid products, mono{0-ethylhexyl)
phthalate (HEHP) and phthallc acid, were quantltated (HPLC/UV monitor) 1n 11
patients. These patients were undergoing maintenance hemodlalysis for
treatment of renal failure. The patients underwent hemodlalysis 3 times/
week, 4 hours/session, and had been receiving treatment ranging from 1 week
to 12 years. The mean estimate of DEHP extracted during a single dialysis
session for the 11 patients was 105 mg (range 23.8-360 mg). Serum choles-
terol, trlglycerldes and AAG concentrations were measured to determine their
04760
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Influence on the extraction of DEHP Into blood. Circulating levels of DEHP
and MEHP (1.9U2.11 w/ml and 1.33^0.58 vg/mi, respectively) during
dialysis I1d not correlate wHh the length of time the patients had been
undergoing dialysis. This, together wHh the observation that blood concen-
trations (if OEHP during 1nterd1alys1s were similar to those 1n nondlalyzed
patients Indicate that these compounds are effectively removed from the cir-
culation between dialysis sessions. This could represent metabolic trans-
formation or sequestration of UpophHU phthalate esters Into fatty tis-
sues. Thi'te was a strong correlation, however, between phthallc acid con-
/
centratlons (5.22*3.94 vq/ml) and the length In years of previous dialy-
sis treatments (rs+0.920, p<0.001). There also was no apparent relationship
between the concentrations of OEHP and either of Us metabolites, Indicating
the need for future metabolic fate and pharmacoklnetlc Investigations. Of
the biochemical factors examined, the sum of the serum cholesterol and trl-
glycerlde concentrations correlated most closely wHh the Teachability of
OEHP, altliough the association was weak (r=+0.565, p~0.1). Thus, although
hemodlalysls patients are exposed to circulating DEHP, the consequence of
long-term systemic exposures to the ester and Us metabolites remains to be
elucidated.
High Risk Subpopulatlons
Although toxic effects of PAE exposure have not been conclusively demon-
strated, 'ndlvlduals who receive exposures above background or environmental
levels, si.ch as those requiring hemodlalysls or blood transfusions, may be
at higher risk for the development of adverse reactions to these compounds.
Parenteral administration of PAEs to these Individuals may prove to be more
toxic because these patients are critically 111 or subject to differences In
their ability to absorb, metabolize and excrete the compounds.
04760
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Summary
DIspHe widespread occurrence of PAEs, Information concerning the
effects of human exposure 1s limited. In one study of acute exposure,
administration of 5 or 10 g DEHP to two adult males did not result In toxic
effects other than mild GI disturbances. Accidental Ingestlon of 10 g DBF,
however, proved more toxic and caused nausea, vertigo, keratltls and toxic
nephritis. Dermal application of BBP on humans did result In Irritative but
not sensitizing effects; however, the application of DEHP did not result In
either effect. Studies on human tissue and cell cultures have demonstrated
Inhibition of cellular growth and decreases In platelet function. However,
chromosomal effects did not occur 1n human leukocytes and fetal lung cells.
In epldemlologlc studies the results have been largely confounded by
exposure to multiple chemicals and lack of quantitative Information on
levels and duration of exposure. One group of Investigators conducted a
morbidity study on 101 workers employed In a DEHP production plant.
Clinical examination and blood analyses revealed no significant differences
between the study group and control groups. No neurologic disease or toxic
ne've damage was Indicated. Although the study was comprehensive 1n scope
1t lacked exposure data prior to a process conversion In the plant. In a
cross-sectional Investigation of 147 persons exposed to a combination of
phthalate plastldzers, the Incidence of polyneurltls Increased with length
of service. HematologU studies revealed a lowering of the number of
platelets and leukocytes, hemoglobin level and blood color Index. Exposure
to multiple agents make It difficult to Interpret these results. Finally In
a prospective cohort study of 221 workers exposed to-DEHP, half of the
expected deaths were observed In the exposed population. However, selection
criteria for Inclusion In the study population were not provided.
04760
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Parenteral administration of PAEs may Involve the greatest risk for
toxic effjcts, especially 1n Individuals requiring blood transfusions or
hemodlalysls. Despite the fact that PAEs may leach Into the contents of
plastic b ood bags or plastic tubes, reports of hepatitis In hemodlalysls
patients
-------
VII. MECHANISMS OF TOXICITY
Introduction
The relationship between the toxlcoklnetks and toxic effects of PAEs
(and their metabolites) has not been fully elucidated because of the
relatively expedient clearance and minimal tissue accumulation of these
compounds. However, Investigation of the mechanisms of phthalate toxlclty
has been promoted In part by Interest In their carcinogenic potential and
their widespread use and environmental disposition.
Interactions
Concern over the ability of PAEs to alter biologic responses to
pharmacologlc agents and xenoblotlcs has stimulated research Into the pos-
sibility that the blotransformatlon of these chemicals may be modified by
the acid esters. DEHP and D8P have been found to Interact with the toxicHy
of other compounds 1n a synerglstlc or antagonistic manner. Carbon
tetrachlorlde was found to act synerglstlcally with DEHP by producing
extensive necrosis of parenchymal cells 1n rat liver (Seth et a!., 1979}.
DEHP significantly (p<0.05) Increased barbiturate-Induced sleeping time In
male mice {Rubin and Jaeger, 1973). A synerglstlc effect was noted when
DEHP or DBP (applied prior to the application of organophosphate
Insecticides) Increased the mortality of female house flies. When applied
simultaneously, DEHP or DBP reduced the toxUHy of organophosphate
Insecticides to house flies (Al-Badry and Knowles, 1980). Antagonism was
noted between the effects of OBP and zinc-Induced testlcular atrophy (Cater
et al., 1977). Methylenedloxyphenol compounds and paraoxon Inhibited DEHP
hydrolysis by rainbow trout liver In vitro (Melancon and Lech, 1979).
Foster et al. (1980) did not find antagonistic effects between testlcular
zinc levels and the two phthalates DEP and DMP. In rats Initiated with
04770
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dlethyln trosamlne, the administration of a chollne supplemented diet
containing DEHP did not result 1n Increased hepatic preneoplastlc foci. The
administration of a chollne deficient diet containing DEHP Inhibited the
appearance of preneoplastlc fod. Chollne deficiency has been shown to be a
promoter of hepatic preneoplastlc foci but the mechanism of DEHP's
ant1-pronot1ng effect Is unclear, according to the Investigators (Deangelo
and Garr?tt, 1983). There were no synerglstlc or antagonistic Interactions
found foi B8P 1n the available literature.
The interaction between ethanol and DEHP has been studied by Agarwal et
al. (198;'b). After a single oral or l.p. dose of DEHP to mice, the ethanol-
Induced sleeping time was Increased while hepatic alcohol dehydrogenase
activity was Inhibited. Repeated administration, however, produced effects
that differed with the route of administration. When DEHP was given In
repeated oral doses, the ethanol-lnduced sleeping time was decreased, but
Increase; In the activities of both alcohol and aldehyde dehydrogenases were
observed. Repeated l.p. doses, however, resulted 1n an Increase 1n the
sleeping time with decreased alcohol dehydrogenase activity. The authors
concludec that DEHP effected changes In the pharmacologlc response to
ethanol Dy altering the activities of alcohol dehydrogenase and aldehyde
dehydrogtnase. .In. vitro assays with mouse liver preparations revealed that
MEHP Inhibited alcohol dehydrogenase activity. Furthermore, both MEHP and
DEHP 1nh bUed to a statistically significant (p<0.05) degree the activities
of both Mgh and low Km aldehyde dehydrogenase activities.
Enzyme Irdudnq Properties
Most studies of the mechanisms of toxIcHy have researched the effects
of PAEs on enzyme systems and metabolites. Pollack and Shen (1984) used
04770 VII-2 07/02/91
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antlpyrlne metabolism as a model for metabolic clearance of drugs. Ant1-
pyrlne's metabolism was Increased In normal and renal failure rats (Sprague-
Dawley rats In which renal failure was Induced by a two-step nephrectomy)
after treatment with DEHP. The plasma clearance was Increased and elimina-
tion half-life of antlpyrlne decreased upon DEHP administration. An
Increase In the liver weight and cytochrome P-450 content was also noted as
evidence of Induction of hepatic mlcrosomal enzymes by DEHP. Renal failure
rats appeared to undergo a more marked Increase 1n antlpyrlne clearance than
did control animals after DEHP treatment.
Changes In hepatic enzyme activities are associated with liver enlarge-
ment and occur In animals exposed to PAEs (Seth, 1982). One change that has
been observed consistently following oral or 1.p. administration of DEHP Is
a decrease In hepatic sucdnate dehydrogenase (SDH) activity occurring
specifically 1n the perlportal zones {Seth, 1982).
One target site for PAE effects on the liver 1s mitochondria. Results
of \n_ vitro studies have Indicated that several PAEs produce Inhibition of
mitochondria! respiration. It has been suggested that PAEs are electron and
energy transport Inhibitors, and that they can cause uncoupling of oxldatlve
phosphorylatlon (Seth, 1982). Since DEHP Inhibited the activities of
succlnlc dehydrogenase (SDH) and adenoslne trlphosphatase (ATPase) 1n rat
heart, lung, kidney and gonads as well as the liver, suppression of
energy-linked reactions may be a generalized effect of DEHP. The enzymatic
alterations may not be related to the physical presence of DEHP since
effects were present several days after final treatment, by which time the
plastldzer would have been excreted from the body.
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DEHP ias also been shown to affect carbohydrate metabolism. Decreased
levels of glycogen were reported 1n the livers of mice, rats and ferrets
receiving DEHP {Seth, 1982). Marked depression of glucose and glycogen
levels was found 1n the livers of rats fed diets containing 2 or 4% DEHP
(Sakural ;t al., 1978). Glucogenesls and glycogenolysls are also Inhibited
by DEHP. However, no quantitative conclusion on the Inhibition of the
reaction .) upon hepatic enzymes, Upld peroxldatlon and hepatic sulfhydryl
content In rats. The authors concluded that the PAEs Interfered with b1o-
transformetlon mechanisms of hepatic mlcrosomal drug-metabolizing enzymes.
After a single oral or 1.p. treatment of DEHP was administered to rats, the
activity of am1nopyr1ne-N-demethylase and aniline hydroxylase was Inhibited.
When DEHP was given In repeated doses, the results showed Increases In these
enzymes with oral administration but decreases with l.p. Injection. The
activity of benzo[a]pyrene hydroxylase and concentrations of cytochrome
P-450 wens also Increased 1n rats that were treated orally with DEHP. The
differences 1n the effects from oral and l.p. administrations may be attrib-
utable to variations 1n the physical state and metabolism of DEHP after
Introduction of the compound Into the Intestine and the peritoneal cavity.
Studies by Seth et al. (1981) also Indicated that the activities of the
liver am1iopyrtne-N-demethylase and aniline hydroxylase vere Inhibited by
l.p. administration of DMP, DEHP and DBP to rats. Hepatic tyroslne amlno-
transferase activity was unchanged after a single administration but was
Increased when the PAEs were given dally for 7 days. The authors concluded
04770 VII-4 08/05/88
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that these results are supportive of previous observations that PAEs prolong
barbiturate sleeping time by Interference with the metabolic disposition of
these pharmacologlc agents.
Walseth et al. (1982} demonstrated contrasting results of PAE treatment
on rat liver and lung. DBP administered l.p. resulted 1n significant
Increases In hepatic cytochrome P-450 but reduced lung concentration by
30X. DMP and DEHP were less effective In this regard. DBP treatment also
altered the enzymatic pathways of benzo[a]pyrene (B[a]P) metabolism In "liver
mlcrosomes while all PAEs tested decreased pulmonary metabolism of B[a]P.
These authors did not detect a relationship between carbon chain length of
PAEs and effects of mlcrosomal enzyme activities.
While DEHP Is associated with Increases 1n the activity of hepatic
mlcrosomal enzymes, Khawaja and Oallner (1982) determined that liver protein
synthesis In vivo was decreased after administration of the compound In the
diet of rats. Although liver weight and protein content Increased, the
capacity for treated livers to synthesize protein was reduced. The accumu-
lation of protein was explained as a result of reduced degradation or
decreased export of liver proteins.
There were no available data concerning the enzyme Inducing properties
of EBP and OEP.
Cellular Effects
Ekwall et al. (1982) assayed 29 plastlclzers Including DMP, OEP, DBP,
DEHP, BBP and three other PAEs, for cytotoxldty of HeLa cells. Cyto-
toxlclty was measured by pH changes of the medium using phenol red as the
04770
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Indicator and by microscopic Inspection of the cultures (the HIT-24 system).
A comparison of the results of this Vn y 11ro cytotoxldty test to other
cytotoxlc ty tests demonstrated that as the chain lengths of PAEs Increase,
llpophlllc Hy Increases. A comparison of these ^n vitro cytotoxldty test
results with ^n vivo test results In mice suggest that a basal cytotoxlc
action to mouse tissues 1s responsible for the lethal action of plastldzers
to mice.
The c;'totox1c mechanisms of PAEs may be better elucidated by studies of
subcellulir distribution and activity as opposed to assays of tissue distri-
bution (Bsll, 1982). Bell (1982) discussed a series of experiments con-
ducted In rats, rabbits and pigs that were directed at the Investigation of
PAE effee :s on llpld metabolism. In studies of rats and rabbits that were
fed OEHP, the dlester Impeded cholesterol synthesis by Inhibition of
3-hydroxy-3-methylglutaryl CoA reductase, which catalyzes the second step of
cholestercl synthesis. The effect was neither sex nor species-specific.
Similar Inhibition of cholesterol synthesis was found to occur In the
adrenal glands and testes. Such Impairment of cholesterol synthesis In
these tls-. ues was thought possibly to account for fetal abnormalities found
In the of:spr1ng of phthalate-treated dams, and testlcular atrophy In other
animals. Plasma and liver cholesterol levels were decreased 1n rats fed
either OBf or DEHP. Inhibition of cholesterol synthesis by these esters may
have been the underlying cause for this effect. Experiments with jjn vitro
tissue slices of rats fed DEHP demonstrated that cte novo fatty add
synthesis and esterlf1cat1on are Inhibited In certain tissues after PAE
administration. Phosphollpld synthesis may also be selectively affected
(Bell, 19£2).
04770
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Upon further Investigation Bell and Buthala (1983) discovered that DEHP
Inhibits mlcrosomal acylCoArcholesterol acryltransferase (ACAT) In rats that
received this compound 1n the diet. The biosynthesis of cholesterol from
14C-mevalonate was also Inhibited 1n treated animals Indicating that other
mlcrosomal enzymes are Influenced by DEHP administration. The post-
mevalonate segment of the Dlosynthetlc pathway requires the Involvement of
numerous mlcrosomal enzymes, while cholesterol esterlfIcatlon Is largely
associated with ACAT.
Bell (1982) also 4escr1bed experiments performed orv the effects of DEHP
on mitochondria! function. Administration of DEHP to rats (50, 250 and 500
mg/kg/day In the diet assuming rats consume 5% of their body weight),
rabbits (490 mg/kg/day In diet assuming rabbits consume 4.9% of their body
weight) and pigs (1.6 mg/kg/day) resulted In Increased production of.
palmitic acid by liver mitochondria accompanied by an enhancement of
14C-palm1toyl CoA oxidation. Studies on heart mitochondria demonstrated
that DEHP directly added to an Isolated suspension will Inhibit aden'.ne
nucleotlde translocase. Thus, exchange of extramltochondrial ADP for
1ntram1tochondr1al ATP Is Impeded. Inhibition of translocase was not
observed In the mitochondria Isolated from rats fed DEHP compared with
controls. It was felt that a level of DEHP Insufficient to affect the
enzyme had accumulated 1n the heart during the 10-day feeding period. The
author concluded that Inhibition of heart adenlne nucleotlde translocase may
be related to reports of myocardlal cell death and decreases In spontaneous
heart rate observed 1n rat hearts after DEHP perfuslon (Rubin and Jaeger,
1973; DeHaan, 1971; Petersen et al., 1972-1975; Aronson et'al., 1978). Bell
(1S82) noted that the biochemical transformations observed 1n these
experiments Indicated that the effects of PAEs may result from
04770
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alterations of membrane fluidity. The UpophllU properties of the PAEs
may, therefore, change the membrane environment sufficiently to modify
enzyme responses.
Melnlck and Schiller (1982) studied the effects of DMP, OBP and DEHP on
liver mitochondria Isolated from rats. Active transport of potassium Ions
(K*), resjlratlon rates and succlnate cytochrome c reductase activities
were monitored. D8P was the most effective energy uncoupler as measured by
Interference with Kf uptake Induced by three energy sources. It also led
to a nearly total loss of respiratory control. DMP was less effective In
this regard; MEHP, but not the parent DEHP, was an effective uncoupler of
energy-11n
-------
TABLE VII-1
Cellular Changes In Rat Hepatocytes Induced by
DEHP Administration3
Organelle
Changeb
Peroxlsomes
Protein and phosphollpld
Beta-oxidation enzymes of fatty adds
Carn1t1ne-acetyl transferase
Catalase
Urate oxldase
Mitochondria
Protein and phosphollpld
B.eta-oxidation of fatty adds
Carn1t1ne-acetyl transferase
Carnltlne-octanoyl transferase
Carnltlne-palmltoyl transferase
Dehydrogenase and respiratory
Respiratory control and oxldatlve
phosphorylatlon
Mlcrosomes
Protein and phosphollpld
NADPH-cytochrome c reductase
Cytochrome P-450
Other electron transport enzymes,
hydroxylases, phosphatases
Homogenate
Sterol and squalene synthesis
CoA and carnltlne
Acetyl-CoA and acetyl-carnltlne
Long chaln-acyl CoA and -acyl
carnltlne
Increased several-fold
Increased 2- to 6-fold
Doubled
Decreased 30-40%
Decreased 30-40%
Increased 2- to 3-fold
Doubled
Increased 10- to 30-fold
Tripled
Increased 3- to 4-fold
No or moderate change
No change
Slight Increase (10%)
Increased 40-60%
Increased 40-60%
No change or moderate Increase
Decreased 75%
Increased 5- to 6-fold
Increased 4-fold
Increased 50%
aSource: Gannlng et al., 1984
^Specific activities or amounts on protein basis compared with the control
04770
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TABLE VII-2
Synthesis and Breakdown of Protein and L1p1d 1n DEHP-Treated Rats3
Protein
Peroxlscmes
Catalase
Beta-cxldatlon enzymes
Mltochordrla
Membrane
Beta-cx1datlon enzymes
Mlcrosonal membranes
Total cytoplasmlc
proteins
Amount or
Activity
Decrease
Increase
Increase
Increase
Unchanged
Unchanged
Synthesis
Decrease
Increase
Increase
Increase
Increase
Increase
Breakdown
Control -» Treated
Half-time 1n Days
1.9 -* 5.0
2-3 -» 5.5-6.5
6 -» 25
Decrease
3.5 -» 5.5
2.5 -» 5
Llpld
Hlcrosonral phosphollplds Unchanged
Blood cholesterol
Total Unchanged
HDLb Decrease
LOLC Increase
VLDLd Unchanged
Increase
aSource: Canning et al., 1984
bHDL, high-density Upoproteln
CLDL, low-density llpoproteln
dVLDLf very low-density Upoproteln
04770
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oxidation of CoA-linked fatty acids, In mlcrosomal NADPH-cytochrome c reduc-
tase and cytochrome P-450 levels, In the number of mitochondria and In the
activity of carnitlne-acetyl transferase. Induction of the transferase was
attributed to an Increase 1n peroxlsomal B-oxidat1on. Ganning et al. (1983)
demonstrated that although peroxlsomal and mitochondria! membranes were
Increased, the endoplasmlc retlculum was not changed In amount or appearance.
Increases In the activity of enzymes 1n rat hepatic cytosol have been
found upon application of various peroxlsome prollferators Including DEHP,
DAP and 2,4,5-triphenoxyacetic add among others (Katoh et al., 1984).
Administration of DEHP resulted in the induction of catalase and two long-
chain acyl-CoA hydrolases. An Increase In peroxlsomal 0-oxidatlon was also
signaled by a marked increase 1n palmHoyl-CoA oxidation after ingestlon of
DEHP in the diet.
Primary rat hepatocyte cultures were used to ascertain effects of
various alky! phthalate esters on peroxlsomal enzyme activities (Gray et
al., 1983). The authors concluded that straight-chain phthalates produce
few effects upon rat hepatic peroxisomes. The 2-ethylhexyl ester, e.g.,
MEHP, increased carnitine acetyltransferase activity and palmltoyl CoA
oxidation, and produced Increased numbers of peroxisomes.
The effects of different PAEs upon liver cells have been compared with
those of clofibrate, another peroxlsome proliferator (Lake et al., 1984b).
Lake et al. (1984a) had previously determined that DEHP Is a potent inducer
of rat hepatic peroxisomal enzyme activities. In the more recent study
(Lake et al., 1984b), rats were orally administered DEHP, di-n-octyl
phthalate (OOP), mono-n-octyl phthalate (MOP) or clofibrate for 14 days.
04770
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This resulted 1n liver enlargement. Liver sections from DEHP and cloMbrate
treated animals showed an Increased number of peroxlsomes. Both DEHP and
clofibrate stimulated the activities of peroxlsomal marker enzymes,
Increased mlcrosomal cytochrome P-45Q content and stimulated mlcrosomal
laurlc ac d hydroxylatlon activity. The compounds, OOP and MOP, did not
produce sich effects. The branched chain ester OEHP was thus determined to
exert effects that differed markedly from the straight chain analogue, OOP
and Us iretabollte MOP. In addition, OEHP was shown to Induce forms of
cytochrome P-450 similar to those Induced by clofibrate. Oklta and Chance
(1984) al;o demonstrated that DEHP, like clofibrate, Increased mlcrosomal
laurate hidroxylatlon activities. Potent Induction of the cytochrome P-450
mediated :atty acid u-hydroxylatlon reaction occurred In rats that were
fed a diet containing OEHP.
There may be some species variation In the biochemical actions of PAEs.
Lake et <,1. (1984a) compared DEHP, MEHP and cloflbrate-lnduced hepatic
peroxlsome proliferation 1n two species, rats and hamsters. It uas
discovered that DEHP was much less effective as a peroxlsome prollferator in
hamsters than In rats. Similar results occurred when clofibrate was
utilized. Although all three compounds caused some Increase 1n liver weight
and hepatic peroxlsome numbers, the response was more marked in rats, for
each of the three treatments, dose dependent Increases 1n the peroxlsomal
marker, cyanide-Insensitive palmHoyl-CoA, and In carnltlne acetyltransfer-
ase were loted In the rats. Only small changes In these parameters were
found 1n the hamsters. The species variation In the effects of DEHP may
have been attributable to differences in peroxlsome proliferation or in the
metabolism of OEHP.
04770 VII-12 09/07/88
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The mechanism of carcinogeniclty for DEHP Is not well understood; how-
ever, It has been suggested that DEHP may fall Into the perox1some~prol1fer-
ator class of hepatocarclnogens (Warren et al., 1982). Peroxlsomal prolife-
rating effects and hepatomegaly do not seem to be related to differences 1n
the sensitivity of suckling rats to toxic effects caused by exposure to DEHP
(Dostal et al., 1987a). Changes In relative liver weight and hepatic
peroxlsomal enzyme activities were similar In age groups showing markedly
different changes 1n body weight and survival rates. Similar Increases 1n
activities of both palmitoyl CoA oxldase and carnltlne acetyltransferase
were noted between suckling and adult rats Indicating that suckling rats are
equally If not more sensitive to the peroxlsomal proliferating effects of
DEHP {Postal et al., 1987a) (see Table V-4). The Induction of perloxlsomes
and peroxlsomal enzyme activity as well as hypolipldemlc effects was not
delected In marmoset monkeys exposed eHher orally (2000 mg/kg/day) or l.p.
(1000 mg/kg/day) to DEHP {Rhodes et al., 1986). Also, there was no Increase
In cyanide-Insensitive acyl oxldase, the peroxlsome marker enzyme. The
marmoset appears to be less sensitive to the peroxlsomal proliferating
effects of DEHP. Rhodes et al. (1986) concludes that If marmosets reflect
more accurately the response in man, then low levels of DEHP may not be of
to;<1colog1c significance with regard to hepatocellular carcinoma.
Possible mechanisms for the hepatocarclnogenlc effects of phthlates and
other peroxlsome prollferators have Included the generation of free radicals
from Increased hydrogen perloxlde (HO) production and decreased
catalase activity, and that peroxlsome Inducing chemicals and/or their
metabolites may act as promoters (Gannlng et al., 1984). The production of
the enzyme catalase by peroxlsomes catalyzes the breakdown of hydrogen
04770
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peroxide to water. Hydrogen peroxide Itself or the hydroxyl 1on, that Is
formed from hydrogen peroxide, causes damage to DNA and chromosomes
(Turnbull and RodrUks, 1985). PAEs, such as DEHP, exhibit hypollpldemlc
activities common to several peroxlsome pro!Iferators that Include liver
enlargement that Is not accompanied by frank hlstologlc Hver damage,
proliferation of smooth endoplasmlc retlculum and an Increase 1n the number
of hepat c peroxlsomes (Cohen and Grasso, 1981). Warren et al. (1982)
hypothesised that If DEHP acts similarly to other peroxlsome prollferators,
the compound may Initiate neoplastlc transformations of hepatic parenchymal
cells by Increasing Intracellular reactive oxygen species, which could cause
DNA damage. Peroxlsome prollferators modify peroxlsomal enzyme profiles
such thct fatty add B-oxIdatlon, H2°?' peroxldlzed llpofusln and
peroxlsomil uMcase levels are Increased and Increased catalase activity 1s
smaller with respect to the Increased peroxlsome volume. That Is, catalase
activity Is Increased, but to a much lesser extent than the activities of
H_0. gene~at1ng oxldases.
Turnbill and Rodrlcks (1985) proposed a possible mechanism of
cardnogeildty for DEHP based on a peroxlsome proliferation hypothesis
(Figure Vll-l). As discussed 1n Chapter III, the Initial step 1n metabolism
of orally administered OEHP Is hydrolysis to yelld MEHP. MEHP then under-
goes w- and w-1-oxidation. One of the ^-oxidation products may then
undergo 3-ox1dat1on, which 1s Important since In this step of DEHP
metabolism hydrogen peroxide Is generated. Excess Intracellular levels of
hydrogen peroxide may be detrimental to the cell. In addition, hydrogen
peroxide :an react with DNA, causing alteration and liberation of DNA bases
and sugar-phosphate backbone breakage (Turnbull and Rodrlcks, 1985).
04770 VII-14 07/02/91
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(/I
£ e
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eu
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CO wi —
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4* e
04770
VII-15
07/28/88
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In a more recent report RodMcks and Turnbull (1987) compared and
summarized the differences between peroxlsomes found 1n various mammalian
species. The most extensive studies on proliferation of peroxlsomes and
Inductlor of peroxlsomal enzymes have been 1n male rats. Species differ In
their morphologic characteristics of peroxlsomes. Humans, as well as other
species lack the enzyme uric acid oxldase (urlcase) since the central
crystallcld core Is absent from the peroxlsome. When comparing peroxlsomal
data, th»re seem to be only slight differences between species; however,
there are even some differences within a species relating to age and
gender. Of the species and sexes tested, male rats are the most sensitive
to chem1:ally Induced peroxlsomal proliferation. Quantitative measurement
of the sjecies differences Is not available. However, the authors speculate
that H may be due to differences 1n absorption, metabolism or Inherent
differences 1n hepatic susceptibility (RodMcks and Turnbull, 1987).
The possible DNA-blndlng activity of DEHP has been Investigated by Albro
et al. |1983a). Ethylhexyl-labeled DEHP, but not ring-labeled DEHP, was
found to be associated with the DNA from the livers of rats. The authors
determined that the radioactivity was not a result of absorption, Intercala-
tion, attachment to RNA or hlstones, an Impurity 1n the labeled DNA, or
artlfactial binding from the sample preparation. The source of the 14C
may have been carbonyl phosphate, which Is a precursor for urea and pyrlmi-
dine basi;s. von Oanlken et al. (1984) concluded that DEHP did not bind
covalently to hepatic DNA 1n rats and mice exposed to the labeled PAEs
through dietary administration. Radioactivity associated with the DNA was
attributed to the biosynthetlc Incorporation of radlolabeled breakdown
products, such as 2-ethylhexanol.
04770 VII-16 07/02/91
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Mechanisms of Reproductive ToxIcUy
Gonadal toxlclty 1n rats has been linked to the adverse effects of
phthalates upon testlcular zinc concentrations. Upon administration of DBP
or DEHP urinary excretlor of zinc was enhanced and the testlcular zinc con-
tent decreased (Cater et al., 1977; Foster et a!., 1980; Thomas et al.
1982). Cater et al. (1977) concluded that after oral administration, DBP 1s
metabolized by nonspecific esterases In the GI tract to the monobutyl
phthalate (MBP) prior to absorption Into the bloodstream. The monoester or
another metabolite of DBP may act as a chelatlng agent by removing the zinc
from the testes. Testlcular zinc deficiency Is, therefore, the possible
causative factor leading to testlcular atrophy. Z1nc depletions have been
noted In both the testes and prostate glands of rodents following oral, s.c.
and l.p. PAE exposures. It has been hypothesized that the testlcular
effects of orally administered dlesters are mediated by the monoesters and
alcohols produced during dlester hydrolysis 1n the GI tract {Gray and
Becmand, 1984). Thomas et al. (1982) provided s.c. and l.p. Injection data
that demonstrated the action of DEHP upon the depletion of endogenous
goriadal zinc was not a function of the Interference of the Intestinal
absorption of the divalent zinc 1on.
Further Investigations of the mechanism of testlcular Injury Indicated
that the testlcular Injury Induced by DBP does not appear to result from the
accumulation of metabolites or the formation of covalent adducts In testl-
cular tissue (Gangolll, 1982). Oral administration of 14C-OBP (lactation
of »*C not stated) did not show evidence of accumulation of radioactivity
In the gonads. Also, testlcular atrophy did not appear to be mediated by an
Interference In androgen synthesis or the availability of gonadotroplns.
04770
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The DBP-Induced testlcular Injury was not reversed by treatment with
testosterone or pregnant mare serum (Gangolll, 1982).
Summary
Research Into the mechanisms of PAE toxldty 1n animal tissues has
Indlcatec that the PAEs may Interfere with the normal enzymatic or metabolic
processes. Investigators have found that PAEs exert their toxic effects by
modifying the physical state of membrane-1tplds and, therefore, change
membrane fluidity. The mechanisms by which these alterations occur has not
been clearly delineated. In the liver, phthalates alter the structure and
metabolism as characterized by Increases 1n the number of peroxlsomes,
mHrochondrla and enzymes of fatty add oxidation. Studies primarily on
DEHP Indicate that llpld and protein metabolism are Inhibited. These
effects en carbohydrate metabolism are also associated with depressions 1n
the energy coupling systems of the liver, Including the mitochondria.
Inhibition of cholesterol synthesis In various organs occurs when phthalates
Inhibit
-------
VIII. QUANTIFICATION OF TOXICOLOGIC EFFECTS
Introduction
The quantification of toxicologlc effects of a chemical consists of
separate assessments of noncarclnogenlc and carcinogenic health effects.
Chemicals that do not produce carcinogenic effects are believed to have a
threshold dose below wh'ch no adverse, noncarclnogenlc health effects occur,
while carcinogens are assumed to act without a threshold.
In the quantification of noncarclnogenlc effects, a Reference Dose
(RfD), [formerly termed the Acceptable Dally Intake (ADI)] Is calculated.
The RfD Is an estimate (with uncertainty spanning perhaps an order magni-
tude) of a dally exposure to the human population (Including sensitive
subgroups) that Is likely to be without an appreciable risk of deleterious
health effects during a lifetime. The RfD Is derived from a no-observed-
adverse-effect level (NOAEL), or lowest-observed-adverse-effect level
(LQAEL), Identified from a subchronic or chronic study, and divided by an
uncertainty factor(s) times a modifying factor. The RfD Is calculated as
follows:
RfD =
(NOAEL or LOAEL)
[Uncertainty Factor{s) x Modifying Factor]
mg/kg bw/day
Selection of the uncertainty factor to be employed In the calculation of
the RfD Is based upon professional judgment, while considering the entire
data base of toxicologlc effects for the chemical. In order to ensure that
uncertainty factors are selected and applied In a consistent manner, the
04780
VIII-1
07/02/91
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U.S. EPA (1991) employs a modification to the guidelines proposed by the
National Academy of Sciences (NAS, 1977, 1980) as follows:
Standard Uncertainty Factors (UFs)
Lse a 10-fold factor when extrapolating from valid experimental
results from studies using prolonged exposure to average healthy
humans. This factor 1s Intended to account for the variation
1n sensitivity among the members of the human population. [10H]
Lse an additional 10-fold factor when extrapolating from valid
results of long-term studies on experimental animals when
results of studies of human exposure are not available or are
Inadequate. This factor Is Intended to account for the uncer-
tainty In extrapolating animal data to the case of humans.
[IDA]
Use an additional 10-fold factor when extrapolating from less
than chronic results on experimental animals when there Is no
useful long-term human data. This factor 1s Intended to
account for the uncertainty In extrapolating from less than
chronic NOAELs to chronic NOAELs. [10S]
Use an additional 10-fold factor when deriving an RfD from a
LOAEL Instead of a NOAEL. This factor Is Intended to account
for the uncertainty 1n extrapolating from LOAELs to NOAELs.
[10L]
Modifying Factor (MF)
Use professional judgment to determine another uncertainly
factor (MF) that Is greater than zero and less than or equal to
10. The magnitude of the MF depends upon the professional
assessment of scientific uncertainties of the study and data
base not explicitly treated above, e.g., the completeness of
the overall data base and the number of species tested. The
default value for the MF 1s 1.
The uncertainty factor used for a specific risk assessment Is based
principally upon scientific judgment rather than scientific fact and
accounts for possible Intra- and interspedes differences. Additional
considerations not Incorporated In the NAS/OOW guidelines for selection of
an unceralnty factor Include the use of a less than lifetime study for
deriving an RfD, the significance of the adverse health effects and the
counterbalancing of beneficial effects.
04780
VIII-2
07/02/91
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from the RfD, a Drinking Water Equivalent Level (DWEL) can be calcu-
lated. The DWEL represents a medium specific (I.e., drinking water)
lifetime exposure at which adverse, noncarclnogenic health effects are not
anticipated to occur. The DWEL assumes 100% exposure from drinking water.
The DWEL provides the noncarclnogenic health effects basis for establishing
a drinking water standard. For Ingestlon data, the DWEL 1s derived as
follows:
DWEL
(RfD) x (Body weight 1n kg)
Drinking Water Volume 1n a/day
mg/i
where:
Body weight = assumed to be 70 kg for an adult
Drinking water volume = assumed to be 2 l/day for an adult
In addition to the RfD and the DWEL, Health Advisories (HAs) for expo-
sures of shorter duration (1-day, 10-day and longer-term) are determined.
The HA values are used as informal guidance to municipalities and other
organizations when emergency spills or contamination situations occur. The
HAs are calculated using an equation similar to the RfD and DWEL; however,
the NOAELs or LOAELs are identified from acute or subchronlc studies. The
HAs are derived as follows:
HA =
(NOAEL or LOAEL) x (bw)
(UF) x ( i/day)
mg/s.
Using the above equation, the following drinking water HAs are developed
for noncarclnogenic effects:
1.
2.
3.
4.
04780
1-day HA for a 10 kg child ingesting 1 l water per day.
10-day HA for a 10 kg child ingesting 1 i water per day.
Longer-term HA for a 10 kg child Ingesting 1 1 water per day,
Longer-term HA for a 70 kg adult ingesting 2 l water per day.
VIII-3
03/30/88
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The 1-day HA calculated for a 10 kg child assumes a single acute
exposure to the chemical and 1s generally derived from a study of <7 days
duration. The 10-day HA assumes a limited exposure period of 1-2 weeks and
Is generally derived from a study of <30 days duration. The longer-term HA
Is der1v;d for both the 10 kg child and a 70 kg adult and assumes an
exposure period of -7 years (or 10% of an Individual's lifetime). The
longer-term HA 1s generally derived from a study of subchronlc duration
(exposure for 10% of animal's lifetime).
The L.S. EPA categorizes the carcinogenic potential of a chemical, based
on the overall welght-of-evldence, according to the following scheme:
Group A: Human Carcinogen. Sufficient evidence exists from
epidemiology studies to support a causal association between
exposure to the chemical and human cancer.
Group 8: Probable Human Carcinogen. Sufficient evidence of
carclnogenlclty In animals with limited (Group Bl) or inade-
quate (Group B2) evidence 1n humans.
Group C: Possible Human Carcinogen. Limited evidence of
carclnogenldty 1n animals In the absence of human data.
G'oup 0: Not Classified as to Human Carclnoqenlclty. Inade-
qjate human and animal evidence of cardnogenldty or for which
nD data are available.
G'oup E: Evidence of Noncarclnoqenlclty for Humans. No
evidence of cardnogenldty 1n at least two adequate animal
t?sts in different species or In both adequate epldemlologlc
aid animal studies.
If tcxlcologlc evidence leads to the classification of the contaminant
as a known, probable or possible human carcinogen, mathematical models are
used to calculate the estimated excess cancer risk associated with the
Ingestlon of the contaminant In drinking water. The data used In these
04780 VIII-4 07/02/91
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estimates usually come from "lifetime exposure studies using animals. In
order to predict the risk for humans from animal data, animal doses must be
converted to equivalent human doses. Thl.s conversion Includes correction
for noncontlnuous exposure, less than lifetime studies and for differences
In size. The factor that compensates for the size difference Is the cube
root of the ratio of the animal and human body weights. It 1s assumed that
the average adult human body weight Is 70 kg and that the average water
consumption of an adult human 1s 2 l of water per day.
For contaminants with a carcinogenic potential, chemical levels are
correlated with a carcinogenic risk estimate by employing a cancer potency
(unit risk} value together with the assumption for lifetime exposure from
1nc;est1on of water. The cancer unit risk Is usually derived from a linear-
ized multistage model with a 95% upper confidence limit providing a low dose
estimate; that Is, the true risk to humans, while not Identifiable, Is not
Hkely to exceed the upper limit estimate and, In fact, may be lower.
Excess cancer risk estimates may also be calculated using other models such
as the one-hit, Welbull, logH and probH. There Is little basis In the
current understanding of the biologic mechanisms Involved In cancer to
suggest that any one of these models Is able to predict risk more accurately
than any other. Because each model Is based upon differing assumptions, the
estimates derived for each model can differ by several orders of magnitude.
The scientific data base used to calculate and support the setting of
cancer risk rate levels has an Inherent uncertainty that Is due to the
systematic and random errors 1n scientific measurement. In most cases, only
studies using experimental animals have been performed. Thus, there 1s
04780
VIII-5
07/02/91
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uncertainty when the data are extrapolated to humans. When developing
cancer risk rate levels, several other areas of uncertainty exist, such as
the Incomplete knowledge concerning the health effects of contaminants 1n
drinking .later, the Impact of the experimental animal's age, sex and
species, 1 he nature of the target organ system(s) examined and the actual
rate of e) posure of the Internal targets 1n experimental animals or humans.
Dose-response data usually are available only for high levels of exposure
and not for the lower levels of exposure closer to where a standard may be
set. Whei there 1s exposure to more than one contaminant, additional
uncertainty results from a lack of Information about possible synerglstlc or
antagonistic effects.
Noncarcinogenic Effects
PAEs
-------
OEHP. A second adult male subject given an oral dose of 10 g of OEHP exper-
ienced mild gastric disturbances and moderate catharsis (Shaffer et a!.,
1945). Accidental Ingestlon of 10 g of DBP by a young adult male produced
nausea, vertigo and signs of keratltls and toxic nephritis (Lefaux, 1968).
A single prospective cohort study was Identified 1n the literature. Thless
et al. (1978b) reported that among 221 workers exposed to DEHP, only half of
the expecte4 deaths were observed In the exposed population. Although the
analysis was not conclusive, no Increased risk of adverse health effects was
attributed to exposure to DBP In this group of workers. £n vitro studies of
human tissue and cell cultures revealed that PAEs Inhibited cellular growth
and decreased platelet function but did not produce chromosomal damage In
human leukocytes or fetal lung cells. The greatest risk for toxic effects
from PAE exposure appears to be among Individuals receiving blood transfu-
sions or hemodlalysis due to extraction of PAEs from plastic blood bags or
plastic tubing used In these treatments. However, reports of hepatitis In
hemodlalysis patients and necrotlzlng enterocolHIs In Infants given blood
transfuslor could not be attributed definitively to PAE exposure.
Species differences occur with respect to metabolism of PAEs. Several
species of animals have been determined to excrete glucuronlde conjugates of
MEHP (the major metabolite of DEHP) upon exposure to DEHP with the exception
of rats (Tanaka et al., 1975; Williams and Blanchfleld, 1975; Albro et al.,
1982). The role that glucuronlde conjugation may play In the sensitivity
between species to toxic endpolnts Is not known; therefore, studies with
rats will still be considered with other test species for quantification of
toxicologic effects.
04780
VIII-7
07/31/91
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Thert 1s no common toxic effect that PAEs as a group of compounds have
been shoeen Identified for PAEs, the HAs, OWELs and cancer risk levels are
calculated for Individual PAEs rather than for the group of compounds
generlcally.
Studies Considered for Noncardnoqenlc Quantifications — DEHP. DEHP
has been studied more extensively than any other PAE, In part because H Is
the most widely used plastldzer. Enlarged liver and testlcular atrophy are
the two most commonly observed effects of DEHP 1n rats. Mangham et al.
(1981) conducted a short-term test to examine the testlcular and hepatic
effects cf DEHP. DEHP was administered orally by gavage to Wlstar rats at a
dose level of 2500 mg/kg/day for 7 and 21 days. After 7 or 21 dally doses,
weight of the testes was decreased, and hlstopathologlc changes were found
In 50-80} of the seminiferous tubules of each male rat. Treatment for 7 or
21 days produced marked liver enlargement In rats of both sexes and
decreased activity of succlnate dehydrogenase 1n males. Also, body weight
gain was significantly decreased In males. The testlcular effects observed
are cons'stent with results of an earlier study by Gray et al. {1977} 1n
which testlcular atrophy occurred within the first 2 weeks of treatment at a
04780 VIII-8 07/02/91
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dietary level of 2% DEHP (-1440 mg/kg/day). Lake et al. {1975} reported
Increased liver weights In Wlstar rats administered 2000 mg/kg DEHP [236
mg/kg/day assuming 0.013 kg/day Intake (Lehman, 1959)] for periods of 4, 7,
14 and 21 days. The Investigator did not examine any reproductive organs.
In a recent study, MHchell et al. (1985) observed similar results when
groups of male and female Wlstar albino rats were administered diets con-
taining 50, 200 and 1000 mg/kg/day DEHP for 3, 7, 14 and 28 days. There was
a total of 90 treated rats and 60 control rats. H1stopatholog1c examina-
tions were performed on the major abdominal organs at all time points. The
livers of male rats were significantly enlarged 3 days after treatment with
10CO mg/kg/day OEHP. After 14 days significant liver enlargement was noted
at the 50 and 200 mg/kg/day doses 1n male rats. There were no significant
differences In testes weight when control animals were compared with experi-
mental animals. Further details were not given. Liver cells from male rats
showed marked proliferation of peroxlsomes after 3 days treatment with 200
or 1000 mg/kg/day. Treatment with 50 mg/kg/day showed Increased numbers of
peroxlsomes after 14 days. Female rats, however, showed only Increased
number of peroxlsomes after 14 days treatment with 1000 mg/kg/day. Prolife-
ration of the smooth endoplasmic retlculum In both males and females
occurred at all doses In a dose-dependent manner. Biochemical changes such
as effects on DNA, catalase activity and laurate hydroxylase activity were
also noted In all dose groups.
SubchronU oral studies have been conducted with DEHP on rats (Shaffer
et al., 1945; Harris et al., 1956; Nlkonorow et al., 1973; Gray et al.,
1977; Mitchell et al., 1985; Cater et al., 1977), mice (NTP, 1984a), and
04780
VII1-9
07/02/91
-------
dogs (Hcrris et al., 1956). The study 1n which adverse effects were
observed at the lowest level of exposure Is that of Mitchell et al. (1985).
Mitchell et al. (1985) fed male and female WUtar albino rats diets
contalnirg 50, 200 and 1000 mg/kg/day DEHP for 9 months. Necropsy of the
thoracic, abdominal and other regions was carried out. The Hvers were
sub^ectet to extensive hlstologlc, electron microscopic and biochemical
examlnat'on. Significant liver enlargement was observed In male rats at all
dose lev;ls. In addition, body weights of both male and female rats were
significantly reduced. Electron microscopy revealed an Increase In
peroxlsomal proliferation at all dose levels and an Increase In number of
Tysosome; at 200 and 1000 mg/kg/day.
In tie study by Gray et al. (1977), male and female CO rats were fed
dietary levels of 0, 0.2, 1 and 2% OEHP In the diet for 17 weeks. Dally
doses calculated from food consumption data corresponded to 143, 737 and
H40 mg/S,g/day for males, respectively, and 154, 797 and 1414 mg/kg/day for
females, respectively. Body weight, food consumption, clinical signs of
toxlclty, serum biochemistry, urlnalysls and hematology were monitored.
Gross and microscopic pathologic examinations were performed on all rats at
the end of the study. Effects were observed at all levels of exposure.
Significantly Increased absolute and relative liver weights were observed In
all expo led groups. Both males and females fed either 1 or 2% DEHP had a
significantly reduced packed cell volume compared with controls. At the
0.2% levd, liver weight was Increased in both sexes and spermatogenesis was
decrease< in males.
Carpenter et al. (1953) conducted chronic toxlclty testing in rats,
guinea p'gs and dogs. Groups of 32 male and 32 female Sherman rats were fed
04780 VIII-10 07/02/91
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dietary levels of 0.04, 0.13 and 0.4% OEHP. Mean dally Intakes were 20, 60
and 200 mg/kg/day. During the first year, male and female rats were housed
together until the females became pregnant. Parental rats were maintained
on their respective diets for 2 years. Offspring of the rats fed 0.4% DEHP
were maintained at this dietary level for 1 year. After 1 year, parental
rats were reduced In number to a maximum of 8/sex/group. No effects were
observed at the 0.04 or 0.13% levels. At the 0.4% level, decreased body
weight and Increased liver and kidney weights were observed at the end of
the first year, but no significant effects on fertility were observed.
While this study provides Information on a chronic NOEL, the low survival
among controls, which experienced 70.3% mortality from causes such as lung
Infections, postpartum complications, peritonitis, abdominal abscess and
intestinal Intussusception over the 2-year period, should be noted.
Guinea pigs (23 male and 23 females/group) were also fed DEHP In the
diet at levels of 0.04 and 0.13% for 1 year (Carpenter et a!., 1953). This
experiment showed no effects at the 0.04% level (-19 mg/kg/day) and
Increased liver weight at 0.13% (64 mg/kg/day). These results appear to
Indicate that the guinea pig was slightly more sensitive to the effects of
DEHP 1n this study. A group of four dogs given capsules 5 times/week
containing 0.03 mt/kg/day for 19 doses, then 0.06 ma./kg/day for 240
doses showed no significant effects (Carpenter et al., 1953).
Harris et al. (1956) reported similar results In a 2-year rat study.
Groups of 43 male and 43 female rats were fed diets containing 0.1 or 0.5%
DEHF. No compound-related effects on mortality were observed; however,
survival over the 2-year study period was very low with 85-95% mortality.
04780
VIII-11
08/16/88
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No effects were observed at the low dietary level (-50-80 mg/kg/day), which
1s consistent with the findings of Carpenter et al. (1953). At the higher
level, focd consumption decreased after the first year. Increased liver and
kidney weights were observed In high-dose rats sacrificed at 3 and 6 months.
The dally Intake at the higher dose, calculated for the first 6 months, was
-300-400 rrg/kg/day. Testlcular atrophy was not reported by Carpenter et al.
(1953) or Harris et al. (1956).
The oral NOAEL for reproductive effects of DEHP appears to be near the
NOAEL for chronic toxic effects. Tomlta et al. (1982a) reported that a
single oril dose of 0.05 ml/kg administered by gavage to mice on day 7 of
gestation was associated with a decrease 1n body weight of viable fetuses;
however, ro abnormal fetuses were observed. Since the density of DEHP Is
0.985 g/nu, the 0.05 ml/kg dose Is equivalent to 49 mg/kg. Using the
dose-response curve for resorptlons and deaths, the authors calculated the
NOEL for fetal lethality to be 64 mg/kg. Shlota and Nlshlmura (1982)
administered DEHP 1n the diets of ICR-ICL mice on days 0-18 of gestation.
At the 0.05% level (70 mg/kg/day)t the only effect observed was retarded
ossification. This effect was thought to be related to general and
under-development of the fetuses rather than teratogenlc activity, since no
Internal inomolles were observed. At a dietary level of 0.1% (190 rag/kg/
day) the rumber of resorptlons and dead fetuses were Increased, although the
statistical significance of this increase was marginal (p=0.05). At 0.25%
(410 mg/kj/day), an Increased number of malformations were observed In
addition lo Increased resorptlons and dead fetuses, decreased maternal and
fetal welchts, and retarded ossification. At 0.4% and 1.0% (830 and 2200
mg/kg/day}, all fetuses were dead or resorbed.
04780
VIII-12
09/15/88
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More recently DEHP was evaluated for developmental toxldty In Fischer
344 rats and CO-1 mice (Tyl et al., 1988). Dietary levels of OEHP were
administered on gestatlonal days 0-20 to rats at 0, 0.5, 1.0, 1.5 or 2.0%
and on gestatlonal days 0-17 to mice at 0, 0.025, 0.05, 0.10 or 0.15%.
Corresponding levels In mg/kg/day were 0, 356, 666, 856, 1054 and 0, 44, 91,
190, 292 1n rats and mice, respectively. Tyl et al. (1988) concluded that
DEHP was not teratogenic at any dose tested in Fischer 344 rats. However,
treatment did produce .maternal and other embryofetal toxldty at 1.0, 1.5
and 2.0%. An embryofetal NOEL In rats was determined to be 0.5% (356
mg/kg/day}. In mice, doses {0.10 and 0.15%) that produced maternal and
embryofetal toxldty also increased Incidence of malformations. A dose of
0.05% (91 mg/kg/day) DEHP produced Increased Incidence of malformations
without maternal or embryofetal toxldty. An embryofetal NOEL In mice was
determined to be 0.025% (44 mg/kg/day} OEHP.
A study by NTP (1984a) tested CD-I mice using a newly developed testing
scheme designated "Fertility Assessment by Continuous Breeding". Results of
this study were similar to those of Shlota and Nishlmura (1982); however,
the NTP study focused on fertility effects rather than teratogenidty. In
the first phase of this test, groups of 20 male and 20 female mice were fed
diets containing 0.01, 0.1 and 0.3% DEHP for 7 days prematlng and for 98
days of continuous mating, after which they were maintained for 21 days with
no treatment. Daily Intakes of DEHP were not calculated by the authors.
However, if one assumes the same dally Intake rate as that calculated for
the low-dose CD-I mice In the cardnogenldty bloassay by Kluwe et al.
(1982a) of 735 mg/kg/day (averaged for males and females) for a 0.3% dietary
level, daily DEHP Intakes for the lower dietary levels of 0.01 and 0.1%
04780
VIII-13
07/02/91
-------
would be -24 and 243 mg/kg/day, respectively. It should be noted that these
calculations do not account for differences 1n food consumption due to preg-
nancy, a je of mice or any additional differences between the two studies.
At the 0.3% level, complete suppression of fertility was observed. At the
0.1% lev«'l, fertility was decreased and various reproductive parameters were
significantly decreased. These parameters Included number of Utters per
pair, anJ number of live pups per Utter, proportion of pups born alive,
number of male pups born alive, live pup weight of females and adjusted live
pup weight of males. A second phase of this study used the mice from the
continuous breeding phase. In this phase control ma'les were mated to the
0.3%-treeted females and control females were mated to 0.3%-treated males.
In addlt'on, control males were bred to control females to serve as the con-
trol groi.p for the second phase. Results of this phase of testing revealed
that the decreased fertility was attributable to effects of DEHP In both
males anc females.
Quantification of Noncarclnoqenlc Effects — DEHP.
Assessment of Acute Exposure Data and Derivation of 1-day HA -- Liver
enlargemtnt, testlcular atrophy In males, depressed weight gain and death
have all been observed after oral administration of single doses of DEHP to
rats. LI) s for DEHP have been measured in a variety of species and range
from 26 g/kg (rats) to 34 g/kg (rabbits). In rats, neonates and sucklings
are more sensitive to the weight gain and lethal effects of DEHP than are
adults. Oostal et al. (1987a) administered five successive doses (gavage in
corn oil) of 0, 10, 100, 1000 or 2000 mg/kg/day to six groups (9-10
pups/groip) of rats, 6-86 days old. For neonates and sucklings, doses of
2000 mg/kg/day were lethal and 1000 mg/kg/day caused depressed weight gain
04780
VIII-14
05/16/91
-------
1n all groups and Increased mortality 1n sucklings 14 days old. Adults (86
days old) were less sensitive to these effects, with no Increase In
mortality at any dose and effects on weight observed only at the 2000
mg/kg/day dose level. At 100 mg/kg/day, sucklings and adults exhibited
Increased I1ver-to-body weight ratios.
Effects of acute oral exposure to D£HP on the liver have been studied by
Mitchell et al. (1985) and Mangham et al. (1981). Mitchell et al. (1985)
administered DEHP 1n the diet of rats (4/sex/group) at nominal doses of 50,
200 and 1000 mg/kg/day. H1stopatholog1c, biochemical cytogenetlc analyses
we-e conducted on days 3, 7, 14 and 28, and at 9 months of dosing.
Indications of hepatotoxldty (Increased liver weight, decreased hepatic
gljcose-6-phosphatase activity) were first observed 1n the 50 mg/kg/day dose
males at 14 days of treatment. Mangham et al. (1981) observed decreased
teitlcular weight, microscopic changes 1n the seminiferous tubules, enlarged
11*er, decreased activity of sucdnate dehydrogenase and decreased body
weight after seven doses of 2500 mg/kg/day.
In addition, DEHP can cause reproductive and developmental toxlclty.
Tyl et al. (1988) observed fetotoxlclty In mice and rats at doses of 91 and
666 mg/kg/day, respectively. Mice were dosed on days 0-17 of gestation
while rats were dosed on days 0-20. NOAELs for reproductive and
developmental effects of 44 mg/kg/day (mice) or 357 mg/kg/day (rats) were
Identified. These observations are supported by the work of TomHa et al.
(1982a). Mice were administered single doses of 50 »l DEHP/kg (-49
mg/kg) or 100 vl DEHP/kg (-99 mg/kg) to pregnant dams at day 7 of
gestation and decreased fetal weights at birth were observed.
04780
VIII-15
05/16/91
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For exposure of 5 days or less, developmental effects In mice and liver
enlargement In rats are the most sensitive endpolnts of toxUHy. From the
studies described previously, 1t Is not possible to determine whether a dose
of 44 mc/kg/day, the NOAEl for developmental effects, would cause liver
effects 1n rats, observed at 100 but not 10 mg/kg/day. Therefore, the NOAEL
for live- enlargement of 10 mg/kg/day from Dostal et al. {1987a} was
selected as the basis for the 1-day HA for DEHP, derived as follows:
HA
Omq/kq/day x 10 kq
100 x 1 I/day
where:
10 mg/kg/day
10 kg
100
1 l/day
= NOAEL based on lack of liver enlargement (Dostal et
al., 1987a)
= assumed weight of a child
= uncertainty factor, according to U.S. EPA and
ODW/NAS guidelines for use with a NOAEL From an
animal study
= assumed water consumption by a child
Assessment of Short-Term Exposure Data and Derivation of 10-day HA —
Effects en the liver appear to be the most sensitive endpolnt of toxlclty
for 10-dey exposure. Mitchell et al. (1985) observed liver effects in rats
after 14 days at doses as low as 50 mg/kg/day, a dose similar to the NOAEL
for developmental toxldty In mice (44 mg/kg/day}. A NOAEL for the observed
liver effects {decreased glucose-6-phosphatase activity, Increase In liver
weight, increase In hepatocyte I1p1d content) was not Identified. This
number 1:; consistent with the 1-day HA If 1t were adjusted for a 10-day
exposure period. However, the Mitchell et al. (1985) study examined other
organs 1r addition to the liver and provides a better estimate of a 10-day
04780
VIII-16
07/02/91
-------
exposure. The 10-day HA 1s also protective of developmental toxldty
Identified at 91.07 mg/kg/day In mice and 666.39 mg/kg/day In rats by Tyl et
al. (1988). Thus, the Mitchell et al. study Is chosen to derive the 10-day
HA as follows:
10.day HA s50mq/kq/daY x 10 kq = Q^
1000 x 1 a/day
where:
50 mg/kg/day = LOAEL based on liver enlargement (Mitchell et al.,
1985)
10 kg
1 l/day
assumed weight of a child
1000 = uncertainty factor, according to U.S. EPA and
OOW/NAS guidelines for use with a LOAEL from an
animal study
assumed water consumption by a child
Derivation of Longer-term HA — Subchronlc oral studies have been
conducted with DEHP, however none of the studies Identify a NOAEL. Mitchell
et al. (1985) observed significant liver enlargement In male rats
administered 50, 200 or 1000 mg/kg/day for 9 months. No clear progression
of hepatotoxU effects was observed from 3-, 7-, 14- or 28-day time points.
This 1s supported by the subchronlc study by Gray et al. (1977) where liver
weights were Increased In both sexes of rats and spermatogenesls was
decreased 1n males (143 mg/kg/day dose level In males; 154 mg/kg/day dose
level 1n females).
Deriving the longer-term HA based on the LOAEL of 50 mg/kg/day Is
protective of the reproductive (NTP, 1984a) and developmental toxldty (Tyl
et al., 1988) observed 1n mice at doses of 243 and 91 mg/kg/day, respec-
tively. The reproductive study by NTP (1984a) showed no effects on
Q4780
VIII-17
07/02/91
-------
fertility In mke fed 0.01% (24 mg/kg/day) In the diet for 7 days prematlng
and 98 cays continuous breeding. The next highest dietary level of O.TX
(240 mg/>g/day) significantly reduced fertility. Tyl et al. (1988) observed
fetotoxUHy In mice and rats at 91 and 666 mg/kg/day. NOAELs for reproduc-
tive and developmental effects of 44 (mice) or 357 (rats) mg/kg/day were
Identified.
Therefore, based on a LOAEL for hepatotoxlclty In rats the longer-term
HA values are calculated as follows:
50 mq/kq/day x 10 kg
Longer-term HA = = 0.5 mg/l,
1000 x 1 l/day
(child)
where:
50 mc/kg/day « LOAEL based on liver enlargement (Mitchell et al.,
1985)
10 kc
1 l/cay
= assumed weight of a child
1000 = uncertainty factor, according to U.S. EPA and
ODW/NAS guidelines for use with a LOAEL from an
animal study
assumed water consumption by a child
50 mq/kq/day x 70 kg
Longer-term HA = = 1.75 mg/l
1000 x 2 I/day
(adult) (rounded to 2 mg/l)
where:
50 mc/kg/day = LOAEL based on liver enlargement (Mitchell et al.,
1985)
70 kc
2 t/cay
= assumed weight of an adult
1000 = uncertainty factor, according to U.S EPA and ODW/NAS
guidelines for use with a LOAEL from an animal study
assumed water consumption by an adult
04780
VIII-18
07/02/91
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Assessment of Long-Term Exposure Data and Derivation of a DHEL — No
data were available on the effects of chronic human exposure to DEHP.
Carpenter et al. (1953) reported no effects 1n rats exposed for 2 years to
dietary levels of 0.04% and 0.13%, equivalent to -20 and 60 mg/kg/day,
respectively. There was low survival of both control and treated rats dur-
ing the second year. However, results with other species and studies sup-
po*t these results. Carpenter et al. (1953) also reported that no effects
we-e observed 1n guinea pigs exposed to 0.04% {-19 mg/kg/day) 1n the diet
for 1 year; however, Increased liver weights were reported. Increased liver
weight was also observed In guinea pigs exposed to 0.13% DEHP (64 mg/kg/
day). No hlstologU effects on liver tissue were observed. The suggested
LOAEL Is -19 mg/kg/day. A study by Harris et al. (1956) showed no effects
In rats fed 0.1% DEHP (50-80 mg/kg/day) for 2 years. Again, low survival In
all groups places limitations on Interpretation of the results of this
study. A recent reproductive study showed no effects on fertility in mice
fed 0.01% DEHP 1n the diet for 7 days prematlng and 98 days continuous
breeding (NTP, 1984a). Assuming food consumption was similar to that
reported by Kluwe et al. (1982a) for mice, dally Intake was calculated to be
24 mg/kg/day. The next highest dietary level of 0.1% significantly reduced
fertility. The approximate dally Intake for this dietary level was calcu-
lated to be 240 mg/kg/day. Also, Shlota and NUhlmura (1982) reported only
reduced ossification In mouse fetuses born to dams fed 70 mg/kg/day on days
0-18 of gestation. Tyl et al. (1988) determined an embryofetal NOEL to be
356.74 mg/kg/day In Fischer 344 rats and 44.07 mg/kg/day 1n CD-I mice. In
light of these results and the fact that no NOAEl was Identified, lower than
the lowest LOAEL observed, -19 mg/kg/day reported by Carpenter et al. (1953)
was selected for use In calculating the DUEL (U.S. EPA, 1991).
04780
VIII-19
07/02/91
-------
Uslnc this IOAEL, the DWEL would be derived as follows:
where:
19 mg/kg/day
RfD • — = 0.019 mg/kg/day
1000 y
(rounded to 0.02 mg/kg/day)
19 mc/kg/day = LOAEL derived from oral exposure to guinea pigs
(Carpenter et al., 1953)
1000
« uncertainty factor, according to U.S. EPA and
OOW/NAS guidelines for use with a LOAEL from a
subchronlc animal study
DWEL
2 I/day
where:
0.02 mg/kg/day = RfO
70 kc = assumed weight of an adult
2 l/cay * assumed water consumption by an adult
The -day HA, 10-day HA, longer-term HA and DWEL values calculated for
DEHP and the effect levels used In the derivations are summarized In Table
VIII-1.
Studies Considered for Noncardnoqenlc Quantification
B8P.
ToxicHy of BBP Is limited to a few studies. The most commonly observed
effects
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04780
VIII-21
07/31/91
-------
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04780
VIII-22
07/31/91
-------
liver and kidney weights were significantly Increased. In addition, the
Incidence of proximal tabular regeneration of the kidney 1ncr-:^sed In a
dose-related manner beginning at the 0.625% dose level. At the 2.5% and 5.0%
levels, effects Included decreased body weight, slightly decreased food
consumption, decreased weights of testes, epldldymus, seminal vesicles and
thymus, hlstologlc atrophy of testes and accessory sex organs, and decreased
bone marrow cellularlty. Dally Intakes of BBP were not calcu- lated.
However, dally food consumption and body weight were estimated from the
figures presented by Agarwal et al, (1985a). At the lowest dietary level of
OJ>25%, rats weighing' -250 g consumed 15 g of food per day or 60 g food/kg
bw/day. The dally Intake of BBP was calculated to be 375 mg/kg bw/day.
Since body weights and food consumption were not affected at 1.25%, the same
food consumption and body weight were used and the dally intake was
calculated to 750 mg/kg bw/day. Food consumption and body weight were
decreased at 2.5 and 5.0%, respectively. Again, approximating from the
figures In Agarwal et al. (1985a), 200 g rats at the 2.5% level consuming
10 g food/day and 150 g rats at the 5.0% level consuming 5 g food/day, the
daily Intake was 1250 and 1667 mg/kg bw/day, respectively.
Lake et al. (1978) administered 160, 480 or 1600 mg/kg/day BBP by
gastric intubation for 14 days to six male Sprague-Dawley rats per group.
Biochemical or morphologic changes In the liver were not observed at 160
mg/kg/day. Activities of ethylmorphlne N-demethylase and cytochrome oxldase
were significantly Increased at the 480 and 1600 mg/kg/day BBP. Significant
liver enlargement was observed at 1600 mg/kg/day in addition to
ultrastructural changes, such as gross dilation of the rough endoplasmlc
04780
VIII-23
07/02/91
-------
retlculum and Increased number of peroxlsomes. Effects on testes weights
were not observed 1n the 160 or 480 mg/kg/day animals; however, 1600
mg/kg/day BBP produced marked depression of both absolute and relative
testes weights as well as severe testlcular atrophy. Testlcular atrophy was
observed in 1/3 animals administered 480 mg/kg/day.
A second study was conducted to confirm the testlcular effects. Both
Sprague-Dawley and Mlstar Albino rats were treated with 480 and 1600
mg/kg/day BBP for 14 days. A significant depression 1n either absolute or
relative liver and testes weight was observed In both strains of rats at
1600 mg/l;g/day BBP. Hlstologlc examination revealed testlcular atrophy In
both strains (1600 mg/kg/day) with the extent of the lesions being more
severe 1i, the Sprague-Oawley strain. At 480 mg/kg/day BBP, 1/6 had testlcu-
lar atrophy, whereas the Wlstar albino strain revealed no hlstologlc changes.
are few oral long-term BBP studies. In a final report, NTP (1985)
conductei toxlclty and mating trial studies 1n F344 rats. The toxlrlty
portion rfas conducted as a dose range-finding study to establish a no effect
level and the dose response curve for BBP. Rats were administered
concentrations of either 0, 0.03, 0.09. 0.28, 0.83 or 2.50% (0, 17, 51, 159,
470 and 1417 mg/kg/day) BBP 1n the diet for 26 weeks. Powdered BBP was
mixed 1r to standard rodent meal diet. Because of the manner 1n which the
BBP was administered considerable waste and spillage was found especially at
the highest dose level. Therefore, the dose conversion for the highest was
based on a 554 food consumption rate/mg rat body weight. There were 15 male
animals in each dose group, starting at 6 weeks of age. Throughout the
04780
VIII-24
07/02/91
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study, body weight gain was significantly depressed at the 2.5% BBP level
when compared with the controls. There were no deaths attributed to BBP
toxlclty. All the rats given 2.5% BBP had small testes upon gross necropsy
at the 26-week termination. Five of 11 had soft testes, and 1/11 had a
small prostate and seminal vesicle. At 0.83%, significantly (p<0.05)
Increased absolute liver weight, llver-to-body weight, Hver-to-braln weight
ratios and Increases In mean corpuscular hemoglobin were noted. In the
0.03, 0.09, 0.28 and 0.83% BBP dose groups there were no grossly observable
effects on male reproductive organs. The kidneys of six animals 1n the 2.5%
grcup contained focal' cortical areas of 1nfarct-l1ke atrophy. In addition,
testlcular lesions were also observed at the 2.5% dose level. Lesions were
characterized by atrophy of seminiferous tubules and aspermla. The other
treatment groups showed no evidence of abnormal morphology In any other
organs.
Hlstopathologlc changes were also seen at the 2.5% BBP level after 10
weeks of exposure 1n the mating trial portion of this study. After hlsto-
pathologlc examination, testlcular lesions were characterized by atrophy of
seminiferous tubules and a near total absence of mature sperm production.
When 10/30 females successfully mated with the 2.5% treatment level males,
none were pregnant at necropsy. The Investigators concluded that the data
suggest a depression In male reproductive organ weights by either a direct
or Indirect toxic effect after 2.5% BBP administration. BBP at 0.83% 1n the
diet did not result 1n any treatment-related effects.
The only other Information on the subchronlc effects of BBP Is taken
from an unpublished study by Monsanto (1972). Rats fed diets containing BBP
04780 VIII-25 07/02/91
-------
at levels of 0.25 {125 mg/kg/day) and 0.50% (250 nig/kg/day} for 90 days
showed no toxic effects. A dietary level of 1.0% {500 mg/kg/day) BBP
resulted 1i Increased liver weight. Levels of 1.5 (750 mg/kg/day) and 2.0%
{1000 mg/lig/day) BBP were associated with Increased liver weight and a
decrease 1i growth rate. No effects were observed In dogs administered BBP
1n capsules at levels equal to 1.0, 2.0 and 5.0% of the diet. No further
details of this study were available for review.
Quantl Mcatlon of Noncarclnogenlc Effects -- BBP.
Assessment of Acute Exposure Data and Derivation of the 1-day HA — No
Information was available on the effects of BBP In humans. The only studies
available >n acute oral toxldty In animals used lethality as the toxic end-
point or were Inadequate for deriving a 1-day HA. Therefore, lack of
sufficient data preclude the derivation of a 1-day HA for BBP. It Is
recommended that the 10-day HA of 20 mg/i be adopted as a conservative
estimate f>>r the 1-day HA.
Assessnent of Short-Term Exposure Data and Derivation of a 10-day
HA — Information presented In a 14-day study was used to approximate the
10-day HA values. Agarwal et al. (1985a) administered BBP to male F344 rats
In the dltt for 14 consecutive days at dose levels of 0.625, 1.25, 2.5 and
5.0%. Effects observed beginning at the 0.625% level were significantly
Increased liver and kidney weights. Dose-related hUtopathologlc changes
{proximal tubular regeneration) were also noted 1n the kidney beginning at
the 0.625% level. Using approximations of food consumptions and body weight
obtained from figures presented In this study, the dally Intake at 0.625%
level was :alculated to be 375 mg/kg/day.
04780
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In male.,Sprague-Dawley rats administered 160, 480 or 1600 mg/kg/day 8BP
for 14 days by gastric intubation, biochemical or morphologic changes In the
liver as well as effects on testes weights were not observed 1n the 160
mg/kg/day dose group (Lake et a!., 1978). However, at 480 mg/kg/day
activities of ethyl morphine N-demethylase and cytochrome oxldase were
significantly Increased and testlcular atrophy was observed in 1/3
Sprague-Dawley rats 1n the first portion of this experiment. In the second
portion, the 480 mg/kg/day dose Induced testlcular atrophy 1n 1/6
Sprague-Dawley rats, whereas the Wlstar albino strain revealed no such
effects (Lake et a!., >978).
When comparing the two studies Lake et al. (1978) Identifies a NOAEL of
160 mg/kg/day. It Is questionable whether 480 mg/kg/day represents a NOAEL;
however, Agarwal et al. (1985a) observed significant Increases In liver and
kidney weights and kidney pathology at 375 mg/kg/day, which represents a
LOAEL. It Is therefore recommended that the NOAEL of 160 mg/kg/day
Identified In the Lake et al., (1978} study be used 1n deriving the 10-day
HA. Although the method of treatment was gavage in the study by Lake et al.
(19"?8) and diet In the study by Agarwal et al. (1985a), treatment-related
effects across similar dose ranges, Including Hver effects 1n both studies
In two sensitive strains of rats, support use of 160 mg/kg/day as NOAEL In
rat> given BBP orally for 14 days.
The 10-day HA 1s calculated as follows:
10-day
160 mq/kq/day x 10 kg
100 x 1 i/day
= 16 mg/l (rounded to 20 mg/l)
04780
VIII-27
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where:
160 mt|/kg/day
10 kg
100
1 l/diy
NOAEL based on the absence of liver and testkular
effects from animal data (Lake et a!., 1978}
assumed weight of a child
uncertainty factor, according to U.S. EPA and
ODW/NAS guidelines for use with a NOAEL from an
animal study
assumed water consumption by a child
Assessment of Longer-term HA — Long-term exposure to BBP causes
adverse (ffects to the testes of male rats. The only study available for
t
the derivation of longer-term HAs 1s the 26-week feeding study conducted by
NTP {1985). Male F344 rats consuming a dietary level of 2.5% BBP exhibited
testUular lesions characterized by atrophy of seminiferous tubules and
aspermla. The corresponding dose from data given, assuming 5% food
consumption/day and 200 g body weight, Is 1417 mg/kg/day. At this level
rats also experienced significantly depressed body weight gains and
significant Increases In the organ-to-body weight ratios In the brain, right
kidney, light testes and liver. Rats given dietary levels of 0, 0.03, 0.09,
0.28 and 0.83V. BBP for 26 weeks exhibited no grossly observable effects on
male reproductive organs. Corresponding doses assuming -300 g bw and -17 g
of food :onsumpt1on/day from data presented 1n the report are 0, 17.0, 51.0,
159 and 470 mg/kg/day, respectively. At 0.83%, the effects noted were
significantly (p<0.05) Increased absolute liver weight. Increased
Hver-to-body weight and Hver-to-braln weight ratios and Increases 1n mean
corpuscular hemoglobin. Liver-to-body weight ratios significantly (p<0.05)
Increased for the brain, right kidney and liver at the 2.5% level; however,
llver-to-braln weight ratios did not significantly (p<0.05) Increase. The
it T T T -"Id
-------
differences may have been due to the reduced weight gain and testlcular
effects at 2.5% BBP. The liver may be a more sensitive endpolnt than the
testes since liver effects were observed at a lower level (0.83%) than
testlcular effects (2.5%). Therefore, 0.28% or 159 mg/kg/day will be used
as a NOAEL to derive the longer-term HAs as follows:
Longer-term HA
(child)
159 mg/kq/day x 10 kg ,, Q ,
inn/x i o/da'v— -15.9 mg/8,
luu x i i/oay (rounded to 20 mg/4)
where:
159 mg/kg/day = NOAEL based on the absence of Increased liver
weights In rats (NTP, 1985)
10 kg
100
1 i/day
= assumed weight of a child
= uncertainty factor, according to U.S. EPA and
ODW/NAS guidelines for use with a NOAEL from an
animal study
= assumed water consumption by a child
Longer -ter. HA - 159 .qAq/day x 70 kq
(adult) tuu
^
x * l/ady (rounded to 60 mg/i)
where:
159 mg/kg/day
70 kg
100
2 i/day
= NOAEL based on the absence of Increased liver
weight In rats (NTP, 1985)
= assumed weight of an adult
= uncertainty factor, according to U.S. EPA and
ODW/NAS guidelines for use with a NOAEL from an
animal study
= assumed water consumption by an adult
04760
VIII-29
07/31/91
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Assessment of Long-Term Exposure Data and Derivation of a DUEL -- NTP
(1985) Is also the only available study for the derivation of the DWEl (U.S.
EPA, 1991). The DWEL Is derived as follows:
Step 1 - RfD Derivation
RfQ
uuu
^ Q<159 mg/kg/day
(rounded 0,2 mg/kg/day)
where:
159 mg/kg/day = NOAEL derived from orally exposed rats (NTP, 1985)
1000
uncertainty factor, according to U.S. EPA and
OOW/NAS guidelines for use wHh a NOAEL from animal
data, for less than lifetime exposure and to protect
sensitive members of the human population
Step 2 - DWEL Derivation
DWEL = —
kq
2 i/day
where:
0.2 mg/kg/day = RfD
70 kg
2 l/day
assumed weight of an adult
assumed water consumption by an adult
The 1-day HA, 10-day HA and DWEL values calculated for BBP and the
effects levels used In calculation are summarized 1n Table VIII-1.
Studies Considered for Noncardnogenlc Quantification — DBP. No
Informatlcn was found 1n the available literature on the effects of DBP In
humans anl Information on effects in animals 1s limited. The teratogenlc
effects o: PAEs following oral administration were studied by Nlkonorow et
04780
VIII-30
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-------
al. (1973). In this study female WUtar rats were administered 120 and 600
rag/kg/day DBP In olive oil for -3 months and during mating. Upon
confirmation of conception the administration of DBP was discontinued. On
day 21 the uteri and fetuses were removed. Results of this study Indicated
that fetal weight was significantly (p<0.05) reduced at 600 mg/kg/day DBP.
No detectable differences were observed In the number of sternum
ossification foci, the development of the bones at the base of the skull,
pa** on the front and hind legs, or rib fusion 1n fetuses from treated rats
at either dose level when compared with the control animals.
Cater et al. (1977) found that DBP Induced testlc'ular atrophy In young
{3-4 weeks old) male Sprague-Dawley rats. DBP was dissolved In corn on and
administered by gavage In doses of 500, 1000 and 2000 mg/kg/day for 14 days,
while control animals received corn oil In a volume of 5 ml/kg. Testes
we'ghts were measured on days 4 and 6 for 500, 1000 and 2000 mg/kg/day doses
of OBP. In addition body weight and relative liver, kidney and testes
weights were measured on days 3, 7, 10 and 14 at 2000 mg/kg/day. The
Initial effect was a progressive reduction In weight of the testes. At 4
days, however, 500 mg/kg/day DBP did not have an effect on testes weight. A
significant (p<0.05) reduction 1n the relative testes weight occurred within
6 days at 500 mg/kg/day and within 4 days at 1000 (significance p<0.01) and
2000 (significance p<0.001) mg/kg/day. By 14 days, the reduction at 2000
mg/kg/day amounted to 60-70% of the original weight. Since there was also a
decrease 1n body weight, the authors used "relative testes weight" and found
that on this basis there was still a significant loss of testes weight.
There was a nonstatlstlcally significant Increase In liver weights.
Hlstopathologlc examination of testes tissue after 4 days of 2000 mg/kg OBP
exposure revealed a diminution of both spermatocytes and spermatogonla.
04780
VIII-31
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In a detary study DBP was fed to male and female Fischer 344 rats at 0,
0.6, 1.2 Jnd 2.5% for 21 days (CMA, 1986). Corresponding dose levels were
0, 624, 1234 and 2156 mg/kg/day for males and 0, 632, 1261, and 2107
mg/kg/day for females. Absolute and relative liver weights were
slgnlfIcartly Increased In both male and female rats at all treatment
levels. 4ale rats fed 2.5% DBP had severe testlcular atrophy and signifi-
cantly lower testes weight. Samples of liver from rats administered the
2.5% level showed "moderate" peroxlsomal proliferation. In addition lauMc
acid 11- and 12-hydroxylase Increased In males given 0.6, 1.2 and 2.5% and
In females given 2.5%. Cyanide-Insensitive palnvUoyl CoA oxidation
Increased at 1.2 and 2.5% In males and 2.5% In females.
Smith (1953) studied the effects of feeding DBP to groups of 10 male
5-week-old Sprague-Dawley rats, weighing 55-65 g. Rats were fed dietary
levels of 0, 0.01, 0.05, 0.25 and 1.25% DBP for 1 year. The dietary Intakes
for DBP vere 0, 5, 25, 125 and 600 mg/kg/day, respectively, estimated from a
figure depicting dally Intake In mg/kg In Smith (1953). Survival rates were
not repotted for the three lowest dose groups. In the group fed 1.25% DBP,
half (presumably 5/10) of the animals died during the first week of the
experlmert while the remaining animals gained weight comparable with
controls It was not Indicated whether the deaths were thought to be
treatmen .-related. Necropsies were performed when rats showed marked weight
loss or signs of severe Infection. Animals alive at the end of 1 year were
sacrlf1c;d and examined for gross pathologic changes. While 1t was stated
that several organs were sectioned and stained, the results of hlstologlc
evaluatljn were not reported. Of the animals surviving, no adverse effects
on growth, survival, gross pathology or hematology were observed among those
04780
VIII-32
07/02/91
-------
fed diets containing 0.01,, 0.05 or 0.25% DSP. The dally Intake of food and
plastlclzer (mg/kg bw/day) decreased as the rats Increased 1n size. No
changes In hematologk parameters or gross pathology were observed at any
dose level.
Shlota and Nlshlmura (1982) found retarded ossification 1n mice fed
diets of 80, 180, 370, 660 and 2100 mg/kg/day DBF on days 0-18 of gestation.
At the 660 mg/kg/day level, reduced fetal weight and retarded ossification
were observed. Among rats fed diets of 2100 mg/kg/day, decreased maternal
weight was observed along with reduced weight In the fetuses, retarded
ossification and neural tube defects In the fetuses. The authors concluded
that delayed ossification was related to the general underdevelopment of the
fetuses. The maximum nonembryotoxlc dose as stated by the authors would be
370 mg/kg/day D8P.
Quantification of Noncardnogenlc Effects — DBP.
Assessment of Acute Exposure Data and Derivation of the 1-Day HA --
No information was found In the available literature on the acute toxldty
of DBP to humans. Cater et al. (1977) found that DBP Induced testlcular
atrophy 1n young (3-4 weeks old) male Sprague-Oawley rats. DBP was
administered by gavage In doses of 500, 1000 and 2000 mg/kg/day for 14
days. Effects of treatment on body weight and relative liver, kidney and
testes weights were measured on days 3, 7, 10 and 14 at 2000 mg/kg/day. In
addition, testes weights were measured on days 4 and 6 for 500, 1000 and
again for 2000 mg/kg/day. At 4 days of 500 mg/kg/day treatment testes
weights were not affected. Liver weights Increased but were not
statistically significant. Treatment at 4 days of 1000 and 2000 mg/kg/day
04760
VIII-33
05/16/91
-------
significantly reduced testes weight and at 2000 rog/kg/day diminished both
spermatocytes and spermatogonla. By 14 days 2000 mg/kg/day reduced the
testes to 60-70% of the original weight.
In another study, CHA (1986) reported significant Increases In absolute
and relative liver weights after 21 days of exposure to doses of 624 and 632
mg/kg/day In male and female Fischer 344 rats, respectively.
Cater et al. (1977) Identified a NOAEL of 500 mg/kg/day for testlcular
effects in male rats. Liver weights Increased but were not statistically
s1gn1f1cait. After 21 days of exposure, CMA (1986) reported significantly
Increased absolute and relative liver weights. It appears that for a 1-day
exposure testes are the most sensitive organ and, therefore, the NOAEL of
500 mg/kg'day will be used to derive the 1-day HA as follows:
HA = 500 mq/kq/day x 10 kq ^
100 x 1 I/day
where:
500 mg/kg/day = NOAEL based on the absence of decreased testes
weight from animal data (Cater et al., 1977)
10 kg
100
1 I/day
= assumed weight of a child
= uncertainty factor, according to U.S. EPA and
OOW/NAS guidelines for use with a NOAEL from an
animal study
= assumed water consumption by a child
Assessment of Acute Exposure Data and Derivation of the 10-Day HA —
Cater et al. (1977) observed decreased spermatocytes and spermatogonla as
well as significantly reduced testes weight after 4 days exposure to 2000
mg/kg/da> DBP. In addition, testes weights were significantly reduced at
04780
VIII-34
07/02/91
-------
500 mg/kg/day at 6 days and 1000 mg/kg/day at 4 days of exposure (LOAEL =
500 mg/kg/day). CMA (1986) reported significant Increases In absolute and
relative liver weights after 21 days of exposure to -600 mg/kg/day In rats.
However, Smith (1953) reported a NOAEL of 125 mg/kg/day for growth,
survival, gross pathology or hematology after 1 year exposure of 08P to male
rats. In light of these data, 125 mg/kg/day appears to be a reasonable
estimate of a NOAEL after 10 days of exposure and will be used to derive the
10-day HA In addition to the longer-term HAs and DWEL.
The 10-day HA Is calculated as follows:
10-day
125 mq/kq/day x 10 kg
100 x 1 i/day
12.5 mg/s.
(rounded to 10 mg/i)
where:
125 mg/kg/day = NOAEL based on the absence of Increased mortality
and hematologlc effects (Smith, 1953)
10 kg
100
1 l/day
= assumed weight of a child
= uncertainty factor, according to U.S. EPA and
ODW/NAS guidelines for use with a NOAEL from an
animal study
= assumed water consumption by a child
Derivation of Lonqer-Term HA — The only study available for the
derivation of longer-term HAs Is by Smith (1953). Male rats fed diets
containing 5.0, 25 and 125 mg/kg/day for 1 year experienced no adverse
effects on growth, survival, gross pathology or hematology. At a level of
600 mg/kg/day DBP, half of the animals died. The remaining animals gained
weight as did the controls. The limitations of this study, such as few
animals of one sex, the lack of animal survival data, animal Infections and
0*780
VI11-35
07/31/91
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the 50% survival rate among the high-dose group, combined with the lack of
mlcropath>1og1c examination, must be noted In Interpreting these results.
HA -
(rounded to 10 mg/t)
where:
125 mj/kg/day = NOAEL In male rats based on the absence of
Increased mortality and hematologic effects (Smith,
1953)
10 kg = assumed weight of a child
100 = uncertainty factor, according to' U.S. EPA and
ODW/NAS guidelines for use with a NOAEL from an
animal study
1 l/day = assumed water consumption by a child
Longer-term HA . 125 mq/kq/daY x 70 kq = ^
(adul 10D * * l/day (rounded to 40 mg/i)
where:
125 rrg/kg/day = NOAEL In male rats based on the absence of
Increased mortality and hematologlcal effects
(Smith, 1953)
70 kc = assumed weight of an adult
100 = uncertainty factor, according to U.S. EPA and
OOW/NAS guidelines for use with a NOAEL from an
animal study
2 l/cay = assumed water consumption by an adult
Assessment of Lonq-Term Exposure Data and Derivation of a DUEL —
Smith (I'i53) is also the only available study for the derivation of the DWEL
(U.S. EP/., 1991). The DWEL is derived as follows:
VIII-36
07/31/91
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RfD .
12'5
. 0.125 rcg/kg/day
(rounded to 0.1 mg/kg/day)
where:
125 mg/kg/day
1000
NOAEL In male rats based on the absence of
increased mortality and hematologlc effects (Smith,
1953)
uncertainty factor, according to U.S. EPA and
OOW/NAS guidelines for use with a NOAEL from a
subchronlc animal study
where:
0.1 mg/kg/day
70 kg
2 l/day
nun 0.1 mq/kq/day x 70 kg _ ,.
DWEl = 2 g/dav * 3'5
* * {rounded to 4 mg/i)
= RfD
= assumed weight of an adult
= assumed water consumption by an adult
Studies Considered for Noncardnoqenlc Quantification — PEP. No
information was available on the effects of QEP \n humans. Information on
DEP toxidty in animals 1s limited. In a 2-year study (Food Research Labor-
atories, Inc., 1955), groups of 30 rats (15/sex) were fed 0.5, 2.5 or 5.0%
levels of DEP (250, 1250 or 2500 mg/kg bw/day, respectively) In the diet.
No effects were observed at levels of 0.5 or 2.5%. OEP at the 5.0% dose
level resulted In a small, but significant decrease In the growth rate of
the rats without any effect on food consumption. Thus, 5.0% DEP appeared to
affect the efficiency of food conversion to body mass. No Information was
available on the numbers of rats surviving (42% or more of each sex
survived) the 2-year study period and hlstopathologlc examination was
performed only on the 5.0% dose group. Statistical analysis was only
-------
conducted on organ weights and excluded statistically higher rats from the
respective group averages. Also, as part of this study, 13 young mongrel
dogs were fed DEP In the diet at levels of 0, 0.5, 1.5, 2.0 and 2.5% for 1
year. Pr >blems were encountered with palatabllHy of DEP 1n the diet. As a
result, tie dogs received varying exposures to DEP before each dog attained
stablUza :1on at the highest tolerated dietary level. Accordingly, three
dogs were maintained at 0.5%, one each at 1.5 and 2.0%, and three at the
2.5% level. The average weekly Intakes of DEP were computed and found to be
0.8, 2.4, 3.5 and 4.4 g/kg/week 1n order corresponding to Increasing dietary
level. N) effects were noted In dogs as a result of DEP exposures.
Brown et al. (1978) also studied the long-term oral toxldty of DEP 1n
rats. Groups of 15 CD strain rats of each sex were given diets containing
0, 0.2, 1.0 or 5.0% DEP for 16 weeks. The authors estimated the mean
Intakes to be 0, 150, 770 and 3160 mg/kg/day In males and 0, 150, 750 and
3710 mg/1-g/day In females, respectively. Autopsies and hlstologk exami-
nations i'ere conducted at the end of 16 weeks. No changes 1n behavioral
patterns or clinical signs of toxldty were observed. Female rats fed diets
containing 1% DEP and both sexes fed diets of 5% DEP gained significantly
less welcht than the controls. Mean food consumption of rats of both sexes
given 5% DEP and females given 1% DEP was significantly lower than that of
control rats. In order to Judge whether palatabHUy was the possible cause
1n decreased weight gain, a paired-feeding study was conducted. Test rats
fed 5% DIP consumed more food (total) and gained less weight than controls.
Weights of the brain, heart, spleen and kidney were significantly lower 1n
male and female rats fed 5% DEP. Female rats given 5% DEP showed a
statistically significant Increase In full caecum weight. There were no
04780
V1II-38
07/02/91
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significant changes In the absolute weights of any organs below the 5% DEP
dietary level. Relative weights of the brain, liver, kidney, stomach, small
Intestine and full caecum were significantly higher 1n both sexes at the 5%
dietary level when compared with the controls. These changes were attrib-
uted to the compound-related effect on growth rate since dose-related
changes In gross or microscopic pathology were not observed. No other
effects were observed.
Quantification of Noncardnoqenlc Effects ~ DEP.
Assessment of Acute Exposure Data and Derivation of the 1-Day HA —
There were no satisfactory studies for the derivation of a 1-day HA.
Assessment of Acute Exposure Data and Derivation of the 10-Day HA —
There were no satisfactory studies for the derivation of a 10-day HA.
Assessment of Longer-Term HA — There Is only one subchronlc study
appropriate for the derivation of longer-term HAs. Brown et al. (1978) fed
CD rats diets containing 0.2, 1.0 and 5.0% DEP (150, 770 and 3160 mg/kg/day,
male; 150, 750 and 3710 mg/kg/day, females) for 16 weeks. Female rats fed
1% DEP and both sexes fed 5% DEP gained significantly less weight than the
controls. A paired-feeding study showed this weight difference was not from
palatabllHy. Both the liver and kidneys were hlstologlcally normal at all
DEP dietary levels. Relative kidney weights at the 5% dose level were 0.67
and 0.69 g/100 g bw In males and females, respectively, compared with
control values of 0.57 and 0.62 g/100 g bw In males and females, respec-
tively. Although slight but significant (p<0.05) changes were seen In
females at the 1% level, the use of multiple T tests for the comparisons
04780
VIII-39
05/16/91
-------
(without correction) and the small magnitude of the changes Indicates that
the "\% feeding level (750 mg/kg/day) represents a NOAEL 1n this study.
Therefore, the NOAEL (750 mg/kg/day, females) determined from the Brown et
al. (1978) study will be used to derive the longer-term HAs as follows:
750 mg/kg/day x 10 kg
Longer-term HA
(child)
100 x 1 i/day
75 mg/S.
(rounded to 80 mg/a)
where:
750 mc/kg/day
10 kg
1 i/d
-------
Assessment of Lonf-terni Exposure Data and Derivation of a DWEL —
There are two possible long-term studies for derivation of a lifetime DWEL.
The Brown et al. (1978) 16-week study as described for the longer-term HA 1s
also considered 1n deriving the DWEL. In a 2-year dietary study, Food
Research Laboratories, Inc. (1955) observed similar results at 5.0% DEP as
1n the Brown et al. (1978) study. They reported a NOEL at 2.5% or 1250
mg/kg/day. Deficiencies 'in the reporting of the study reduce confidence 1n
the use of this data, since complete Mstopathologles were not conducted and
no Information was available on the number of rats surviving the 2-year
study by Food Research'Laboratories, Inc. (1955). Therefore, the NOAEL (750
mg/kg/day, females) determined from the Brown et al. (1978) study will be
used to derive the lifetime DWEL (U.S. EPA, 1991).
Step 1 - RfD Derivation
RfD
750 mg/kg/day
1000
0.75 mg/kg/day
(rounded to 0.8 mg/kg/day)
where:
750 mg/kg/day = NOAEL In orally exposed rats based on lack of
kidney and weight gain effects (Brown et al., 1978)
1000
= uncertainty factor, according to U.S. EPA and
ODW/NAS guidelines for use with a NOAEL from a
subchronlc animal study
Step 2 - DWEL Derivation
m 0.8 mq/kq/day x 70 kq _ '
2 I/day
where:
0.8 mg/kg/day = RfD
70 kg = assumed weight of an adult
2 l/day = assumed water consumption by an adult
04780
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Studies Considered for NoncardnogenVc Quantification — PHP. No
Information was available on the effects of DMP 1n humans. The only studies
available on acute oral toxldty 1n animals used lethality as the toxU end-
point. Tre only long-term oral data was from an unpublished review article
(Lehman, 1955).
Quantification of Noncardnoqenlc Effects — PHP. The 1-day. 10-day
or longer-term HAs and a lifetime DWEL for OHP cannot be derived due to
Insufficient Information.
Carcinogenic Effects
There are very few animal carcinogenic studies on PAEs considering the
number of esters. The available human studies are Inadequate due to the
small numters of subjects studied and the lack of quantitative Information
on levels and duration of exposure. The human studies were designed to
assess todc effects caused by PAEs. However, there Is adequate data to
consider !>EHP to be a Group 82 compound (I.e., probable human carcinogen)
based on significant Increases 1n liver tumor responses 1n rats and mice of
both sexe; . B8P has been classified as a Group C compound (I.e., possible
human can inogen) based on mononuclear cell leukemia In female rats. OBP,
DEP and DMP are classified as Group 0 (I.e., not classifiable) since
pertinent data regarding cardnogenlclty was not located 1n the available
literature (U.S. EPA, 1986; These classifications have all been verified
by the CR/VE Work Group.
Studlts Considered for Carcinogenic Quantification — DEHP. In an NTP
study (19ii2a), 50 male and 50 female Fisher 344 rats per group were fed 6000
04780
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or 12,000 ppm DEHP In the diet for 103 weeks. Similarly, groups of 50 male
and 50 female B6C3F1 mice were given 3000 or 6000 ppm OEHP In the diet for
103 weeks. In this study rodent meal was provided in such a way that
measured food consumption actually represented significant spillage and
waste rather than true food Intake. For this reason a standard food
consumption rate of 13% of mouse and 5% of rat body weight was used 1n the
dose conversion. Corresponding dose levels are 300 and 600 mg/kg/day for
rats and 390 and 780 mg/kg/day for mice (low and high dose, respectively).
Doses were those estimated to be maximally and one-half maximally tolerated
1n preliminary 90-day Subchronlc feeding studies. The animals were -6 weeks
old when the study began, and survivors were sacrificed at 105 weeks. All
animals were necropsled, and a hlstologU examination of tissues was made.
Treeted animals were compared with 50 matched controls of each sex.
Median survival times were >104 weeks for all groups. Body weight loss
was evident In low- or high-dose animals In each treatment group.
A statistically significant {p<0.05 or better) Increase 1n the Incidence
of hepatic neoplasms In both rats and mice treated with DEHP was found as
described In Table VIII-2. Hepatic tumors are described as hepatocellular
carcinomas and neoplastlc nodules 1n rats and hepatocellular carcinomas and
hepatocellular adenomas In mice. Metastasis of liver carcinoma to the lung
1n mice was found 1n 5/50 high-dose males, 7/49 low-dose males, 7/50 high-
dose females and 2/50 low-dose females (NTP, 1982a).
Carpenter et al. (1953) evaluated the chronic toxlclty of OEHP 1n
Sherman rats. The untreated control group and each treatment group con-
04780
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slsted of 32 males and 32 female animals, which were 60 days old when treat-
ment began. Dosage groups were given 400, 1300 or 4000 ppm DEHP in the diet
for 1 year. After 1 year of treatment, each group was reduced to a maximum
of eight males and eight females, and treatment was continued for another
year until sacrifice of the survivors.
A filial (F.) generation of animals was produced from 'animals In the
control and 4000 ppm groups, which had been given the appropriate diet for
120 days. Each Utter was reduced to two males and two females when the
pups had reached 15 days of age, and 32 males and 32 females In the F
generation were assigned to each of the control and 4000 ppm groups. All
surviving F, animals were sacrificed after being maintained on control or
4000 ppm diets for 1 year.
All animals were subjected to necropsy and hlstopathologlc examination.
Only 40-47% of the animals In each group, Including FI animals, survived
1 year. Of the animals allowed to be on study for 2 years, 61-71% died
before termination of the study at 2 years. Lung Infection was diagnosed as
the primary cause of death.
No malignant tumors were observed In this study. One to five rats in
each group (males and females combined} had tumors with no treatment-related
trend In evidence; however, the tumor types were not Identified.
A carcinogenic effect of DEHP was not evident 1n this study. However,
thH study Is weakened by the fact that of the 32 animals of each sex In
each group of the study {excluding the FI animals, all of which were
allowed to survive 1 year), only eight were allowed to survive beyond 1 year
04780
VIII-45
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of treatmj'nt. Furthermore, mortality was high with respect to all groups.
Hence, an Insufficient number of animals were available for a lifetime
feeding study of DEHP cardnogenlcHy 1n rats.
Carperter et al. (1953) also Investigated the toxldty of DEHP 1n long-
term studies In guinea pigs and dogs. Groups of 23 or 24 guinea pigs of
each sex vere fed 1300 or 4000 ppm DEHP 1n the diet for 1 year until termi-
nation of the study. Four dogs were dosed 5 days/week by oral administra-
tion of DEHP In capsules at a dosage of 0.03 ml/kg for the Initial 19
doses followed by 240 doses at 0.06 ma/kg. The dogs were sacrificed at
the end cf the 1-year dosing period. Pathologic evaluation of the guinea
pigs and dogs did not reveal a carcinogenic effect of DEHP. However, the
treatment and survival periods for these animals were considerably below
their lifetimes.
Since the animal evidence 1s considered by U.S. EPA to be sufficient and
there 1s 10 human data, according to the U.S. EPA Guidelines for Carcinogen
Risk Assessment, OEHP Is classified as a B2 carcinogen (U.S. EPA, 1986).
This classification was verified (10/07/87) by the CRAVE Work Group (U.S.
EPA, 1991 .
Quant flcatlon of Carcinogenic Effects — DEHP. The risk calculation
1s based on the liver tumor data from the NTP study (1982a) on DEHP.
Hepatocel lular carcinoma and hepatocellular adenoma Incidence were reported
1n both nale and female rats and male and female mice. * However, male mice
were the most sensitive group. The combined Incidence of hepatocellular
carcinoma; and adenomas In male mice (see Table VIII-2) was 14/50 for con-
04780
VIII-46
08/08/91
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trol animals, 25/48 for 390 mg/kg/day animals and 29/50 for 780 mg/kg/day
animals.
NTP (19823), Kluwe et al. (1982a), U.S. EPA {19875} and IARC (1982)
concluded that these results provide sufficient evidence of d1(2-ethyl-
hexyl) phthalate-lnduced carclnogenlclty In rats and mice. This conclusion,
however, 1s disputed. Northrup et al. (1982) claim that the NTP (1982a)
results are equivocal since the MTD was exceeded 1n some treatment groups,
Incidences of liver tumors varied within different control groups of the
same species and sex; and treated animals may have been malnourished.
Northrup et al. (1982) also claimed that the rodent data cannot be used to
predict carcinogenic risk In humans because DEHP 1s metabolized differently
1n rats than In humans. In response, Kluwe et al. (1983) noted that the MTD
was not exceeded since there were no compound-related effects on survival,
the Incidence of Hver tumors was Increased 1n OEHP-treated animals
regardless of the control data used and the differences In metabolism
between rodents and humans would not affect the carcinogenic response In
rodents. More recently, Turnbull and RodMcks (1985) concluded that using
NTP (1982a) data to estimate OEHP-lnduced carcinogenic risk to humans will
probably overestimate actual risk. This conclusion was based on the
differences between rodents and primates 1n the metabolism of DEHP, a
nonlinear relationship between the administered dose of DEHP to the dose of
the "proximate carcinogenic species" In rodents, the fact that the
"proximate carcinogenic species," which 1s hypothesized to Induce cancer, Is
produced to a greater extent In rodents than In primates and that there are
differences In target-site sensitivity between humans and rodents for liver
tumors 1n general.
VIII-47
08/08/91
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The dose-response data used 1n the potency calculations Included rats
with el .her hepatocellular carcinomas or neoplastlc nodules and mice with
either lepatocellular carcinomas or adenomas 1n the NTP (1982a) bloassay.
Hale ant! female response data from the rat and mouse were used to calculate
i
a q-j* falue (Table VIII-3). The oral slope factors were 3.18x10~' and
4.52x10"' (mg/kg/day)"1 for male and female rats, and 1.41xlO~3 and
1.03x10"* (nig/kg/day)"1 for male and female mice. The value of
1.41x10"* represents the most sensitive response and hence 1s selected as
the potency value for DEHP. This value and the following risk/concentration
calculations should be viewed as Interim since 1t would appear that
metabol sm and pharmacoklnetU considerations should be accounted for 1n the
dose response analysis. The examination of these factors has been done by
Turnbul I and RodMcks (1985). but has not been further evaluated 1n this
documen;. These refinements will be further evaluated before the risk
values are put to final use. The upper-bound estimate of the cancer risk
due to the Ingestlon of 2 1 of water for a 70-year lifetime with a
concent-atlon of contaminant 1s 4.0xlO~7 {wg/l)~l. Since risk 1s
assumed to be linear with dose 1n this range, risk factors of 10"*, 10"s
and 10~s correspond to 300, 30 and 3 vq/i, respectively.
Studies Considered for Carcinogenic Quantification — BBP. A bloassay
was performed to evaluate the carcinogenic potential of BBP 1n rats and mice
(Kluwe et al., 1982b; NTP. 1982b). Dietary levels of 6000 and 12,000 ppm
(780 and 1560 mg/kg/day) BBP were each fed to groups of 50 male and 50
female F344 rats and 50 male and 50 female B6C3F1 nice. Untreated groups of
50 mains and 50 females of each species were used as controls. The female
-------
-------
TABLE VIII-3
Cancer Risk Calculations
Animal Dose
(mq/kg/day)
Animal/Se::
Rat/male
Rat/female
Mouse/mail?
Mouse/fem
-------
-------
rats and Doth sexes of mice were maintained on these diets for 103 yeeks;
however, ingestlon rates and average weights were not available from the
study. The male rats at both dose levels experienced high mortality within
the first 30 weeks of the study, at which time the male rat study was
term1nate< . No chronic or carcinogenic effects were observed 1n male or
female m1:e. Among female rats, however, an Increase In mononuclear cell
leukemia uas observed at the higher dose level.
Quant f1cation of Carcinogenic Effects — B8P. The available data
meets the criteria for limited animal evidence based on mononuclear cell
leukemia In female rats. Hence BBP 1s considered to be a Group C, possible
human carcinogen according to U.S. EPA Guidelines for Carcinogen Risk
Assessment This classification has been verified (08/26/87) by the CRAVE
Work GrouD (U.S. EPA, 1991). A bloassay was performed by the NTP (1982b) to
evaluate the carclnogenUHy of BBP In both rats and mice. The male rats at
both dose levels experienced high mortality within the first 30 weeks of the
study due to apparent Internal hemorrhaglng; all male rats were terminated
at 30 weeks. Among female rats a statistically significant Increase In
mononuclear cell leukemia was observed at the high-dose level by comparison
with boti concurrent controls and historical controls. The conclusions
reached Dy the peer review group of this study Indicate that BBP was
"probably" carcinogenic 1n female rats. Although the Increase In leukemia
was stat stlcally significant, the biological relevance of this finding was
questioned due to the background Incidence of mononuclear cell leukemia In
Fischer 344 rats. The NTP 1s currently repeating the rat portion of the
cancer bloassay for BBP. Testing began In June, 1989 (NTP, 1991).
04780
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Studies Considered forCarcinogenic Quantification— DBF. Pertinent
data regarding the carclnogenlcHy of DBP could not be located 1n the
available literature. According to U.S. EPA guidelines DBP 1s classified as
Group D, not classifiable. This classification was verified (08/26/87) by
the CRAVE Work Group (U.S. EPA, 1991).
Studies Considered for Carcinogenic Quantification — D€P. Pertinent
data regarding the carclnogenlcHy of DEP could not be located 1n the
available literature. According to U.S. EPA guidelines DEP 1s classified as
Group D, not classifiable. This classification was verified (08/26/87) by
the CRAVE Work Group (U.S. EPA, 1991).
Studies Considered for Carcinogenic Quantification -- PHP. Pertinent
data regarding the carclnogenlcHy of DMP could not be located 1n the
available literature. According to U.S. EPA guidelines DMP 1s classified as
Group D, not classifiable. This classification was verified (08/26/87) by
the CRAVE Work Group (U.S. EPA, 1991).
Existing Criteria and Standards
The American Conference of Industrial Hyglenlsts has set a TLV of 5
, as an 8-hour TWA, for DEHP, DBP, DEP and DMP (ACGIH, 1985).
The RfD Work Group verified the following RfDs: 0.02 mg/kg/day for DEHP
(01/22/86); 0.2 mg/kg/day for BBP (06/15/89); 0.1 mg/kg/day for DBP
(01/22/86); and 0.8 mg/kg/day for OEP (07/16/87). These assessments are all
available on IRIS (U.S. EPA, 1991). Quantitative data are not available for
DMP. The CRAVE Work Group has verified the following cancer classlflca-
04780
VIII-51
08/08/91
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Hons: Group B2 for DEHP (10/07/87}; Group C for BBP (08/26/87); and Group D
for DBP, D£P and DMP (08/26/87). The oral slope factor for DEHP, the only
one of thsse five phthalates to have a quantitative cancer risk Assessment,
1s 4xlO~7 (yg/i)"1. These assessments are also available on IRIS
(U.S. EPA 1991).
Interactionswith Other Chemicals
PAEs lave been shown to Interact with other compounds In a synerglstlc
or antagonistic manner. Carbon tetrachloMde, barbiturates and
organophoiphate Insecticides (applied following PAE exposure) were shown to
act synerglstlcally with PAEs (Seth et al., 1979; Rubin and Jaeger, 1973;
Al-Badry and Knowles, I960}. Antagonistic effects were noted between PAEs
(and testlcular zinc levels), methylenedloxyphenyl compounds, paraoxon and
simultaneously applied organophosphate Insecticides (Cater et al.. 1977;
Foster et al., 1980; Melancon and Lech, 1979; Al-Badry and Knowles, 1980).
DEHP has been shown to Increase antlpyrlne metabolism In rats, possibly by
Inducing hepatic mlcrosomal enzymes (Pollack and Shen, 1984). Interaction
between i)EHP and ethanol In rats has been studied by Agarwal et al.
(1982a). DEHP produces changes 1n the pharmacologlc response to ethanol by
altering ihe activities of alcohol dehydrogenase and aldehyde dehydrogenase.
Agarwjl et al. (1982b) examined the effects of DEHP administration on
phenobarbUal-lnduced sleeping time 1n rats. The authors concluded that
PAEs Interfere with blotransformatlon mechanisms of hepatic mlcrosomal drug-
metabolizing enzymes. The effects of DEHP on the activity of various
enzymes differed between oral and Intraperltoneal exposure routes.
04780
VIII-52
08/08/91
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Special Groups at R1sk
Patients receiving blood transfusions or hemodlalysls constitute a
high-risk subpopulatlon for PAE exposure. This group rr.ay receive excessive
quantities of PAEs during transfusion or hemodlalysls due to leaching of PAE
plastlclzers from plastic blood bags or plastic tubing.
Hlllman et al. (1975) studied the occurrence of necrotUIng enterocoll-
t1s and OEHP tissue concentrations 1n Infants who had received treatment
using arterial catheters containing DEHP. Higher DEHP content was found In
catheterlzed Infants with necrotlzlng enterocolHIs than In Infants that had
been catheterIzed but did not develop this disease. While the study did not
show a causal relationship, It did demonstrate that DEHP accumulated In the
tissues of critically 111 Infants.
Gibson et al. (1976) estimated that the amount of DEHP delivered to a
patient during hemodlalysls ranged from 1.5-150 mg for dialysis lasting 15
minutes to 5 hours. Another study suggested that exposure to dlethyl
phthalate during hemodlalysls may be linked to development of hepatitis
(Neergaard et al., 1971). However, evidence of the causal relationship was
not conclusive.
It 1s also possible that workers In the manufacture of PAEs or In the
plastics Industry constitute a high-risk population. However, little
Information 1s available for these groups. The only prospective cohort
sttdy looked at workers exposed to OEHP for periods of 3 months to 24
years. This study did not demonstrate any compound-related Injury or
disease. Therefore, the degree of risk to workers cannot be quantified
(Thless et al., 1978b).
04780
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9. REFERENCES
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Agarwal, O.K., S. Agarwal and P.K. Seth. 1982a. Effect of d1-(2-ethyl-
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Agar^al, O.K., H.H. Lawrence, L. 0. Nunez and J. Autlan. 1985c.
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Albro, P.M., J.T. Corbett, J.L. Schroeder and S.T. Jordan. 1983a. Incorpor-
ation of radioactivity From labeled d1-(2-ethylhexyl) phthalate Into DNA of
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Bell, P.P. 1982. Effects of phthalate esters on I1p1d metabolism in vari-
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Bell, P.P. and D.A. Buthala. 1983. Biochemical changes 1n liver of rats
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