•:CAO-CIN-.jCC9
Un,i.d States August. 1963
Environmental Protection . 3t ' inm
Agency Revised August. 1991
Research and
Development
DRINKING WATER CRITERIA DOCUMENT FOR
PHTHALIC ACID ESTERS (PAES)
Prepared for
OFFICE OF WATER
Prepared by
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Cincinnati, OH 45268
DRAFT: 00 NOT CITE OR QUOTE
NOTICE
This document I* a preliminary draft. It has not been formally released
by the U.S. Envlroraental Protection Agency and should not at this stage be
construed to represent Agency policy. It Is being circulated for comments
on Its technical accuracy and policy Implications.
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DISCLAIMER
This document has been reviewed In accordance with the U.S.
Environmental Protection Agency's peer and administrative review policies
and approved for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
11
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FOREWORD
Section 1412 (b)(3)(A) of the Safe Drinking Water Act, as amended in
1986, requires the Administrator of the Environmental Protection Agency to
publish maximum contaminant level goals (MCLGs) and promulgate National
Primary Drinking Water Regulations for each contaminant, which, in the
Judgment of the Administrator, may have an adverse effect on public health
and which Is known or anticipated to occur in public water systems. The
MCLG is nonenforceable and is set at a level at which no known or antici-
pated adverse health effects in humans occur and which allows for an
adequate margin of safety. Factors considered in setting the MCLG include
health effects data and sources of exposure other than drinking water.
This document provides the health effects basis to be considered in
establishing the MCLG. To achieve this objective, data on pharmacoklnetics,
human exposure, acute and chronic toxicity to animals and humans, epidemi-
ology and mechanisms of toxldty are evaluated. Specific emphasis Is placed
on literature data providing dose-response Information. Thus, while the
literature search and evaluation performed In support of this document has
been comprehensive, only the reports considered most pertinent In the deri-
vation of the MCLG are cited In the document. The comprehensive literature
data base in support of this document Includes Information published up to
1986; however, more recent data may have been added during the review
process.
When adequate health effects data exist. Health Advisory values for less
than lifetime exposures (1-day, 10-day and longer-term, -10% of an Indi-
vidual's lifetime) are Included In this document. These values are not used
In setting the MCLG, but serve as informal guidance to municipalities and
other organizations when emergency spills or contamination situations occur.
Tudor Davis, Director
Office of Science and
Technology
James Elder, Director
Office of Ground Water
and Drinking Water
11
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DOCUMENT DEVELOPMENT
Linoa R. Papa, Document Manager
Environmental Criteria and Assessment Office, Cincinnati
U.S. Environmental Protection Agency
Helen H. Ball., Project Officer
Environmental Criteria and Assessment Office, Cincinnati
U.S. Environmental Protection Agency
Authors
Battelle Columbus Laboratories
Columbus, Ohio
Assessment
Contract #68-03-3229
U.S. Environmental Protection Agency
Linda R. Papa
Annette M. Gatchett
Annie M. Jarabek
Environmental Criteria and Assessment
Office. Cincinnati
U.S. Environmental Protection Agency
Scientific Reviewers
Editorial Reviewer
Judith Olsen
Environmental Criteria and
Office, Cincinnati
Richard A. Carchman
Professor
Medical College of Virginia
William B. Pelrano
Annette Gatchett
Rita S. Schoeny
Cynthia Sonlch-Mullln
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
John L. Egle Jr.
Department of Pharmacology and
Toxicology
Medical College of Virginia
Document Preparation
Technical Support Services Staff: Bette L. Zwayer,
Jacqueline L. Bohanon, Environmental Criteria and
Cincinnati
Klmberly Davidson,
Assessment Office.
iv
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TABLE OF CONTENTS
Page
I. SUMMARY 1-1
II. PHYSICAL AND CHEMICAL PROPERTIES II-l
INTRODUCTION II-l
PREPARATION II-l
ANALYTICAL METHODS II-6
USES AND INDUSTRIAL SOURCES 11-6
DISTRIBUTION '. II-7
FATE AND TRANSPORT 11-7
ADSORPTION 11-14
SUMMARY 11-15
III. TOXICOKINETICS III-l
INTRODUCTION III-l
ABSORPTION III-l
DEHP III-l
BBP IH-5
DBP III-5
DEP IH-6
DMP III-6
DISTRIBUTION III-6
OEHP II1-6
BBP 111-15
DBP 111-16
DEP 111-17
DMP IH-19
METABOLISM 111-19
DEHP 111-19
BBP III-36
DBP 111-37
DEP 111-39
DMP Ill-39
EXCRETION 111-40
DEHP 111-40
BBP 111-46
DBP HI-47
DEP 111-47
DMP IH-48
SUMMARY 111-48
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TABLE OF CONTENTS (cent.)
Paae
IV. HUMAN EXPOSURE '. IV-1
[To be provided by the Office of Drinking Water]
V. HEALTH EFFECTS IN ANIMALS ' V-l
INTRODUCTION V-l
SHORT-TERM ANIMAL TOXICITY V-l
DEHP V-7
8BP V-21
D8P V-24
DEP V-25
DMP V-26
LONG-TERM TOXICITY V-26
DEHP V-26
BBP V-40
QBP V-44
DEP V--45
DMP V-47
REPRODUCTIVE EFFECTS V-47
DEHP V-47
BBP V-67
DBP V-69
DEP V-73
OMP V-75
MUTAGENICITY V-76
DEHP V-77
BBP V-79
DBP V-86
DEP V-86
DMP V-86
CARCINOGENICITY V-86
DEHP V-87
BBP V-95
OBP' V-99
DEP V-99
DMP V-99
SUMMARY V-99
vl
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TABLE OF CONTENTS (cont.)
Page
VI. HEALTH EFFECTS IN HUMANS VI-1
INTRODUCTION VI-1
CLINICAL AND CASE STUDIES VI-1
DEHP VI-1
8BP VI-4
DBP VI-4
EPIDEMIOLOGIC STUDIES VI-5
HIGH RISK SUBPOPULATIONS VI-13
SUMMARY VI-14
VII. MECHANISMS OF TOXICITY VII-1
INTRODUCTION VII-1
INTERACTIONS VII-1
ENZYME INDUCING PROPERTIES VII-2
CELLULAR EFFECTS.. VII-5
MECHANISMS OF REPRODUCTIVE TOXICITY VII-17
SUMMARY VII-18
VIII. QUANTIFICATION OF TOXICOLOGIC EFFECTS VIII-1
INTRODUCTION VIII-1
NONCARCINOGENIC EFFECTS VIII-6
Studies Considered for Noncarclnogenlc
Quantification « OEHP VIII-8
Quantification of Noncarclnogenlc Effects — OEHP. . . . VIII-14
Studies Considered for Noncarclnogenlc
Quantification — BBP VIII-20
Quantification of Noncarclnogenlc Effects -- BBP .... VIII-26
Studies Considered for Noncarclnogenlc
Quantification — DBP VIII-30
Quantification of Noncarclnogenlc Effects — DBP .... VIII-33
Studies Considered for Noncarclnogenlc
Quantification — DEP VIII-37
Quantification of Noncarclnogenlc Effects « DEP .... VIII-39
Studies Considered for Noncarclnogenlc
Quantification — OMP VIII-42
Quantification of Noncarclnogenlc Effects — OMP .... VIII-42
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TABLE OF CONTENTS (cent.)
Paqe
CARCINOGENIC EFFECTS VIII-42
Studies Considered for Carcinogenic
Quantification -- OEHP VIII-42
Quantification of Carcinogenic Effects -- OEHP VIII-46
Studies Considered for Carcinogenic
Quantification -- B8P VIII-48
Quantification of Carcinogenic Effects -- BBP VIII-50
Studies Considered for Carcinogenic
Quantification — DBP VIJI-51
Studies Considered for Carcinogenic
Quantification — OEP VIII-51
Studies Considered for Carcinogenic
Quantification — OMP VIII-51
EXISTING CRITERIA AND STANDARDS VIII-51
INTERACTIONS WITH OTHER CHEMICALS VIII-52
SPECIAL GROUPS AT RISK VIII-53
IX. REFERENCES IX-1
vlll
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LIST OF TABLES
No. Title Page
II-l Chemical and Physical Properties of Various PAEs II-3
II-2 Production of Individual Phthallc Add Esters In the
United States from 1977-1984 II-8
II-3 Henry's Law Constants for PAEs 11-12
III-l Hydrolysis of Phthallc Acid Esters by Rat Intestinal
Contents III-2
III-2 Estimation of Intestinal Absorption of Phthallc Acid
Esterb 1n Rats III-4
III-3 Distribution of Orally Administered Phthallc Acid Esters . III-7
III-4 Distribution of 14C-OEHP In Rats Injected l.p. on
Either Day 5 or 10 of Gestation 111-13
III-5 Distribution of 14C-DEP in Rats Injected l.p. on
Either Day 5 or 10 of Gestation 111-18
III-6 Synthetic Metabolism of Phthallc Add Esters 111-25
III-7 Summary of Biliary, Fecal and Urinary Excretion of
DBP or DEHP In Rats 111-41
III-8 MEHP/DEHP Ratios and Biological Half-Lives of DEHP and
MEHP at 6 Hours After Administration 111-44
V-l Summary Table of Short-term Toxldty Studies of PAEs
In Mammals V-2
V-2 Dosage. Survival and Mean Body Weight of Rats Fed Diets
Containing D1-(2-ethylhexylJphthalate (DEHP) for 14 Days . V-9
V-3 Dosage, Survival and Mean Body Weight of Mice Fed Diets
Containing D1-(2-ethylhexylJphthalate (DEHP) for 14 Days . V-10
V-4 Summary of Short-term Effects of DEHP on Weight,
Morphology and Biochemical Constituents of Liver V-15
V-5 Effects of OEHP on Llpld and Protein Metabolism
Relating to Hepatotoxldty V-20
V-6 Summary Table of Long-term Toxldty Studies of
PAEs In Mammals V-27
1x
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LIST OF TABLES
No. Title Page
V-7 Long-Term Effects of DEh'P on Blocnemlcal Constituents
Relating to Hepatotoxlclty V-41
V-8 Mean Terminal Organ Weights In Male Rats After 26 Weeks. . V-42
V-9 Summary of Teratogenldty and Reproductive Effects
of Phthalates V-48
V-10 Summary of Genotoxlclty Tests of
Phthalatlc Acid Esters V-80
V-ll Incidences of Animals with Neoplastlc Lesions In the
NTP CarclnogenlcHy Bloassay of DEHP V-89
V-12 Summary of the Carcinogenic Effects of DEHP on the
NTP Bloassays and Interpretation of These Findings .... V-92
V-13 Incidences of Female Rats with Tumors of the
Hematopoletlc System In the NTP CarclnogenlcHy
Bloassay of BBP V-97
V-14 Summary of the Carcinogenic Effects of BBP In the NTP
Bloassays and Interpretation of These Findings V-98
VII-1 Cellular Changes In Rat Hepatocytes Induced by
DEHP Administration VII-9
VII-2 Synthesis and Breakdown of Protein and Llpld In
DEHP-Treated Rats VII-10
VIII-1 Summary of Data Used to Derive HA and OWEI Values
for DEHP, BBP, DEP, DMP and OBP VIII-21
VIII-2 Preliminary Results of a 2-Year CarclnogenlcHy
Bloassay of DEHP In Rats and Mice VIII-44
VIII-3 Cancer Risk Calculations VIII-49
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LIST OF FIGURES
No. Title Page
II-l SUctures of Various PAEs II-2
II-2 Preparation of Phthallc Acid Enters II-5
III-l Routes of Metabolism of DEHP 111-21
III-2 The Mean Plasma Concentration-Time Curves of DEHP and Its
Investigated Metabolites 1n Rats Infused with 50
mg/kg DEHP II1-23
III-3 The Hean Plasma Concentration-Time Curves of DEHP and Us
Investigated Metabolites In Rats Infused with 500
mg/kg OEHP 111-24
III-4 Routes of Metabolism of MEHP In Rats 111-28
VII-1 Schematic of the Peroxlsome Proliferation Hypothesis . . . VII-15
xl
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LIST OF ABBREVIATIONS
ACAT Cholesterol acryltransferee
ADP Adenosine dlphosphate
AHP Phthallc anhydride
ATP Adenosine tMpnosphate
BBP Butyl benzyl phthalate
BOP Butyloctyl phthalate
BPBG Butylphthalyl butylgylcolate
BSP Bromosulfophthaleln
bw Body weight
CoA Coenzyme A
CRAVE Carcinogen Risk Assessment Verification Endeavor
DAP Dlallyl phthalate
DBOP Dlbutoxyethyl phthalate
DBP Olbutyl phthalate
DCP Olcyclohexyl phthalate
DDP Dllsodecyl phthalate
OEHA D1-(2-ethylhexyl)ad1pate
OEHP D1(2-ethylhexyl) phthalate
OEN D1ethyln1trosam1ne
DEP Dlethyl phthalate
DHP Dlhexyl phthalate
DIB Dllsobutyl phthalate
DID Dlsodecyl phthalate
OIOP 011sooctyl phthalate
DMEP Olmethoxyethyl phthalate
OMP Dimethyl phthalate
OnBP 01-n-butyl phthalate
DnOP 01-n-octyl phthalate
DNP Dlnonyl phthalate
n-OP Oecyl phthalate
DTP D1-tr1decyl phthalate
OWEL Drinking Water Equivalent Level
FSH Follicle stimulating hormone
GC/ECD Gas chromatography/electron capture detector
GGT Gamma glutamyl transpeptldase
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LIST OF ABBREVIATIONS (cont.)
GI Gastrointestinal
GLC Gas-liquid chromatography
HA Health Advisory
n-HP Hexyl phthalate
HPLC High pressure liquid chromatography
l.a. Intra-arterlal
1.m. Intramuscular
1.p. Intraperltoneal
IRIS Integrated Risk Information System
l.v. Intravenous
LH Lutelnlzlng hormone
LOAEL Lowest-observed-adverse-effect level
HBP Monobutyl phthalate
MEHP Hono(2-ethylhexyl) phthalate
MTD Maximum tolerated dose
NCI National Cancer Institute
NOAEL No-observed-adverse-effect level
NOEL No-observed-effect level
NTP rfatlonal Toxicology Program
PA Phthallc add
PAEs Phthallc add esters
RAM Phthalamlde
PB Phenobarbltol
P.O. per oral
PVC Polyvlnyl chloride
RfD Reference dose
s.c. Subcutaneous
SDH Sucdnate dehydrogenase
SER Smooth endoplasmlc retlculum
SGOT Serum glutamlc oxalacetlc transamtnase
SGPT Serum glutamlc pyruvlc transanrtnase
TLC Thin-layer chromatography
xin
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I. SUMMARY
Phthalic add esters (PAEs) are primarily used as plaslldzers in
polyvinyl chloride- resins. These compounds are environmentally ubiquitous
due to their widespread use and ease of extraction: PAEs have been detected
In soil, water, air, and food Indicating widespread potential For human
exposure. Their presence has also been detected" "In human tissues.
PAEs generally occur as colorless liquids characterized by low water
solubility, high solubility in oils and organic solvents and, for the higher
molecular weight compounds, low volatility. Although phthalate has three
isomers (ortho, meta, and para positions), the term phthallc acid esters
generally refers to esters formed from the ortho phthallc acid Isomer. This
document will be primarily concerned with the ortho Isomer compounds.
PAEs are rapidly absorbed from the Intestine, skin, peritoneum, blood,
and lungs. A large percentage of the dlesters are hydrolyzed to monoest«»rs,
although the intact compounds are found in excretory products. Distribution
studies indicate that PAEs and their metabolites are found mainly In adipose
tissue, liver, kidney and intestine. Accumulation and retention of these
compounds is minimal. Most dialkyl phthalates are metabolized to their
corresponding monoesters; however, short-chain alkyl phthalates such as
dimethyl phthalate (OMP) may be metabolized to phthallc add. In most
species, glucuronlde conjugates are formed with the monoester; however, rats
appear to be unable to form glucuronlde conjugates of mono(2-ethylhexyl)
phthalate (MEHP) while forming glucuronlde conjugates with monobutyl
phthalate (MBP). PAEs and their metabolites are eliminated through the
urine, feces, and bile.
04710 1-1 07/02/91
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Acute animal toxlcity of PAEs is low and generally tends to be Inversely
related to the molecular weight of the compound. Subchronlc and chronic
toxlcity Includes decreased body weight and Increased liver and kidney
weights. Target organs of PAEs Include the testes and the liver, although
these effects have not been observed with all PAEs. Testlcular atrophy has
been associated with exposure to d1(2-ethylhexyl) phthalate (OEHP], butyl
benzyl phthalate (BBP), and blbutyl phthalate (D8P). Hepatic effects
commonly reported Include enlargement of the liver, effects on the
mitochondria, and decreased succlnate dehydrogenase activity. Reproductive
effects have been reported following exposure to PAEs during mating and
gestation. DEHP has been shown to decrease fertility and reproductive
performance In mice. Decreased fertility was attributable to effects In
both males and females. PAEs are generally regarded as nonmutagenlc. Two
PAEs have been tested In 2-year carclnogenesls bloassays performed by the
National Toxicology Program. DEHP was found to cause Increased incidence of
hepatocellular carcinomas In both rats and mice. There was limited evidence
that BBP Induces leukemia In female rats. The rat portion of the NTP
bloassay on BBP 1s currently being repeated.
Information on the effects of PAEs In humans, particularly for oral
exposures, Is limited. A single dose of 5 or 10 g of DEHP caused mild GI
effects In one Individual. Accidental Ingestlon of 10 g of OBP caused
nausea, vertlgt), keratltls, and toxic nephritis. Dermal exposure to most
PAEs does not cause Irritation or sensltlzatlon. Studies of human tissues
and cell cultures demonstrated Inhibition of cellular growth and decreases
In platelet function but did not Induce chromosomal damage. In
epldemlologlc studies the results have been largely confounded by exposure
to multiple chemicals and lack of quantitative Information on levels and
04710 1-2 08/08/91
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duration of exposure. Only two stuoies repor-.ecj :o aa:e lOenufy soec^fic
phthalate exposure. However, lac'< of exposure data and limited cnemical
details result In a relatively weak data base. The highest risk group In
humans appears to be among patients receiving blood transfusions or
hemodlalysls due to leaching of PAEs from plastic blood bags or plastic
tubing. Hepatitis 1n hemodlalysls patients and necrotlzlng enterocoUtls In
Infants given blood tranfuslons or umbilical catheters were related to PAE
exposures, but a causal relationship could not be conclusively demonstrated.
Researchers have Investigated several possible mechanisms of PAE
toxIcHy; however, there Is no conclusive evidence on any one mechanism.
Mechanistic studies have Indicated that PAEs may be Interfering with the
normal enzymatic or metabolic processes that occur at the cellular level.
However, the exact processes Involved In these alterations have not been
clearly delineated. It has been suggested that PAEs exert their toxic
effects by altering the physical state of membrane llplds, thereby changing
membrane fluidity. In the liver, PAEs act to Increase fatty acid metabolism
by Inducing peroxlsomes, mitochondria and enzymatic activities. PAEs may
become associated with hepatic ONA as a result of blosynthetlc Incorporation
of PAE metabolites Into the genetic material. Gonadal toxldty of PAEs In
males has been related to the antagonistic effect of PAEs upon endogenous
testicular zinc levels. Testlcular lesions may result from morphologic
changes of Sertoll cells Induced by PAE exposure. It would appear that
different mechanistic processes are operating on the various target organs.
The 1-day and 10-day HAs for OEHP were derived based on the dose
producing noncarclnogenlc effects in animals after oral administration. The
04710 1-3 07/02/91
-------
1-day and 10-day HAs for DEHP for a 10 "kg child are 1 mg/i and 0.5 mg/l.
respectively. The recommended longer-term HAs are 0.5 mg/i and 2 mg/i
for a 10 kg child and 70 kg adult, respectively. A lifetime DWEL based upon
a LOAEL for guinea pigs administered DEHP in the diet was determined to be
0.7 mg/a.
Lack of sufficient data preclude the derivation of a 1-day HA for B8P.
It Is recommended that the 10-day HA (20 mg/i} be adopted as the 1-day
HA. The 1-day and-10-day HA for BBP for a 10 kg child are 20 mg/i. The
longer-term HAs were based on a NOAEL derived from orally exposed rats. The
longer-term HAs are 20 mg/l for a child and 60 mg/i for an adult. A
lifetime DWEL based on the same study as the longer-term HAs was determined
to be 7 mg/4.
The 1-day HA of 50 mg/l for OBP Is based on a NOAEL for testlcular
effects In rats. The 10-day HA and the longer-term HAs were based on a
NOAEL derived from orally exposed rats. The corresponding 10-day HA and
longer-term HA for a child Is 10 mg/l. The longer-term HA for an adult 1s
40 mg/l. A lifetime DWEL based on the same study as the longer-term HAs
was determined to be 4 mg/a.
Lack of sufficient data precludes the derivation of a 1-day and 10-day
HA for OEP. The recommended longer-term HAs are 75 mg/l for children and
300 mg/l for adults, based on a NOAEL tn rats after oral exposure. A
lifetime DWEL based on the same oral rat data was determined to be 30 mg/l.
Lack of sufficient data precludes the derivation of 1-day and 10-day
HAs, longer-term HAs or DWEL for DMP.
04710 1-4 07/30/91
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There is sufficient evidence to classify OEHP as a 82. prooaole numan
carcinogen (I.e., inadequate evidence from human studies and sufficient
evidence from animal studies). Questions have been raised concerning tne
mechanisms of OEHP cancer Induction and doslmetry. A re-evaluation of DEHP
may be performed when more Information becomes available. The drinking
water risk levels of 10"*, 10"5 and 10~6 for DEHP are 300, 30 and 3
ng/l, respectively.
There Is limited evidence to classify B8P as a Group C possible human
carcinogen. Pertinent data regarding the carclnogenlclty of OBP, OEP and
OMP are nonexistent. Under the U.S. EPA guidelines DBP, OEP and DMP should
be placed 1n Group 0, not classified as to human cardnogenlclty.
04710 1-5 08/08/91
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II. PHYSICAL AND CHEMICAL PROPERTIES
Introduction
Phthalate acid esters, commonly referred to as PAEs, are colorless
liquids characterized by low volatility, low solubility In water, -and high
solubility In oils and organic solvents. Structures for the compounds
considered In this document are listed In Figure II-l. Table II-l
summarizes the pertinent chemical and physical properties for various
phthalic add alkyl and aryl esters Including the PAEs of particular
Interest reviewed In this document.
Preparation
The reaction of phthallc add (benzene dlcarboxyllc add) with a
specific alcohol to produce the desired phthallc add ester 1s a common
method of preparation. PAEs are often manufactured Industrially fr.om
phthallc anhydride rather than the add. Figure II-2 illustrates the
preparation of phthallc acid esters. Manufactured esters frequently contain
mixtures of various Isomers and Impurities {U.S. EPA, I960). Commercially
produced PAEs are usually >99% pure with a residual maximum acidity of
0.01%. The remaining Impurities may be mixtures of terephthalic acid.
maleic anhydride or dlesters of Isophthallc acid (U.S. EPA, 1978). The term
phthalate ester In this document refers to an ester formed from the ortho
phthallc add Isomer. Other PAEs formed from the meta and para phthallc
acid Isomers are generally referred to as Isophthalates and terephthalates,
respectively. Since the ortho phthallc add Isomers are the most prevalent
and extensively studied PAEs, this document will address those Isomers.
04720 II-l 09/07/88
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DEHP
BBP
0 CH2CH3
C-OCH2tH(CH2}3CH3
C-OCH2CH{CH2)3CH3
0
2-0(CH2)3CH3
-0-CH2
DBP
C-0(CH2)3CH3
C-0(CH2)3CH3
0
DEP
-0-CH2CH3
-0-CH2CH3
DMP
C-0-CH3
-0-CH3
FIGURE II-1
Structures of Various PAEs
04720
II-2
06/07/88
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1AB11 11-1
Cheated 1 and Physical Properties of Various PAEs
ro
o
Phlhaldle
At !U fster
Butyl beniyl*
(BBP)
Butyloclyl
(BOP)
Butyl
phlhalylbulyl
gylcolale (BPBG)
Olallyl (DAP)
Olbuloiyethyl
-. (OBOP)
LJ Dl-n-butyl1
(DBP)
Ol-n-oclyl
(OnOP)
Dtcyclohexyl
(OCP)
Olethyl (DfP)t
OI-2-ethylhexyl1
( Of HP )
Dlhexyl (OIIP)
Dllsobutyl (DIB)
Dtlsooclyl (010P)
Molecular
rorauld*
CHW.
NO
C,flH2406
C,4H140«
C20«3006
C16H2204
C24H3fl04
C20H2604
C,2H,404
C24H380,
C20H3004
C,6H?204
C24"3B°4
Molecular
Height*
312.39
334.0°
336.42
246.28
366.50
278.38
390.62
330.46
222.26
390.62
334.50
278.38
385.57
CAS
Number*
85-68-7
NO
85-70-1
131-17-9
117-83-9
84-74-2
117-84-0
84-61-7
84-66-2
117-81-7
84-75-3
84-69-5
27554-26-3
ttltCS
Number4
11199900
NO
1105350
C242000
1101750
1108750
1119250
1108690
1110500
1103500
111 1000
1112250
1113000
Botltny
Polnle
3/0*
225"
219
156-175*-'
2709
340
?20-248*-l
212-218* -?
296
23,f-?
3509
295-298c
3109
Me 11 Ing
Polnl
CO
-35
<-50*
<-359
65*
NO
-359
-25
58-65'
-4*
-46'
-509
NO
NO
Dens 1 1 yc
NO
NO
NO
NO
NO
1.0426"
NO
1.383
1.1175
0.9843"
NO
1.0490
NO
Specific
Gravity0
'•»"
0.991'
1.097
1.120
1.063
1.0465
0.978
1.148*
1.118*
0.985
1.010-1.0169
1.040
0.981
Vapor
(on)
(25-C)
1.1x10'*
NO
NO
NO
NO
9.7xlO'»
1.44x10-0
NO
2.?xlO'«
a.6xio~*
1.9x10'*
NO
7.4xlO'«
Udler
Solubility
2 6911
NO
120b
I00b
300b
1I.211
3.0)
Insoluble
10BOd
0.34d
0.241
Insoluble
0.09d
toy Oi tdnol/
UdU-i
I'di Itllun
5 56
NU
NU
NO
NU
4 /911
9.87
NO
2.24«l
9.61
NU
NO
NU
O
10
CO
CO
-------
IABIE 11-1 (conl.)
ro
o
Phlhalale Molecular
Aitil Ister formula*
Dlnelhoxyelhyl Cl4H|a°a
(OM1P)
DttKlhyl (BMP)1 C10M|0°4
Olnonyl (ONP) C2b»4204
Molecular
Height*
282.32
194.20
4)8. be
CAS
Number*
117-82-8
131-11-3
84-76-4
0HCS
Number4
1114000
TllSJbO
II 10000
Boiling
Point6
m
3409
282
20S-2209
Melting
Point
-M
0-2'
NO
Density'
1.047
1 . I90S
NO
Sped Me
Gravity6
1.171
1.192'
0.9799
Vapor
Pressure™
NO
2.2x10"'
NO
Uater
Solubility
'*'
4000"
Insoluble
lug Oi Idiiul/
Udler
Cat lit Inn
CoetfUltnl1-'
NU
1 4/"
Nil
'NIOSH. 198S
bU.S. IPA. 1980
'Ueait. 1983
dHoward et dl.. 1985
eU.S. tPA. 198?
'Hark et al.. 198?
(1) At S33 Pa (4.0 on Hg)
(2) Al bbb Pa (S.O on Hy)
VHiMley. 1981
»0ean. 1979
tpttthalttei of prladry concern tn this document
Jll.S. EPA. I981a
NO > No data
oo
00
-------
2ROH
2ROH
ortho phthallc add
alcohol
phthtallc add ester
FIGURE II-2
Preparation of Phthallc Add Esters
Source: U.S. EPA (1980)
04720
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03/25/88
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Analytical Methods
Analysis for specific PAEs Is complicated due to matrix Interferences
and limited analytical methods and analysis procedures. To preserve samples
for analysis, containers must be refrigerated at 4°C and protected from
light (40 CFR Part 136). This will reduce matrix Interferences caused by
evaporation and photosensitlvHy.
PAEs are readily soluble only In organic solvents. Ease of solvent
extraction Increases with Increasing molecular weight of the organic solvent
(Leah, 1977). Since phthalates are a component of many plastic and rubber
products, contamination of laboratory apparatus and solvents may occur
requiring a sample clean-up procedure. Clean-up procedures require sample
extraction by either a floMsl! or alumina column (40 CFR Part 136).
Extracted PAEs are separated and quantified by gas chromatography with an
electron capture detector {40 CFR Part 136).
Uses and Industrial Sources
PAEs are used primarily to Impart flexibility to plastics. The final
products may contain as much as 50% PAEs by weight (Kluwe, 1982b). DEHP is
the most commonly used plasticizer In polyvinyl chloride (PVC) products,
which Include syringes, dialysis tubing and other medical devices (Kluwe,
1982b). OEHP may constitute as much as 40% of the plastic material In blood
storage bags and medical tubing (Sjoberg et al., 198Sb). PVC resins are
also used In the production of high temperature electric wire, cable
Insulation, flooring material, swimming pool liners, furniture upholstery,
wall coverings, seat covers for cars, footwear and packaging materials
(Graham, 1973). NonplastUlzer uses Include pesticide carriers, cosmetics,
fragrances, munitions, industrial oils and Insect repellents (USITC, 1983).
04720 11-6 09/07/88
-------
In 1984. the UnUed States produced 1179 million pounds of PAEs (USITC.
1985). Table II-2 Indicates various PAEs and their corresponding production
figures. Annual production on a worldwide scale Is estimated to be between
3 and 4 billion pounds (U.S. EPA, 1980).
Distribution
PAEs enter the environment as a result of release during production, use
and disposal. Due to their widespread use PAEs are available as potential
contaminants to the environment. The U.S. EPA (1980) Ambient Water Quality
Criteria for Phthalate Esters cites publications detailing numerous
detections of PAEs In the soil (Ogner and Schnltzer, 1970), water (Ewlng and
Chlan, 1977; Corcoran. 1973; Kites and Bleman, 1972). air (Mathur, 1974),
fish (Mayer, 1976; Stalling et al., 1973) and biologic tissues (Nazir et
al.t 1971; Rubin and Sniffer, 1976; Jaeger and Rubin, 1970). In a U.S. EPA
monitoring survey of U.S. surface waters DEHP and DEP were found with the
greatest frequency (levels not provided In the report) out of 89 base
extractable compounds (Ewlng and Chlan, 1977). However, because of their
low water solubility, PAEs tend to partition to soil, sediment and biota in
an aqueous environment (Gledhlll et al.. 1980).
Fate and Transport
PAEs are removed from the environment mainly by biologic degradation.
Biodegradatlon of PAEs Is the primary method of removal from soil, water and
sediment. Phthalate esters have been reported to be metabolized In water by
mixed and pure cultures of microorganisms. The rates of degradation vary
widely depending upon environmental conditions, such as temperature, pH,
amount of dissolved oxygen and the structure of the phthalate
04720 II-7 07/03/91
-------
TABLE II-2
Production of Individual Phthallc Acid Esters In the
United States from 1977-1984
Ester Volume Produced
(million pounds)
BBP-
DBP
DEP
OOP
DMP
Dloctyl
DEHP
Other dloctyl
phthalates
DIOP
DTP
DID
101-510
22.21
17.75
1-10
8.64
251.1
301.12
1-10
21.79
145.82
Year
1977
1984
1984
1977
1984
1982
1984
1977
1984
1984
Reference
U.S. EPA. 1985
USITC, 1983
USITC, 1985
U.S. EPA, 1985
USITC, 1985
USITC, 1983
USITC, 1985
U.S. EPA, 1985
USITC, 1985
USITIC, 1985
04720
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09/07/88
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(Hattori et al., 1975). Tne degradation of phthalate esters by pure culture
Isolated from natural water, activated sludge and soil have been studied by
several Investigators (Taylor et al., 1981; Kurane et al.. 1979a,b;
Engelhardt et al., 1975. 1977; Engelhardt and Wallnofer. 1978; Klausmeler
and Jones, 1960; Perez et al., 1977; Ohta and Nakamoto, 1979). Several
authors have studied the blodegradatlon of phthalate esters by mixed
microorganisms. Thus, activated sludge, domestic wastewater and natural
river water have been used as mlcroblal Inoculum to study the blodegradatlon
of phthalate esters (O'Grady et al., 1985; Saeger and Tucker, 1973b, 1976;
Sasaki, 1978; Sugatt et al., 1984). Tabak et al. (1981) observed 100%
degradation of DMP, OEP, DBP and BBP In 7 days with unaccllmated
microorganisms from domestic wastewater. On the other hand, OEHP and OOP
required 21 days of acclimatization with microorganisms before 90%
blodegradatlon In 7 days occurred (Tabak et al., 1981). Similarly, the
mineralization of >8554 occurred with various phthalates In 28 days with both
activated sludge and river water (Saeger and Tucker, 1976; Sugatt et al.,
1984). The metabolic pathway data Indicate that phthalate esters first
und'ergo enzymatic hydrolysis to form the monoester, followed by further
hydrolysis to phthallc acid. The phthallc acid Is further degraded to
carbon dioxide and water (U.S. EPA, 1978; Saeger and Tucker, 1976).
Saeger and Tucker (1973a,b. 1976) and Gledhlll et al. (1980) concluded
from their river die-away and activated sludge studies that phthalate
plastldzers, as a class, undergo rapid primary degradation and
mineralization by bacteria commonly found 1n the environment. In a
simulated lake microcosm, Gledhlll et al. (1980) observed >95% primary
degradation of BBP 1n 7 days (CQ=1 mg/l). The blodegradatlon half-life
for BBP In this natural water system was <4 days. The length and
04720 II-9 09/07/88
-------
configuration of tne alfcyl esier cnalns significantly influences trie
blodegradation rate of phihalates In freshwater ecosystems, wnereas
acclimation of mlcrooes appears to have IHtle effect (HattoM el al., 1975,
Johnson et al., 1984). In freshwater systems, phthalates such as OHP and
DEP are expected to degrade faster than tne larger and more complex
phthalate esters (Johnson et al., 1984; HattoM et al., 1975). However, In
relatively clean ocean water, -14-20% degradation of OEP and DMP phthalate
was measured after 14 days, while the larger phthalates were decomposed >3G%
during the same period. The degradation of all the phthalate esters were
much higher with impure ocean water. For example, while 33% of DBP and 14%
of DEP degraded In clean ocean water In 14 days, the degradation was 100% \r\
5 days for DBP and 68% In 14 days for OEP with Impure ocean water. Hattorl
et al. (1975) observed 100% decomposition of DEP after S days and 100%
decomposition of DNP after 8-11 days 1n river water Initially spiked with 25
mg/i of the ester. OEHP degraded only -40% after 2 weeks In river water.
The higher degradation 1n Impure water was attributed to the presence of
higher concentrations of nutrients. Longer chain phthalate estsrs
decomposed faster than OHP and OEP In clean ocean water, a finding not
further explained (HattoM et al.. 1975).
In aquatic sediments under anaerobic conditions, blodegradatlon of short
chain alkyl esters appears to be slow and degradation of the longer'chain
esters has been observed to be very slight or undetectable (Johnson et al.,
1984; Johnson and Lulves, 1975; Horowitz et al., 1982; Shelton et al.,
1984). Johnson and Lulves (1975) observed 61 and 98% anaerobic mineraliza-
tion of DBP In 14 and 30 days, respectively. Under the same conditions, no
detectable degradation of OEHP was measured after 30 days. Johnson et al.
(1984) measured 10% anaerobic mineralization of radlolabeled OEHP after 28
04720 II-10 08/05/88
-------
days and <1% mineralization of OIOP. Optimal degradation of long cnain
phthalates occurred at high concentrations In nutrient-rich aquatic
sert-.ments with temperatures above 22°C. Such environmental conditions are
typical of sewage treatment ponds, wetlands, eutrophlc likes and enriched
streams during summer. Winter conditions, particularly at northern
latitudes and environmentally realistic (low, <1 vg/l) concentrations
would adversely affect blodegradatlon (Johnson et al., 1984).
Volatilization and leaching are two common modes of PAE transport
through the environment. When PAEs are used as plastlclzers In polymers,
the link between the plasticizer and the polymer Involves a physical
Interaction rather -than a chemical reaction. The polar groups of PAEs
adhere to the residual free PVC dlpoles, but are not chemically bound.
Thus, the PAEs are potentially free to be removed by volatilization and
leaching. For example. Atlas et al. (1982) measured the mass-transfer
coefficient of DBP to be 0.104 cm/hour In stirred (200-300 rpm) seawater
free of Interfering organic contaminants at 23°C. At a depth of 4.5 cm, the
volatilization half-life of DBP has been calculated to be 30 hours following
the method of Ollllng (1977). Henry's Law constants (H) for some PAEs, are
listed In Table II-3. Lyman et al. (1982) generalized volatility according
to ranges of (H). The Information presented In Table II-3 suggests that
volatilization from water would not be a rapid but still a possibly
significant removal process for these PAEs. Volatilization of OEHP (27.1%)
and OEP (4.5X) from PVC occurred after heating PVC material for 24 hours at
87°C over activated carbon (Darby and Sears, 1969). In another study.
Graham (1973) found that the air Inside new automobiles contained <0.72
wg/l phthalates due to volatilization from plastics (upholstery,
seatcovers, automobile mats, automobile tops).
04720 11-11 08/05/88
-------
TABLE II-3
Henry's Law Constants for PAEs*
Compound Constants
atm»m3/mol
DMP 1.3x10"'
DEP 5.5x10''
OBP 2.9xlO~-
OOP 2.3xl
-------
The rate of environmental leaching 1s affected by the formation of
various complexes. Evidence suggests that complexatlon of phthalates 1n
natural water with organic substances may be one mode of transport of
phthalates (Khan. 1980; Ogner and Schnltzer, 1970; Matsuda and Schnltzer,
1971). Phthalate esters have been observed readily Interacting with fulvlc
add, a widely occurring humlc substance found In soils and waters. The
phthalates appear to adsorb to the surface of the fulvlc add molecule
rather than react with It. The fulvlc add-phthalate complex Is very
soluble In water; thus, mobility of otherwise Insoluble phthalate esters Is
modified. Extent of solublllzatlon appears to vary with phthalate size.
Equivalent quantities of fulvlc add will solublllze 4 times as many
equivalents of dl(2-ethylhexyl) phthalate as of dl-n-butyl phthalate
(Matsuda and Schnltzer, 1971). Hydrated phthalates, for example, are more
readily extracted from PVC tubing than nonhydrated forms (Wlldbrett, 1973).
Theory suggests that Immigrating water molecules Into the tubing adhere to
the unsolvated phthalates molecules, which are ultimately responsible for
the plastldzlng effect In PVC. This prevents the molecules from adhering
to residual free PVC dlpoles and therefore permitting mobility (WUdbrett,
1973). Furthermore, dlalkyl phthalates and the widely occurring humlc and
fulvlc acid form a stable, soluble complex that allows transport In water.
Surfactants are used for solublllzlng phthalates from stream beds and
landfills (Ogner and Schnltzer. 1970). Very little OEHP 1s extracted by
water because of OEHP's low solubility In water. Ethanol significantly
Increases the amount of OEHP extracted, while pH has little or no effect.
The mean DEHP concentration extracted from ethanol solutions of 5, 10. 40
and 70% were 2, 6, 29.8 and 322.7 mg/J. respectively (Lawrence and Tuell,
1979).
04720 11-13 08/05/88
-------
Hydrolysis does nor. appear to play an important role In tne removal of
PAEs from the environment. Glendnill et al. (1980) ooserved >5% hydrolysis
of 1 mg/l n-butyl benzyl phthalate In 28 days. Wolfe et al. (1980)
estimated second-order rate constants for alkaline hydrolysis of phthalates
at pH 10-12 and 30°C. Rate constants varied with the size and complexity of
the phthalates and ranged from 1.1x10"* M"1 sec"1 for OEHP to
6.9xlO~a M~l sec"1 for OMP. Thus, corresponding estimated half-lives
at pH 7 range from 3.2-2000 years.
Experimental data regarding oxidation and photolysis of PAEs in water
were not located 1n the available literature. However, calculated
predictions Indicate that these processes would not be environmentally
Important (Mabey et al.. 1982; Callahan et al., 1979).
Adsorption
Sullivan et al. (1982) studied the adsorption of OBP and DEHP onto clay
minerals, calclte and sediment samples from seawater. Results Indicate that
adsorption increases with increased salinity or decreased solubility of
phthalates. Adsorption onto the clay minerals and calclte appeared to be a
reversible process, whereas adsorption onto sediments was Irreversible.
This suggests that marine sediments may act as a final repository of PAEs
(Sullivan et al.. 1982). Mabey et al. (1982) calculated sediment-water
partition coefficients for phthalates, Indicating adsorption Is likely for
all PAEs with adsorption tendency Increasing wHh size and branching of the
ester chain. Sediment adsorption coefficents range from 98 for OMP to
>150,000 for OBP and the larger PAEs including BBP. Gledhlll et al. (1980)
04720 II-H 09/07/88
-------
observed significant partitioning of 8BP to sediments in a simulated lake
microcosm. The average ratio of this compound measured in sediments versus
water was 571:1.
The contention that phthalates will be absorbed significantly onto
sediments In aquatic ecosystems Is supported by the observation that
phthalates are commonly found In bottom sediments from both streams and seas
at levels ranging from <0.1-316 ng/i {Glam et al.. 1978).
Summary
PAEs are colorless liquids at standard temperature and pressure. They
are characterized by low volatility, low solubility In water and high
solubility in oils and organic solvents. PAEs are formed by the reaction of
phthallc acid wUh a specific alcohol. Industrially manufactured. PAEs,
however, are often formed from phthallc anhydride rather than the add.
Analysis of PAEs Is complicated due to matrix Interferences and limited
analytical methods and analysis procedures. The most precise analytical
method Is by gas chromatography wUh an electron capture detector.
PAEs are produced by reacting phthallc anhydride with an excess amount
of the corresponding alcohol(s) In the presence of an esterfIcatlon
catalyst. The commercial products are usually >99% pure. Total U.S.
production volume of PAEs amounted to .1179 million pounds in 1984. They are
used predominantly as plastlclzers for polyvlnyl chloride resins. To a
lesser extent, PAEs are used as plastlclzers for other vinyl resins,
04720 11-15 06/07/88
-------
cellulose es:er plasiics, synioe.tic ela-stome'rs and other .polymers.
Nonplastidzer uses include pesticide carriers, cosmetics, fragrances,
munitions, Industrial oils and Insect repellants.
Biodegradatlon of PAEs Is the primary method of removal from the
environment. PAEs are reported to be metabolized in the aquatic environment
by a variety of pure microorganisms and degraded by mixed microbial
systems. The microbial degradation rates vary widely depending upon
environmental conditions such as temperature, pH, amount of oxygen present
and the phthalate structure. BlodegradabllHy of phthalates in freshwater
decreases with Increasing size and complexity of the phthalate ester
chains. Under anaerobic conditions blodegradatlon of short-chain esters Is
possible but slower than aerobic conditions, while degradation of the
longer-chain esters under anaerobic conditions Is very slight or
undetectable. Hydrolysis, oxidation and photolysis are not expected to be a
significant removal mechanism of PAEs.
Volatilization and leaching are two common modes of PAE transport
through the environment. Estimated Henry's Law constants suggest that
volatilization from water would not be rapid but could possibly be a
significant removal process. The rate of leaching Is affected by the
formation of various complexes. Complexatlon with the widely occurring
humic and fulvlc substances causes solublllzatlon of PAEs In water, thus
modifying their mobility.
Results from sediment absorption studies in saltwater suggest that
adsorption increases with Increased salinity or decreased solubility of
04720 11-16 09/07/88
-------
PAEs. Adsorption onto clay minerals and calcUe appears to be a reversible
process, whereas absorption onto sediments may act as a final repository of
PAEs. Calculated sediment water partitioning coefficients Indicate
absorption Is likely for all PAEs, while absorption tendency Increases with
the size and complexity of the ester chain.
04720 11-17 06/07/88
-------
III. TOXICOKINETICS
Introduction
The route of administration of PAEs can affect the eventual absorption,
distribution, metabolism and elimination of these compounds. Orally admin-
istered PAEs are hydrolyzed In and absorbed from the GI tract as the mono-
ester form. Hydrolysis Is greater for lower molecular weight esters than
for higher molecular weight esters. Once absorbed, PAEs or their metabo-
lites are distributed throughout the body. Initially the majority of these
compounds accumulate in the liver. Deposition of PAEs Is mostly in fat, the
GI tract, kidneys and liver. PAEs are primarily excreted In the urine;
however, elimination through feces and bile also occurs.
Absorption
Most orally administered PAEs are hydrolyzed In and absorbed from the GI
tract as monoesters (Pollack et al., 1985a). Absorption also occurs after
dermal, l.p., l.v. or inhalation exposures. In \j± vitro experiments with
DEHP, DBP, DEP and DHP In the presence of intestinal preparations from rats,
ferrets, baboons and humans, the dlesters were hydrolyzed to their mono-
esters. In these studies, DEHP required the longest time for jm vitro
hydrolysis to the monoester by intestinal preparation (Lake et al., 1977).
Alkyl-chaln length and PAE concentration have been shown to affect hydroly-
sis rates (Table III-l).
DEHP. Intestinal absorption of DEHP and DBP by rats (strain not
specified} after administration by oral gavage has been estimated from
analysis of urinary excretion products (Kluwe et al., 1982a). Absorption of
DEHP appeared to be less complete than that of DBP with only 40-50% of the 3
04730 III-l 09/08/88
-------
TABLE III-l
Hydrolysis of Phthallc Add Esters by Rat Intestinal Contents3-1*
Compound0
DMP
DBP
DEHP
DEHP
Concentration
(mg/ml)
1
1
1
0.1
Portion Metabol
(*)
60
80
20
100
1zedd
aSource: Kluwe, 1982a
bThe chemicals were Incubated for 16 hours at 37eC under an N2 atmo-
sphere In 20% (v/v) suspensions of gut contents In phosphate-buffered
Ringers solution containing 1% (w/v) D-glucose.
CDMP = Dimethyl phthalate; OBP = dl-n-butyl phthalate; DEHP = a1(2-ethyl-
hexyl) phthalate.
dPercent metabolized In 16 hours
04730 IH-2 07/25/88
-------
or 1000 mg/kg dose recovered In urine, respectively. However, >90% of the
dose appeared 1n the urine of rats following the 1ngest1on of 10 or 2000 ppm
(0.5 or 100 mg/kg/day, respectively, assuming 5% food consumption and 350 g
rats) via feed (Table III-2) (Kluwe, 1982a).
Esterases that are capable of hydrolyzlng dlester phthalates have been
found 1n rat Intestinal mucosal cells as well as extracellularly In the
Intestinal contents (Rowland, 1974; Rowland et al.. 1977). Wallln et al.
(1974) demonstrated that a small portion of orally administered DEHP may be
absorbed from the GI tr,act as the Intact compound.
Albro et al. (1982) and Albro (1986) observed an absorption threshold
for a series of single oral doses of DEHP In Fischer rats. Animals received
1.8-1000 mg/kg of 14C-OEHP In cottonseed oil. As the dosage Increased, a
threshold (121 yg/g DEHP In the liver) was reached above which a steady
Increase In the amount of unhydrolyzed DEHP or Intact dlester reached the
liver. This may be due to saturation of esterases In the GI tract. Dosages
below this threshold result In absorption of hydrolyzed dlesters.
Administration of DEHP In the diet resulted In Intact DEHP reaching the
liver at dietary levels exceeding 4300 ppm (430 mg/kg/day calculated using
the authors' assumption of 10X food consumption). In contrast to the
results observed In rats, Albro et al. (1982) did not detect an absorption
threshold In either CD-I or B6C3F1 mice administered <1000 mg/kg of DEHP.
Rhodes et al. (1986) reported that the excretion profile and tissue
levels of radioactivity following oral administration of 14C-OEHP
demonstrated considerably reduced absorption In the marmoset compared with
the rat. The urinary metabolite pattern In the marmoset was qualitatively
04730 111-3 09/08/88
-------
TABLE III-2
Estimation of Intestinal Absorption of Phthallc Add Esters In Rats3
Compound
DBP
DEHP
Doseb
60 mg/kg
270 mg/kg
2310 mg/kg
3 mg/kg
1000 mg/kg
10 ppmd (0.5 mg/kg/day)
2000 ppmd (100 mg/kg/day)
T1mec
(days)
2
2
2
4
1
NR
NR
Percentage
Excreted 1n
90
90
90
40
50
>90
>90
of Dose
Urine
aSource: Kluwe, 1982a
bOral gavage, unless specified otherwise
cPer1od of sample collection, between exposure and termination
Concentration Incorporated Into feed
NR = Not reported
04730 III-4 07/25/88
-------
similar to, but quantitatively different from, that In the rat. Following
an oral dose of 2000 mg/kg (In corn oil), marmoset tissue Is exposed to
approximately equivalent levels of DEHP and metabolites to that expected for
rat tissues following an oral dose of 200 mg/kg (In corn oil). This
suggests that DEHP Is not readily hydrolyzed by marmoset Upases and
therefore not readily absorbed by this species.
The Intestinal absorption of OEHP has been studied In two human
subjects. In one human subject, 4.5% (as metabolites) of a single oral dose
of 10 g DEHP was recovered In the urine after 24 hours. Similarly, a second
subject received 5 g DEHP orally, and 2% (as metabolites) of the dose was
recovered 1n the urine after 24 hours (Shaffer et al., 1945).
BBP. Data regarding the absorption of BBP could not be located In the
available literature. Systemic effects observed after oral exposure to BBP,
consistent with those observed following exposure to other PAEs, Indicate
that absorption of BBP does occur.
DBP. Intestinal absorption of DBP 1n rats (strain not specified) has
been estimated following oral gavage administration (see Table III-2).
Greater than 90% of the 60 mg/kg to 2310 mg/kg range of DBP dosages was
found In the urine (as the parent compound or Its metabolite) within 2 days
after gavage administration (Kluwe, 1982a). Kaneshlma et al. (1978) also
found a small amount of Intact DBP In the bile of rats given oral doses of
the compound. However, U Is hypothesized that In most cases PAEs are
absorbed from the Intestine as monoesters rather than dlesters. Absorbtlon
following the administration of DBP by other exposure routes Is not well
documented.
04730 III-5 09/08/88
-------
PEP. Data regarding the absorption of DEP could not be located In the
available literature. Systemic effects observed after oral exposure to OEP
(consistent with those observed following exposure to other PAEs) Indicate
that absorption of DEP does occur.
PHP. Reports of the absorption of PHP are limited to one Russian
article on the dermal absorption of DMP for rats and humans. For rats,
maximum levels 1n the blood were reached In 0.5 hours after application to
the skin. The metabolites phthallc add and monomethyl phthalate were found
1n urine, organs (not' otherwise specified) and blood. Similar results were
reportedly observed In human volunteers; however, the experimental details
were not provided 1n the abstract (Glelberman et al., 1978).
Distribution
Once absorbed, PAEs or their metabolites are distributed to various
tissues and organs. Kluwe (1982a) has provided a thorough overview of this
topic. In general, orally administered PAEs are blotransformed In the
Intestine to the monoester. Initially these compounds accumulate In various
organs, predominantly 1n the liver. These materials are excreted almost
completely within days demonstrating that Uttle long-term accumulation
occurs. In the case of humans with compromised kidney function who are on
hemodlalysls, phthallc acid does accumulate (Pollack et al., 1985b). As
Table III-3 Indicates, most of the orally administered PAEs are found In
adipose tissue. GI tract, kidney or liver.
DEHP. A factor essential In understanding differences In PAE disposi-
tion Is the role of the route of administration. Distribution of labeled
PEHP has been studied after Intravenous administration. Daniel and Bratt
04730 II1-6 07/02/91
-------
TABLE III-3
Distribution of Orally Administered Phthallc Acid Esters3
Compound Species
DBP rat
rat
DEHP rat
rat
Dose
(rag/kg)
60
270
500
800
T1meb
(days)
1
1
2
1
1
4
Repository Organs
Intestine, adipose,
liver, kidney, muscle
Liver, kidney, adipose
None
Intestine, stomach,
liver, kidney, adipose
Liver, kidney, adipose,
muscle, testls
Adipose
aSource: Kluwe, 1982a
bT1me between administration and examination
04730
I1I-7
07/25/88
-------
(1974) found 60-70V. of a single l.v. dose of radlolabeled DEHP (emulsified
In olelc acid) In the Hver, lungs and spleen of rats. The compound and
metabolites disappeared from the blood rapidly and were detected In these
organs within 2 hours. Subsequently, an elimination half-life of 1-2 days
from these distribution sites was estimated.
Examination of tissues from two deceased" patients who had received
transfusions of blood stored 1n PVC blood bags, revealed DEHP In the spleen,
liver, lung and abdominal fat at concentrations ranging from 0.025 mg/g (dry
weight) In the spleen* to 0.270 mg/g (dry weight) In the abdominal fat
(J.aeger and Rubin, 1970).
Tissues were also analyzed for BGBP (butyl glycolylbutyl phthalate) and
DEHP, using the isolated perfused rat liver technique. BGBP was recovered
from the perfusate as a water-soluble metabolite. DEHP was cleared from the
perfuslon medium after 60 minutes. Upon analysis of the liver, -90% of the
total recoverable dose remained as unmetabollzed compound. The
investigators concluded that DEHP is accumulated by the liver primarily as
the unmetabollzed parent compound (Jaeger and Rubin, 1970).
When whole body autoradiography techniques were utilized In mice after a
single l.v. Injection of 14C-DEHP (2.293 yg) using sterile mouse plasma
as the solubillzlng substance, radioactivity was detected in the kidney and
liver initially, followed by accumulations In the urine, bile and
Intestine. After 168 hours (7 days), radioactivity was found In the
intestinal lumen (due to secretion of the compound from the liver Into the
bile), but no radioactivity was detected in the spleen or lung (Waddell et
al.. 1977).
04730 III-8 09/08/88
-------
Llndgren et al. (1982) also Investigated the distribution of labeled
DEHP with whole body autoradlography. C57B1 mice received 14C-OEHP by
oral Intubation (soybean oil vehicle) or Injection (absolute ethyl alcohol
vehicle In the tall vein). Although DEHP administered i.v. was labeled at
either the carbonyl group (dose level 3.6 mg DEHP/kg) or at the position of
an alcohol (2-ethylhexyl-l-1*C) (dose level 9.6 mg DEHP/kg), the distribu-
tion of the compound was similar with both labeled forms. Within 4 hours of
a single l.v. Injection, high levels of activity were found In the gall
bladder. Intestinal contents, urinary bladder, liver, kidney and brown fat.
Lower levels were observed In the white fat, myocardium, muscles, blood,
bone, cartilage, testicles and nervous system. The concentrations of DEHP
remained high In the gall bladder. Intestinal contents, urinary bladder and
brown fat 24 hours after the single Injection. In mice that were pretreated
with either DEHP, sodium phenobarbltal or 3-methylcholanthrene before
receiving oral doses of labeled DEHP once dally for 5 consecutive days, the
concentration of 14C-DEHP In the brown fat was higher than levels found in
mice treated with DEHP alone. Further, mice orally dosed with DEHP and then
sacrificed at Intervals between 5 and 30 days retained the carbonyl-14C-
DEHP (but not the 2-ethylhexyl-l-14C-DEHP) 1n the skin, cartilage and
tendons. Low concentrations of DEHP, labeled at either site, were observed
In the bone. The authors state that the mechanisms that may underlie
accumulation are unresolved. Llndren et al. (1982) attributed high levels
in brown fat to Induction of mixed function oxldases causing an Increased
production of OEHP metabolites with affinity to brown fat.
A single dose of labeled DEHP administered l.v. 1n saline solution In
mice resulted In accumulation primarily In the lungs with lesser amounts
04730 III-9 09/08/88
-------
occurring in the brain, fat, heart and blood. There was no apparent
preference for fatty tissue (Dllllngham and Autlan, 1973).
In experiments performed by Tanaka et al. (1975). ^-C-DEHP (500 mg/kg
as a 25% solution) was administered p.o. (solublUzed 1n Tween 80) or 1.v.
(as a dispersion prepared by sonlcatlon of DEHP In saline) to groups of male
Wlstar rats. DEHP was labeled with l«C at the carbonyl carbon. After
oral dosing, liver and kidney concentrations of the compound reached a
maximum In 2-6 hours with peak blcv; levels occurring after 6 hours.
Detection of the radioactivity In the IWer after the first hour following
the 1.v. Injection revealed that 70-80% of the original dose was deposited
1n the liver. These Initial radioactivity levels 1n the liver decreased to
50% after 2 hours and 0.17% after 7 days. The results from both p.o. and
1.v. administrations demonstrated high levels of radioactivity occurring in
the Intestine, and lesser amounts In other organs and tissues. However, the
testicles and brain showed Uttle affinity for the compound.
In a study performed by Olshl and Hlraga (1982), Wlstar rats received a
single oral dose of 25 mmol/kg (9.77 g/kg) DEHP by gastric Intubation
(vehicle not stated). The animals were then examined after 1, 3, 6, 24, 48
and 96 hours. Blood and tissue sample analyses revealed that the concentra-
tions of DEHP and Its hydrolysis product, MEHP. reached peak levels within
6-24 hours after dosing. The peak concentrations In the heart and lungs
occurred within 1 hour, while fat levels of OEHP and MEHP Increased for 2
days. The highest ratio of MEHP/DEHP (mol %) was found In the testes
(-210%) while all other tissues sampled exhibited a ratio of -113% or less.
04730 111-10 09/08/88
-------
Minimal amounts (<1 yg/g) of both compounds were detected In the kidney
and brain. The lung contained only MEHP while low levels of DEHP were found
In the spleen.
The effects of "C-DEHP In the diet were Investigated so that tissue
accumulation of DEHP In rats could be examined (Daniel and Bratt, 1974).
Groups of 24 female rats were fed diets containing either 1000 or 5000 ppm
DEHP (50 or 250 mg/kg/day, assuming 350 g rats consume 0.05 kg food/day) for
35 and 49 days, respectively. Radioactivity was then monitored In the liver
and abdominal fat after the food was consumed. The labeled compound was
found to Increase In these tissues until steady-state concentrations were
achieved. Steady-state levels were reached after 1 week In the liver
tissues and after 2 weeks In adipose tissue. Upon cessation of OEHP
administration, radioactivity In the liver was decreased below the level of
detection within 3 weeks. The levels In adipose tissue remained at nearly
one-third of the steady-state concentrations after 3 weeks (Daniel and
Bratt, 1974).
Jacobson et al. (1977} also demonstrated that DEHP or Us metabolites
achieve steady-state levels In experiments using rhesus monkeys. The
animals received transfusions of blood contaminated with DEHP to yield doses
ranging from 6.6-33 mg/kg. The compound or Us metabolites were retained 1n
trace amounts (liver, testls, heart and fat) for <14 months after treatment.
As pointed out by both Daniel and Bratt (1974) and Jacobson et al. (1977),
there Is a steady-state concentration that Is reached, after which DEHP (or
metabolites) Is then rapidly eliminated from the organs or tissues through
various routes, thus preventing significant accumulation over long periods
of exposure.
04730 III-ll 09/08/88
-------
Transfer of OEHP and Us metabolites from maternal to fetal tissues has
been Investigated by Singh et al. (1975). In the study, one group of 13
pregnant Sprague-Dawley rats was Injected l.p. with a single 5 ma/kg (250
mg/kg bw) carboxy-labeled l«C-DEHP dose on day 5 of gestation. A second
group of 10 pregnant rats was Injected with a single 5 ml/kg 14C-OEHP
dose on day 10 of gestation. One rat Injected on day 5 of gestation was
asphyxiated by an overdose of ether 72 hours after the ^C-DEHP Injec-
tion. The remaining rats were asphyxiated at 24-hour Intervals (one rat/24-
hour Interval) through day 20 of gestation. Rats Injected on day 10 of
gestation were sacrificed every 24 hours through day 20 of gestation.
Radioactivity was detected In the maternal blood, placentas, amnlotlc fluid
and fetal tissue. None of the fetal tissue levels exceeded the maternal
blood levels. Less than 1% of the Injected dose was detected 1n the fetal
tissue at any of the measured times. Concentrations of radioactivity
diminished quickly In amnlotlc fluid and maternal blood as Indicated In
Table III-4. The half-life for DEHP was calculated as 2.33 days. Fetal
concentrations ranged from 5.9xlO"« to 4xlO"6 mol/kg. Specific
metabolites were not Identified In this paper. This Investigation
demonstrates that l4C-1abeled material (14C-DEHP and Its metabolites) Is
distributed to the developing rat fetus throughout organogenesls. Further,
the authors conclude that the presence of DEHP and It metabolites may act
directly on embryonic tissues to Induce developmental effects.
Bratt and Batten (1982) observed clear species and sex differences In
the tissue retention of DEHP. Rats and marmosets were given 1960 mg/kg/day
of **C-OEHP (14C ring labeled) orally for 14 days. In the rat, the
females retained higher concentrations of the l4C-rad1olabel In the liver
and kidney (286 and 176 wg/g tissue, respectively) than the males (216 and
04730 111-12 08/05/88
-------
to
o
CM
Ul
CO
00
TABLE I1I-4
Distribution of "C-DEHP In Rats Injected l.p. on EUher Day 5 or 10 of Gestation3
Gestation
Day
B
9
10
11
12
13
14
15
16
17
18
19
20
DEHP
Maternal
Blood
95,098
70,285
46.297
22.439
20.326
27.993
26,837
11,656
10.401
8.003
6.000
6.402
4,192
Injection Day 5b
Placenta
NR
NR
NR
NR
1180
1386
1672
1215
2732
1723
2075
1327
1146
Amnlotlc
Fluid
2577
1171
200
539
375
683
225
254
346
352
239
182
232
Fetal
Tissue
470
556
641
890
176
579
437
453
1453
2223
1802
1938
1641
DEHP
Maternal
Blood
114.656
118.338
67.243
30,278
31.813
26.510
19.139
24,793
15.491
6.022
Injection Day 10b
Placenta
NR
NR
4447
3023
5224
7551
3940
3821
2914
3085
Amnlotlc
Fluid
6186
983
616
660
1022
965
562
733
458
275
Fetal
Tissue
13.136
5.461
1.263
1.224
2.550
2.971
2.519
4.705
1.526
726
aSource: Singh et al.. 1975
bTotal counts (dpm) In tissues assuming total blood In the rat to be 7X of Its body weight.
NR = Not reported
-------
115 yg/g tissue, respectively). In addition, male rats retained 36 yg
DEHP/g tissue In the testls. A similar pattern was observed 1n the
marmoset. Female marmosets retained 47 and 35 yg DEHP/g In the liver and
kidney, respectively, whereas male marmosets retained 29 and 15 yg DEHP/g
In the liver and kidney, respectively. Testls concentrations reached 8
wg/g tissue In the marmoset. The rats of both sexes retained higher
tissue concentrations of l4C-rad1olabel than did the marmosets.
Similar results were also reported by Rhodes et al. (1986) In a compari-
son of the blood and 'tissue levels of DEHP and Its metabolites In the rat
and marmoset. The animals were administered 2000 mg/kg/day of 14C-DEHP
(labeled In the phenyl ring) for 14 days. The level of l4C-rad1olabel In
the marmoset tissues was only 10-20% that of the rat. In both the rat and
marmoset the liver retained the highest level of 14C-rad1olabel.
Llndgren et al. (1982) examined the distribution of 14C-DEHP admin-
istered to pregnant C57B1 mice at gestation day 8 and 16 by oral Intubation.
Dose levels administered at day 8 of gestation corresponded to 7.7 mg
DEHP/kg (2-ethylhexyl-l-14C) and 2.9 mg DEHP/kg (carbonyl-14C). Dose
levels administered at day 16 of gestation corresponded to 4.8 and 1.8
DEHP/kg of (2-ethylhexyl-l-14C) and (carbonyl-l4C) labeled DEHP, respec-
tively. Uptake of the 14C labeled DEHP was not quantified. Whole body
autoradlography revealed that at early gestation (8 days), uptake occurred
1n the yolk sac with high concentrations of {carbonyl-i4C) labeled DEHP In
the gut at 4 hours after treatment. Twenty-four hours after administration
of (2-ethylhexyl-l-14C) labeled DEHP, activity In the neuroepHhellum was
observed. At late gestation (16 and 17 days), accumulations of either 14C
04730 111-14 07/25/88
-------
labeled DEHP were also high In the yolk sac. The fetuses on days 16 and 17
of gestation were found to have high concentrations (levels not quantified)
of either *«C labeled DEHP In the renal pelvis, urinary bladder and Intes-
tinal contents. Lesser amounts (levels not quantified) were detected In the
liver and the mineralized portions of the fetal skeleton. Some skeletal
uptake of DEHP was also noted.
The placental transfer of DEHP was examined In guinea pigs by Klhlstrom
(1983). The author utilized a placental perfuslon technique and determined
that the solution employed as a perfuslon medium may affect the placental
transport of DEHP. The level of DEHP administered was not reported. The
maternal liver uptake and total placental uptake of DEHP was calculated
after a constant plasma concentration was reached by catheter Infusion Into
the vena JugulaMs. Maternal hepatic uptake was estimated to be 41% of the
dose, while total placental uptake was -13-15% of the dose. A significant
difference (p<0.001) of 0.2U0.09 and 0.47^0.10 ppm of the total dose/mg was
found between the DEHP concentrations 1n the fetal plasma and that of the
final perfuslon media, respectively. Indicating that the compound was dis-
tributed In the fetal tissues. In addition, placental tissue concentrations
of DEHP were much larger (7.5±2.5 ppm of total dose/mg tissue) than the
levels 1n the perfuslon plasma (0.47±0.10 ppm of total dose/mg plasma),
Indicating that the greater part of the DEHP taken up by the placental
tissues does not enter Into the fetal circulation.
BBP. The distribution of 8BP has been studied In rats following both
1.v. and oral administration. Elgenberg et al. (1986) evaluated the
disposition of BBP after an l.v. dose of 20 mg/kg to male F1scher-344 rats.
04730 111-15 07/25/88
-------
Brain, lung, liver, kidney, spleen, testes, small Intestines, renal fat,
muscle (thigh) and skin (abdominal) were removed and examined. 8BP was
rapidly distributed to the tissues and eliminated. The Initial half-life
was <30 minutes and the terminal half-life was 4.5-7.3 hours. Elimination
from the blood and fat was mono-exponential whereas elimination by the
kidney, muscle, skin and small Intestine followed a blexponentlal decay
curve. Since BBP Is rapidly metabolized (see Metabolism Section) and
eliminated. It Is not sequestered In fat.
Lake et al. (1978) observed similar results In male Sprague-Oawley rats.
Oral doses of 16, 160 or 1600 mg/kg were administered by oral Intubation.
At the end of 5 days animals were sacrificed and examined. Radioactive
residues were present 1n the liver, kidney, small Intestine and total gut
contents. However, the residues present were <1% of the administered dose.
There was no evidence of tissue accumulation.
DBP. Tanaka et al. (1975, 1978) compared the distribution of
*»C-DBP (labeled In the carbonyl moiety) and 14C-DEHP wHh male Wlstar
rats after single l.v. or p.o. doses. Few differences were observed In the
distribution pattern of DBP compared with that of DEHP. Following 1.v.
administration DBP did not accumulate In the liver to the same extent as
DEHP. After 1 hour 14C levels In the liver were 6% of the total l.v. dose
for DBP while DEHP had been detected at 76% of the total l.v. dose.
Retention of DBP In the heart, lung and spleen 24 hours after oral or l.v.
exposure appeared to be shorter than DEHP In these organs. Affinity for
adipose tissue appeared to be higher following l.v. or oral administration
for DEHP than for DBP after 24 hours.
04730 111-16 09/08/88
-------
The distribution of DBP has been studied with rats administered the com-
pound In the diet or by gavage (corn oil vehicle). Williams and Blanchfleld
(1975) added 1000 mg/kg of OBP-7-14C to the diets of 24 male Wlstar rats
for 12 weeks. No substantial accumulation of DBP or MBP was detected at 4,
8 or 12 weeks In any of the organs and tissues. Four hours after a single
(0.27 or 2.31 g/kg "C-DBP) Intubated dose was administered to rats, the
label was detected throughout the body. Yet, within 48 hours the tissues
and organs contained only traces of radioactivity. Clearance of the labeled
D8P was more rapid at the lower dosage.
PEP. Singh et al. (1975) Investigated the maternal-fetal transfer of
carboxy-labeled 14C-OEP 1n rats. Thirteen pregnant Sprague-Dawley rats
were Injected l.p. with a single 1.0116 ml/kg (51 mg/kg bw) l*C-OEP dose
on day 5 of gestation. Another group of 10 rats was Injected with the same
amount of 14C-DEP on day 10 of gestation. The group of rats Injected on
day 5 of gestation were sacrificed by an overdose of ether 72 hours after
the 1*C-DEP Injection and then at 24-hour Intervals through day 20 of
gestation. Rats Injected on day 10 of gestation were sacrificed In the same
manner every 24 hours through day 20 of gestation. As with DEHP, radio-
activity was detected in maternal blood, placentas, amnlotlc fluid and fetal
tissue at both gestations! stages (5 and 10 days) and <1% of the Injected
dose was detected In the fetal tissue at any of the measured times. The
concentrations of radioactivity diminished quickly In maternal blood as
Indicated In Table III-5. Based on a first-order excretion curve, the
half-life for DEP was calculated to be 2.22 days. Fetal concentrations
ranged from 1.5xlO~«-2.8xlO~« mol/kg. Specific metabolites of DEP were
not Identified In this study. OEP and Us metabolites were present In the
04730 111-17 07/25/88
-------
TABLE II)-5
Distribution of "C-OEP In Rats Injected l.p. on Either Day 5 or 10 of Gestation3
I
co
ro
en
CO
CD
Gestation
Day
B
9
10
11
12
13
14
15
16
17
18
19
20
DEIIP
Hater nal
Blood
29.811
27.180
19.422
9.204
14.376
6.258
7,491
4.806
3.804
3.444
3.661
2,479
2.848
Injection Day 5D
Placenta
NR
NR
NR
447
390
412
572
613
1245
1313
1347
831
965
Amnlot 1c
Fluid
11
21
61
131
198
128
158
231
167
176
166
122
116
Fetal
Tissue
333
562
372
145
124
107
210
228
507
672
947
916
1185
DEliP
Maternal
Blood
50.145
41,497
33.645
27.423
27.081
18.546
11.257
6.068
3.892
2.674
Injection Day 10b
Placenta
B71
1434
1549
1424
824
799
800
1775
1473
656
Amnlot Ic
Fluid
471
824
428
390
898
526
583
364
257
101
Fetal
Tissue
317
216
332
333
887
664
1008
1820
1695
1176
aSource: Singh et al.. 1975
bTotal counts (dpm) In tissues assuming total blood In the rat to be 7X of its body weight.
NK = Not reported
-------
developing rat fetus during organogenesls. Singh et al. (1975) suggest that
the presence of DEP and Us metabolites may act directly on embryonic
tissues 1n the Induction of teratogenldty.
PHP. Data regarding the distribution of DMP could not be located In
the available literature.
Metabolism
Olalkyl phthalates are hydrolyzed to monoesters In the Intestine and
other organs and tissues before and after absorption. The rate of hydroly-
sis Is greater for the lower molecular weight esters such as OMP and DBP
(see Table II-l for weights). Although both ester linkages of PAEs can be
hydrolyzed to produce phthallc add, only small fractions of the long-chain
alky! phthalates undergo such complete conversion. The metabolic profile!
of single doses of phthalates may differ from profiles after multiple
exposures; some phthalates such as DEHP and HEHP have been shown to Induce
their own metabolism. Thus, duration of exposure, the dose level admin-
istered and the status of the animal with respect to the metabolic pathway
of peroxlsomal proliferation must be considered when evaluating studies on
the metabolism of phthalates.
QEHP. Numerous studies have focused upon the metabolic profile of
DEHP. Albro et al. (1973) Identified the first step In the rat metabolism
of orally administered DEHP In rats as the conversion of the dlester to the
monoester, HEHP. Two distinct alcohol Intermediates are formed by «- and
u-1 oxidation of the monoester sldechaln. Just as Is the case for DBP
metabolism as noted by Albro and Moore (1974), oxidation of these alcohols
0473Q ILI-19 08/05/88
-------
s In the generation of carboxyllc acid (which can be further oxidized
to a ketone.) Figure III-l shows a number of products that can be formed
from metabolism of orally Ingested DEHP In rats. Albro et at. (1983b)
postulated that oxidation of the aliphatic side chain of OEHP or MEHP may
Involve placement of the hydroxyl group at positions more distant than u-l
from the terminal methyl group. Based upon the discovery of highly polar
metabolites In the urine of rats, given two gavage administrations of DEHP
or MEHP. the authors then hypothesized that attacks by oxygen species may
occur concurrently at two sites, or that an oxidized metabolite may receive
a second oxidation.
SJoberg et al. (1985a) supported the Albro et al. (1983b) hypothesis and
further studied the four major metabolites of OEHP. The four metabolites
studied were MEHP. mono-(5-carboxy-2-ethyl pentyl) phthalate, mono-(2-ethyl-
5-oxohexyl)phthalate and mono-(2-ethyl-5-hydroxyhexyl) phthalate and the
metabolites will be referred to as MEHP, Met V. Met VI and Met IX, respec-
tively. The primary metabolite MEHP was studied separately. Thirty-four
male Sprague-Oawley rats (40 days old) were administered a single cannulated
Infusion over a 3-hour period of either 5. 50 or 500 mg/kg bw DEHP. Blood
samples were drawn 1, 2. 3. 3.5. 4. 6. 8, 11. 14. and 24 hours after the
Infusion. Elimination patterns of DEHP and MEHP were similar In the groups
administered 5 and 50 mg/kg DEHP. Plasma concentrations of DEHP were much
higher at all times than MEHP. and MEHP plasma levels were much higher than
Met V. VI and IX. Plasma concentrations of Met V. VI and IX could not be
detected 6 hours after the Infusion of 5 mg/kg DEHP. SJoberg et al.
04730
HI.20 09/08/88
-------
LJ
o
Cll,
-Cll - (Cllj) j-Cllj
«
OEIIP
C,,j
V
hydrolysis
Ic-o-cii2-cii -
o tjiz
Oil
(anno-(2-elhyI-5-hy
-------
(19853) suggested that the parallel decrease observed In the plasma concen-
trations of DEHP, MEHP and the metabolites IX and V (Figures 1II-2 and
III-3) Indicate that the elimination of DEHP Is the rate-limiting step In
the depositions of these metabolites. The shape of the plasma concentra-
tion-time curve also Indicated that the elimination of MEHP was rate-limited
by Its formation. The Investigators stated this was verified by the obser-
vation that the clearance of MEHP when given separately was higher than that
of the parent compound.
Although several species of animals have been observed to excrete
glucuronlde conjugates of MEHP upon exposure to DEHP, rats are an exception
(Tanaka et al.. 1975; Williams and Blanchfleld, 1975; Albro et al., 1982).
Table III-6 Illustrates the rat's inability to excrete the MEHP glucuronlde,
but not MBP derivatives in comparison to other mammals.
Studies performed by Lake et al. (1984a) demonstrated that a single
orally administered dose of 14C-DEHP at 100 or 1000 mg/kg was metabolized
to a greater extent In the rat than \n the hamster. Although similar
amounts of radioactivity were recovered in the urine and feces of both
species, fecal extracts contained only unchanged DEHP In the hamster while
tn the rat, -50% of the radioactivity occurred as metabolites {specific
metabolites not Identified), possibly including MEHP. After 96 hours only
negligible amounts of radioactivity were present In either the liver, kidney
or total gut contents of both species.
Lake et al. (1976) examined urine samples from rats and ferrets treated
orally with 14C-DEHP. In ferrets the compound was hydrolyzed to MEHP and
04730 111-22 07/25/88
-------
100
o
E
c
o
6
««
*»
CL
A
Infusion
12
Time (hours)
FIGURE III-2
The Mean Plasma Concentration-Time Curves of DEHP and Us Investigated
Metabolites In Rats Infused with 50 mq/kg DiHP
Source: SJoberg et a!., 1985a
04730
111-23
07/25/88
-------
5000
1000
€
V.
100
o
m
a 10
Infusion
12
Time (hours!
16
FIGURE III-3
The Mean Plasma Concentration-Time Curves of OEHP and Us Investigated
Metabolites 1n Rats Infused with 500 mg/kg D£HP
Source: SJoberg et al., 1985a
04730
111-24
07/25/88
-------
TABLE III-6
Synthetic Metabolism Of Phthallc Add Esters*
Compound
DBP
Species
rat
rat
Route
gavage
gavage
Dose
500 mg/kg
60 mg/kg
Conjugated Metabolites
MBP-glucuron1de
MBP-glucuron1de
guinea pig
hamster
'Source: Kluwe. 1982a
NR = Not reported
NR
NR
NR
NR
derivatives
MBP-glucuron1de
derivatives
MBP-glucuronlde
derivatives
DEHP rat
ferret
monkey
human
various
gavage
l.v.
l.v.
various
600 mg/kg
NR
94-171 mg
None
MEHP glucuronlde
derivatives
MEHP glucuronlde
derivatives
MEHP glucuronlde
derivatives
04730
111-25
07/25/88
-------
ultimately excreted 1n the urine as free and glucuronlde conjugated MEHP
derivatives. Metabolism In the rat also produced MEHP derivatives; however,
glucuronlde conjugates of MEHP were absent from the urine Indicating the
rat's Inability to excrete glucuronlde conjugates of MEHP. Otherwise, the
authors stated that the two species metabolized DEHP similarly. In
addition, Rhodes et al. (1986) demonstrated that the metabolism of DEHP by
marmoset monkeys Is comparable with that of other primates and shows the
same characteristic differences from the rat as other primate species.
In another study, Lake et al. (1977) compared species metabolism of DEHP
and other PAEs 1n hepatic tissue preparations. The authors compared male
Sprague-Oawley rats, male albino ferrets and male olive baboons. In the
liver homogenates from baboons and ferrets the order of hydrolysis of the
tested dlester PAEs to their monoester forms was DEHP < DBP < DEP < DMP.
The rates of hydrolysis In preparations from rats were In the order of OEHP
< DBP < DMP < DEP. The rates of hydrolysis for DEHP were slower (statis-
tical analysis not reported) than the other PAEs 1n all three species
examined. Dlester hydrolase activity In liver homogenates generally
Increased In the order ferret, rat, baboon. The authors stated that the
baboon, rat and ferret would be suitable for assessing toxlclty In man since
the results show species similarities In their hydrolysis of PAEs.
Lhuguenot et al. (1985) Investigated the metabolism of DEHP and MEHP
after multiple administration In rats. Male Alderley Park (Wlstar derived)
rats were gavaged with dally doses of 50 or 500 mg/kg DEHP or MEHP In corn
oil for 3 consecutive days. Rats were grouped three/dose group for each
04730 III-?6 08/05/88
-------
chemical. Urine samples were collected from each animal at 24-hour Inter-
vals for 4 days. Water-soluble conjugates were not detected In rat urine
after DEHP or MEHP administration. A novel metabolite (XII), however, was
detected In the urine after administration of both compounds (Figure III-4).
The proportion of the dally doses excreted In the urine reached a steady-
state within 48 hours of multiple exposure to DEHP or MEHP. At 50 mg/kg
DEHP and MEHP there were essentially no changes In the metabolic profile
when expressed as a percentage of total metabolites. At the 500 mg/kg dose
level quantities of metabolites I and V were Increased 6.4- and 2.5-fold,
respectively. When Individual metabolites were expressed as percentages of
total metabolites at the 500 mg/kg dose level, metabolites I and V Increased
with time while the proportion of metabolites IX and VI decreased with time.
Multiple dosing with 500 mg/kg MEHP Increased the quantity of metabolite I
4.1-fold. Small decreases In metabolites VI and IX were also observed.
After 3 consecutive days of 500 mg/kg MEHP treatment, hepatic peroxlsomal
0-oxldatlon (as measured by the enzyme palmHoyl-CoA) Increased 4-fold. The
authors concluded that the metabolism of metabolite V Is by peroxlsomal
Q-oxldatlon of the w-oxldated MEHP since at the 500 mg/kg dose level there
was a 4-fold Increase of peroxlsomal B-oxIdation and hence an Increase in
metabolite I. A 2-fold decrease In peroxlsomal w-1 oxidation products was
also observed. The Increase in u-oxldatlon In the absence of an Increase
1n w-1 oxidation, may Imply the Involvement of a cytochrome P-450 with a
high specificity for w-hydroxylat1on. Lhuguenot et al. (1985) also
confirmed that MEHP Is metabolized by the same pathways as OEHP In rats.
Peroxlsomal B-oxIdatlon enzyme system Is Important since peroxlsomes contain
enzymes Involved In fatty acid B-oxldatlon. which generates hydrogen
peroxide. Turnbull and Rodrlcks (1985) and Rodrlcks and Turnbull (1987)
04730 111-27 07/25/88
-------
'COOH
(j-| -oildKkm
'C-0-CHrCH-CH^H-CM,
0 |X CHfHj OH
O-IIYPROXY)
OOM
I
II
O
COOH
C-0-CH1-CH-«CHJ))-CHJ
0 |v CHjCOOH
(5-KETO)
0 CMJCMJ
MOtP(XI)
C00»l
, 01,01,
(3-CARBOXYLIC ACIU)
COOH
C-0-
0 X
.COOH
O v
C1SCKI
(6-liVDROXY)
B-oiUaUon
CC-CH,-,,-*,,,,-
(S-CARBOXVLIC ACID)
|-O-CH,-CH- CHj-enmncHj-COOH
0 XII CHjCH,
FIGURF III-4
Routes of Metabolism of ME IIP In Rats
(metabolites are numbered according to Figure 111-1)
Source: Adapted from Lhuguenol et al. (19BS). Albro et al. (1973) and Albro and Hoore (1974)
-------
hypothesized that the process of hydrogen peroxide formation by the peroxl-
somes Is responsible for DEHPs carcinogenic effects. For a more detailed
explanation of the mechanisms Involved see Chapter VII.
Short et al. (1987) demonstrated that monkeys have a lower capacity to
metabolize DEHP by B-oxldatlon than rats. Fischer 344 rats (12 males/group)
were fed diets containing 1000. 6000 or 12.000 ppm OEHP (50, 300 or 600
mg/kg/day assuming 350 g rats consume 5% of their body weight). Three
subgroups were formed for each dose and received the above diets for 0, 6 or
20 days. They received a similar dietary level of 14C-carbonyl labeled
DEHP for 1 day. Cynomolgus monkeys (2 males/group) received 100 and 500
mg/kg/day by gavage for 21 days. Each monkey then received a single dose of
14C-DEHP followed by three additional dally dosages of OEHP. In rats,
urinary elimination of metabolite 1 was constant with all dietary levels on
day 0; however, It Increased with all dose levels by day 6. The metabolites
are numbered according to Figures III-l and III-4. The Increased percentage
of metabolite I In the urine persisted for 20 days. In contrast, metabolite
V Increased with dietary level on day 0 but decreased with dietary level
from days 6 to 20. Urinary levels of MEHP and metabolite X In monkeys
appeared to Increase with repeated doses of OEHP. There was no Increase In
the conversion of metabolite X to metabolites V and I. The output of
metabolites IX and VI was unchanged or slightly decreased suggesting little
change In the u-1 oxidation pathway.
In a second portion of this study, Short et al. (1987) did not find any
treatment-related evidence of hepatic peroxlsomal proliferation In monkeys
at OEHP levels <500 mg/kg/day. Whereas, exposure to similar levels (11,
04730 111-29 09/08/88
-------
105. 667, 1223 and 2100 mg/kg/day) of DEHP 1n rats produced hepatic
peroxlsomal proliferation. It Is difficult to compare exposure levels since
monkeys were administered bolus doses and rats were administered feed.
However, Short et al. (1987) stated that the doses are In a comparable
range. The authors concluded that urinary levels of metabolite I serve as a
useful marker to detect peroxlsomal Induction activity and hence may serve
as a marker for making Interspecles comparisons. As a result rats may not
provide a good basis for predicting the possible carclnogenlclty 1n higher
primates If peroxlsome proliferation Is Indeed the mechanism or one of the
mechanisms of action.
Schmld and Schlatter (1985) found that a single oral dose of DEHP taken
by two volunteers (30 mg each) was excreted In the urine as OEHP metabolites
within 24 hours. Only 11 and 15% of the dose was eliminated as metabolites
In the urine with the remainder most likely eliminated In the feces (details
not provided). The urinary metabolites [derivatives of mono(2-ethylhexyl)
phthalate] were enzymatlcally hydrolyzed and methylated for Identification.
The quantitative distribution of conjugated and free metabolites determined
was by gas liquid chromatography-mass spectrometry. Twelve metabolites were
detected, the four major ones being free and conjugated forms of the
monoester (MEHP) and Us 5-carboxyllc add (metabolite V), 5-keto (metabo-
lite VI) and 5-hydroxy (metabolite IX) derivatives (see Figures III-l and
III-4). These same few metabolites were also observed In rats (SJoberg et
al., 1985a.d, 1986a). thus Indicating that humans and rats share common
metabol'i tes. Two of the major metabolites Involved oxidation of the
terminal (C-6) methyl group to the corresponding acid, a blotransformatlon
process that also occurs In the rat (Watts, 1985).
04730 111-30 09/08/88
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Rowland (1974) found that Wlstar and Sprague-Dawley rat Intestinal and
caecum contents degraded OEHP. However, there were differences between the
two strains. The authors stated that OEHP was degraded to a single metabo-
lite, which was Identified as MEHP. Hlstar rats were fed diets of 2% w/w
l*C carboxyl labeled DEHP and were found to degrade DEHP at the rate of
-1300 yg/g Intestinal contents/16 hours or 700 ug/g caecal contents/16
hours. The maximum rate of degradation occurred at pH 7.0, which Is the
approximate pH values of the Intestines and caecum 1n rats. When a mixture
of the antibiotics tetracycllne hydrochlorlde, neomycln sulphate,
chloramphenlcol and streptomycin sulphate were added to the Incubation
mixture at 2 mg/mt each, the breakdown of DEHP by the caecal contents was
reduced from -700 to 300 wg/g caecal content/16 hours. The mixture of
antibiotics had a similar effect on OEHP degradation In the contents of the
small Intestines (Rowland, 1974).
In the same experiment, degradation of OEHP by caecal and Intestinal
contents Increased -3-fold In Sprague-Dawley rats fed 2% w/w DEHP when
compared with rats fed a standardized diet. The Increased rate of OEHP
metabolism by the small Intestine was >60% In the DEHP-fed rats as compared
with only 16% by the Intestinal content of rats fed only the standardized
diet. Addition of the mixture of antibiotics tetracycllne hydrochlorlde,
neomycln sulphate, chloramphenlcol and streptomycin sulphate at 2 mg/mi
each had no effect on the rate of DEHP breakdown by the caecal or Intestinal
contents. Changes In the mlcroblal flora of the alimentary tract were
compared with the controls In the Sprague-Dawley rats. In both regions the
total number of bacteria were lowered by a factor of 10 in the DEHP-fed
rats. The mlcroblal flora In the proximal small Intestines of OEHP-fed rats
consisted of blf Idobacterla, bacteroldes and lactobadlH. whereas the
04730 111-31 09/08/88
-------
proximal small Intestines of the controls harbored a wide variety of
bacterial types. The differences 1n the mlcroblal flora between the caecum
and the distal region of the small Intestines of DEHP-fed rats and controls
were negligible.
Rowland (1974) concluded that gut flora "play only a minor role" 1n the
metabolism of DEHP In the alimentary tract of Sprague-Oawley rats since In
the presence of antibiotics the small Intestines and caecum still metabo-
lized DEHP at -50% of the control rate. The author also Found that the
Increase In rate of DEHP degradation and change In mlcroblal flora In the
alimentary tract did not occur In the Wlstar rats fed DEHP. No explanation
was given for these findings and no other data was provided for the rate of
metabolism of DEHP-fed Wlstar rats. Thus, the results support the hypothe-
sis that the Increased rate of DEHP degradation Is due to enzyme Induction
In the mucosal cells of the Intestine.
In another study Rowland et al. (1977) confirmed that phthalate esters
are metabolized to the corresponding monoester by the GI contents In rats
and In cultured human feces. However, the rates of hydrolysis were greatest
In the presence of rat small Intestine contents and much slower with caecal
or stomach contents. The percentage of metabolized OEHP by 16 hours post-
treatment In the stomach, small Intestine, caecum and human feces were
1.0^0.2, 22.U0.5, 6.9il.O and 0.6*0.2, respectively. Each value represents
the mean _+ SEM of four Incubations.
The metabolism of 14C-labeled DEHP In the tissue homogenates of young
(45 day old) and old (630 day old) male Sprague-Dawley rats has been studied
by Gollamudl et al. (1983). The metabolite MEHP was Identified In liver,
04730 II1-32 09/08/88
-------
kidney and lung homogenates Incubated In a mixture of 0.5 pCi
14C-labeled DEHP and a concentration of unlabeled compound to yield a
final concentration of 1 mM DEHP. An unidentified metabolite was present In
the homogenates of the 630-day-old rats but not 45-day-old rats. The forma-
tion of MEHP was decreased by 14% (p<0.001) (expressed as dpm/mg protein) In
the liver homogenates of the old rats. In contrast, the lung and kidney
homogenates did not show any significant change In MEHP formation (measured
as dpm/mg protein).
Pollack et al. (1985a) Investigated the differences In the route of
administration of DEHP In rats following single or multiple Injections by
1.p., 1.a. or p.o. administration, and found that the formation of
monoethylhexyl phthalate (MEHP) from DEHP was route-dependent. Following
p.o. administration of DEHP, 80% was converted to MEHP, while only 1% MEHP
was seen following single doses by 1.a. or l.p. Agarwal (1986) stated that
1.p. administration converts DEHP to MEHP much slower because of the limited
hydrolyzlng capacity of visceral organs.
Gollamudl et al. (1985) Investigated the rates of DEHP hydrolysis In the
tissues of adult Sprague-Dawley male and female 45-day-old rats, fetuses on
day 19 of gestation, newborns within 12 hours of parturition, pregnant dams
and the placenta on day 19 of gestation. Placenta and fetuses were removed
on day 19 of gestation for the hydrolysis study. Tissues from the fetuses
of each rat were pooled. Neonates were sacrificed within 12 hours of
birth. The conversion of DEHP to MEHP by the liver and for placenta
preparations (analyzed as organ-to-whole-body metabolism) was significantly
(p<0.05) less active In the placenta < fetus < neonate than In the adult
male and female rats and pregnant dams, whereas the conversion by the lung
04730 111-33 09/12/88
-------
and kidney preparations (analyzed as organ-to-whole-body metabolism} was In
the order of fetus < neonate < placenta < adult female < adult male <
pregnant dams. The tissues oF the fetuses and neonates showed significant
(p<0.05) OEHP hydrolysis activity.
Peck et al. (1979) examined humans receiving DEHP-laden platelet concen-
trates. Urinary metabolites deluded MEHP, eight oxidized derivatives of
the monoester (the predominant species being mono-?-ethyl-3-carboxyl-propyl
phthalate) or 5-ethyl-1sohexanol monoester of phthallc acid and trace
amounts of Intact DEHP (Peck and Albro, 1982). In contrast to the rats
Inability to excrete the MEHP glucuronlde conjugates (Tanaka et al.. 1975;
Albro et al., 1982; Williams and Blanchfleld, 1975), -90% of the metabolites
were excreted In the human urine as glucuronlde conjugates, while the
remaining -1054 was excreted In feces (Peck et al.( 1979).
Both primates and humans exhibited similar metabolic profiles. Peck and
Albro (1982) described OEHP metabolism In studies with African green
monkeys. The experiments simulated human blood transfusion exposures to
OEHP by Impregnating PVC plastic strips with i*C-carbonyl labeled DEHP.
The strips were Immersed 1n plasma, which In turn was Infused Into the
monkeys. The predominant metabolic products In the urine Included the
5-ethyl-lsohexanol monoester of phthallc acid and MEHP. As 1n the
previously described study by Peck et al. (1979) with humans, >90y. of the
urinary metabolites In the monkeys were glucuronlde conjugates.
Evidence submitted by Albro et al. (1982) Indicated that mice, guinea
pigs and hamsters also excrete glucuronldes of MEHP following single oral
04730 111-34 09/12/88
-------
exposures to {carbonyl labeled) [7-l*C]-DEHP (cotton seed oil vehicle).
In each species these conjugates of DEHP metabolites comprised at least 64%
of the urinary metabolites detected.
von Danlken et al. (1984) found that when rats (F344) and mice (NMRI)
were pretreated with OEHP (10 g/kg) In the diet for 2-3 weeks and then given
radlolabeled 14C-DEHP by gavage (In olive oil), the metabolism of the
subsequent dose of 14C-carboxyl labeled DEHP was Increased. Exhaled
14CO. from the degradation products of 14C-DEHP was generated over a
shorter time period fbr pretreated animals as compared with nonpretreated
rats. Liver DNA was Isolated 16 hours after treatment with 14C-carboxyl-
ate labeled DEHP and analyzed for radioactivity. No evidence for covalent
binding of 14C-labeled DEHP or metabolites to liver DNA In either species
was detected.
In studies where 200 mg/kg 14C-carbonyl labeled DEHP was administered
Intravenously to groups of rats, the blood levels of radioactivity were used
to estimate the blphaslc disappearance of DEHP. The half-life values corre-
spond to 9 and 22 minutes (Schultz and Rubin, 1973). Within 1 hour, 8% of
the dose was detected as water soluble metabolites In the liver, Intestinal
contents and urine. After 24 hours, 54.6% of the dose was found as the
water soluble metabolites In the Intestinal tract, excreted feces and urine.
Only 20.5% was recovered In organic extractable form.
Rubin (1976) Injected rats Intravenously with an emulsified form of DEHP
(dose not reported) resulting In blexponentlal disappearance of blood OEHP.
Blood half-lives of 3.5 and 35 minutes were determined. However, when the
04730 111-35 08/05/88
-------
DEHP was solubHizec wltnout surfactant, disappearance was monoexponentlal
wHh a half-life of 19 minutes. Further studies in humans with OEHP
solubilized without surfactant also yielded a monoexponential rate of
compound disappearance where the mean half-life was calculated at 28 minutes.
BBP. Only one study was found regarding the metabolism of BBP.
Elgenberg et al. (1986) Identified the major urinary metabolites after rats
were administered oral doses of 2, 20, 200 and 2000 mg/kg BBP. Urinary
metabolites consisted of monophthalate (HP), monophthalate-glucuronide
(MP-glucuronide) and "unidentified" metabolites. At 200 mg/kg BBP the
amount of free HP and the ratio of free to conjugated HP was greater than at
2 and 20 mg/kg. At 2000 mg/kg BBP there was a shift to primarily fecal
elimination (72%) with only 22% of the dose excreted In the urine.
Four hours after l.v. administration of 20 mg/kg BBP, rats excreted 55%
of the total l*C dose In the bile and 34% of the total 14C dose in the
urine (Eigenberg et al., 1986). Biliary metabolites were Identified as
large quantities of monobutyl phthalate glucuronlde (MBuP-glucuronldeJ and
monobenzyl phthalate glucuronlde (HBeP-glucuronlde), (26 and 13% of the
dose, respectively), trace amounts of free HBuP and MBeP (1.1 and 0.9% of
the dose, respectively) and unidentified metabolites (14% of the dose).
Elgenberg et al. (1986) concluded that the BBP-treated rats major
urinary metabolites were HP and HP-glucuronlde, 1n contrast to the rats
Inability to excrete the glucuronlde conjugates of MEHP upon exposure to
OEHP (Tanaka et al., 1975; Williams and BlanchMeld, 1975; Albro et al.,
1982). As the oral dose of BBP Increased, there appeared to be a decrease
04730 111-36 09/08/88
-------
in tne ra:io of MP-glucuronlde to unconjugated MP meiaooHf.es. Ar'-.e-- i.v.
administration, reduced amounts of glucuronloe were ooserved.
DBP. The urinary metabolites of orally administered 08P were studied
by Albro and Moore (1974). Doses of 0.2 mi DBP (599 mg/kg/day assuming
0.350 kg rats) were administered by gavage to adult male CD rats at 24-hour
Intervals. Urine samples were collected <48 hours after the Initial
dosing. 08P was converted to stx metabolic products with a total of 24.6%
of the phthalate moiety recovered 48 hours after the first feeding and 24
hours after the second feeding. DBP was detected, to a lesser extent
(0.1%), as the Intact ester In rat urine. Each metabolite could be resolved
by HPLC; however, the complete structures could not be Identified.
Metabolism of DBP was characterized largely by hydrolysis of one ester bond
and terminal (w) and subtermlnal (w-1) oxidation to primary and
secondary alcohols, which were ultimately oxidized to acid and ketone
species, respectively.
Urinary metabolites of l«C-DBP In the rat, guinea pig and hamster were
determined as MBP, the MBP glucuronlde, phthallc acid, unchanged DBP and
w- and w-1 oxidation products of MBP (Tanaka et al.. 1978). Hydrolysis
of DBP was found to occur primarily In the liver with some contribution from
the Intestinal mlcroflora. Similarly, Williams and Branchfleld (1975)
Identified the urinary meta-bol-1 tes of a sVngle oral dose of l*C-DBP in the
rat to be phthallc acid. MBP and two other methylated metabolites.
In experiments by Foster et al. (1982), the major urinary metabolite
detected In both species after p.o. treatment (no vehicle stated)
04730 II1-37 09/08/88
-------
of male Sprague-Qawley rats and DSN hamsters witn 14C-labeled 08P (2 q) or
HBP (800 mg) was the MBP glucuromde. Most of the MBP {17.4% In rais and
6.3% In hamsters) and metabolites (as measured by HPLC) were excreted as
glucurontde conjugates (47.8% \n rats an-: 06.9% In hamsters) and not as tne
free acid. Further studies Indicated that after oral administration of D8P
or MBP, the levels of free unconjugated MBP In the urine were 3- to 4-fold
higher In the rat than in the hamster. Intestinal esterase activities were
comparable 1n the two species, but testlcular Q-glucuron1dase activity was
significantly higher (p<0.001) In the rat than In the hamster. The
Increased level of B-glucuronldase activity In the testlcular tissue of rats
suggests that the levels of free MBP available to hamsters testes would be
much lower than In the case of the rat. MBP produced cell Injury to
cultured sertoll and germ cells much more effectively than OBP (Gray, n.d.).
Increased free MBP may account for species susceptibility to MBP- or
DBP-lnduced testlcular damage. In conclusion, the' authors state that the
major urinary metabolite of MBP or OBP Is the MBP glucuronlde and not the
free acid.
Rowland et al. (1977) examined the rate of DBP hydrolysis to the mono-
ester In suspensions of human feces or raw-gut contents from Wlstar rats.
The rates of hydrolysis were greatest In the presence of rat small Intestine
contents and much slower with caecal or stomach contents. The percentages
of metabolized OBP In 16 hours by the stomach, small Intestine, caecum and
human feces were 0.5*0.07. 80.8*2.3. 22.9±1.3 and 6.2i.0.2. respectively. At
concentrations of '£200 yg/mi DBP was metabolized -70% 1n 30 minutes.
Both the age and sex of the rats Influenced the rates of OBP metabolism.
Young male rats, 26 days old, metabolized OBP more slowly than 33-day-old
male rats or 26-day-old female rats 30 minutes after exposure.
04730 111-38 09/08/88
-------
Female rats between 33 and 40 aays old metaoollzed 15.7^2.5 and 25.5-3 3%
D8P In 30 minutes, respectively, whereas male rats at tne same ages me:aoo-
lized 34.5*2.4 and 34.4±2.2, respectively.
PEP. One study was Found regarding the metabolism of OEP. Rowland et
al. (1977) examined the rates oF hydrolysis oF OEP to the monoester In sus-
pensions oF human Feces or raw-gut contents From Wlstar rats. The specific
monoesters Formed, however, were not Identified. The rates of hydrolysis
were greatest In the presence of rat small Intestine contents and much
slower with caeca! or stomach contents. The percentages of metabolized OEP
In 16 hours by the stomach, small Intestine, caecum and human feces were
2.5^0.2, 36.4+2.1. 11.5+0.5 and 3.0^0.1, respectively.
PHP. Kaneshima et al. (1978) studied the effects of a single oral
dose of 500 mg/kg i4C-OMP In 50% ethanol upon the biliary excretion of
rats. Several metabolites were detected. An extract of the bile contained
DBP. M8P, phthallc acid and an unidentified substance. A glucuronide of MBP
and traces oF other glucuronldes were also discovered upon Further analysis
using TLC.
Albro and Moore (1974) studied the urinary metabolites of OMP. Adult
male CD rats were administered 0.1 mi PHP (17 mg/lcg/day assuming 0.350 kg
rats consume 5% body weight) by gavage at 24-hour Intervals. Urine samples
were collected <48 hours following the Initial dosing of OMP. A sample of
urine obtained after 24 hours contained the following metabolites: 14.4%
free phthallc acid, 77.5% monomethyl phthalate and 8.1% dimethyl phthalate
intact. The metabolites were identified by GLC and TLC.
04730 lfl-39/ 08/05/88
-------
Rowland et al. (1977) examined tne rate of W? nydrolysis to tne mcno-
ester in suspensions of human feces or raw-gut contents from Vlistar ra'.j.
The rates of hydrolysis of DMP to the monoester were greatest in tne
presence of rat small intestine contents and much slower with caecal or
stomach contents. The percentages of metabolized OMP in 16 hours by the
stomach, small Intestine, caecum and human feces were 21.2^1.1, 61.U0.9,
15.9^0.4, 8.3^0.2, respectively.
Excretion
The PAEs and their metabolites are eliminated from the body .through
urinary, fecal and biliary excretion routes. The greater part of the
metabolites of the administered esters are excreted in the urine. Most
studies of excretion have utilized the compounds OEHP and OBP as is shown in
Table III-7.
DEHP. Excretion of phthalates has been predominantly studied using
DEHP. Schultz and Rubin (1973) found -13% of a single oral dose of 200
mg7kg 14C-carbonyl labeled DEHP (In corn oil) In the 'organic solvent
extracts in the urine, feces and large intestine contents of rats. The
urine contained 62% in water extracts. Daniel and Bratt (1974) reported
that upon a single oral exposure to 2.9 mg/kg i«C-carbonyl labeled OEHP,
rats excreted 42% and 57% of the dose In the urine and feces, respectively,
in 7 days. In another portion of the study, rats were fed 1000 ppm DEHP (50
mg/kg/day assuming rats weighing 180 g consume 0.05X of their body weight)
for 7 days and then given a single oral dose of 2.9 mg/kg 14C-labeled
o
DEHP. Rats excreted 57% of the radioactivity In the urine and 38% in the
feces in 4 days. 811 lary-cannulated rats excreted 14 and 9% of the 2.6
mg/kg i*C-OEHP labeled dose In 4 days In urine and feces, respectively.
04730 111-40 09/08/88
-------
TABLE III-7
Summary of Biliary. Fecal and Urinary Excretion of OBP or DEHP In Rats3
Compound
OBP
DEHP
Dose
60 mg/kg
500 mg/kg
2.31 g/kg
50 mg/kg
2.6 mg/kg
1.0 g/kg
10 ppmd
2000 ppmd
50 mg/kg
T1meb
24 hours
6 hours
48 hours
5 hours
4 days
4 days
NR
NR
7 days
Exposure
Route
gavage
gavage
gavage
l.v.
gavage
gavage
feed
feed
l.v.
Port
Bile
40
5
NR
10
14
NR
NR
NR
NR
1on of Dose. %c
Feces
5
NR
5
NR
. 56
40 (8)
4 (0)
9 (6)
28
Urine
88
NR
82
NR
42
60
96
91
49
aSource: Kluwe. 1982a
bT1me of collection post treatment
"•Metabolites 1n parentheses
^Concentration Incorporated Into feed
NR = Not reported
04730
111-41
07/25/88
-------
14C-car;cp^'. 'a^e'^c D:-? •.; «'.s:ar -a-.j -- .-a:
the jnne and feces and only
-------
i";': ?'. = '9S2, s'. aC'-ec '.fie e;;e:;. cr" I:-1? eoos-"? ~' T»:'T
Tnree grojps cr male F'scne' 34i rats received :«C-OEHP ;ir. cov.or, ;;?e:
o'l) o> gavage a: one of tn.-ee oosage "levels ("• 8. 18 or 180 mg/icg D./oay
•DEHF) for 10 oays. After an imnal 4-day acclimation period, excretion «as
found to oe independent of the dose indicating tnat tne overall elimination
mechanism was not affected by the doses given. Rats receiving 180 mg/kg/day
OEHP eliminated l*C at a rate 100 times that of rats receiving 1 8
mg/kg/day DEHP.
Tissue half-lives of DEHP and MEHP were determined In male Wlstar rats
after a single oral administration of DEHP (25 mmol/kg or 9.8 g/kg) by
gastric Intubation (Olshl and Hlraga. 1982). Both compounds disappeared
exponentially from the blood, liver, testes, heart, spleen, lung and
epldldymal fat with t. . values of 23-68 hours for MEHP and 8-156 hours
o '' ^
for DEHP (Table III-8). The longest half-lives for both DEHP and MEHP
occurred In the epldldymal fat followed by disappearances from the testes
for MEHP and the liver for DEHP. In the present study MEHP exhibited the
long half-life and the highest ratio of MEHP/DEHP 1n the testes. in
addition, with the exception of epldldymal fat and Igngs, the biologic
naif-life of MEHP was slightly longer than DEHP.
In studies with African Green monkeys described by Peck and Albro
(1982), l.v. administration of 14C-DEHP resulted- in an accumulation of
>50% of the Injected -3 mg dose In the urine after 5 hours. Within 24
hours. >70X of the dose was excreted In the urine. Fecal excretion was
found to account for >5X of the administered dose after 4 days.
04730 111-43 07/02/91
-------
TABLE III-8
MEHP/DEHP Ratios and Biological Half-Lives of DEHP anfl MEHP at b Hours
After Administration*
Blood
Liver
Testes
Heart
Spleen
Lung
Epldldymal fat
MEHP/DEHP Ratio6
(mol%)
113±23
79*17
210i4.8
46±0.57
I/
d/
87±24
Biological
MEHP
23.8
31.9
49.9 (6
-------
Sr.coss 5'. a" '"30:1, s'.^o'.e: \~e ::mDd"s ". ' • ? ;ris--c; ;.< • -a-. ;s ;• l:-:
trie Wls:ar rat ano tne marmoset .iion
-------
urine after 48 nouns. On tKe basis of this the investigators est'maisc 3
half-life of 12 hours, and concluded that accumulation of DEHP in the bociy
is unlikely to occur. Unfortunately, fecal analysis that could have
supported ihis hypothesis were not performed. Generally these data compare
well with those of Peck et al. (1979) for human patients that received
Infusions of DEHP-contaminated blood.
Peck et al. (1979) followed the excretion of DEHP and Us metabolites
from two patients receiving DEHP-laden platelet concentrates. In one
Individual, 57% of 94.7 mg DEHP that was Infused over 4 hours was detected
In the urine 8.5 hours later. A second subject received 174.3 mg DEHP in
1.5 hours. Within 24 hours of administration, over 60% of the dose was
recovered In the urine.
BBP. BBP was rapidly excreted after single oral doses of 2, 20, 200
or 2000 mg/kg to male Fischer 344 rats (Elgenberg et al., 1986). This
phthalate undergoes extensive enterohepatlc circulation. The majority of
the dose (-75%) was eliminated In the urine and -20% eliminated in the
feces; >92% of the dose was excreted by the fourth day. At 2000 mg/kg there
was a shift to primarily fecal elimination (72% of the dose after 4 days).
Elgenberg et al. (1986) stated that Increased fecal elimination at the
highest dose may be due to Incomplete absorption during enterohepatlc
circulation. Four hours after a single 1.v. Injection (20 mg/kg BBP) 55% of
the total dose was excreted Into the bile and 34% In the urine.
04730 II1-46 09/08/88
-------
lake et al (1973) found similar results when single oral ooses or '6.
160 ana 1600 mg/kg 8BP was administered to male Sprague-Oawley rats.
Approximately 80% of the administered dose was excreted In the urine within
5 days; most of the remainder was excreted 1n the feces (-17%).
DBP. After single oral doses of 0.1 and 0.13 g/kg D8P-7-14C to male
Wistar rats, 96% of the radioactivity was excreted in the urine. Most of
the original dose (80-90%) was recovered in the urine (Williams and
Blanchfield, 1975). Tanaka et al. (1978) found that 08P was rapidly
absorbed, metabolized and excreted after either oral or V.v. administration
to rats. Within 48 hours. >90% of the administered DBP was excreted in the
urine, regardless of the exposure route. Excretion In the feces was minimal
(Tanaka et al.. 1978).
Kaneshlma et al. (1978) also looked at excretion of 14C-r.adlolabel
(position of label not reported) In the bile after l.v. or oral administra-
tion of 14C-DBP to rats. About 10% of a 50 mg/kg dose-was recovered In
the feces (though H could be the bile; the paper was not clear) within 5
hours after Injection and 4.5% of a 500 mg/kg dose was detected within 6
hours after Ingestlon. The results of these studies upon excretion have led
to postulations that hepatobl1lary excretion of DBP metabolites may be
saturated at high doses or they may occur only after a specific period of
time following absorption (Kluwe. 1982a).
PEP. Data regarding the excretion of OEP could not be located In the
available literature.
04730 111-47 09/08/88
-------
PHP. In a OMP metaoollte study t>y Albro and Moore (1974). 14.4% free
•phthalic acid. 77.5% monomethyl phthalate and 8.1% OMP as Intact compound
were excreted in the urine after 24 hours. These values are mole
percentages of recovered phthalate.
Summary
The fate of PAEs 1n the body has received considerable attention.
Administered doses have been found to be rapidly absorbed from the Intes-
tine, skin, peritoneum,' blood and lungs. A large percentage of the dlesters
are hydrolyzed although It 1s not uncommon to find low levels of "the Intact
compounds present In the excretory products. However, hydrolysis of the
dlesters- appears to be Inversely proportional to their alkyl-chaln length
and concentration. Both dose- and time-dependent quantitative differences
In the profile of DEHP urinary metabolites were observed In rats exposed to
DEHP or MEHP for 1, 2 or 3 days. The results suggested that DEHP and MEHP
are metabolized by similar routes and stimulate their own metabolism by
Inducing u-oxidatlon (cytochrome P-450-med1ated u-hydroxylat1on) and
peroxlsomal B-oxIdatlon. Thus, the duration of exposure, the dose level
administered and the status of the animal with respect to peroxlsomal
proliferation Is Important when evaluating metabolic studies on PAEs.
Once absorbed. PAEs or their metabolites are deposited throughout the
body. Retention or accumulation of PAEs Is minimal. Orally administered
PAEs are deposited primarily In the liver, Intestine, muscle and adipose
tissue. However, accumulation In many of these tlssires may be a function of
the excretion of the compound. Several studies confirm placental transfer
as well as fetal tissue uptake.
04730 111-48 09/08/88
-------
Tne route of exposure and structure of PAEs ana tneir metaoo". • :es
Influence theu body distribution. Following i.v. administration D3P c.d
not accumulate in the liver to the same extent as OEHP. In addition.
retention of OBP In the heart, lung and spleen after oral or i.v. exposure
appeared to be shorter than DEHP. Few differences were observed in the
distribution pattern of DBP compared with that of OEHP. Information on the
distribution of 8BP, DEP and OMP is either limited or not available. The
study of BBP, DEP and DMP as a function of the routes of administration, as
well as the pharmacoklnetics and disposition of biologically relevant
metabolites, such as MEHP, remain Important areas to Investigate.
Metabolism of PAEs 1s governed by their molecular weight and alkyl-chaln
length. Dialkyl phthalates are hydrolyzed to monoesters In the Intestine
and other organs following absorption. The rate of hydrolysis is greater
for the lower molecular weight esters than for the higher molecular weight
esters. Only a small fraction of long-chain alkyl phthalates undergo
complete hydrolysis. The hydrolyzed monoesters form glucuronide conjugates
in many species. Species differences in PAE conjugation has been observed;
for example, DEHP 1s glucuronated in man and monkey, whereas this does not
occur In the rat.
PAEs and their metabolites are eliminated from the body through the
urinary, fecal and biliary excretion routes. Though urinary excretion Is
quantitatively the major route of removal, feces can also be of Importance.
Most PAEs are excreted as a monoester metabolite (glucuronide conjugate)
with a small portion being unchanged parent compounds. Rats are an apparent
exception in their Inability to form and excrete glucuronide conjugates of
MEHP.
84730 111-49 09/08/88
-------
Following multiple oral admini s tration of- 105. 667 or 1223 mg/kg/cay
DEHP. the average amount of recovery In the urine of rats ranged from 88-96%
of the administered doses. After single oral doses of 100 mg/kg ldC-DEHP.
both rats and monkeys eliminated -30% of the dose In the urine during the
first 24 hours. Intact OEHP was not detected In the urine; however. 20% was
recovered In the feces of rats and 34.3% was recovered in the feces of
monkeys. In one human patient receiving 174.3 mg OEHP. >60% of the dose was
recovered in the urine within 24 hours. Only 10-15% of an oral 30 mg dose
of DEHP was excreted In the urine of human volunteers. Unfortunately fecal
analysis was not performed. Fischer 344 rats rapidly excreted -75% in urine
and -20% In the feces of single oral doses of 2, 20 or 200 mg/kg BBP. At
2000 mg/kg BBP there was shift to fecal elimination (-72% of the dose after
4 days). After single oral doses of -0.1 g/kg DBP to Wlstar rats. 80-90% of
the dose was recovered In the urine. Within 24 hours after an oral dose of
17 mg/kg/day DMP, CD rats excreted the majority of the dose in the urine
with 8.1% (mole percentage) as Intact compound. Data regarding the
excretion of DEP could not be located in the available literature.
04730 111-50 08/05/88
-------
IV. HUMAN EXPOSURE
Text to be provided by the Office of Drinking Water
04740 IV-1 08/27/86
-------
V. HEALTH EFFECTS IN ANIMALS
Introduction
Since the early 1970s, PAEs have been the subject of extensive toxlco-
logtc research. The overall effects of PAEs have been reviewed by several
authors (Autlan, 1973; Peakall, 1975; Thomas et a!., 1978; Thomas and
Thomas, 1984). A national conference on the potential health threats of
PAEs was held 1n 1972, the results of which were published In the January
1973 Issue of Environmental Health Perspectives. A subsequent Issue of
Environmental Health Perspectives (1982) was devoted to recent research on
phthalate esters following a conference sponsored by the National
Toxlcologlcal Program (NTP) and the U.S. Interagency Regulatory Llason. Tfte
U.S. EPA (1980) published an Ambient Water Quality Criteria Document for
PAEs, which summarized literature published through 1979 and developed water
quality criteria for ambient water. In 1982, the International Agency for
Research on Cancer (IARC) published monographs on several PAEs and related
compounds suspected of causing cancer. The Consumer Products Safety
Commission reported estimated possible Increased risk of cancer to children
exposed to DEHP In children's products such as pacifiers, teethers, squeeze
toys, plastic baby pants and vinyl fabrics covering playpen pads (CPSC.
1983, 1985). The majority of tox1c1ty studies have focused on DEHP since
this compound accounts for -40% of the phthalates produced for commercial
use. Limited Information on toxlclty Is available for several other PAEs.
Short-Term Animal ToxIcUy
Based on data accumulated from several studies, the acute toxlclty of
PAEs Is considered, qualitatively, to be rather low. All oral, dermal and
l.p. LD s are >1.Q g/kg bw (Table V-l). Oral LD50 values reported for
04750 V-1 07/03/91
-------
l.p. LTJrQs are >1.0 g/kq bw (Table V-l). Oral LQ,Q values reoor:ed for
PAEs range from 1.0 g/kg bw for DEP to 34 g/kg bw for DEHP (see Taole v-lj.
Generally, the acute oral toxlcHy of the PAE tends to decrease witn
Increasing molecular weight. For any of the tested PAEs, acute toxlcHy may
also vary with species tested. The oral LD50S for OEHP ranged from 26
g/kg bw 1n rats to 34 g/kg bw 1n rabbits (Autlan. 1973).
Dermal LO-..S appear to be approximately twice the oral LO^.s. The
high dermal L0,-s may result, In part, from reduced absorbtlon of the
administered compounds.. In the case of low molecular weight PAEs, dermal
exposure may be decreased by compound volatilization. As a group, PAEs
produce little Irritation when placed 1n contact with the skin of animals or
Humans.
The LD5Q ovalues for PAEs administered 1.p. ranged from 0.7-20 g/kg,
again Indicating low acute toxlclty for these compounds. Toxlclty of PAEs
Is generally greater following l.p. Injection than following oral
administration. For example, comparison of the oral and l.p. LO.Q values
for the same species Indicated that DMP administered l.p. was approximately
twice as toxic (on a mg/kg basis) as when administered orally (Autlan,
1973]. Oral administration of >4 g/kg bw of bttylbenzyl phthalate (8BP) to
rats proved fatal (Mallette and Von Hamm, 1952). It was unclear as to
whether the compound was administered In mineral oil or..propylene glycol.
The authors reported that animals died between 4 and 8 days after treatment.
showing weight loss, apathy and leukocytosls. H1stolog1c examination of the
organs revealed toxic splenltls and degenerative lesions of the central
nervous system with congestive encephalopathy, myelln degeneration and gllal
prolIferatton.
04750 V-2 07/03/91
-------
1ABLE V-l
Suomary lable of Short term loxlctty Studies of PAIs In Hamnals
O
00
Exposure/Route PAE
Oral DIHP
01 HP
01 HP
01 HP
OEHP
DIHP
DIHP
01 HP
01 HP
01 IIP
01 HP
DIHP
01 HP
01 HP
01 HP
Of HP
OEHP
HBP
HBP
HBP
BHP
BBP
Critical Effect
No effect on Mortality
No effect on Mortality
No effect
Diarrhea
No deaths
No deaths
Death In 8/10
No deaths
L°M>
L«50
l°50
LD&O
Progressive hepatic
changes. Increase In
relative liver weight.
biochemical changes.
cellular changes
Hepatic changes, blo-
chenlcal changes, cellular
changes
Hepatic enlargement
(1.5 X control)
No effect on liver weight
Decreased body weight
gain; a NOAH In Mies
Identified at 71.06
and 126.31 mg/kg/day
In females
>DM>
i "so
IUSO
Norldllty with toxic
splenttls. degeneration
of CNS
No effects on heniopolellc
system
Species
Rat. Mscher 344
House. B6C3M
Rat
Rat
Rat
House
Hat
Rat
Rat
Rabbit
House
Guinea pig
Rat. albino
Ulslar
Rat. Ulstar
Rat
Rat
Rat. F344
Hat. Mscher 344
House. B6C31 1
House. B6C3EI
Rat. unspecified
strain
Rats, Msiher 344
Dose/line
0.8-20 g/kg/slngle
1.25-20 g/kg/slngle
25.0 g/kg/slngle
110 g/kg/slngle
15.8 g/kg/slngle
34. 5 g/kg/slngle
79.5 g/kg/stngle
5 9 g/kg/slngle
26.0 g/kg
34.0 g/kg
33.5 g/kg
26.3 g/kg
2 g/kg/day for 4.
7. 14 or 21 days
2.5 g/kg/day for 7
or 21 days
0.3 g/kg/day
(0.5X In diet)
for 49 days
0.06 g/kg/day
(0.1X In diet)
for 35 days
38 84. 77.06 154.13.
308.25 and 616.50
mg/kg/day In males;
31.83. 63.16. 126.31.
252.63 and 505.25
mg/kg/day In females/
14 days
2 33 g/fcg
4.17 g/kg
6 16 g/kg
l.B g/kg/day
for 4-8 days
0 625 (3/5 my/kg/day)
and 1 25X ( /5U mg/kg/
Reference
NIP. I982a
NIP. 19B2a
Krauskopf. 19/3
Krauskopf. 19/3
Krauskopf. 19/3
Krauskopf. I9n
Krauskupf. 19/3
Krauskopf. 19/3
Autlan. 19/3
Autlan. 19/3
IARC. I9H2
IAHC. 1982;
Krauskopf. 1973
Lake et al..
19/5
Nangham et al..
1981
Daniel and
Bratt. 19/4
Daniel and
Brail. 19/4
NIP. I982a
IAHC. 1982
IAHC. 1982
IAKC. I9H?
Hal let 1 and
Von Hdnin. 1952
Aijarwal el al .
1985a
day) for 14 ddys
-------
I ABU VI (conl.|
o
u>
liposure/Roule
Oral (conl.)
OerMl
Inhalation
l.p lnjt.-t.llan
PAl
DBP
DIP
OIP
DIP
OIP
ONP
ONP
DNP
ONP
ONP
ONP
Of HP
OBP
DNP
OfP
OBP
Of HP
Of HP
Of HP sonicated
Into plaiM
01 HP sonicated
Into plaseu
Of HP after blood
pressure held
al shock level
30 alnutes
Of HP
01 HP
Critical Iffecl
AbnorMl biochemical
changes, growth depression
Increased absolute and
relative liver weights;
peroitsoM) proliferation
at 2.SX as well as
testlcular atrophy;
LOAfl Identlfed al 0 6X
"SO
leaporary distress
"SO
"SO
"SO
"SO
"so
Mo effect
No effect
"SO
"SO
"SO
"SO
Height loss. Increase
In gaeM globulin
"so
"so
"SO
10,00
Mortality ?/b
"so
Hepjlotoilc effect
Species
Rats. Wtstar
Rats. f344
Rabbit
Rabbit
Rat
Nouse
Rat
Guinea pig
Rabbit
Nice
Dogs
Guinea pig
Rabbtt
Rabbit
Rabbtt
Rat
Nouse
Rat
Rat
Rat
Rat
Nouse. ICR
Rat. albino
Oose/Me«
0 SX (0 2S g/kg)
powder In diet for
34 days/tOAfl
0.6. 1.2 and 2.SX |k?«.
1234 and 2IS6 ng/kg/day
for Mies; 632. 1261
and 210? ag/kg/day for
lew let}/?) days
1.0 g/kg
3.35 g/kg/B days
9.16 g/kg/s Ingle
1. 2 g/kg
2. g/kg
2. g/kg
4. g/kg
I- g/kg
0. -1.4 g/kg
10.0 g/kg
20.9 g/kg
11.9 g/kg
22. IS g/kg/s Ingle
0.4 mq/m* for
10 weeks
14.2 g/kg
49.2 g/kg
200 Mg/kg
400 ag/»
a- 13 e^/kg
1 31 M/kg*
for 1? week*
S M/tg/day on days
). S and 10 of 2?-day
lest period
Reference
Nurakaal and
NlthtyaiM. I90b
CNA. I 98k
Aullan. 1913
Bllckensdorfer
and lenpleton.
1930
Krauskopf. 1913
Aullan. 19)3.
Krauikopf. 19/3
Aullan. I9?3
Aullan. 19/3.
Krauskopf. 19(3
Krauskopf. 1913
Krauskopf. 19/3
Aullan. 19/3
Autlan. I9»l
Aullan. 19/3
Autlan. 1*13
Ovoskln el al .
1969
Aullan. 1913
Aullan. 1913
Rubin and
Chang. 19/8
Rubin and
Chang. 19(8
RuUtn and
Chang. 19(8
1 jwrem e
el al . 19/i
Sf Uavlavd
el al . 14(1
-------
lABtf VI (conl
t
•ft
1 ipoiure/Roule
1 p Injection
(tool |
1 ¥. Injection
1 m.
PAl
BBP
DBP
DBP
OBP
DIP
01 P
DIP
OtP
DNP
OMP
ONP
DIP
OINP solublltied In
nonlonlc detergent
OBP
Critical fffect
10*0
iDso
i«so
Growth depression.
peritonitis
10 jg
10 jQ
Sow temporary distress
No effect
ID™
JW
lOjJ
Respiration stimulated
Initially and then
lOjo *"
LOU
Spec tes
Mouse
Mouse
Rat
House. Swtss
Webster
House
•at
Rabbit
Guinea pig
House
House
Rat
Oogs
flat. Htstar mtlt
Rat
Dose/ llw
3.16 g/kg
4.0 g/kg
3.19 g/kg
0 * g/kg/day for
6 weeks
2 0 g/kg
5.64 g/kg
2.24 g/kg bw/6 days
1.6B g/kg/B days
1.40-3.99 g/kg
1.6-3.6 g/kg
4.02 g/kg
0.2B g/kg/s Ingle
250-300 •g/kg
8.0 g/kg
Reference
Aullan. 1)13
Autlan, 1)13
Autlan. 1)13
Calley et al..
1)66
Auttan. l)?3
Auttan. 11/3
Bltckenidorfcr
and feopleton.
I930
Bllckensdorfer
and lewpleton.
IS 30
Autlan. 198?
Aullan. 19/3
Aullan. 19/3
Bllrkensdorfer
and leap lei on,
1930
Schulti el al..
I9JS
Aullan. l«)3
«0ose represents the 1050 calculated for .Ut receiving I. p. Injections 5 days/week.
1040 f *«* toed the sane for 3 weeks lalnlMua of 10 weeks of dosing).
Mints were calculated each neck unltl the
-------
PAEs range from 1.0 g/kg bw for OEP to 34 g/kg bw for DEHP (see Table V-'. )
Generally, the acute oral toxldty of the PAE tends to decrease witn
Increasing molecular weight. For any of the tested PAEs, acute toxldty may
also vary with species tested. The oral LD-Qs for DEHP ranged from 26
g/kg bw In rats to 34 g/kg bw 1n rabbits (Autlan, 1973).
Dermal LD,Qs appear to be approximately twice the oral LD5Qs. The
high dermal LD,ns may result, In part, from reduced absorbtlon of the
administered compounds. In the case of low molecular weight PAEs, dermal
exposure may be decreased by compound volatilization. As a group, PAEs
produce Uttle Irritation when "placed In contact with the skin of animals or
humans.
The LD5Q values for PAEs administered l.p. ranged from 0.7-20 g/kg,
again Indicating low acute toxldty for these compounds. Toxldty of PAEs
Is generally greater following l.p. Injection than following oral
administration. For example, comparison of the oral and l.p. LD5Q values
for the same spedes Indicated that DMP administered l.p. was approximately
twice as toxic (on a mg/kg basis) as when administered orally (Autlan,
1973). Oral administration of >4 g/kg bw of butylbenzyl phthalate (8BP) to
rats proved fatal (Mallette and Von Hamm, 1952). It was unclear as to
whether the compound was administered In mineral oil or propylene glycol.
The authors reported that animals died between 4 and 8 days after treatment,
showing" weight loss, apathy and leukocytosls. Hlstologlc examination of the
organs revealed toxic splenUVs and degenerative lesions of the central
nervous system with congestive encephalopathy, myelln degeneration and gllal
prolIferatIon.
04750 V-6 07/03/91
-------
As will be discussed In more detail In Chapter VII, Mechanisms of
Toxlclty, the toxic effects of phthalate esters are thought to be caused by
monoester metabolites. Acute toxldty studies of various phthalate esters
are summarized In Table V-l. Krauskopf (1973) reported Information on the
levels of various PAEs that do not cause death or adverse effects. The
Information, however, was largely taken from unpublished data. It 1s
presented here to supplement the available Information on LD.gS but should
be Interpreted cautiously.
DEHP. Range flndVng tests performed by the National Cancer Institute/
National Toxicology Program (NCI/NTP) as part of the OEKP carclnogenesls
bloassay provided some Information on nonlethal levels of DEHP. In these
tests, no fatalities occurred within 14 days following the administration of
single oral doses of 0.8-20 g/kg of DEHP to groups of five male and five
female rats or single oral doses of 1.25-20 g/kg of OEHP to groups of five
male and five female B6C3F1 mice. Doses were administered In corn oil by
gavage (NTP. 1982a).
Lawrence et al. (1975) studied the short-term toxlclty of a number of
PAEs to determine the Intraperltoneal LD5Qs. Groups of 10 male ICR mice
were administered a range of dally doses for 5 days/week. An apparent
LD,Q was calculated for each week. This dosing schedule continued until
the mice had been Injected for at least 10 weeks, and the apparent LD,Q
remained constant for 3 consecutive weeks. After the first week, the LD^g
was 38.35 ml/kg for DEHP. In the second week, the LO,- was reduced to
6.40 ma/kg. By the end of 12 weeks, the LD5Q was reduced to 1.37
mi/kg for DEHP. Cumulative toxlclty factors (the ratio of acute LD •
04750 V-7 09/12/88
-------
chronic LD50) was 27.99 for DEHP, Indicating Increased toxIcUy (much
lower LO, s) over time. Other PAEs had cumulative toxlclty factors
ranging from 2.04-4.01. Indicating that cumulative toxlclty was minimal over
the test period. Neither the Implication of the high cumulative toxlclty
factors for DEHP nor the reasons for these results, when compared with the
other PAEs, are clear. It Is possible that very high doses of DEHP prevent
the body from eliminating the compound and metabolites to the same degree as
occurs when lower doses are repeatedly administered. It Is also not known
1f oral doses would lead to the same or similar results (Lawrence et al.,
1975).
A 14-day range finding study using animals fed diets containing DEHP was
conducted as part of the NTP Cardnogenesls Bloassay (1982a). The survival
and mean body weight responses are presented In Tables V-2 and V-3 for both
rats (F344) and mice (B6C3F1). It can be seen In Table V-2 that rats of
both sexes (5/group) exposed to 100,000 ppm (616.50 mg/kg/day In males,
505.25 mg/kg/day In females) DEHP experienced high mortality (40% males, 80%
females). Significant changes In body weight relative to controls were seen
at 25,000, 50,000 and 100,000 ppm (154.13. 308.25 and 616.50 mg/kg/day) In
males (-29%, -94% and -197%, respectively), and at 50,000 and 100,000 ppm
(252.63 and 505.25 mg/kg/day) 1n females (-165% and -171%, respectively).
As shown In Table V-3, at 50,000 and 100,000 ppm (63.50 and 127.00
mg/kg/day) DEHP, 20 and 100% mortality, respectively, was observed In male
mice. Changes In weight relative to controls were dose dependent, ranging
from -69 to -315% (6300-100,000 ppm) 1n males and -50 to -675% (6300-100,000
ppm) In females. Qualitatively similar responses were seen In female mice
both with respect to survival and body weight change.
04750 V-8 09/12/88
-------
TABLE V-2
Dosage, Survival and Mean Body Heights of Rats Fed Diets Containing
D1-l2-ethylhexyl)phthalate (DEHP) for H Days3
Dose (ppm)
(mgAg/day)b
Survival0
Mean Body Weights (g)
Initial Final Gain
Weight Changed
Males
6
12
25
50
0
,300
,500
,000
000
(0)
(38.84)
(77.06)
(154.
(308.
,13)
.25)
100,000 (616.50)
5/5
5/5
5/5
5/5
5/5
3/5
122,
123,
123.
123.
123,
168.4
174.0
175.6
155.
126,
,2
,2
45.0
123.4
79.6
50.6
52.2
31.8
2.8
-43.B
+12
+16
-29
-94
-197
Females
6
12
25
50
100
0
,300
,500
.000
.000
,000
(0)
(31.
(63.
(126.
(252.
(505.
83}
16)
31)
63)
25)
5/5
5/5
5/5
5/5
5/5
1/5
101
101
101
101
101
101
.2
.0
.1
.0
.0
.0
116
133
121
117
90
90
.8
.4
.0
.6
.8
.0
15
32
20
16
-10
-11
.6
.4
.0
.6
.2
.0
+108
+28
+6
-165
-171
aSource: NTP, 1982a
Assuming that adult rats consume an amount of food equivalent to 5% of
their body weight each day. (Average Initial body weight for males =
123.30 g; females = 101.05 g)
cNumber surviving/number per group
dWelght change relative to controls =
Height Gain (Dosed Group) - Height Gain (Control Grouo)
Height Gain (Control Group)
x 100
04750
V-9
09/12/88
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TABLE V-3
Dosage, Survival and Mean Body Weights of Mice Fed Diets Containing
D1-(2-ethylhexyl)phthalate (DEHP) for 14 Days3
Dose (ppm)
(mg/kg/day)b
Survival
Mean Body Weights (g)
Initial Final Gain
Weight Changed
Males
6
12
25
50
0
,300
,500
,000
,000
(8,
(15,
(31,
(63,
00}
88)
75)
50)
100,000 (127.00)
5/5
5/5
5/5
5/5
4/5
0/5
25.4
25.
25,
25,
25.4
25.4
28,
26,
26
23
20
19.8
2.6
0.8
0.6
-2.4
-5.4
-5.6
-69
-77
-192
-308
-315
Females
6
12
25
50
100
0
,300
.500
,000
,000
.000
(5
(11
(23
(46
(93
.86)
.63)
.25)
.50)
.00)
5/5
5/5
5/5
5/5
1/5
0/5
18
18
18
18
18
18
.6
.6
.6
.6
.6
.6
19
19
19
19
14
14
.4
.0
.8
.8
.7
.0
0
0
1
1
-3
-4
.8
.4
.2
.2
.9
.6
-50
*50
*50
-588
-675
aSource: NTP (1982a)
bAssuming that adult mice consume an amount of food equivalent to 5% of
their body weight each day. (Average Initial body weight for males =
25.4 g; females = 18.6 g)
cNumber surviving/number per group
dWe1ght change relative to controls =
Weight Gain (Dosed Group) - Weight Gain (Control Group)
Weight Gain (Control Group)
x 100
04750
V-10
09/12/88
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Rhodes et al. (1986) compared morphologic and biochemical changes and
toxic effects after 14 days of OEHP exposures to rats and marmoset monkeys.
Groups of 10 adult male and female Wlstar albino rats and five male and
female 12- to IB-month-old marmosets were administered single dally oral
doses of 2000 mg/kg bw DEHP 1n corn oil for 14 consecutive days. In addi-
tion, groups of five 24-month-old male marmosets were administered single
dally l.p. Injections of 1000 mg/kg bw DEHP 1n corn oil for 14 consecutive
days. Treated rats experienced testlcular atrophy, hepatomegaly and a
significant reduction (p<0.05) 1n body weight gain. Marmosets body weight
was reduced with both treatments; however, changes 1n'organ weight were not
detected. Hepatic peroxlsomes and peroxlsomal enzymes were Induced In both
male and female rats. Hypotrlglycerldemlc and hypocholesteremlc effects
were observed only In male rats. Oral and l.p. administration of OEHP to
marmosets did not Induce peroxlsomes and peroxlsomal enzyme activity or the
hypollpldemlc effects. Rhodes et al. (1986) concluded that the data
Indicated that the Interrelationship of hepatomegaly, peroxlsomal Induction
and hypollpldemlc effects Is complex and appears to be dose- and species-
dependent. Marmosets metabolize DEHP differently than rats, which may
explain why marmosets are less sensitive to the effects of peroxlsome
prollferators.
Short et al. (1987) observed similar results. Male cynomolgus monkeys
were administered 100 or 600 mg/kg/day DEHP by gavage for 21 days. On day
22 each monkey received a single dose of i4C-DEHP followed by three dally
doses on days 23-25. There were no treatment-related changes In relative
liver weight, palmltoyl CoA oxidation, carnltlne acetyl-transferase or
lactic add 11- and !2-hydroxylat1on. In the comparative rat study animals
04750 V-ll 07/03/91
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wore fed diets containing 11, 105, 667, 1223 and 2100 mg/kg/day DEHP for 21
days. There was metabolic, biochemical and morphologic evidence of
peroxlsomal proliferation at doses comparable with those In the monkey.
Peroxlsomal proliferation was thought to be the result of a relationship
between DEHP treatment and the formation of metabolite I [numbered according
to the Albro et al. (1973) system. See Chapter III, Figure III-2]. Urinary
levels of metabolite I In monkeys were low compared with levels found In the
rat. Short et al. (1987) stated that rats do not provide a good basis for
predicting results of DEHP exposure In higher primates.
Although not a normal route of environmental exposure, the possibility
of exposure to PAEs from medical devices such as blood bags and plastic
tubing has prompted studies of Injection exposures. DEHP may constitute as
much as 40% of the plastic material In blood storage bags and medical
tubings (Sjoberg et al., 1985b). The type of vehicle or preparation of DEHP
used In administration may Influence the pharmacoklnetlc pattern observed.
Due to the limited solubility of DEHP 1n blood and blood products, the total
dose given to animals would be relatively small and. In general, no acute
toxlclty would be expected (U.S. EPA. 1980). Rubin (1976) has suggested,
however, that pulmonary effects may occur when surfactant-solublUzed DEHP
Is administered l.v. This type of pulmonary pathology, characterized by an
Inflammatory state, has been referred to as "shock lung" or "wet lung"
(Rubin, 1975).
In earlier studies, DEHP was mixed by sonlcatlon .Into the collected
plasma of donor rats (unspecified strain) at concentrations <10 mg/mi
04750 V-12 07/03/91
-------
(Rubin and Chang, 1978). The plasma was then returned to the original
packed cell volume, resulting In whole blood OEHP concentrations of <5
mg/ma. Similarly prepared DEHP-free blood was used For treatment of
controls. In one set of experiments, 40-80 ml of DEHP-treated blood was
exchanged with the rats' own blood, resulting In received doses <400 mgAg.
All nine control rats survived, while In DEHP-treated rats, a dose-related
Increase 1n lung edema and 1n lethality was observed with an LD5Q of -200
mg/kg. At 400 mg/kg, all of the six rats tested died. Necropsy revealed
severe lung hemorrhage and edema. In the second set of experiments, rats
/
were bled until their blood pressure dropped to 50 mm Hg. This pressure was
maintained for 30 minutes, then an equal volume of donor blood containing
1.25 mg/mt. DEHP was relnfused. All five control rats survived. Lung
weights were elevated In the control rats, but the lungs were not grossly
hemorrhaglc. Two of the six rats receiving 7.7-13.0 mg/kg DEHP died within
90 minutes of the transfusion. In all six rats receiving OEHP, lungs were
grossly hemorrhaglc. The authors concluded that sensitivity to DEHP was
greatly Increased In animals whose blood pressure was held at shock levels.
The LD- for male Wlstar rats receiving l.v. Injections of DEHP solu-
blUzed In a nonlonlc detergent was 250-300 mg/kg (Schultz et al., 1975}.
The primary effect was a respiratory distress syndrome progressing to death
from respiratory failure. The overt signs and morphologic alterations
observed with DEHP/detergent treatment were not observed In control animals.
Mangham et al. {1981} studied the oral effects of DEHP on the liver and
testes. In this study, groups of six male and six female Wlstar rats were
administered dally doses of 2500 mg/kg/day DEHP by gastric Intubation In a
04750 V-13 07/03/91
-------
corn oil vehicle for 7-21 days. DEHP produced pronounced liver enlargement
at 7 or 21 days In both sexes of rats. The activity of sucdnate dehydro-
genase was decreased In male rats administered DEHP for 7 or 21 days. No
effect on this enzyme occurred In females. Hlstopathologlc changes were not
present 1n the livers of rats treated with DEHP. although ultrastructural
studies revealed proliferation of smooth endoplasmlc retlculum (SER), an
Increase In the number of mlcrobodles (peroxlsomes) and mitochondria!
changes. Effects of DEHP on the liver are summarized 1n Table V-4. Mangham
et al. (1981) also noted a significant decrease (p<0.001, student's t-test)
In the weight of the testes (relative to body weight) after 7 and 21 days.
Treatment also resulted In bilateral tubular atrophy after 21 days.
Mitchell et al. (1985) observed similar results when groups of four male
and four female Wlstar albino rats were administered nominal doses of 50,
200 or 1000 mg/kg/day DEHP In the diet for 3. 7, 14. 28 days or 9 months.
Hlstopathologlc examinations were performed on the major abdominal organs at
all time points. The livers of male rats were significantly (p<0.05)
enlarged at all time points with 1000 mg/kg/day OEHP. With the two lower
dose groups liver enlargement was noted only at 14 days and 9 months In male
rats; however, It was significant (p<0.05) only at the 1000 mg/kg/day dose.
There were no significant differences In testes weight when control animals
were compared with experimental animals. Further details were not given.
Liver cells from male rats showed marked proliferation of peroxlsomes after
3 days of treatment with 200 or 1000 mg/kg/day. Treatment with 50 mg/kg/day
resulted 1n Increased numbers of peroxlsomes after 14 days. Proliferation
of the smooth endoplasmlc retlculum In both males and females occurred at
all doses 1n a dose-dependent manner. DEHP administration caused an Initial
04750 V-14 07/03/91
-------
TABLE V-4
Suomary of Short-Jem [Meets of 01 HP on Weight. Morphology and Blochenlcal Constituents of Liver4
Species/Strain
Oil /CO
Ral/NR
Rat
Rat/albino
Ulslar
Rat/Sprague-
Dawley
Haas ler /Syr Ian
Haaster /Chinese
Partially hepatec-
tontied rals/
Spraque-Dawley
lerret/NR
Monkey/rhesus 0
Honkey/rhetus H
Ral/Ulstar
Route
diet
diet
diet
oral
Intubation
oral
Intubation
oral
Intubation
oral
Intubation
diet
diet
l.v.
diet
Dose/
Treatment Duration
0.2. 1 and 2X/17 weeks
(100. SOD and 1000
•g/kg/day)b
SOOO ppu/7 weeks
|?70 *g/kg/day)b
0.3SX/3 Months
|!7i Mg/kg/day)b
2000 Mg/kg/21 days
1000 *g/kg/day/14 days
1000ng/kg/day/14 days
1000 ag/kg/day/14 days
0 SI/10 weeks
(250 Bg/kg/day)0
1200 ag/kg/dayc/
14 Monthi
74.80 BNi/12 Months
20.52 og/12 nunths
2 and 4X/4 weeks
(1000 and 2000
Principal findings
liver enlargement
Liver enlargeaent
Liver enlargeoent
Liver enlargement, proliferation of SIR.
Increase In number of •Urobodles and swelling
of •! tochondr la with shortening of crtstae,
decreased SDH activity, altered aniline
hydroxylaie activity, decrease In glucose-b-
phosphalase activity
Increase In liver weight. pal*1toyl-CoA oxi-
dation. enoyl-CoA hydratase. and carnltlne
acetyltransferase (p
-------
I ABU V-4 (cont.)
o
*.
«j
tn
Species/Strain
Route
Dose/
Treatment Duration
Principal findings
Reference
Rat/Wlstar
Rat/Sprague-Dawley
Rat/albtno Ulstar
oral
Intubation
l.w.
diet
diet
2500 mg/kg/day//-2l days
0. 5. 50 or 500 mg/kg bw
50 mg/kg/day/28 days
200 mg/kg/day/28 days
Pronounced liver enlargement at 7 or 21 days Hangham
In both sexes; succlnate dehydrogenase et a I., 19BI
decreased In male rats at 7 or 21 days;
proliferation of SER; Increase In number of
mtcrobodles and mitochondria! changes
Increase In liver weights and number of liver SJOberg et
peroxlsomes; no differences In serum enzymes al., I985b
or BSP clearance values
Decreased glucose-6-phosphatase activity In
females. Increased llpld content In liver;
I GAEL of 50 mg/kg/day Identified based on
liver enlargement at 14 days of exposure.
Decreased glucose-6-phosphatase activity In
females
Mitchell
et a I.. 1985
Rat/albtno
Nice/albino Wtstar
Rats/Sprague-Dauley
(CD)
1000 mg/kg/day/28 days
diet 320 mg/day/30 days
diet 60 mg/day/30 days
oral 10 mg/kg/bw/5 days to
intubation 6-10. 14-18. 16-?0.
21-25. 42-46. 86-90
day old rats
100 mg/kg/bw/5 days to
6-10. 14-18. 16-20.
21-25. 42-46. 86-90
day old rats
Decreased glucose-6-phosphatase. Cytochrome
P-450 Increased after 3 and 7 days, decreased
to control levels after 14 days and Increased
after 28 days; significantly Increased (p<0.05)
liver weights at 3. 7. 14 and 28 days
Significant Increase In liver weight; signifi-
cantly reduced serum cholesterol levels: sig-
nificant Increase In carnlllne acetyllrans-
ferase and glycerophosphate activity
Significant Increase In liver weight and
carnltlne acetyltransferase and glycerophos-
phate activity
Increased activities of hepatic peroxtsomal
eniymes palmltoyl CoA oxldase and carnltlne
acetyltransferase In all age groups
Significant Increases In relative liver weights
In all but 1-week-old rats; Increased absolute
liver weight In all but 1-week-old rats; signifi-
cantly Increased activity of hepatic peroxlsomal
eniymes palmltoyl CoA oxldase and carnltlne
acetyllransferase In all age groups
Nalr and
Kurup. 1986
Nalr and
Kurup. 1986
Dostal et
al.. 198/a
CO
oo
-------
1ABLE V-4 (cent.)
Species/Strain
Route
Dose/
Treataeni Duration
Principal findings
Reference
Rals/Sprague-Oawley
(CD)
oral
Intubation
Rats/yistar
Nonkeys/marmosel
oral
Intubation
oral
Intubation
I.p.
2000 mg/kg bw/S days
to 6-10. 14-18. 16-20.
?1-?S. 4?-4b. 86-90
day old rats
?000 mg/kg/14 days
2000 mg/kg/14 days
1000 mg/kg/14 days
Significantly decreased body weight gain In rats
ages 6-10. 16-20 and 21 -25 days old. death In
66-70X of rats ages 14-18 days; significant In-
creases In relative liver weights In all age
groups; Increased relative kidney weight In 21
day or older rats; significantly Increased
activity of hepatic peroxtsoaal enzymes,
palnltoyl CoA oxldase and' carnltlne acetyl
transferase In all age groups
Death In rats ages 6-25 days old; significantly
decreased body weights In 42-46 and 86-90 day
old rats; significantly Increased relative
kidney weight In 42 day or older rats
Hepatomegaly, testlcular atrophy and reduced
body weight gain. Induction of hepatic peroxl-
tunes and peroxlsonal enzymes; hypoUpldemlc
effects
No effect on peroxlsomal eniyme activity;
reduced body weight gain
No effect on peroxlsomal eniyme activity;
Identified as a IOAEL based on reduced body
weight gain.
Dostal el
al.. 19B7a
Rhoades
el al.. 1986
Rhoades
el »!••
'Source: Adapted from Seth. 1982
DAssumtng rats consume 5* of their body weight
cNean dally Intake
NR - Not reported
CO
00
-------
Increase 1n mitosis. DNA synthesis was significantly elevated (p<0.05) in
all treated males at 3 days. Changes 1n llpld content and distribution were
observed at all dose levels. Loss of glycogen was observed at 1000
mg/kg/day starting at 7 days, and llpofusdn accumulation after 28 days at
200 and 1000 mg/kg/day. Biochemical changes were also noted as summarized
In Table V-4.
Lake et al. (1975) examined liver effects after oral administration
(gavage) of 2000 mg/kg (236 mg/kg/day) DEHP for periods of 4. 7, 14 and 21
days. Increased relative liver weights and a number of biochemical and
ultrastructural changes were noted. Komltowskl et al. (1986) also observed
ultrastructual changes after a single l.p. dose of DEHP. Six-week-old
Syrian golden hamsters were administered either 0. 30. 300 or 3000 mg/kg
DEHP. The Investigators did not observe gross or hlstopathologlc changes.
However, ultrastructural changes such as Increased number and size of
peroxlsomes were demonstrated In the high-dose group. The same type of
changes were less pronounced In the middle-dose group. In the low-dose
group only variability 1n peroxlsomal size and shape occurred.
SJoberg et al. (1985b) Investigated the effect of DEHP on the liver of
young male Sprague-Dawley rats after repeated l.v. Infusions. Emulsions of
DEHP were administered every other day on six occasions In dally doses of 0,
5, 50 or 500 mg/kg bw DEHP to groups of 6, 6, 6 and 5 40-day-old male
Sprague-Dawley rats, respectively. Infusions were administered every other
day on six occasions. Cannula were surgically Inserted Into the jugular
veins of the rats 2 or 3 days before administration. DEHP emulsion was
Infused for 3 hours at a rate of 1.0 mi/hour. Blood samples were drawn 7
04750 V-18 07/03/91
-------
and 17 minutes after the l.v. Injection throughout the experiment. A
significant dose-related Increase 1n liver weights (p<0.0001) and number of
liver peroxlsomes (p<0.0051) was observed. However, smaller mitochondria
occurred In the livers of both control and treated animals, but were more
common In the DEHP-treated groups. There were no differences In serum
enzymes or BSP clearance values 1n treated animals when compared with
control animals. The kidneys appeared normal. Although the relationship
between dose and effect has not been established, the author concluded from
the above results that measures be taken to reduce the exposure to DEHP
through l.v. transfusion exchange.
Effects of DEHP on llpld and protein metabolism are summarized 1n Table
V-5. Rats receiving 0.554 (250 mg/kg/day assuming rats consume 5% of their
body weight) DEHP In a normal protein diet showed accumulation of phospho-
llplds, decrease 1n cholesterol and trlglycerlde contents 1n liver and
plasma and a rise In fatty acid levels In plasma (Reddy et al., 1976). The
Importance of the altered llpld concentrations In the body Is not clear at
present. Although the effects of PAEs on protein metabolism have not been
studied, protein content In the liver has been shown to Increase In DEHP-
treated rats. The Increase In liver protein content has been attributed to
a decrease In protein breakdown.
Recent studies Indicate that PAEs may cause adverse effects when trans-
ported to the developing organism by milk. Groups of seven nursing rat pups
were randomly assigned to five dams (at birth). The dams were gavaged with
2000 mg/kg bw DEHP for 21 days. On day 21, three of the pup livers were
examined. Parmar et al. (1985) observed a decrease 1n body weight gain and
changes In enzyme activities of aniline hydroxylase, ethylmorphlne,
04750 V-19 09/12/88
-------
471
TABLE V-5
Effects of OEHP on Llpld and Proietn Netabollsa Relating to Hepatotoxlcllya
r\>
Species
Rat
Rat
Rat
Rat
Rat, aouse
Rat. Mute
Rat
Rat
Rat
Route
oral
oral
l.p.
oral
oral
oral
oral
oral
oral
Dose/Exposure Length
O.S or l.OX In diet/
10 or 18 days, respectively
(250 or SOO ag/kg/day)°
2.S onol./lOO g dlet/?l days
(490 ag/kg/day)D
S ag/kg bw/on days 1. 5 and 10
1. 2 or « In dlel/4 weeks
(500. 1000 or 2000 ag/kg/day)D
0.5. 2.0 and 4X In dlel/1-4
weeks (250. 1000 and 2000
ag/kg/day)6
M w/w /2 weeks
(1000 ag/kg/day)D
0.5X In dlet/7 days
(250 ag/kg/day)b
1000 ag/kg
Principal findings
Inhibition of llpld biosynthesis
Inhibition of llpld biosynthesis
Decrease In cholesterol content
Decrease In trlglycertdes and Increase
In phosphollptds
Decrease In serua cholesterol; prolif-
eration of peroxlsoaes; Increase of
catalase and carnltlne acetyltransferase
Induction of enzymes of fatty acyl-CoA
0-oxldatlon
Increase In altochondrlal and alcrosoaal
phosphollptds; Increase In protein
content
Increase In protein content (decrease In
protein degradation)
Decreased glycogen In liver
Reference
Bell and Nailr.
1976
Bell et al..
1977
Srlvastava
et al.. 197B
Sakural
et al.. 197B
Reddy et al..
1976
Osual and
Hashlaoto. 1978
Vanaglta
et al.. 1979
PI Hal and
Seth. 1978
i
Mitchell
et al.. 1985
'Source: adapted froa Seth. 1982
DAssuatng rats consume SX of their body weight
00
00
-------
N-demethylase and arylhydrocarbon hydroxylase, and decreased levels of
cytochrome P-450 1n 21-day-old rats. Oostal et al. (1987b) observed an
Increase In hepatic peroxlsomal enzymes palmltoyl CoA oxldase and carnltlne
acetyltransferase In rat dams and their suckling pups exposed to DEHP. Rats
were orally administered 5 dally doses of 2 g/kg bw DEHP on days 2-6, 6-10
or 14-18 of lactation. At all three stages of lactation relative liver
weight was Increased as well as palmHoyl CoA oxldase and carnHlne acetyl-
transferase activity In both treated dams and their suckling pups. Plasma
cholesterol and tryglycerlde concentrations were decreased by 30-50% In DEHP
treated dams at all three stages of lactation. Although mammary gland
weights were decreased In treated dams, the Investigators attributed these
results to decreased food consumption An the 0£HP-treated rats.
BBP. Agarwal et al. (1985a) Investigated the effects of BBP on the
hematopoletlc system 1n a 14-day dietary study 1n F344 rats fed levels of
0.0, 0.625, 1.25, 2.5 and 5.OX 88P (0.0, 375, 750, 1250 and 1667 mg/kg/day,
respectively). At the 0.625% and 1.25% levels, liver and kidney weights
were significantly (p<0.05) Increased. In addition, the Incidence of
proximal tubular regeneration of the kidney Increased In a dose-related
manner beginning at the 0.625% dose level. At the 2.5% and 5.0% levels,
effects Included decreased weight of the testes, epldldymus, seminal
vesicles and thymus. hlstologlc evidence of atrophy of the testes and
accessory sex organs. The authors reported no significant effects on the
circulating blood components or blood clotting ability. Effects on the
partial thromboplastln time were Increased, but not significantly; however,
mean values and large variability were observed at the 2.5 and 5.0% BBP
levels. Bone marrow cellulaHty was significantly (p<0.05) reduced at 2.5
04750 V-21 07/03/91
-------
and 5.0% BBP In a dose-related manner. These authors conclude that
prolonged exposure to BBP could alter the development of blood components
and lead to a deficit in clotting ability.
Hale and female Fischer 344 rats were fed 0, 0.6. 1.2 or 2.5% BBP for 21
days (CMA, 1985). Corresponding dose levels were 0, 639. 1277 and 2450
mg/kg/day for males and 0. 679, 1346 and 2628 mg/kg/day for females,
respectively. Relative liver to body weights significantly Increased
(p<0.001 1n both males and females except for p<0.01 at 0.654 1n female rats)
In all treatment groups. However, absolute liver weights were significantly
Increased (p<0.01 1n males, p<0.01 at 1.2% and p<0.001 at 2.5% 1n females)
only at the 1.2 and 2.5% dietary levels. Significantly reduced testes
weight (p<0.001) and testlcular atrophy occurred In the 2.5% treatment
group. Relative kidney weights were higher In BBP-treated rats; however,
the differences were not dose-related. In both males and females, total
cholesterol concentrations were lower than the controls, but there was no
dose relationship. Treatment with BBP at all dose levels of male rats and
at 2.5% of female rats significantly (p<0.01 In males at 0.6% and p<0.001 at
all other dose levels) Increased cyanide-Insensitive palmltoyl Co-A
oxidation. Male rats were more sensitive than females with respect to
Increases 1n 11- and 12-hydroxylatlon of laurlc add.
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. Significantly Increased activities of ethylmorphlne
N-demethylase (p<0.05) and cytochrome oxldase (p<0.01) were observed 1n the
04750 V-22 07/03/91
-------
480 and 1600 mg/kg/day BBP treatment groups. In addition, significant
(p<0.001) liver enlargement was observed at 1600 mg/kg/day BBP as were
Increases In mlcrosomal cytochrome P-450 (p<0.05) content and cytosollc
alcohol dehydrogenase (p<0.001). Liver sections from animals at the highest
dose revealed ultrastructural changes such as gross dilation of the rough
endoplasmlc retlculum and Increased number of peroxlsomes. Administration
of 1600 mg/kg/day BBP also produced marked depression of both absolute and
relative testes weights as well as severe testlcular atrophy. Effects on
testes weights were not observed In the animals given 160 or 480 mg/kg/day;
however, testlcular 'atrophy was observed In 1/3 of the animals administered
480 mg/kg/day BBP.
A second study was conducted to confirm the testlcular effects. Both
Sprague-Dawley and Vhstar albino rats were gavaged with 480 and 1600
mg/kg/day BBP for 14 days. A significant depression (p<0.001) 1n either the
absolute or relative testes weight was observed In both strains of rats at
1600 mg/kg/day. Additionally 1600 mg/kg/day BBP significantly reduced
(p<0.05) the growth rate and Increased the absolute (p<0.05) and relative
(p<0.001) liver weights In both strains of rats. Relative liver weights
were also significantly (p<0.001) Increased In Wlstar rats at 480 mg/kg BBP.
Hlstologlc examination of the testes revealed testlcular atrophy In both
strains (1600 mg/kg/day) with the extent of the lesions being more severe In
the Sprague-Dawley rats. At 480 mg/kg/day BBP. 1/6 Sprague-Dawley rats
showed a degree of testlcular atrophy, whereas the Wlstar albino strain
revealed no hlstologlc changes.
04750 V-23 07/03/91
-------
DBP. Galley et al. (1966) found that weight gain retardation and
peritonitis occurred In Swiss Webster mice that had received dally l.p.
Injections of 250 or 500 mg/kg DBP for 6 weeks. Testlcular atrophy occurred
In the DEHP-treated rats (see Reproductive Section). No clear hematologlc
differences were found between control and experimental test groups.
In a dietary study DBP was fed to male antf female Fischer 344 rats at 0,
0.6, 1.2 and 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, respectively. Absolute and relative liver weights
were significantly Increased In both male and female'rats at all treatment
levels. Male 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 laurlc
acid 11- and 12-hydroxylase Increased In males given 0.6, 1.2 and 2.5%.
Cyanide-Insensitive palmltoyl CoA oxidation Increased at 1.2 and 2.5% In
males and 2.5% In females.
Murakami and Nlshlyama (1986) fed male Wlstar rats powdered diets
containing 0, 0.5 or 5% DBP, MBP, PA or DEHP. Corresponding levels were 250
mg/kg and 2500 mg/kg (assuming 0.05 kg food consumption and a 350 g rat).
The relative weights of liver, kidney, testicle and spleen were signifi-
cantly Increased In the 5% DBP group. Ultrastructural examination of liver
cells revealed Increased numbers of peroxlsomes, lysosomes and mitochondria
(5% DBP). Only hepatocytes of animals In the 5% dose group were examined.
Marked spermatogenlc damage and testlcular atrophy occurred at 5% DBP. The
succlnate and pyruvate dehydrogenase activities 1n liver mitochondria were
04750 V-24 07/03/91
-------
significantly decreased at both the 0.5 and 5X DBP levels. The Investi-
gators concluded that the adverse effects of DBP at least on the liver may
be caused by the direct action of Intact DBP entering the liver.
PEP. Bllckensdorfer and Templeton (1930) studied the toxic properties
of DEP In rabbits, guinea pigs and dogs. Rabbits were administered 2 cc/kg
bw (2.24 g/kg) l.p. for 8 successive days. No abnormal conditions or
"paralysis" was observed, although during and after the period of adminis-
tration there was some temporary distress. Similar results occurred when
rabbits were fed 3 cc/kg (3.35 g/kg) DEP by stomach-tube for 8 successive
days. The rabbits appeared normal during feedings and for 2 weeks following
the last administration. The authors had not yet developed a satisfactory
quantitative method for urine analysis; however, they did estimate quantita-
tively (methods not reported) that >50% was excreted by the kidneys. In the
same experiment, guinea pigs administered 1.5 cc/kg (1.68 g/kg) l.p. for 8
successive days showed no permanent 111 effects at any time during or after
treatment. The authors did not explain what was meant by no permanent 111
effects or the length of observation after treatment. Dogs were adminis-
tered 0.25 cc/kg (0.28 g/kg) DEP In a physiologic salt solution by Injection
Into the femoral vein. Respiration was first stimulated and then
paralyzed. Traces of OEP were detected In urine samples taken after the
Injection began. The authors stated that "considerable" quantities of DEP
(2 cc/kg bw 1n rabbits) may be taken without causing any damage. However,
they also stated that since DEP Is rapidly excreted by the kidneys, func-
tional damage to the. kidneys may cause sufficient DEP accumulation 1n the
blood leading to "nervous system damage".
04750 V-25 07/03/91
-------
PHP. Krauskopf (1973) summarized data on the LOcrjS for several
PAEs. some of which came from unpublished reports. LDcns for DMP ln
guinea pigs, mice, rabbits and rats were 2.4, 7.2, 4.4 and 6.7-6.9 g/kg,
respectively. In one acute oral study, mice and dogs Ingested a single dose
of 1-4 and 0.7-1.4 g/kg DHP, respectively, without observable effects.
Details of the study were not reported (Krauskopf, 1973).
Christian (1985) also summarized LD5Q results. Rats, mice, rabbits,
guinea pigs and chicks were orally administered undiluted DMP. The LD5Qs
were 6.9, 7.2, 4.4, '2.4 and 8.5 ml/kg (8.2, 8.6, 5.2, 2.9 and 10.1 g/kg),
respectively. Animals were observed for 6 days following treatment.
Details of the study were not reported.
Long-Term Toxldty
Long-term toxlclty has been evaluated for several phthalate esters.
Results of these long-term studies 1n mammals are discussed as follows and
are summarized 1n Table V-6. The primary target organs of PAE toxldty are
the liver and the testes. Other organs and cellular systems have also been
shown to exhibit toxic responses following exposure to PAEs. Examples of
these Include lungs, kidneys and blood platelets (reviewed In Thomas and
Thomas, 1984). These complex responses are probably not related to any
single active moiety of the PAEs.
DEHP. The oral toxldty of DEHP has been Investigated by numerous
authors. One of the earliest oral studies of DEHP was reported by Shaffer
et al. (1945). In this study, groups of five male albino rats, weighing
120-150 g, were fed dietary levels of 0.375, 0.75, 1.5 and 3.0% DEHP for 90
04750 V-26 07/03/91
-------
TABLE V-6
Sumary Table of Long-tern loxlclty Studies of PAEs In Manuals
o
«JD
oo
00
Exposure/Route Critical Effect Compound Species
Oral Mortality 75*. pathological DEHP rat. Ulstar
changes In digestive tract
Increase In liver weight Of HP rat
lestlcular Injury DEHP rat. unspecified
(within 2 weeks)
Testicular Injury Of HP rat. unspecified
Tesltcular degeneration, Of HP rat. albino
tubular atrophy
Slight decrease In growth DEHP rat
No effect level OEHP rat
Body weight slightly decreased DEHP rat. F344
In Mies; NOAEl for lesttcular
atrophy
Testlcular atrophy; pituitary DEHP rat. F344
hypertrophy trend toward
Increase. Itver clear cell
damage
Slight depression of weight DEHP muse. B6C3M
gain In females only
No Increased mortality when DEHP mouse. B6C3F1
compared to controls but slight
decreased weight gain In fenales
No. /Sex
lOf
ION
10F
ION
NS
NS
SN
S
SN
SOF, SON
SOF. SON
SOf, SON
SOF. SON
Dose/Time
3.40 g/kg/day for
90 days by gavage
In olive oil
0.34 g/kg/day for
90 days
0.6 g/kg/day for
90 days
0.12 g/kg/day for
90 days
0.9 and 1.9 g/kg/day
for 90 days
0.4 g/kg/day for
90 days
0.2 g/kg/day for
90 days
6000 ppm (322 mg/kg/day
In males; 394 mg/kg/day
In females) for 24
months
12.000 ppm (674 mg/
kg/day In males;
774 mg/kg/day In
females) In diet
for 24 months
3000 ppm (674 mg/
kg/day In males;
799 mg/kg/day In
females) In diet
For 24 months
6000 ppm (132S mg/kg/
day In males; 1821
ng/kg/day In females)
Reference
Nlkoronow
el al.. 1973
Nlkoronow
et al.. 1973
Cater et al..
1977
Cater et al..
1977
Shaffer
et al.. 194S
Shaffer
et al.. 1945
Shaffer
et al.. 194S
NTP. 1982a;
Kluwe et al..
19B2a
NTP. 19B2a;
Kluwe et al..
19B2a
NTP. 1982a;
Kluwe et al..
1982a
NTP. 1982a;
Kluwe et al..
19B2a
only; tesllcular atrophy and
chronic IntlanroalIon of the
kidney In males
In diet for 24 months
-------
TABLE V-b (cent.)
en
o
i
ro
CD
ca
oo
Exposure/Route Critical Effect
Oral (cont.) lestlcular damage, cessation
of spermatogenesls. reduced
renal function (females)
Growth retardation, testtcu-
lar daouge. cessation of
spermatogenesls. Increased
liver weight
Decreased spermatogenesls,
Increased liver weight
Mild toxic effect
No effect level
Significantly Increased liver
weights In males only; per ox 1-
somal proliferation
Significantly Increased liver
weights; peroxtsomal prolife-
ration; significantly decreased
glucose-6-phofphatase activity
activity In males only; Increased
number «f lysosomes
Significantly reduced body weight
alterations In thyroids; signi-
ficantly Increased liver weights;
peroxlsomal proliferation; reduc-
tion In glucose-6-phosphatase
activity; Increased number of
lysosomes
Increased liver and kidney
weights
Ho adverse effects
Increased liver and kidney
weight (after 90 days); de-
Compound
Of HP
DEHP
DEHP
DEHP
DEHP
DEHP
DEHP
; DEHP
DEHP
DEHP
DEHP
Species
rat. CD
| Sprague-Dawley
derived)
rat. CD
(Sprague-Dawley
derived)
rat. CD
(Sprayue-Dawley
derived)
dog. unspecified
breed
dog. unspecified
breed
rat. Ulstar
albino
rats. Ulstar
albino
rats. Ulstar
albino
rat. Sherman
rat. Sherman
rat. Ulstar
No. /Sex Dose/Time
15E. 15N 1440 mg/kg/day male;
1414 mg/kg/day female
for 11 weeks
15f. 1SH 131 mg/kg/day male;
191 mg/kg/day female
for 17 weeks
ISf. 1SN 143 mg/kg/day male;
154 mg/kg/day female
for 11 weeks
IN S g/kg/day by
stomach tube for
14 weeks
If 0.1 g/kg/day mixed
In diet for 14 weeks
4f . 4N SO mg/kg/day for
9 months
4f . 4N 200 mg/kg/day for
9 months
4f . 4N 1000 mg/kg/day for
9 months
32F . 32H -?00 mg/kg/day for
up to 24 months
3?F. 32N 60 mg/kg/day for up
to 24 months
43F. 43N range of 300-400 mg/
kg/day for 24 months
Reference
Gray et al..
1911
Gray el al.,
1911
Cray et al..
1911
Harris
et al.. 1956
Harris
et al.. 1956
Hit che 11
el al.. 1985
Mitchell
et al.. 198S
Mitchell
et al.. 1985
Carpenter
et al.. 1953
Carpenter
et at.. 1953
Harris
et al.. 1956
creased body welghts/LOAEL
-------
TABLE V-6 (cont.)
en
o
Exposure/Route Critical Effect Compound
Oral (conl.) No advene effects OIHP
Mortality (30X). DEHP
Increased liver enlargement.
Increased kidney weight.
decreased body weight
No significant adverse DiHP
effects
Statistically significant In- DEHP
crease In relative liver weights
In females
Statistically significant In- OIHP
crease In relative liver weights
In females
No effect BBP
Increased liver weights; BBP
mild decrease In growth
Species
rat. yistar
rat. Ulstar
dog. both sexes.
2 pure blooded
cocker spaniels,
2 wire-haired
terriers
guinea pigs
guinea pigs
rats
rats
No./Seic Dose/Time
43F . 43N range of SO -80 mg/
kg/day for 24 months
20F. 20N 0.19 g/kg/day for
1? months
0.03 mi/kg/day (30
mg/kg/day) first 19
doses and then U.Ob
mi/kg/day (60 mg/kg/
day) for 240 doses
23f . 23H 19 mg/kg/day for
1 year
241 . 24N 64 mg/kg/day for
1 year
NS 0.25X (125 mg/kg/day)
and O.SOX (250 mg/kg/
day) In diet for 90 days
NS 1.00, 1.50 and 2.00X
1500. 750 and 1000 mg/
Reference
Harris
et al.. 1956
Nlkonorow
et al.. 1973
Carpenter
et al.. 1953
Carpenter
et al.. 1953
Carpenter
et al.. 1953
Krauskopf.
1973
Krauskopf.
1973
OB
CO
No effect BBP
Depressed weight gain; de- BBP
creased mean organ weights
In heart, right kidney, lungt.
seminal vesicle and right
testes; kidneys contained focal
cortical areas of Infarct-llke
atrophy; atrophy of seminiferous
tubules
Mortality (SOX In first week) DBP
rats/f-344
rats/f-344
15 males/
group
15 males
kg/day) In diet for
90 days
0.03. 0.09. 0.28.
0.83X (17. 51. 159.
470 mg/kg/day) for 26
weeks In the diet
2.SOX (2875 mg/kg/day)
for 26 weeks In the
diet
NTP. 1985
NIP. 1985
rat, Sprague-Oawley ION
1.25X (600 mg/kg/day)
In diet for 12 months
Smith. 1953
-------
I HDL I V-0 (lOfll. )
I
CO
o
Exposure/Route
Oral (cent.)
l.p. Injection
Inhalation
Critical Effect Compound
No adverse effects OBP
No effects at O.S and 2.5X: DEP
decreased growth rate at 5. OX
No adverse effects DEP
Reduced weight gain In DEP
females only
Reduced weight gain In both DEP
sexes; Increased relative
weights of the brain, liver.
kidney, stomach, small
Intestine and full caecum
No effects on growth at 2. OX DNP
level; effects on growth at
4.0 and 8. OX; chronic nephritis
at 8. OX; NOAEL Identified at
1000 mg/kg/day
LOso DBP
L050 OEP
LD50 ONP
Dose-related Increase In OBP
gamma globulin
Species No. /Sex Dose/Time
rat. Sprague-Dawley ION 0.25X (125 mg/kg/d. y)
In diet for 12 months
rats, albino 15N. 15E/ 0 . 5X ( 250 mg/kg/daj ) .
group ?.5X (I2SO mg/kg/d. y)
and 5. OX (2500 mg/lg/
day) for 2 years
rats/CD 15N. 1SF ISO mg/kg/day for
16 weeks In diet
rats/CD 15N. 15F 770 mg/kg/day In
males and 750 mg/kg/
day In females for
16 weeks In diet
rats/CD 15N. 15F 3160 mg/kg/day In
males and 3710 mg/kg/
day In females for
16 weeks In diet
rats lOF/group 2.0. 4.0 and 8. OX
(1000. 2000 and 4000
mg/kg/day) for 2 years
mouse. ICR ION 0.85 ml/kgb
(0.89 g/kg)
mouse. ICR ION 1.39 mi/kgb
(1.46 g/kg)
mouse, ICR ION 1.18 ml/kgb
(1.24 g/kg)
rat. albino 60N 0.098. 0.256 and
0.98 mg/rn* (0.12.
0.33 and 1.2 mg/
kg/day )a contin-
uous for 93 days
Reference
Smith. 1953
Food Research
Lab.. 1955
Brown et al..
1978
Brown et al.,
1978
Brown et at..
1978
Lehman. 1955
Lawrence
et al.. 1975
Lawrence
et al.. 1975
Lawrence
et al.. 1975
Nen'shlkova,
1971
CO
o
CD
CO
'Assuming a default of 50X absorption factor
DDose represents the chronic LOjo calculated for mice receiving l.p. Injections 5 days/week. LD50 values were calculated each week
until the LDso remained the same for 3 weeks (minimum of 10 weeks of dosing).
NS • Not specified
-------
days. The author determined approximate dally Intakes of 0.2, 0.4. 0.9 and
1.9 g/kg bw/day DEHP, respectively. A fifth group served as a control. At
the three highest levels a slight decrease In growth was "somewhat retarded"
relative to the controls. Quantitative data was not collected. At 1.5 and
3.0% DEHP, tubular atrophy and degeneration of the testes were observed. No
deaths occurred In any of the treated animals and the blood cell counts,
hemoglobin concentrations, and differential white cell counts remained
normal. The authors concluded that no adverse effects from oral administra-
tion would occur at -0.2 g/kg bw/day or less; however, a slight retardation
In growth occurred at 0.4 g/kg bw/day.
Nlkonorow et al. (1973) administered DEHP 1n olive oil by gavage to
groups of 10 male and 10 female Wlstar rats weighing 90-120 g for 3 months
at levels of 340 and 3400 mg/kg/day. The higher dose level resulted 1n 75%
mortality. Pathologic examination of the dead animals revealed congestion
of the small Intestine and loss of mucosa In the stomach and endometrltls.
The mean liver weight of animals treated with the lower dose level Increased
relative to that of the controls. These authors also reported that a dally
dietary dose level of 0.36% (180 mg/kg/day assuming rats consume 5% of their
body weight) DEHP for <12 months resulted In 30% mortality In groups of 20
female and 20 male Wlstar rats. Relative to the controls, significant liver
enlargement and decreased body weight occurred In rats administered 0.36%
OEHP 1n feed.
Gray et al. (197?j reported the effects of a 17-week dietary Intake of
0, 0.2%, 1.0% or 2.0% OEHP on groups of 15 female and 15 male CD (Sprague-
Dawley-deMved) strain rats. Mean dally Intakes of DEHP, calculated from
food consumption data, were 143, 737 and 1440 mg/kg bw/day for male rats fed
0475Q V-31 07/03/91
-------
0.2, 1.0 and 2.0% DEHP and 154, 797 and 1414 mg/kg bw/day for female rats
fed 0.2, 1.0 and 2.OX DEHP. respectively. At the two highest dose levels,
the rate of body weight gain and food Intake were reduced; however, a paired
feeding study showed that the effect on body weight gain was not entirely
due to decreased food consumption. Renal concentrating and diluting ability
were reduced In the females receiving 2.0% DEHP. At 1.0% or 2.0% dose
levels, the relative testes weights were significantly (p<0.001) decreased
and hlstopathologlc examination revealed severe seminiferous tubular atrophy
and cessation of spermatogenesls. At 0.2% DEHP, the testls weight was not
reduced, but there was hlstologlc evidence of decreased spermatogenesls.
Significantly Increased relative liver weight (p<0.001 at all levels In
males; p<0.05 at 0.2%, p<0.01 at 1.0% and p<0.001 at 2.0% In females)
occurred at all treatment levels. Absolute weights of most other organs
(brain, heart, spleen, kidneys, adrenals) were decreased at the 1.0 and 2.0%
levels, but relative weights (organ we1ght:body weight) were Increased.
Because effects on the liver and testes were observed at all dietary levels
tested In this study, the NOAEL for DEHP In rats 1s below the lowest doses
tested, 143 or 154 mg/kg/day for males and females, respectively. Cater el
al. (1977) also found testlcular effects at similar dose levels In a 90-day
feeding study conducted by BIBRA.
Mitchell et al. (1985) administered DEHP In the diet of Wlstar albino
rats (4/sex/group) at nominal doses of 50, 200 or 1000 mg/kg/day DEHP for 3,
17, 14, 28 days or 9 months. Effects at earlier time points are described
In Table V-4. Hlstopathologlc examinations were performed on the major
abdominal organs. By 9 months the body weights of both sexes treated with
1000 mg/kg/day were significantly reduced. Lesions were also seen In the
04750 V-32 07/03/91
-------
thyroids of rats treated with 1000 mg/kg/day. Liver weights were signifi-
cantly (p<0.05) Increased at all dose levels In males and at the 200 and
1000 mg/kg/day dose levels In females. Hlstologlc examination revealed
marked centrllobular eoslnophllla and Increased number of llpofuscln
deposits In the hepatocytes In both male and female rats (1000 mg/kg/day).
Proliferation of the smooth endoplasmlc retUulum occurred at all doses In a
dose-dependent manner. Marked peroxlsomal proliferation was apparent after
treatment with 200 or 1000 mg/kg/day. The Increased numbers of peroxlsomes
at SO mg/kg/day were less pronounced, with males exhibiting greater
Increases than females. Glucose-6-phosphatase activity was reduced In both
sexes at all dose levels. However, decreases were significant at the 200
and 1000 mg/kg/day level In males and only at the 1000 mg/kg/day level In
females. Loss of this endoplasmic retlculum enzyme activity may be Indica-
tive of hepatotoxlclty (Mitchell et al., 1985). Increased number of lyso-
somes were observed In both sexes of animals at 200 and 1000 mg/kg/day DEHP,
however the Increase was less marked at the 200 mg/kg/day level.
Carpenter et al. (1953) conducted one of the first long-term oral multi-
generation toxlclty studies on DEHP using Sherman rats, guinea pigs and
dogs. In the rat study, 32 males and 32 females constituting the parental
(P.) generation were fed diets containing 0.04%, 0.13% or 0.4% DEHP for 2
years. The dally Intakes of DEHP were calculated to be the following: 0.20
g/kg bw/day for the first year and 0.19 g/kg bw/day for the 2-year period at
the 0.4% DEHP level; 0.06 g/kg bw/day for both periods at the 0.13% level;
and 0.02 g/kg bw/day for both periods at the 0.04% level. In addition, -80
first filial generation (F^ rats were fed -200 mg DEHP/kg/day for 1 year.
Numbers of rats per group surviving the 2-year test period were not
04750 V-33 07/03/91
-------
specified. However, It was reported that 70.3% mortality occurred In the
parental (P.,) controls. This figure was 9.3% higher than mortality among
the -200 and 60 mg/kg/day treated groups and 5% higher than that for the 20
mg/kg/day group. It was unclear how the percentages were calculated. No
Increases 1n mortality were associated with DEHP In the diet 1n either the
(P.|) or the progeny (F^) test groups.
Mean weights of the liver and the kidneys of the 0.4% (P^ group
sacrificed after 365 days and of (F.,) rats (also fed 0.4% OEHP) were
significantly (p
-------
In the female guinea pigs front treated groups than 1n the control animals.
The liver weights In females, as percentage of body weight, were 3.07X,
3,43% and 3.49X for the control. 0.04X and 0.13% groups, respectively.
However, combining the data for both sexes removed the significance at the
0.04% dietary level but not at 0.13%. The authors concluded that the effect
was not related to DEHP concentrations since the Increase In liver weight
did not appear to be dose-related. A 'no effect" dose (NOEL) of DEKP in
guinea pigs (for 1 year) was estimated to be -0.06 g/kg bw/day.
Carpenter et al. (1953) also studied dogs after 1 year of exposure to
OEHP. Four pure-blooded cocker spaniels and four wire-haired terriers were
randomly separated by breed and sex Into two groups. The dogs In one group
served as controls. The second group was administered OEHP In gelatin
capsules at 0.03 tu/kg/day, S days/week for the first 19 doses and then
0.06 mt/kg/day for 240 doses. The mean weight gain of doge receiving 0.06
OEHP was 0.78 kg as compared with 0.31 kg In th« controls. The two groups
were never statistically (p
-------
records, body weight, and IWer, testes, kidney, lung, brain, stomach, heart
and spleen weights were examined. Food consumption of the 0.5% DEHP group
was -75% that of the control group by the end of the first year. At that
time the DEHP Intake of the 0.1% dietary group ranged between 0.05 and 0.08
g/kg bw/day and that of the 0.5% group between 0.3 and 0.4 g/kg bw/day with
the higher amounts consumed during the first 6 months. Hlstopathologlc
studies were also conducted on selected tissues and organs. The study was
terminated after 24 months. Significant Increases In liver and kidney
weights were noted at the 0.5% dose level at 3 and 6 months but not at 1 and
2 years. The liver,and kidney weights did not differ significantly (analy-
sis not provided) In any of the groups, but the authors pointed out that
this may have been due to the small number of rats that remained after these
longer periods. During the 2-year test period, 85-95% of the rats died. Mo
unusual organ or tissue pathology was noted. The average body weight of the
0.5% DEHP group was -50 g less than the 0.1% and control groups at the end
of 1 year. Body weight averages of the three groups at 2 years were
similar. The authors reported no adverse effect on mortality with
Increasing percentages of DEHP 1n the diet. However, they did not report
the DEHP consumption of Individual survivors. The results of this study, at
least for the first year, appear to be consistent with those of Carpenter et
al. (1953) In that no effect levels and doses producing liver and kidney
enlargement were comparable. However, high mortality In all groups prevent
statistical analysis of results reported 1n the 2-year study by Harris et
al. (1956).
Kluwe et al. (1982a) reported on the non-neoplastlc effects observed In
male and female Fischer 344 rats and B6C3F1 mice during the 2-year NCI/NTP
carclnogenesls bloassays on DEHP. Details of the experimental procedures
Q475Q V-36 07/03/91
-------
for this study are given In the section titled "Cardnogenlclty" later In
this chapter. In male Fisher 344 rats fed diets containing 6000 and 12,000
mg/kg of DEHP and female rats fed 12,000 mg/kg, body weight gain was
slightly decreased throughout the latter 78 weeks of the study. This
decrease was also found In female mice treated with either 3000 or 6000
mg/kg diet of DEHP but did not occur 1n male mice treated at these levels.
Treated male and female rats consumed slightly less food than did control
rats, but food consumption In mice was largely unaffected. Mean dally
Intake of DEHP calculated from the food consumption data was 322 and 674
mg/kg bw/day for low- and high-dose male rats, 394 and 774 mg/kg bw/day for
low- and high-dose female rats, 674 and 1325 mg/kg bw/day for low- and
high-dose male mice, and 799 and 1821 mg/kg bw/day for low- and high-dose
female mice. No other clinical signs of toxldty were observed 1n either
rats or mice. Survival was not significantly (p<0.05) affected In male or
female rats or In male mice. In the low-dose female mouse group, however,
survival was significantly decreased (p<0.05) with most deaths occurring
after 75-90 weeks of treatment. The authors felt that these deaths were not
due to DEHP because pathologic changes In tissues were not observed
microscopically, and deaths were not observed at the higher DEHP dose.
Several nonneoplastlc lesions were associated with DEHP treatment.
Among male rats receiving 12,000 mg/kg diet (high-dose) of DEHP, seminif-
erous tubular degeneration and testlcular atrophy occurred In 90% of the
animals compared with an Incidence rate of 2% In controls. The tubules In
the affected animals were devoid of spermatocytes and germinal epithelium
and only Sertoll cells were found lining the basement membrane. These
lesions occurred In only 5% of the male rats receiving 6000 mg DEHP/kg and
were not significantly Increased. Another effect observed 1n male rats
04750 V-37 07/03/91
-------
fed 12,000 mg/kg DEHP 1n the diet was hypertrophy (cytoplasmlc enlarge-
ment) of the cells 1n the anterior pituitary. This effect occurred In 45%
of the animals compared with 2% In controls and none In low-dose males.
Cellular hypertrophy of this sort probably occurs as a secondary effect of
atrophy of the seminiferous tubule epithelium and may be Indicative of
anterior pituitary hyperactlvlty (Kluwe et al., 1982a). A dose-related
Increase In the number of animals with foci of clear cell changes 1n the
liver was observed among both male and female rats; however, palrwlse
comparison of controls to low- or high-dose groups did not show slgnlflcent
differences.
In male mice Ingesting 6000 mg/kg diet (high-dose). Incidence of semi-
niferous tubular degeneration was Increased (p<0.05) compared with controls,
and the lesion was similar to that In rats. Chronic Inflammation of the
kidney was significantly (p<0.05) Increased In the high-dose male mice.
Although nonneoplastlc effects were observed at the low-dose levels, the
carcinogenic effects of DEHP preclude Identifying a NOAEL for effects on the
liver.
Immature rhesus monkeys (6 months old,
-------
(BSP), by kinetic compartmental analysis, and by routine light microscopy of
liver tissues. For hepato-splenlc ratio determinations the abdomen of each
monkey was scanned 45 minutes after a lechnetlum-labeled sulfur colloid
Injection. The hepato-splenlc ratio was determined by detecting the mean
counts as measured over the liver and the spleen. The tests were repeated
3, 6, 12, 17 and 26 months following the beginning of transfusions. The BSP
calculations were done by computer analysis of the plasma disappearance
curve following a single BSP Injection. Heasurements were also made of the
following serum chemistries: SGPT, SGOT, lactic add dehydrogenase, blll-
rubln and alkaline phosphatase. These were made prior to the start of the
experiment and at 4-month Intervals. The results of this study showed that
abnormalities In liver function persisted <14 months following cessation of
transfusion therapy. DEHP was detected In liver tissue <14 months after the
last blood transfusion 1n an amount equivalent to 0.8X of that Infused.
Blood chemistry levels remained normal; however, the authors believed the
time period between their measurements was too long to allow detection of
transient changes. The work of Kevy et al. (1978) 1s Important since It
demonstrates DEHP effects through an exposure route applicable to humans.
DEHP was also tested In rhesus monkeys (2 or 3/group) given repeated
transfusions (for 1 year) of plasma containing this chemical (Jacobson et
al., 1977). Total DEHP doses ranged from 7-33 mg. DEHP was detectable In
the liver of these animals for as long as 5 months after the cessation of
exposure to DEHP. This treatment did not Induce cancer; however, abnormal
liver hlstopathologlc effects and function (such as, decreased BSP
clearance] were observed. Horphologlc changes Included hyperplasla and
vacuolatlon of Kupffer cells, foci of parenchymal necrosis and chronic
Inflammatory cell Infiltrate.
04750 V-39 09/12/88
-------
Effects on energy and carbohydrate metabolism have been observed after
DEHP exposure. Lake et al. (1976, 1977) reported that a 14-month dietary
Intake of 1200 mg/kg/day DEHP produced the following effects 1n male albino
ferrets: marked liver enlargement; decreased body weight; and decreased
activities of succlnate dehydrogenase, aniline 4-hydroxylase and glucose-
6-phosphatase. Mitchell et al. (1985) reported similar results 1n rats
administered 1000 mg/kg/day DEHP (Table V-7).
BBP. In a final report, NTP (1985) conducted a concomitant toxlclty
and mating trial study (discussed In the Reproductive Effects, BBP Section)
1n F344 rats. For the toxlclty study, male rats (15/group) were adminis-
tered concentrations of either 0, 0.03, 0.09, 0.28. 0.83 or 2.50% BBP In the
diet for 26 weeks. Using data presented In the report these dietary levels
correspond to 0, 17, 51, 159, 470 and 1417 mg/kg/day. respectively. In this
study powdered rodent meal was provided 1n such a way that measured food
consumption at the highest dose level could Include significant waste and
spillage rather than true food Intake. For this reason a standard food
consumption rate of 5% rat body weight was used 1n the 2.5% dose
conversion. Throughout the 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 terminal kill. Five of 11 had
soft testes, and 1/11 had a small prostate and seminal vesicle. In the
0.03, 0.09, 0.28 and 0.83% BBP dose groups there were no grossly observable
effects on male reproductive organs. Terminal mean organ weight values
significantly decreased (p<0.05) for the heart, right kidney, lungs, seminal
vesicles and right testes In the 2.5% treatment group, whereas significant
Increases (p<0.05) occurred In the brain at 0.03 and 0.09% BBP (Table V-8).
04750 V-40 07/03/91
-------
TABLE V-7
Long-Term Effects of DEHP on Biochemical Constituents Relating to Hepatotoxlclty
Species/
Strain
Route
Dose/Time
Principal Findings
Reference
Ferret/NR
diet
Rat/albino diet
Ulstar
1200 mg/kg/dayV
14 months
50 mg/kg/day/
9 months
200 mg/kg/day/
9 months
1000 mg/kg/day/
9 months
Absence of glycogen. decreased SDH
activity, aniline 4-hydroxylase and
glucose-6-phosphatase
Increased number of perox 1 somes;
laurate acid hydroxylase Induction
Increased number of perox 1 somes;
decreased glucose-6-phosphatase
activity In males only; laurate acid
hydroxylase Induction; D-D-galactostdase
Induction In females
Increased number of peroxIsomes;
decreased glucose-6-phosphatase
activity In males and females; decreased
cytochrome P-450; laurate acid and B-D-
galactosldase Induction
Lake et al..
1976. 1977
Mitchell
et al.. 1985
*Mean dally Intake
NR - Not reported; SDH
succlnate dehydrogenase
-------
f\>
TABLE V-8
Mean Terminal Organ Weights In Hale Rats After 26 Weeksa«b
Dose Level
(X)
Control
0.03
0.09
0.28
0.83
2.50
Number of
Surviving
Animals
12
14
14
14
IS
11
Brain
1.966*0.108
2.067*0.067C
2.08U0.044C
2.030*0.086
2.033*0.070
1.916*0.132
Heart
1.182*0.230
1.179*0.109
1.226*0.145
1.139*0.126
1.140*0.141
0. 796*0. I18d
Right Kidney
1.277*0.394
1.281*0.145
1.365*0.114
1. 249*0.137
1.422*0.173
1. 021*0. 192d
Liver
12.181*1.840
13.017*0.744
13.759*1.428
12.599*1.247
14. 261*1. 353C
11.782*2.511
Lung
1.570*0.204
1.726*0.200
1. 745*0. 171C
1.648*0.133
1.638*0.164
1. 147*0. 150d
Seminal
Vesicles
1.387*0.364
1.654*0.387
1. 798*0. 179«
1.489*0.426
1.538*0.343
0. 979*0. 345d
Right Testes
1.517*0.151
1.581*0.062
1.619*0.108
1.559*0.100
1.594*0.093
0.457*0.088''
'Source: Adapted from NTP (1985) draft report
D0rgan weights In grams (mean»standard deviation)
cS1gn1f1cant1y greater (p<0.05) compared with controls: Ounnett's t-test
^Significantly less (p<0.05) compared with controls: Dunnett's t-test
00
o
•s.
CO
CD
-------
At 0.83% the effects noted were significantly (p<0.05) Increased absolute
liver weight. Increased llver-to-body weight and llver-to-braln weight
ratios and Increases In mean corpuscular hemoglobin. Hematologlcal effects
at 2.5% BBP Included decreased red cell mass, which the authors state Is
Indicative of deficient hemoglobin synthesis, reduced values for hemoglobin.
total RBC and hematocrlt. The kidneys of six animals In the 2.5% group
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 0.03,
0.09. 0.28 and 0.83% treatment groups showed no evidence of abnormal
morphology In any other organs. Collectively, the effects associated with
feeding BBP at 2.5% Included depression of body weight gain, growth
retardation, decreased testlcular size, suppression of male reproductive
capacity and alterations 1n hematology values.
In an addendum to the NTP (1985) final report, evaluation of the data
revealed a significantly reduced total marrow cell count 1n the 2.5% dose
group (NTP. 1986). The change 1n total cell count was comprised primarily
of significant decreases In neutrophll, metamyelocytes, bands, segmenters,
lymphocytes and Ieasoph1l1c rubMcytes. The total marrow cell counts,
metamyelocyte and leasophlllc rubMcyte counts were also significantly
decreased 1n the lowest dose group (0.03%). No statistically significant
differences were noted In the middle dose groups (0.09, 0.28 or 0.83%) when
compared with controls. The addendum states that decreased total marrow
cell count In the 0.03 and 2.5% dose groups represent change of uncertain
meaning In light of the systemic effects noted In the middle dose groups.
Trend analysis by the Terpstra-Jonckheere test revealed significantly
04750 V-43 07/03/91
-------
(p<0.05%) decreasing trends In all of the previously mentioned parameters as
well as an increasing trend for monocytes at 0.03 and 2.5%.
Krauskopf (1973) presented data on BBP from an unpublished long-term rat
and dog study done by Monsanto (1972). No effects were observed In rats
administered levels of 0.25 (125 mg/kg/day) and 0.50% (250 mg/kg/day) BBP In
the diet for 90 days. Liver weights were Increased In animals fed diets
containing 1.0, 1.5 or 2.0% (500, 750 or 1000 mg/kg/day, respectively) for
90 days, and a mild decrease In growth rate was reported for the 1.50 and
2.00% groups. No other hematologlc, hlstopathologlc or urlnalysls effects
were observed. Dogs were given gelatin capsules containing BBP at doses
equivalent to 1.0, 2.0 or 5.0% of the dally diet (10, 20 and 50 g/kg) for 90
days. No deaths occurred, and weight gain was not affected at the 1.0 and
2.0% dose levels. The group fed 5.0% gained less weight Initially because
they refused to eat; however, the use of capsules restored normal eating.
No effects were found at any dose level on hematologlc parameters,
urlnalysls or liver and kidney functions (Krauskopf, 1973).
PBP. Smith (1953) studied the effects of feeding DBP to groups of 10
male 5-week-old Sprague-Oawley 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. Body
weights, food and DBP Intakes were measured dally for the first 3 months and
weekly for the remainder of the study. The dietary Intakes for DBP were 0,
5, 25, 125 and 600 mg/kg/day for 0.01, 0.05. 0.25 and 1.25%. respectively.
estimated from data by Smith (1953). Hematologlc parameters (hemoglobin,
total erythrocyte and total and differential leucocyte counts) were assessed
at Intervals of 3, 6 and 9 months. Necropsies were performed when rats
04750 V-44 07/03/91
-------
showed marked weight loss or signs of severe Infection. Animals remaining
at the end of 1 year were sacrificed and examined for gross pathologic
changes. While H was stated that several organs were sectioned and
stained, no hlstologlc evaluation was reported. No adverse effects on
growth, survival, gross pathology or hematology were observed In the animals
fed diets containing 0.01, 0.05 or 0.25% DBP. However, the number of
animals surviving the 1-year period were not reported for the control or
three lowest dose groups. In the group fed 1.25% DBP, half of the animals
(5/10} died during the first week of the experiment. The remaining animals
gained weight proportionate to controls. It was not Indicated whether the
deaths were thought to be treatment-related. The dally Intake of food and
plastldzer (mg/kg bw/day) decreased as the rats Increased In size. No
changes In hematologlc parameters or gross pathology were observed at any
dose level. Results of this study suggest that DBP has low chronic oral
toxlclty. However, this study 1s weakened by the small number of animals
used In the study, the lack of animal survival data, animal Infections, the
few survivors among the high-dose group, and a lack of mlcropatholngk
examination.
PEP. In a 2-year study (Food Research Laboratories, Inc., 1955}
groups of 30 rats (15 of each sex) were fed either 0.5, 2.5 or 5.0% DEP
(250, 1250 or 2500 mg/kg bw/day. respectively, assuming rats consume 5% of
their body weight) In the diet. No effects were observed at levels of 0.5
or 2.5X. 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 consump-
tion. No Information was available on the numbers of rats surviving the
2-year study period. Also as part of this study, 13 young mongrel dogs were
04750 V-45 07/03/91
-------
fed DEP 1n the diet at levels of 0.5, 1.5, 2.0 and 2.5% for 1 year. The
average weekly Intakes of DEP calculated by the Investigators were 0.8, 2.4,
3.5 and 4.4 g/kg/week In order corresponding to Increasing 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. No effects were noted In dogs as a result of
DEP exposures; however, hlstopathologlc examinations were performed only on
the kidney and liver In all the dogs. In addition, the heart, spleen,
pancreas, GI tract, adrenal glands and thyroid glands were
hlstopathologlcally examined In the three dogs of the 2.5% dosage group.
Brown et al. (1978] also studied the long-term oral toxldty of DEP In
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/kg/day In females, respectively. Hater Intake, food Intake and body
weights were measured weekly. Variables monitored In the study Included
body weight, food consumption, water Intake, hematology, urlnalysls, serum
biochemistries, and gross hlstopathology. Autopsies and hlstologlc exami-
nations were carried out at the end of 16 weeks. No changes In behavioral
patterns or clinical signs of toxldty were observed. Female rats fed diets
containing 1% OEP and both sexes fed diets of 5% DEP gained significantly
less weight 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 rule out palatablllty as the possible cause In
decreased weight gain, a paired-feeding study was conducted. Test rats fed
5% DEP consumed more food (total) and gained less weight than controls.
Absolute weights of the brain, heart, spleen and kidney were significantly
04750 V-46 07/03/91
-------
lower In male and female rats fed 5% DEP. Female rats given 5% DEP showed a
statistically significant Increase In "full caecum" weight. There were no
statistically significant changes 1n the absolute weights of any organs
below the 5% DEP dietary level. Relative (to body) weights of the brain,
liver, kidney, stomach, small Intestine and full caecum were significantly
higher In both sexes at the 554 dietary level when compared with the
controls. These changes were attributed 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.
PHP. In a study of Insect repellants by Lehman (1955), OMP was fed In
the diet to 10 female rats per dose group at levels of 2.0. 4.0 and 8.0%
(1000, 2000 and 4000 mg/kg/day assuming rats consume 5% of their body
weight) for 2 years. Details of the study were brief. No effect on growth
was observed at the 2% DHP dietary level. However, at the 4.0 and 8% DMP
levels there was a "slight but significant" (analysis not reported) effect
on growth. It was not stated 1f the effect was an Increase or decrease.
Chronic nephrllls occurred only at the 8.0% DMP level. Mortality rates did
not differ In treated rats when compared with the controls.
Reproductive Effects
Studies have shown that several PAEs have adverse effects on reproduc-
tion. These effects are summarized In Table V-9.
DEHP. Peters and Cook (1973) administered 4 ml/kg (4 g/kg) of DEHP
In saline to pregnant rats (Sprague-Dawley) 1.p. on days 3, 6 and 9 of
gestation. At this dose level, Implantation of embryos was prevented in 4/5
04750 V-47 07/03/91
-------
U8LE W-9
Sunmary of Teralogenlclty and Reproductive Effects or Phthalates
V
0
^^
00
o
to
*x
PO
^^
00
00
Exposure/Route Critical fffecl
Oral Embryonic Mortality
Retarded ossification
Reduced fetal weight.
ossification retardation.
skeletal abnormalities.
decreased maternal weight
No viable fetuses
Decreased fetal weight,
Increased fetal resorptlons;
LOAfl of 340 mg/kg/day
Average body weight of viable
fetuses was significantly lower
than the controls
Dose producing sane Mortality
rate as that of controls
est touted fro> dose-response
curve
Fetal IDjo: estimated fro*
'dose-response curve
NOAEL for gross abnormal It les;
estimated from a dose-response
curve
NOAfl for skeletal abnor-
malities; estimated from a
dose-response curve
No effect on fertility or
reproductive performance
Compound
DfHP
DIHP
DfHP
DIHP
DEHP
DtHP
Of HP
DEHP
DfHP
DEHP
DEHP
DEHP
Spec les
mouse
mouse
mouse
mouse
mouse
rat
mouse
mouse
mouse
mouse
mouse
mouse
Dose
l.OX' (10 g/kg)
0.5Xb (SO g/kg)
O.OM
(10 mg/kg/day)
0.2X
(410 mg/kg/day |
0.4X
(830 mg/kg/day)
l.OK
(2200 mg/kg/day)
340 mg/kg/day
1700 mg/kg/day
49.3 and 98.6
•g/kg
-64 mg/kg
S92 mg/kg
-769 mg/kg
-670 mg/kg
0.01X
(100 mg/kg)
Tine
gestation days 0-16
gestation days 0-18
gestation days 0-18
gestation days 0-18
21 days
21 days
single dose on day 7
of gestation
single dose
on day 7 of gestation
single dose on day 7
of gestation
single dose on day 1
of gestation
single dose on day
of gestation
In diet 7 days
pr nut Ing 98 days
continuous breeding
Reference
Hamano et al.,
1977
Shlola and
Mlsntmura. 1982
Shlola and
Nlshtmura. 1982
Shtota and
Nlshlmura, 1982
Nlkonorow
et al.. 1973
Tomlla et a)..
1982a
Tomtta et al..
198?a
Tomlta et al..
19B2a
Tomlta et al..
1982a
Tomlta el al..
1982a
NTP. 1984a
-------
TABLE V-9 (cont.)
Exposure/Route
Oral (cont.)
Critical Effect
Reduced fertility, decreased
number of Utters, decreased
viable fetuses
Total Infertility0
Reduced fetal body weight at
1.0. I.S and 2. OX: reduced
Compound
DEHP
DEHP
OEHP
Species
muse
mouse
rats
Dose
0.1X
(1 g/kg)
0.3X
O 9/kfl)
0. 0.5. 1.0. l.%5
and 2. OX (357.
Tine
In diet 7 days
prematlng 98 days
continuous breeding
In diet 7 days
prematlng 98 days
continuous breeding
dietary levels admin-
istered on gestatlonal
Reference
NTP, 1984a;
Lamb et al.,
1987
NTP. 19843 ;
Lamb et al..
1987
Tyl et al..
1988
gravid uterine weight at 2.OX;
not teratogentc at any dose
level: Increased number and
percentage of resorptlon, non-
live and affected tnplants per
Utter at 2.OX
Reduced gravid uterine weight OEHP
at 0.10 and 0.1SX; Increased
number and percentage of resorp-
tlons. nonllve and affected
tnplants per Utter at 0.10
and 0.1SX; number of Utters
with affected Inplants Increased
at O.OSX; number and percentage
of fetuses ma 1 formed/Utter
elevated at O.OS. 0.10 and 0.1SX;
embryofetal NOEL determined to be
44.07 mg/kg/day
Significant Increase In Incl- DEHP
dence of resorptlons and dead
fetuses at 1000 and 2000 mg/kg;
dose-related decrease In
average weight and Increase In
Incidence of malformed viable
fetuses; significant only at
1000 and 2000 mg/kg
mice
mouse
666. BS6 and
105S mg/kg/day)
0. 0.02S. 0.05.
0.10 and 0.15X
(44, 91. 191 and
292 mg/kg/day)
days 0-20
dietary levels admin-
istered on gestatlonal
days 0-17
Tyl et al..
1988
0. 250. 500, 1000
and 2000 mg/kg
Intubation In olive
oil on gestation days
7. 8 and 9
Shlota and
Ntma. 1985
-------
1ABLE tf-9 (cant-)
en
o
Exposure/Route
Oral (conl.)
Critical Effect Compound
Retarded body weight gain In a Of HP
dose -dependent Banner (statisti-
cally significant at 5000 and
20.000 ppm; significantly reduced
absolute and relative testls
weight at 20.000 ppm; no signifi-
cant changes In weights of senlnal
vesicles or pituitary; severe
atrophy In seminiferous tubules
and loss of spermatogenesls.
reduced epldldymal spenn density
and Mttllty at 20.000 ppa;
Increased occurence of abnormal
sperm and decreased line content
at 20.000 ppa
Tubular atrophy In testls Of HP
Tubular atrophy In test Is; Of HP
Species Dose
rats 0. 320. 12SO.
5000 or 20.000
ppa (16. 62. S.
250 or 1000
mg/kg/day)d
hamster 4200 mg/kg/day
rat 2800 mg/kg/day
Tine Reference
•ale rats In diet for Agarwal et al.,
60 days 1986
Intubation In corn Gray et al..
oil for 9 days 1982
Intubation In corn oil Gray et al..
un
o
significant Increased urinary
line excretion; significant
decreased testlcular line
concentration
No effect BBP
No effect BBP
No effect BBP
Significant reduction In body BBP
weight gain; significant reduc-
tion In aean organ weights of
right kidney, liver, lungs,
prostate, sealnal vesicles and
right testes; atrophy of semi-
niferous tubules and near total
absence of mature sperm production
rat
rat
rat
rat
0.03X (17 ag/koy
day)
0.28X (159 mg/
kg/day)
0.83X (470 mg/
kg/day)
2.ft (287S mg/
kg/day)
for 9 days 1982
In diet for 10 weeks NTP. 1985
In diet for 10 weeks NTP. 1985
In diet for 10 weeks NTP. 1985
In diet for 10 weeks NTP. 1985
CO
CD
-------
TABlf V-9 (cont.)
I
in
Exposure/Route Critical iffecl Compound
Oral (cont.) Increased liver and kidney BBP
weights
Decreased body weight; slight BBP
decrease In food consumption;
decreased testes. epldtdymus.
seminal vesicle and thynus
weights; hlstologlcal atrophy
of testes and accessory sex
organs
No adverse effects DBP
Fetal weight significantly DBP
reducrd/LOAEL
Retarded ossification OBP
Reduced fetal weight; DBP
retarded ossification
Reduced fetal weight; retarded DBP
ossification; decreased mater-
nal weight; neural tube defects
Embryonic mortality DBP
Species Dose lime
rat 0.625X 14 days In diet
(375 mg/kg/day)
1.25X
(750 mg/kg/day)
rat 2.5X 14 days In diet
(1250 mg/kg/day)
5. OX
(1667 mg/kg/day)
rat 120 mg/kg/day ?1 days
rat 600 mg/kg/day 21 days
mouse 0.05X gestation days 0-18
(80 mg/kg/day)
0.1X
(1BO mg/kg/day)
0.2X
(370 mg/kg/ay)
mouse 0.4X gestation days 0-lfl
(660 mg/kg/day)
mouse l.OX gestation days 0-18
(2100 mg/kg/day)
mouse O.SXD gestation days 0-18
(SO g/kg)
Reference
Agarwal et al.,
198Sa
Agarwal et al..
1985a
Nlkonorow
et al.. 1973
Nlkonorow
el al.. 1973
Shlota and
Nlshlmura. 1982
Shlota and
Nlshlmura. 1982
Shlota and
Nlshlmura. 1982
Htmano et al..
1977
o
\o
r\J
Significant depression of
testes weight; atrophy of semi-
niferous tubules; loss of
spermallds; reduction In num-
ber of primary spermatocytes
and spermalogon la
DBP
mouse
2000 mg/kg
Intubation In corn oil Gray et al..
for 9 days 1982
ca
oo
-------
TABLE V-9 (cont.)
en
o
Exposure/Route
Critical Effect
Compound
Species
Dose
Time
Reference
i
17*
O
lO
Oral (cont.) Significant depression of DBP
testes weight; atrophy of
seminiferous tubules; loss
of spermatlds; reduction In
number of primary spermato-
cytes and spermatogonla
No hlstologlcal changes; OBP
reduction In testes weight
but was not significant
Decreased number of litters. DBP
Utter size and fertility
as well as decreased mean
pup weight at 10 g/kg; NOAEL
at 3 g/kg
No effect on Maternal weight DNP
gain, litter slie or average
pup weight
I.p. Injection Gross or skeletal DEHP
abnormalities
Decreased body weight gain for DEHP
1? weeks after exposure to 5.?
g/kg/day; test Is weight not
affected; no reductions In sperm
numbers at any dose; no Increases
In sperm morphology
•Decreased body weight gain at DEHP
6.0 g/kg; significant decline In
lest Is weight 8 weeks after
exposure to b.O g/kg; signifi-
cant reduction In sperm number
per epldldymls from 4 weeks
after exposure to b.O g/kg; no
Increases In sperm morphology
guinea 2000 mg/kg
pigs
hamsters
CD-I mice
mouse
rat
rats
mice
3000 mg/kg
0.03. 0.3 and
IX (0.3. 3 and
10 g/kg)
3500 mg/kg
5 mt/kg (S g/kg).
10 mt/kg
(10 g/kg)
0. 0.52. ?.b. S.2
g/kg/day
0. O.b. 3.0. 6.0
g/kg/day
Intubation In corn oil
for 1 days
Intubation In corn oil
for 9 days
for 1 days prior to
pairing and for 98
days to breeding
pairs and for an
additional 21 days
gestation days 7-15
days S. 10 and
15 of gestation
S days of exposure
followed by 12 weeks
of observation
5 days of exposure
followed by 12 weeks
of observation
Gray et al..
1982
Gray et al..
1982
NTP. 1984b
Plasterer
et al.. 1985
Singh et al.,
1972
Douglas et
at.. 1986
r>O
CD
CD
-------
TABLE V-9 (cont.)
en
o
I
in
Exposure/Route
l.p. Injection
(cont.)
Critical Effect Compound
Implantation of embryos OEHP
prevented, adverse effects
on parturition (excessive
bleeding. Incomplete expul-
sion of fetus, maternal
deaths)
No teratogenlc effects below DEHP
the abort If ac lent dosage of
8000; average weight of viable
fetuses and Incidence of
resorptlons and dead fetuses did
not differ significantly from the
controls
Gross or skeletal abnormalities DBP
Gross or skeletal abnormalities OEP
Gross or skeletal abnormalities; DMP
fetal deaths
Species Dose
rat 2 mt/kg (2 g/kg)
4 mt/kg (4 g/kg)
mouse 0. 500. 1000.
2000. 4000 and
8000 mg/kg
rat 0.305. 0.610 and
1.02 mt/kg
(0.318. 0.636 and
1.06 g/kg)
rat 0.506. 1.012
and 1.686 mt/kg
(0.566. 1.13 and
1.88 g/kg)
rat 0.338. 0.675
and 1.125 mt/kg
(0.40. 0.80 and
1.33 g/kg)
Time
days 3. 6 and 9
of gestation
gestation days 7, 8
and 9
days 5. 10 and
15 of gestation
days 5. 10 and
15 of gestation
days 5. 10 and
15 of gestation'
Reference
Peters and
Cook. 1973
Shlota and
Mima. 19B5
Singh et al..
1972
Singh et al..
1972
Singh et al..
1972
'Highest concentration used
DLowest concentration used
'Subsequent crossover mating of these mice to controls revealed Infertility In males and decreased fertility In females
dAssumtng rats consume 5* of their body weight
GO
CO
-------
rats. At a dose level of 2 mi/kg, a similar response occurred 1n 3/5
rats. Adverse effects on parturition such as excessive bleeding, and
Incomplete expulsion of fetuses as well as maternal deaths were noted In
dams treated with DEHP. OBP and DMP were also tested by these authors;
however, the adverse effects were less severe than those observed for the
DEHP-treated rats. It was noted that adverse effects prior to gestation day
6 were primarily on Implantation while after this time, the effects were
primarily on parturition.
In a study by Singh et al. (1975), pregnant Sprague-Oawley rats were
Injected 1.p. on day 5 or 10 of gestation with 5 mi/kg (5.6 g/kg)
radlolabeled DEHP and 1.0116 mi/kg (1.13 g/kg) radlolabeled DEP. Results
of this study demonstrated that those PAEs could pass through the placental
barrier to the developing fetus. The data Indicate that the developmental
toxlclty of the PAEs could be the result of the direct effect of the
compound (or Its metabolites) upon developing embryonic tissue.
The teratogenlc effects of PAEs following oral administration were
studied by Nlkonorow et al. (1973). In this study pregnant Wlstar rats were
administered DEHP orally In olive oil at doses of 0.34 and 1.70 g/kg/day for
21 days following confirmation of conception. Results of this study
Indicated that fetal weight was significantly reduced at both dose levels of
DEHP. No detectable differences were observed In the number of sternum
ossification fod. the development of the bones at the base of the skull.
paws on the front and hind legs, or rib fusion 1n fetuses from treated rats
when compared with the control animals.
04750 V-54 07/03/91
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Shlota and Nlshlmura (1982) studied the effects of DEHP and DBP In mice.
DEHP and DBP were administered at dietary levels of 0.05, 0.1, 0.2, 0.4 and
IX by weight to groups of pregnant ICR-ICL mice from days 0-18 of gestation.
Average dally doses, calculated from food Intake and body weight, were 70,
190, 410, 830 and 2200 mg/kg/day for the 0.05, 0.1, 0.2, 0.4 and 1% dietary
levels of OEHP, respectively. Mice were monitored dally for food consump-
tion and weight. On day 18, the mice were sacrificed and uteri were
removed. Implantation sites, resorptlons and dead fetuses were recorded.
Live fetuses were dried of amnlotlc fluid, weighed, sexed and Inspected.
Half of the fetuses, from each litter were examined for skeletal malforma-
tions and the state of ossification. The other half were razor blade
sectioned and examined for Internal abnormalities. Maternal weight gain was
decreased and resorptlon rate was Increased when mice were fed 0.2, 0.4 and
IX DEHP. Intrauterlne deaths generally occurred In the early stages of
conception. At 0.4 and IX DEHP, all Implanted ova died In utero resulting
In no viable fetuses at term. A dose-related decrease In the mean weight of
fetuses alive at term was found In the treated groups. Malformed fetuses
resulted from treatment with 0.2X DEHP. Ihe major malformations In these
fetuses were neural tube defects (exencephaly and myeloschlsls). Indicating
that the PAEs affect neural tube closure In developing embryos. Ossifica-
tion was retarded 1n all treated groups except the one given 0.1X DEHP. The
authors concluded that delayed ossification was related to the general
underdevelopment of the fetuses. Incomplete skull and leg bones also
occurred occasslonally 1n the treated groups. Mlcrodlssectlon of the
fetuses showed no Internal malformations. The authors stated that the
maximum nonembryotoxlc dose In mice would be at least 70"mg/kg/day for DEHP.
04750 V-55 07/03/91
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More recently DEHP was evaluated for developmental toxlcity in Fischer
344 rats and CD-I mice (Tyl et al., 1988). Dietary levels of DEHP were
administered on gestaUonal 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, 357, 666, 856, 1055 and 0, 44, 91,
191, 292 In rats and mice, respectively. Maternal body weight and fetal
body weight were slgnlflcanty reduced (p<0.01) at 1.0, 1.5 and 2.OX DEHP In
rats. Gravid uterine weight In rats was also reduced at 2.0% DEHP. In
mice, gravid uterine weight was reduced at 0.10 and 0.15% DEHP while
maternal relative liver weight was elevated at the same levels. The number
and percentage of resorptlons, nonllve (dead plus resorbed) and affected
(nonllve plus malformed) Implants per Utter were significantly Increased at
2.0% In rats and 0.10 and 0.15% In mice. The number and percentage of
fetuses malformed/Utter were unaffected In OEHP-treated rats; however,
reduced reduced fetal body weight/litter was observed at 1.0, 1.5 and 2.0%.
The number and percentage of malformed fetuses/Utter were elevated (p<0.01)
at 0.05, 0.10 and 0.15% DEHP In male and 0.10 and 0.15% DEHP In female
mice. Treatment-related malformations consisted of open eye, exophthalmla,
exencephaly, short, constricted or no tall, major vessel malformations.
fused or branched ribs and fused or misaligned thoracic vertebral centra.
Tyl et al. (1988) concluded that DEHP was not teratogenU at any dose tested
In Fischer 344 rats. However, treatment did produce maternal and other
embryofetal toxlcity at 1.0, 1.5 and 2.0%. An embryofetal NOEL In rats was
reported as 0.5% (357 mg/kg/day). In mice, doses (0.10 and 0.15%) that
produced maternal and embryofetal toxlcity also Increased Incidence of
malformations. A dose of 0.05% (91 mg/kg/day) DEHP 1n mice produced
Increased Incidence of malformations without maternal or embryofetal
04750 V-56 07/03/91
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toxlclty. An embryofetal NOEL In mice was reported as 0.025% (44 mg/kg/day)
DEHP.
The teratogenlc potential of plasma-soluble extracts of two DEHP-plastl-
clzed PVC plastics 1n Sprague-Oawley rats was Investigated by Lewandowskl et
al. (1980). The extracts were administered Intravenously to pregnant rats
dally on days 6-15 of gestation. Two groups of rats received extracts from
one plastic preparation In doses equivalent to 1.3 and 4.7 mg/kg/day DEHP.
Two additional groups received extracts from a second plastic preparation In
doses equivalent to 1..4 and 5.3 mg/kg/day DEHP. The high doses were thought
to approximate the doses a 60 kg human would receive when undergoing an
exchange transfusion of 21-day-old blood. No significant differences
between controls and treated groups were found In growth rate and behavior
of test animals, fetal weight, number of live and resorbed fetuses or
Incidence of gross external, skeletal or visceral defects among offspring.
TomHa et al. (1982a) tested the teratogenlc and fetotoxlc effects of
DEHP In mice given single oral doses of 0.05-30 ml/kg on day 6, 7. 8, 9 or
10 of gestation. Test animals used were female ddY-Slc(SPF) mice bred to
CBA(SPF) mice. A high-dose experiment was performed using dosages of 1.0,
2.5, 5.0. 7.5, 10.0 and 30.0 ml/kg (0.986, 2.47, 4.93, 7.40, 9.86 and 29.6
g/kg/day) of DEHP. In this experiment mice were dosed on day 6. 7, 8, 9 or
10 of gestation. A low-dose single administration was also performed using
1/600, 1/300 and 1/30 of the LD5Q (0.05, 0.1 and 1.0 ml/kg of DEHP
corresponding to 49.3, 98.6 and 986 mg/kg/day) administered on day 7 of
gestation. For each of the experiments, an untreated control group and an
ethylurethane-treated control (positive control) group were Included. These
04750 V-57 07/03/91
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experiments showed that number of Implantations per pregnant mouse was not
significantly different among control and DEHP treated groups; however, the
number of early and late embryo deaths varied greatly, depending on day and
amount of DEHP administered. Generally, 1n mice receiving DEHP on days 7 or
8 of gestation. Incidence of embryo death was high, whereas Incidence of
embryo death was low In mice treated on days 6, 9 or 10. The Incidence of
early embryo death was higher at the higher doses of DEHP, while the
Incidence of late embryo death was greater at the lower doses. The rate of
gross and skeletal malformations was high In mice receiving 2.5 mi/kg
(2.47 g/kg/day) of DEHP on day 7 of gestation and In mice receiving 7.5
ml/kg (7.40 g/kg/day) on day 8 of gestation. No abnormality was observed
In the groups receiving 0.05 or 0.1 mi/kg on day 7; however, the average
body weight of viable fetuses was significantly lower (p<0.01) than that of
the control group. Treatment-related gross abnormalities Included
exencephaly, open eyelid, club foot, and bent or no tall, and skeletal
malformations such as abnormally shaped thoracic, lumbar, sacral and caudal
vertebrae. The authors calculated the fetal LD5Q to be 592 mg/kg/day
assuming that the specific gravity of DEHP Is 0.986. The dose level
producing a rate of 2% fetal deaths (the mortality rate found In the control
group) was -64 mg/kg/day. The exact values of maximum no-observed-adverse-
effect dose for gross and skeletal abnormalities could not be determined due
to lack of data but are estimated to be <0.80 ml/kg (789 mg/kg/day} and
0.68 ml/kg (670 mg/kg/day), respectively, based on their dose-response
curves.
Autlan (1982) reported results of a study of antlfertlllty effects of
DEHP. Hale mice were Injected subcutaneously with DEHP on days 1. 5 and 10
04750 V-58 07/03/91
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of the study at dose levels of 1, 2. 5, 10, 15, 20. 40. 60. 80 and 100
mi/kg (1. 2, 5, 10, 15, 20, 40, 60, 80 and. 100 g/kg). Seven or more mice
were used for each dose level. A group of 16 mice receiving Injections of
saline served as controls. Hales were bred to virgin females on day 21
following the first Injection. Females were sacrificed on day 12-13 of
gestation, and uterine horns and ovaries were exposed surgically to deter-
mine the number of corpora lutea. Implantations, prelmplantatlon losses,
early fetal deaths and viable fetuses. Incidence of pregnancy was decreased
at all dose levels compared with controls; however, the statistical signifi-
cance of this decrease was not evaluated. Males treated with the lowest
dose of 1 mi/kg per Injection produced progeny 1n 62.5% of the treated
group compared with 87.5% 1n controls. The trend of antlfertlllty appeared
to Increase as the dose administered to male mice Increased. Increased
early fetal deaths and prelmplantatlon losses were also noted In the treated
groups, generally Increasing as the dose administered Increased. The author
noted, however, that the results of this experiment Indicate antlfertlllty
effects but cannot be considered definitive under the experimental condi-
tions employed. Autlan (1982) also noted that In a parallel ^tudy (the
details of which were not given), the effects of lower doses of DEHP on
testlcular structure and function did not show changes In hlstopathologlc
organization or macromolecular contents (nucleic acids and protein) of the
tissue, suggesting that Increased fetal deaths were not a consequence of
testlcular atrophy. Alterations 1n the activity of certain mitochondria!
and lysosomal enzymes of the testlcular tissue were observed after treatment
with OEHP. which may account for changes In the functional ability of the
reproductive system. PAEs have also been shown to cause testlcular atrophy.
A more detailed description of these effects Is given in the section
discussing target organ toxlclty.
04750 V-59 07/03/91
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A recent study of the effects of DEHP on reproduction and fertility was
conducted by NTP (1984a). The NTP (1984a) study was subsequently published
as Lamb et al. (1987). This study employed a reproductive toxicology
testing scheme referred to as "Fertility Assessment by Continuous Breeding".
DEHP was administered In the diet at levels of 0, 0.01, 0.1 and 0.3% (0.1.
1.0 and 3.0 g/kg). Male and female CD-I mice were given continuous dietary
exposure to DEHP during a 7-day premattng period and a 98-day continuous
breeding cohabitation period. A 21-day segregation period with no DEHP
exposure followed. The control group consisted of 40 pairs of mice while 20
pairs of mice were tested at each dose level. Results of the study showed
that fertility was completely suppressed at the 0.3% DEHP level and was
significantly reduced at the 0.1% DEHP level. The fertility Index {number
of fertile/number cohabited x 100) was 0 and 74% for the 0.3 and 0.1%
groups, respectively. The fertility Index value for the control and 0.01%
groups was 100% 1n both cases. Among the 0.1% DEHP breeding pairs that were
successfully mated, fewer litters were produced, numbers of male and female
live pups per litter were decreased, and the proportion of pups born a ""We
per TUter was lower when compared with either the control group (p<0.01) or
the 0.01% group (p<0.01). Also, the proportion of live male pups per total-
live pups per litter and the female live pup weight were Increased In the
0.1% DEHP group compared with the control group (p<0.05). Live male pup
weight adjusted for the total number of pups per litter was significantly
lower at the 0.1% level than at the 0 or 0.01% DEHP (p<0.05).
Because the continuous breeding test showed that DEHP had significant
effects on fertility and reproduction, a second test, the crossover mating
trial, was conducted to determine which sex (male and/or female) was
04750 V-60 07/03/91
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adversely affected. The control males and females from the continuous
breeding test were mated with the high-dose (0.3% DEHP) females and males,
respectively, from the continuous breeding test. Another group of control
males were mated to control females, both from the first test, to serve as
the control for this experiment. Results Indicated that fertility was
significantly reduced (p<0.01) In both the 0.3X DEHP male x control female
and the control male x 0.3X DEHP female groups when compared with the
control male x control female group. Fertility Index values for the three
groups were 20%, 0% and 90%, respectively. The number of detected matlngs
(determined by the presence of a copulatory plug) did not differ signifi-
cantly among the three groups. In the 0.3X DEHP male x control female
groups, the proportion of pups born alive was significantly lower (p<0.05)
than In the control male x control female group; however, the number of live
pups/litter, sex of pups born alive and live pup weight were not signifi-
cantly different. Following the crossover mating trial, male and female
parental mice from the control and 0.3% DEHP groups were subjected to gross
necropsy. Effects on the male reproductive system Included significant
reductions In percentage of motile sperm and sperm concentration 1n the
cauda epldldymus and In weight of the testls. epldldymus and prostate 1n the
0.3X DEHP-treated mice compared with controls. The percentage of morpho-
logically abnormal sperm In the cauda was also significantly Increased.
Hlstopathology revealed extensive destruction of the seminiferous tubules In
the treated males. Decreased plasma testosterone levels and elevated plasma
follicle stimulating hormone (FSH) and lutelnlzlng hormone (LH) levels were
observed In the treated group, although differences were not significant due
to large variation within treatment groups. Among females, no hlstopatho-
loglc effects were observed. The reproductive tract weight In treated
04750 V-61 07/03/91
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females was significantly decreased, but It 1s possible that this was an
artifact of lack of pregnancy 1n this group. Significant Increases 1n liver
weight were also observed for both males and females In the 0.3% DEHP groups.
A study In which groups of rats were fed diets containing 0.2, 1.0 or
2.0% DEHP for 90 days, corresponding to mean dally Intakes of -ISO. 750 and
1500 mg/kg bw/day, showed a decrease 1n the relative testes weight of rats
fed 1.0 and 2.OX DEHP (Gray et al.. 1977). All three treatment levels pro-
duced hlstologlc evidence of testlcular Injury and "castration" cells In the
pituitary. The hlstopathologlc changes 1n the testes were characterized by
a marked reduction In the diameter of seminiferous tubules, presence of a
germinal epithelium that consisted only of Sertoll cells, spermatogonla and
a few spermatocytes, and a cessation of spermatogenesls. Interstitial
tissues and Leydlg cells appeared normal. At the 2% dietary level, testlcu-
lar atrophy occurred within 2 weeks of treatment. A 2-week target organ
experiment showed that, whereas DEP had no discernible adverse effects on
the testes, DBP produced testlcular atrophy, possibly more severe than that
produced by DEHP (Gangolll, 1982).
Cater et al. (1977) summarized an unpublished study on DEHP In which an
unspecified strain and number of rats were fed various dose levels of the
ester for 90 days. At a dally level of 0.2%, DEHP produced testlcular
Injury. When the level of DEHP was Increased to l.OX, testlcular Injury was
noted In 2 weeks. The authors further stated that DEHP and DBP have about
the same potency 1n causing testlcular atrophy 1n rats.- It was noted that
other esters of phthallc add were studied; however, no data were presented.
04750 V-62 07/03/91
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Young rats appear to be more sensitive than older rats to the testlcular
effects Induced by DEHP. Curto (1984) found that 32-day-old male rats given
2000 mg/kg of DEHP for 5 days showed testlcular atrophy and reduced zinc,
DNA and RNA concentrations In the gonads while 62-day-old rats did not.
However, older rats were not completely Insensitive to testlcular effects of
DEHP. IntraperHoneal Injection of 100 mg/kg DEHP every other day for 20
days caused reduced zinc gonadal and prostatlc concentrations In adult rats.
Curto (1984) also studied the reversibility of the effects In 32-day-old
rats. At 1 day post-treatment, testlcular atrophy, -reduced zinc and RNA
concentrations and Increased alkaline phosphatase activity were observed.
All parameters with the exception of testlcular atrophy had returned to
normal at 20 days post-treatment.
Results presented by Hushtaq and Datta (1981) In an abstract also
Indicate that young rats may be more sensitive to the testlcular effects of
DEHP than older rats. These authors studied the effects of DEHP In young
male albino rats ranging In age from 4-12 weeks old. Animals were given
2000 ppm (2000 mg/kg) DEHP dally by oral Intubation for 30 days. The weight
of the testls was decreased by 60-70% 1n the 8-week group accompanied by
several biochemical changes In the testls. Rats In the older groups showed
no decrease In testlcular weight and fewer biochemical changes. Hlstopatho-
loglc studies found severe destruction of the seminiferous tubules 1n
8-week-old rats following DEHP treatment. Similar results were reported by
Gray and Gangolll (1986). Oral administration of 2800 mg/kg/day DEHP for 10
days to 4- and 10-week old Sprague-Dawley rats produced hlstologlc changes
1n the testes along with depression In the weight of the testes, seminal
04750 V-63 07/03/91
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vesicle and prostate. The effects were less marked In the 10-week-old rats.
In 15-week-old rats DEHP had no effect on any of the above organ weights and
no hlstologlc abnormalH1es.
SJoberg et al. (1986b) '.nvestlgated the age-dependent response of the
rat testes to DEHP. Groups of 25-, 40- and 60-day-old Sprague-Dawley rats
were administered dietary dose levels of 1.0 or 1.7 g/kg bw DEHP for 14
days. Body weight gain and testlcular weight were reduced In all groups of
25- and 40-day-old rats given 1.7 g/kg DEHP. Testlcular damage was more
t
severe In 25-day-old rats administered 1.7 g/kg doses and less severe In
25-day-old rats given 1.0 g/kg and 40-day-old rats given both 1.0 and 1.7
g/kg. No changes were observed 1n 60-day-old rats at either dose. Similar
results were reported after gavage administration of 1.0 g/kg/day DEHP to
Sprague-Oawley rats In the same age group (SJoberg et al., 1985c). SJoberg
et al. (1986b) speculate that the causes for the age-dependent variation In
testlcular response may be an age-dependent difference In tissue sensitivity
or differences in absorption, distribution, metabolism and/or excretion.
Olshl and Hlraga (1983) studied the effects of OEHP on lipld composition
of liver, testes and serum in rats fed diets containing 2% DEHP for 9 days.
DEHP Induced changes in lipld and fatty acid composition, which resembled
those caused by a zinc deficiency. In another study Olshl (1984a) studied
the Upld composition of serum and testes In DEHP-treated rats. Altered
liptd metabolism In the testes Is frequently associated with testlcular
atrophy. Olshl (1984a) found that nonesterlfled fatty add Increased and
cholesterol, trlglycerlde, phosphollpld and zinc decreased In the serum
after rats were fed 2% DEHP In the diet. Concentrations of cholesterol and
04750 V-64 07/03/91
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nonesterlfled fatty acid Increased 1n the testes, whereas zinc concentra-
tions decreased. 01sh1 (1984a) concluded that llpld alterations after DEHP
administrations were similar to changes ca.used by a zinc-deficient diet;
therefore, testlcular atrophy caused by DEHP may be related to zinc concen-
trations In the testes and hormonal abnormalities.
Olshl (1986) studied the changes 1n testlcular enzyme activity during
exposures to DEHP. Changes In testlcular cell-specific enzymes appear to be
useful biochemical markers of testlcular Injury. Wlstar rats (30 days old)
were administered 2 g/kg/day DEHP by gavage dally for 10 days. Testlcular
weight gain was significantly reduced after 3 days. Zinc concentrations In
the testes significantly decreased after 6 and 10 days and decreased In the
ventral prostate after 10 days. Concentrations of zinc In the serum and
seminal vesicle were not affected. Activities of lactate dehydrogenase
Isozyme-X (LDH-X), sorbHol dehydrogenase (SDH) and hyaluronldase, which are
associated with postmelotlc spermatogenlc cells, decreased In treated
animals after 10 days. Specific activities of these enzymes Increased 1n
controls during the experimental period. By day 10, all seminiferous
tubules were shrunken In DEHP-treated rats; primary and secondary spermato-
cytes and spermatlds were absent or showed severe degenerative changes. The
specific activities of o-glycerophosphate dehydrogenase (GPDH). B-glucu-
ronldase and y-glutamyl transpeptldase (Y-GTP), Sertoll cell and sperma-
togonlc specific enzymes also significantly Increased (p<0.05) after 10 days
(Olshl, 1986). Parmar et al. (1986) also observed Increases In Y-GTp and
LDH-X and decreases 1n SDH and B-glucuron1dase after treatment with DEHP.
If biochemical changes are detected prior to hlstologlc Changes, they may be
useful as markers for tissue damage. The biochemical changes reported 1n
04750 V-65 07/03/91
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the 01sh1 (1986) study occurred after or simultaneously wHh massive hlsto-
loglc or morphologic changes. The usefulness of this study (Olshl, 1986) 1s
limited since only one dose level was tested and therefore the threshold for
biochemical changes cannot be determined.
Olshl (1984b) also Investigated the relationship of OEHP-lnduced
testlcular atrophy to vitamin A and zinc deficiencies. Zinc deficiencies
have been associated with low plasma vitamin A levels, and vitamin A
deficiencies have been associated with testlcular atrophy and Impaired
spermatogenesls (Smith et al.. 1973; Coward et al., 1966; Mason, 1933).
Young male Wlstar rats were fed diets containing 2% DEHP for 1 week at which
time they were sacrificed. Body weight and testlcular weight were decreased
while liver weight was Increased among DEHP-treated rats. Zinc concentra-
tions were decreased compared with controls In the liver and testls;
Vitamin A concentrations were decreased In the liver and Increased In the
serum and testls of DEHP-treated rats. These results suggest that DEHP-
assoclated testlcular atrophy was not due to dietary or Intrinsic vitamin A
deficiency since DEHP treatment raised testlcular and serum vitamin A levels.
Mangham et al. (1981) compared testlcular effects of DEHP and dlalkyl 79
phthalate (DA79P) In rats. The phthalates were administered orally In corn
oil by gavage at a dose level of 2500 mg/kg/day for either 7 or 21 days.
DEHP caused a significant reduction of relative (to body) weight of the
testes after 7 or 21 days, whereas DA79P reduced the relative weight only
after 21 days of treatment. Similarly, DA79P did not produce changes in
tubules detectable with a light microscope at 7 days, with the exception of
one male where -20X of the germ cells were missing from the seminiferous
04750 V-66 07/03/91
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tubules. Among the 7-day DEHP group, 50-80X of the tubules were affected 1n
each animal. After 21 days of treatment, both DEHP and OA79P produced
bilateral tubular atrophy and 5C-100X of the tubules were affected In all
animals. No effects were observed on Interstitial cells or Sertoll cells
with either compound.
BBP. BBP has also been shown to cause testlcular atrophy. In an NTP
final report, BBP produced testlcular atrophy 1n rats fed a dietary concen-
tration of 2.5X (NTP, 1985). After 1 day of acclimation male Fischer 344
rats (15 animals/group) were fed diets containing 0,' 0.03, 0.28, 0.83 and
2.5X BBP for 10 weeks. Using the data presented In the report, these
dietary levels correspond to 0, 17, 159, 470 and 1417 mg/kg/day. In this
study powdered rodent meal was provided 1n such a way that measuered food
consumption at the highest dose level could Include significant waste and
spillage rather than true food Intake. For this reason a standard food
consumption rate of 5X rat body weight was used 1n the 2.5X dose
conversion. Two untreated females were assigned to each mating trial male
after the 10-week pretreatment. Throughout the 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. There were no
grossly observable abnormalities In the testes at any dose group. In a
corresponding 26-week toxlclty study on BBP, testlcular abnormalities were
observed after 26 weeks suggesting that the effects become more pronounced
after 10 weeks of exposure (see long-term toxlclty section on BBP).
Terminal mean organ weight values significantly decreased In the right
kidney, liver, lungs, prostate, seminal vesicles and right testes of the
2.5X treatment group, whereas the heart significantly Increased at the 2.5X
04750 V-67 07/03/91
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treatment group. Organ-to-body weight ratios significantly Increased In
heart, liver, lung and thymus and significantly decreased 1n the brain and
prostate at the 2.5X level. Hlstopathologlc changes were seen at the 2.5%
BBP level. After hlstopathologlc 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
2.5X treatment level males, none of the females were pregnant at necropsy.
Agarwal et al. (1985a) evaluated the effects of BBP on the male repro-
ductive system In a 14-day dietary study using groups of 10 adult male
Fischer 344 rats fed BBP at levels of 0.0, 0.625, 1.25. 2.5 and 5.0%.
Results of this study showed that the absolute weights of the testls,
epldldymus, prostate and seminal vesicles were significantly reduced
(p<0.05) 1n rats eating the 2.5% and 5.OX BBP diets. The effects were dose-
dependent. Since the overall body weight gain was significantly reduced at
these two dietary levels, expression of the organ weights relative to body
weight reduced the magnitude of the effect, but the decreases In weights of
the testls, epldldymus and seminal vesicles remained significant (p<0.05)
compared with controls. Hlstopathologlc examination of these tissues
revealed that the decreased weights of these organs were associated with
generalized hlstologlc atrophy. The severity of the changes In tissues from
the testls, seminal vesicles and prostate were clearly dose-related with
degenerative changes found at the 2.5X and 5% levels. In the epldldymus,
atrophlc conditions were predominantly due to the necrosis of the tubular
epithelium In the caput (head) portion. Numerous Immature spermatogenlc
cells were found 1n the lumens of the epldldymus. In addition to the
effects on the male reproductive organs, relative {to body weight) liver and
04750
V-68 07/03/91
-------
kidney weights were Increased at all levels of BBP administered. Relative
(to body) weight of the thymus was significantly reduced In the 2.5 and 5.0%
groups. Plasma levels of testosterone were significantly reduced (p<0.05)
In the 5.0% group. The testosterone levels were lower than controls at
2.5%, but the decrease was not significant. Plasma FSH was significantly
Increased (p<0.05) In rats fed 2.5% and 5.0% BBP and plasma LH was Increased
at 2.5%. The LH levels In the 5.0% group could not be determined due to
Insufficient sample volumes.
DBP. The teratogenlc effects of PAEs following oral administration
were studied by Nlkonorow et al. (1973). In this study female Wlstar rats
were administered 120 and 600 mg/kg/day DBP In olive oil for -3 months and
mated. 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 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, paws on the front and hind legs, or rib fusion In fetuses from
treated rats when compared with the control animals.
Shlota and Mshlmura (1982) studied the effects of DBP given to pregnant
ICR-ICL mice on days 0 through 18 of gestation. DBP was administered at
dietary levels of 0.05, 0.1, 0.2. 0.4 and 1.0% corresponding to 80, 180,
370, 660 and 2100 mg/kg/day. At the 660 mg/kg/day dose level the Investi-
gators observed reduced fetal weight In addition to retarded ossification.
Diets of 2100 mg/kg/day decreased maternal weight, reduced fetal weight and
retarded ossification. Fetuses also experienced neural tube defects at the
04750 V-69 07/03/91
-------
2100 mg/kg/day treatment level. The authors concluded that delayed ossifi-
cation was related to the general underdevelopment of the fetuses. The
maximum nonembryotoxlc dose as slated by the authors would be 370 mg/kg/day
DBP.
Cater et al. (1977) found that DBP Induced testlcular atrophy In young
male Sprague-Dawley rats. In this study, the DBP was dissolved In corn oil
and administered by gavage. The doses administered were 500, 1000 and 2000
mg/kg/day while control animals received corn oil In a volume of 5 mi/kg.
The Initial effect was a progressive reduction In weight of the testes. A
significant reduction In the relative (to body weight) testes weight
occurred within 6 days at 500 mg/kg and within 4 days at 1000 and 2000
mg/kg. By 14 days, the reduction at 2000 mg/kg amounted to 60-70% of the
original weight. Since there was also a decrease In body weight, the
authors used "relative testes weight" and found that on this basis there was
still a significant loss of testes weight. Hlstopathologlc examination of
testes tissue demonstrated morphologic damage similar to that produced by
DEHP. Further Investigations by these authors revealed that the DBP
adversely affects zinc metabolism and Increases urinary zinc excretion.
Similar results were observed by Gray et al. (1982) (see Table V-9).
NTP (1984b) conducted a continuous breeding study In male and female
CD-I mice to determine the reproductive and fertility effects when exposed
to DBP. Mice (11 weeks of age) were administered 0, 0.03, 0.3 and 1.0%
(0.3, 3.0 and 10.0 g/kg) DBP In the diet for 7 days prior to pairing and for
98 days to breeding pairs and then for an additional 21 days. In the 1.0%
dose group, mice experienced decreased average number of Utters and litter
04750 V-70 07/03/91
-------
size and fertility was 2554 lower than the controls. There were no
significant differences between the 0, 0.03 and 0.3% dose groups. Only 50%
of the pups In the 1.0% dose group were born alive compared with 99% In the
0, 0.03 ana 0.3 groups. Of the 50% In the high-dose group (1.0%) a
significantly (p<0.01) larger portion of the pups were males. Mean pup
weight decreased In the highest dose group when adjusted for average litter
size. The data Indicate that male fetuses may be slightly more resistant to
the toxic effects of DBP.
A cross mating trial was also performed by NTP (1984b) \n order to
determine whether one or both sexes were adversely affected In the contin-
uous breeding study. The crossover mating trial consisted of three combina-
tions of breeding pairs. These were control males x control females, 1% DBP
males x control females, and control males x 1% OBP females. Animals were
necropsled 26 days after the 7-day crossover mating trial. The proportion
of fertile mice was slgr., 'kantly reduced (p<0.01) 1n control male x 1.0%
DBP females. In addition the number of live pups/litter, the proportion of
pups born alive and the litters per pair were significantly decreased
(p<0.01) 1n control males and 1% DBP-treated females. As 1n the reproduc-
tive study, the proportion of live males per litter (males/total) was
significantly (p<0.01) higher In the 1% treated females and control males.
In the 1% DBP-treated male mice there were no significant differences 1n the
percentage of abnormal sperm. No treatment-related gross or hlstopatho-
loglc lesions were noted In the reproductive organs of treated male and
female mice. Absolute and relative uterine weight were significantly
Increased In the 1.0% DBP-exposed group, perhaps reflecting the production
of fewer and smaller litters.
04750 V-71 07/03/91
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An Increase In urinary excretion of zinc, which Is an essential element
for testlcular function, was observed following DBP treatment (Gunn and
Gould, 1970}. In an experiment using "Zn, treatment with DBP led to a
marked Increase 1n urinary excretion of radioactivity and a decrease of
*5Zn associated with testlcular tissue. The activities of two enzymes
containing zinc (alcohol dehydrogenase and carbonic anhydrase) were also
decreased.
Foster et al. (1980) tested the testlcular effects of a series of
dl-n-alkyl phthalates ranging from GI to Cg In rats. The PAEs were
administered orally at a dose of 7.2 mmole/kg/day (2000 mg/kg/day) for 4
days to young male rats. Results showed that OBP produced testlcular
Injury, whereas both shorter chain compounds (DMP and DEP) and longer chain
compounds were Inactive. Urinary excretion of zinc and depletion of zinc
from the testes were only observed with those compounds producing testlcular
Injury. The reason that these two compounds produce Injury Is not known at
present, but the determining factor does not appear to be related to the
Intestinal hydrolysis rate, since the monoesters of the Inactive compounds
were also Ineffective In producing gonadal Injury. Because these findings
are based on short-term (4-days) tests, the compounds shown to be Inactive
In these tests may In fact cause testlcular Injury with longer exposure.
Johnson and Gabel (1983) evaluated three PAEs In a study Investigating a
new procedure using artificial "embryos" from hydra to detect agents causing
abnormal development. This Is an in vitro teratogen. screening protocol
wherein a bolus of dissociated hydra cells Is monitored for development to
normal adult Individuals. Test compounds can be added to the medium, and
04750 V-72 07/03/91
-------
disruption of development evaluated. In this study, the ratio of the dose
level toxic to adults (A) to the dose level affecting development of
offspring (DJ was reported for mammalian testing, using dose levels from
published reports, and for the new hydra procedure. DMP and DBP each had
A/0 ratios of <3 In mammals and 2 In hydra. DEP had an A/D ratio of 2.5 In
mammals and 2.0 1n hydra. These ratios Indicate that the larger the A/0
ratio, the greater the tendency of the compound to cause developmental
effects without causing toxUHy In adults. For comparison, however, the
A/0 ratio for thalldomlde Is about 60, Indicating teratogenlc effects at
concentrations far be*ow those causing maternal toxlclty.
PEP. In a study by Singh et al. (1972), Sprague-Dawley rats weighing
200-250 g were Injected l.p. with DEP on days 5, 10 and 15 of gestation to
ascertain the effect on the fetus. DEP was administered at three dosage
levels to groups of five female rats/group. The dosage levels were 1/10,
1/5 and 1/3 the acute LD5Q of 5.0579 ma/kg (5.66 g/kg) DEP corresponding
to 0.506, 1.012 and 1.686 ml/kg (0.566, 1.13 and 1.88 g/kg),
respectively. Animals were sacrificed on day 20 of gestation. No resorp-
tlons, dead fetuses or skeletal abnormalities occurred In the untreated
control group. Resorptlons did not occur at the 1.012 ml/kg treatment
level. Treatment levels 0.506 and 1.686 ml/kg DEP produced 44.4 and 3.6%
resorptlons, respectively. The authors did not give any reason for this
finding. There were no gross abnormalities at any treatment level. Fetuses
were significantly (p<0.01) smaller than the untreated controls at all
treatment levels. The number of skeletal malformations were 26.3, 47.1 and
81.3% for the treatment levels 0.506, 1.012 and 1.686 ml/kg OEP, respec-
tively. The skeletal malformations most commonly encountered were elongated
04750 V-73 07/03/91
-------
and fused ribs, absence of tall bones, abnormal or Incomplete skull bones
and Incomplete or missing leg bones. The data Indicate that Incomplete
skull bones may be an Induced developmental defect In which delayed ossifi-
cation 1s secondary to growth and development retardation of the fetus. In
another study, Singh et al. (1975) Injected rats 1.p. on day 5 or 10 of
gestation with either 5 ma/kg (5.6/kg) radlolabeled OEHP or 1.0116 ml/kg
(1.13 g/kg) radlolabeled DEP. Results of this study demonstrated that DEP
could pass through the placental barrier to the developing fetus. The data
Indicate that developmental toxlclty of the PAEs could be the result of the
direct effect of the compound (or Its metabolites) upon developing embryonic
tissue.
Dlethyl phthalate was evaluated to determine the reproductive and
fertility effects after oral administration (NTP. 1984c). CO-1 mice (11
weeks of age) were fed diets containing 0, 0.25, 1.25 and 2.5% DEP for 7
days prior to pairing and for 98 days to breeding pairs and then for an
additional 21 days. Exposure to DEP did not affect the reproductive
performance In mice. The number of litters produced, average litter size,
proportion of male pups and live pups and mean pup weight were unaffected.
The numbers of live pups/litter, however, did significantly Increase at
0.25X DEP.
Since reproductive performance was not adversely affected, a crossover
mating trial was performed (NTP, 1984c). The crossover mating trial
consisted of controls and Utters from high-dose (2.5%) FQ mice. Mice
were weaned at 21 days of age and maintained on the same treatment regime as
their high-dose FQ parents. Mice continuously exposed to DEP via their
04750 V-74 07/03/91
-------
mothers experienced lower body weight when compared with controls. There
were no statistically significant differences In fertility, proportions of
pups born alive, number of live males or females/Utter, or live pup weight
or sex of pups born alive. On the average, the number of litters were
significantly decreased In both treated males x control females and treated
females x control males. Sperm assessment of treated F. parental mice
Indicated no significant differences In the percentage of motile or abnormal
sperm. Sperm concentration, however, did significantly diminish. In the
high-dose males, right, testls weight significantly decreased and prostate
weight significantly Increased when compared with controls. High dose F.
females exhibited decreased pituitary weights.
OMP. Plasterer et al. (1985) studied the developmental toxlclty of
DMP In pregnant CD-I mice. Mice {50/treatment group) were administered 3500
mg/kg OMP by gavage for 8 consecutive days starting on day 7 of gestation.
There was no effect on maternal weight gain, Utter size or average pup
weight. The pups were not examined for malformations. The authors did
state, however, that the dose level may have been below the threshold of
reproductive effects.
In a teratogenlclty study Singh et al. (1972) observed adverse effects
on developing rat embryos and/or fetuses after DMP administration. Female
Sprague-Oawley rats (200-250 g) were Injected 1.p. with 1/10, 1/5 and 1/3
the acute LD5(J of 3.3751 ml/kg (4.01 g/kg) DMP on days 5, 10 and 15 of
gestation. The dosages corresponding to 0.338, 0.675 and 1.125 mi/kg
(0.40, 0.80 and 1.33 g/kg) OMP, respectively, were administered to groups of
five female rats. Animals were sacrificed on day 20 of gestation. No
04750 V-75 07/03/91
-------
resorptlons, dead fetuses or skeletal abnormalities occurred In the
untreated control group. The 0.675 ml/kg DMP treatment level did not
produce any resorptlon sites. However, the 0.338 and 1.125 mi/kg DMP
treatment group produced 33.3 and 32.1% resorptlons. The 0.675 ma/kg
level did show fetal death and the 1.125 mi/kg level showed five fetal
deaths. Gross abnormalities of 9.5, 7.5 and 11.1% were observed at 0.338,
0.675 and 1.125 ml/kg DMP levels, respectively. Fetuses were signifi-
cantly (p<0.01) smaller than the untreated controls at all treatment levels.
The number of skeletal malformations were 25.0, 35.3 and 75.0% for the
treatment levels 0.338, 0.675 and 1.125 ml/kg DMP., respectively. The
skeletal malformations most commonly encountered were elongated and fused
ribs, absence of tall bones, abnormal or Incomplete skull bones and Incom-
plete or missing leg bones. The authors concluded that Incomplete skull
bones may be an Induced developmental defect 1n which delayed ossification
Is secondary to growth and development retardation of the fetus.
Mutagenlclty
Thomas and Thomas (1984) and Hopkins (1983) reviewed the mutagenlcHy
and genotoxlclty of DEHP, Us metabolites and other phthalU add esters.
DEHP and Its metabolites, monoethylhexyl phthalate (MEHP) and 2-ethyl-
hexanol, have been tested extensively In Ames assays with Salmonella typhl-
murlum with and without metabolic activation. Negative results have been
reported by Zelger et al. (1982), Klrby et al. (1983), Kozumbo et al.
(1982), Ruddlck et al. (1981), Simmon et al., (1977), Warren et al. (1982),
and Yoshlkawa et al. (1983). DEHP was also found not to cause reverse
mutation In Escherlchla coll with and without S9 (TomHa et al.. 1982b;
Yoshlkawa et al., 1983). Kozumbo et al. (1982) and Rubin et al. (1979)
04750 V-76 07/03/91
-------
reported that OMP and DEP were mutagenic in strain TA100 of S. typhlmurium
but only in the absence of S9. Seed (1982) reported that DMP, DEP (with and
without S9) and DBP (without, but not with, S9), but not DEHP. dl-n-octyl,
dlisodecyl and diisobutyl phthalates, were found to cause mutation to
8-azaguanine resistance in bacterial suspension assays with S. tvohimurium;
the DEHP metabolite, 2-ethylhexanol, was found to be mutagenic without S9.
Tomita et al. (1982b) reported that HEHP, but not DEHP, yielded positive
results In rec assays with Bacillus subtlHs.
DEHP. The work of Tomita et al. (1982b) Indicate that OEHP, while not
a direct-acting mutagen, can be metabolized to a mutagenic form, MEHP.
HEHP, but not DEHP, was shown to be a direct-acting DNA-damaglng agent in
the Bacillus subtllis rec assay. In the Salmonella reverse mutation assay
MEHP was a direct-acting mutagen for strain TA100 whereas DEHP required
addition of S9 to produce this effect. When administered Ui vitro MEHP was
mutagenic at the hypoxanthine guanine phosphorIbosyl transferase (HGPRT)
locus 1n V79 cells and produced both chromosomal aberrations and sister
chromatld exchanges in these cells, which have little capacity for metabo-
lism of xenobiotlc compounds. DEHP or MEHP was also administered to
pregnant Syrian hamsters on day 11 of gestation, and the transplacentally
exposed fetal cells were cultured. Both DEHP and MEHP Induced mutations at
the HGPRT locus, chromosomal aberrations and morphologic transformation In
the cultured cells.
With two exceptions, in vitro genotoxldty assays have yielded negative
results. DEHP failed to cause an Increase In chromosomal aberrations In
human lymphocytes (Turner et al., 1974), In Chinese hamster flbroblasts (Abe
0475Q V-77 07/03/91
-------
and Sasaki. 1977; IsMdate and Odashlma, 1977), and In CHO cells {Phillips
et al., 1982). DEHP did not cause aneuploldy In human fetal lung cells
(Stenchever et al., 1976). DEHP and Us metabolites (MEHP and 2-ethyl-
hexanol) failed to Induce unscheduled DNA synthesis In primary rat hepato-
cytes (Hodgson et al.. 1982). MEHP was reported to cause an Increase In
chromosomal aberrations and SCE In Chinese hamster V79 embryonic cells
(TomHa et al.. 1982b) and CHO cells (Phillips et al.t 1982).
Chromosomal aberrations were observed 1n embryonic cells 1n a study 1n
which Syrian golden hamsters were treated orally with1 3.75-15 g/kg DEHP on
day 11 of gestation (TomHa et al.. 19B2b). Putman et al. (1983) failed to
observe significant Increases In clastogenlc changes 1n bone marrow cells
taken from male F344 rats treated by gavage with DEHP (0.5-5 g/kg/day) or
MEHP (0.01-0.14 g/kg/day) for 5 days. Positive results were observed In a
dominant/lethal study on ICR mice, where DEHP was administered as a single
Intraperltoneal dose (2/3 LD5_) (Singh et al., 1974).
Agarwal et al. (1985b) evaluated the antlfertllUy and mutagenlc effects
of DEHP In ICR mice. Eight male mice per group were given DEHP by s.c.
Injection at doses of 0.99, 1.97, 4.93 and 9.86 g/kg on days 1, 5 and 10 of
the experiment. Sixteen control animals were given saline by s.c.
Injection. On day 21. each male was housed with a female for 7 days.
Mutagenlclty was evaluated utilizing two Indices: prelmplantatlon
loss/Implants per pregnancy and early fetal deaths/Implants per pregnancy.
The prelmplantatlon loss mutagenlcHy Index was significantly Increased
during the early study segment In the 0.99, 1.97 and 9.86 g/kg groups and
04750 V-78 07/03/91
-------
for the overall study (weeks 1-8) In the 1.97, 4.93 and 9.86 g/kg dose
groups. The early death Index was significantly Increased for all doses at
all study segments.
In experiments with F344 rats, Albro et al. (1982) showed that radio-
labeled OEHP and ME HP (but not ethylhexanol) associated strongly with DMA.
Covalent binding, however, was not demonstrated.
DEHP was one of 10 chemicals recently examined In an International
collaborative study employing a wide range of short-term assays (Ashby et
al., 1985). A total of 69 assays were conducted on DEHP. some of which were
Identical tests performed In different laboratories. Negative results were
consistently (though not universally) observed In assays measuring gene
mutations and structural chromosome aberrations. Negative results were also
reported for unscheduled DNA synthesis and DNA single-strand breakage.
Positive results were observed In four of five cellular transformation
assays and In four of six mltotlc aneuploldy assays. The mechanism of cell
transformation Is not clear, but positive results for this endpoint with
agents that are not mutagenlc Is not without precedent. Aneuploldy Is
believed to be due to desegregation of chromosomes during mitosis, probably
as a result of damage to the spindle fiber proteins. Hence, positive
results for these endpolnts are not Inconsistent with the conclusion that
OEHP Is not mutagenlc.
BBP. BBP was negative In Salmonella typhlmurlum when tested with S9
(Rubin et al., 1979; Kozumbo et al., 1982; Zelger et al., 1982). For more
Information, see Table V-10.
04750 V-79 07/03/91
-------
1ABLE V-10
Summary of Genotoxlctty Tests of Phthalatlc Acid Esters
i
00
Compound Organlsn (assay)
DEHP Bacillus subtllls
(rec assay)
DEHP Salmonella typhlaurlun
(reverse nutation, his)
DEHP Salmonella typhtmurtum
(forward nutation. BAG*)
DEHP Escherlchla coll
(reverse nutation, his)
E xogenous
Activation
System*
PROKARVOTES -
none
nouse
pancreas S9
PROKARVOTES
rat liver S9
(Aroclor)
rat liver S9
(Aroclor)
rat liver S9
(Aroclor)
rat liver S9
(Aroclor)
rat liver S9
(unspecified)
rat liver S9
(Kanaclor 400)
rat liver
(unspecified)
rat liver S9
(Kanaclor 400)
Reported
Results
DMA DAMAGE
negative
pos 1 t 1 ve
- NUTATION
negative
negative
negative
negative
positive
negative
negative
negative
Comments
Tested at SOO yg/dtsk
At 1000 ng/plate with and
without S9 In strains TA98
and TA100
With and without S9 In strains
1A153S. TA1537. TA1538. TA9B
and TA100. Dosage unspecified
In reference
Tested up to 1000 ug/plate
with and without S9 In strains
TA9B and TA100
Tested up to 10.000 ug/plate
In strains TA98. TA100. TA1535
and TA1S37. prelncubatton assay
Tested at S og/plate In
strain TA100
Tested with and without S9 In
strains TA98 and TA100 at 0.1
m/test tube
Tested with and without S9
dose not specified
Tested with and without S9
In strains UP2 uvr Af and
UP? uvr A- at 0.1 nt/test
tube
Reference
T omit a et al..
198?b
Rubin et al..
1979
Simmon et al..
1977
Kozumbo et al..
1982
Zelger et al..
1982
Tomlta et al..
19B2b
Voshlkawa et
al.. 1983
Seed. 1982
Voshlkawa et
al.. 1983
CO
03
-------
TABLE V-10 (conl.)
en
o
00
00
CD
Conpound
BBP
BBP
OBP
DBP
OEHP
0(P
DBP
Organtsa (assay)
Salmonella typhtnurlum
(reverse autatlon. Ms)
Salmonella typhlmuctun
(reverse mutation, his)
Salmonella typhlmurlum
(forward nutatton. BAG**)
Salmonella lyphlnurtua
(reverse nutation, his)
Salmonella lyphlmurlum
(reverse autatlon his)
Salmonella typhlmurlum
(reverse mutation his)
Salmonella tyjihtmurlum
(reverse mutation his)
E xogenous
Activation
System*
PIOKARVOTES
rat liver S9
(Aroclor)
rat liver S9
| Aroclor )
ral and haasler
liver S9
| Aroclor)
ral liver S9
rat liver S9
(Aroclor)
rat liver S9
(Aroclor)
rat and hamster
liver S9
(Aroclor)
rat liver S9
rat liver S9
rat liver S9
rat liver S9
Reported
Results
- NUTATION (cont.)
negative
negative
negative
positive,
weakly
autagenlc
negative
negative
negative
negative
positive
negative
weakly positive
Contents
At 1000 ng/plate with and
without activation In strains
TA9B and TA100
Tested up to 1000 ng/plate
with and without S9 In strains
TA98 and TA100
Tested up to 11.550 vg/plata
In strains TA98. TA100. TA1535
and IAT537 In prelncubatlon
assay
Nulagenlclty observed at
0.09-0.18 iff. Tested with
and without S9
At 1000 vg/plate with and
without activation In strains
TA98 and TA100
Tested up to 1000 yg/pldte
with and without S9 In strains
TA90 and TA100
Tested up to 10.000 wg/plate
In strains 1A98. 1A100. TA1S3S
and 1A1537 In prelncuballon
assay
Tested with and without S9 In
strains TA98 or TA100 at
100. 200. 500. 7SO. 1000 and
2000 pg/pljte
Tested without S9 In strains
TA100 and TA1S35 at 100. POO.
500. 150. 1000 and 2000 ,,g/
plate
Tested with and without S9 In
strain 1A9B at 100. 200. 500.
750. 1000 and ?000 ug/plate
Tested without S9 In strains
TA100 at ?00. 500. 750. 1000
Reference
Rubin et a).,
1979
Koiuabo et al.,
198?
Zelger el al..
1982
Seed. 1982
Rubin el al..
1979
Kor.-' •> el al..
1982
felger el al..
1982
Agarwal el al..
I985c
Agarwal el al.,
1985c
Agarwal et al. .
1985c
Ayarwal el al..
I985c
and ?000
-------
TABLE V-10 (cent.)
CD
o
vO
00
oo
E Kogenous
Compound Organism (assay) Activation
System*
PROKAR VOTES
OMP Salmonella typhlnurlum rat liver S9
(reverse nutation his)
DEP Salmonella typhtmurtum rat liver S9
(reverse nutation, his)
none
none
rat liver S9
(Aroclor)
host -media ted
assay
rat liver S9
(Aroclor)
DEP Salmonella typhlmurlum rat liver S9
(forward nutation. 8AGR) (unspecified)
DNP Salmonella typhlmurluin rat liver S9
(reverse mutation, his) (Aroclor)
none
rat liver S9
(Aroclor)
none
Reported
Results
- NUTATION (cont.)
weakly positive
negative
positive and
negative
positive and
negative
negative
negative
negative
positive.
weakly
mulagenlc
negative
positive and
negative
negative
positive and
negative
Comments
Tested without S9 In strain
TA1535 and TA100 at 100. 200.
500. 750. 1000 and 2000 pg/
plate
At 1000 pg/plate In strains
TA98 and TA100
At 1000 pg/plate without
activation In strain TA100;
negative for TA9B
At 500 and 1000 pg/plate
In strain TA100; negative
In TA98
At 1000 pg/plate In
strain TA100
Urine tested In strain TA100
Tested up to 10.000 pg/plate
In strains TA98. TA100, TA1515
and TA1537 In prelncubatlon
assay
Nutagenlctty observed at
2.2-3.3 nfl. Tested with and
without S9
Tested at 1000 pg/plate
In strains TA98 and TA100
At 1000 pg/plate In strain
TA100; negative for TA9B
Tested up to 6666 pg/plate
In strains TA98. 1AIOO. 1A1535
and 1A1537. prelncubatlon
assay
At 500 and 1000 pg/plate In
strain 1AIOO; negative for TA98
Reference
Agarwal et al..
19B5c
Rubin et al..
1979
Rubin et al.,
1979
Kozumbo et al.,
1982
Kozumbo et al..
1982
Kozumbo et al..
1982
Zelger et al..
1982
Seed. 1982
Rubin et al..
1979
Rubin et al..
1979
Zelger et al..
1902
Kozumbo et al.,
1982
-------
1ABLE V-10 (conl.)
t/1
o
09
CJ
rsj
CO
CO
Compound
ONP
DBP
OEHP
Organism (assay)
Salmonella tvphlmurlum
(reverse mutation, his)
Salmonella typhlmurlum
(forward mutation. 8AGB)
Saccharoroyces cerevlslae
(reverse mutation.
multiple loci)
Syrian hamster embryo cells
(forward mutation. BAG".
61G")
E xogenous
Activation
System*
PROKARVOTES
rat liver S9
(Aroclor)
host -mediated
assay
rat liver S9
(unspecified)
FUNGI
mouse S9
(unspecified)
NANNAL1AN
none
Reported
Results
- NUTATION (cont.)
negative
negative
positive.
weakly
mutagenlc
- NUTATION
negative
CELLS - NUTATION
positive
Comments
At 1000 ng/p1ate with S9
In strain TA100
Urine tested In strain TA100
Nutagentclty observed at 5-10
mN. Tested with and without
S9
Tested up to 100 pg/mt
3.75-1.5 g/kg administered
transplacentally. Dosage
resulting In mutations
unspecified
Reference
Koiumbo et al.,
198?
Korumbo et al.,
198?
Seed, 1982
Shah In and von
Borstel. 1977
Tomlta et al..
1982b
CHRONOSONE EFFECTS
OEHP
Chinese hamster Mbroblast
(CHL) cells (chromosomal
aberrations)
Chinese hamster ovary (CHO)
cells (chromosomal aberra-
tions)
Syrian hamster embryo cells
(chromosomal aberrations)
Human leukocytes
(chromosomal aberrations)
Human leukocytes
(chromosomal aberrations)
none
none
none
none
none
negative
negative
positive
negative
negative
Tested at 4.1 ng/mt
Tested up to 2.0 mN
At 7500 and 15.000 mg/kg
applied transpacenlally
lested up to 60 |ig/mt
lested at 6.0 ug/ml
Ishtdate and
Odashlma. 1977
Phillips et al..
1982
Torolta et al..
19B?b
Stenc he ver
el al.. 19/6
Stenc hover
el al.. 1976
-------
TABLE V-10 (cont.)
Compound
OBP
OEP
DMP
DEHP
DEHP
Exogenous Reported
Organism (assay) Activation Results
System*
Human leukocytes none
(chromosomal aberrations)
Chinese hamster flbroblast none
(CHL) (chromosomal aberrations)
Human leukocytes none
(chromosomal aberrations)
Chinese hamster flbroblast none
cells (chromosomal
aberrations)
Human leukocytes none
(chromosomal aberrations)
Human leukocytes none
(chromosomal aberrations)
Syrian hamster embryo cells none
(morphological transformation)
Nice - Harlan ICR strain
(Incidence of pregnancies.
CHROMOSOME EFFECTS (cont.)
negative
weakly
positive
negative
negative
negative
negative
CELL TRANSFORMATION
positive
MAMMALIAN ]N VIVO TESTS
positive
Comments
Tested at 0.25 ng/ml
Tested at 1.1 mg/mt
Tested at 0.25 Mg/at
Tested at 11.3 mg/mt
Tested at 0.25 pg/mt
Tested at 0.25 ng/mt
At 7500 and 15.000 mg/kg.
transplacental application
At 12.8. 19.2 and 25.6 ml /kg
bw. l.p. reduced Incidence of
Reference
Tsuchlya and
Hat tori. 1977
Ishldate and
Odashlma. 1977
Tsuchlya and
Hattorl. 1977
Ishldale and
Odashlma. 1977
Tsuchlya and
Hattorl. 1977
Tsuchlya and
Hattorl. 1977
T omit a et al..
1982b
Singh et al..
1974
Implantations per pregnancy.
early fetal death)
Mouse (dominant lethal
mutations)
positive
pregnancies. Increase In fetal
deaths, reduced number of Implants
Male mice Injected s.c. with Autlan. 1982
doses ranging from 1.0-100
mi/kg bw. Ant I fertility
effect at 1 rot/kg and Increased
with dose. Mutagenlc Index
Increased with dose.
oo
00
-------
TABLE V-tO (conl.)
VI
O
Compound Organism (assay)
Exogenous Reported
Activation Results Comments
System'
Reference
OEHP
DMP
House (dominant lethal
nutations|
MAMMALIAN IN VIVO 1ES1S (cont.)
positive
House (dominant lethal
nutations)
Rat (nutations In
regenerating hepatocyles)
negative
positive
Hale alee Injected s.c. with
0.99. 1.97. 4.93 and 9.86
g/kg. Statistically signifi-
cant Increase In nutagenlclty
Index Interns of prelnplanta-
ttiin losses, early fetal deaths
and all speroatogenlc stages
Treated I.p. with 1250 t,g/kg
bu and at 1?50 ng/kg bw
applied to skin
Unspecified dose applied
to skin
Agarwal et al..
1985b
Yurchenko and
Glelberrun. 1980
Yurchenko. 1977
*S9 refers to the postaltochondrlal fraction (supernatant from 9000 xg centrifugal Ion) of honwgenates of organs.
used for S9 preparations Is In parentheses.
Inducing treatment of animals
oo
m
-------
DBP. Kozumbo et al. {1982) found the ortho dlester, OBP, to produce a
dose-related mutagenlc response 1n a modified version of the reverse muta-
tion plate Incorporation assay 1n Salmonella (Ames test). This activity,
which was observed only 1n strain TA100, a detector of both base pair and
frameshlft mutagens, was eliminated upon addition of S9. In addition. DBP
showed some evidence of clastogenlc activity In Chinese hamster flbroblasts
(Ishldate and Odashlma, 1977) (see Table V-10).
PEP. DEP Is a direct-acting mutagen for Salmonella typhlmurlum (Rubin
et al., 1979). Seed (1982) found DEP weakly mutagenlc In a forward mutation
assay In Salmonella typhlmurlum (see Table V-10).
PHP. Extracted urine of rats administered 2 g/kg DMP l.p. was found
not to be mutagenlc (Kozumbo et al., 1982). In. vitro assays by these
authors showed that S9-assoc1ated esterases hydrolyzed DMP to the monoester,
which has not been shown to be mutagenlc In the Ames assay, and to methanol
thereby eliminating Us mutagenlc capacity. An abstract by Yurchenko and
Glelberman (1980) Indicates that PHP Is not positive In a mouse dominant
lethal test. Rubin et al. (1979) and Kozumbo et al. (1982) found DMP to be
a direct-acting mutagen for Salmonella typhlmurlum. In a forward mutation
assay DMP was weakly mutagenlc In Salmonella typhlmurlum conducted In liquid
suspension (Seed, 1982). For more Information see Table V-10.
CarclnogenlcUy
The most conclusive Information on the carclnogenldty of PAEs was
obtained from bloassays performed by the NTP. Hllbourn and Hontesano (1982)
reviewed the results from carclnogenldty testing of PAEs conducted prior to
04750 V-86 07/03/91
-------
the NTP bloassays and concluded that all the studies were limited with
respect to study design or reporting, making the results Inconclusive.
DEHP. Cardnogenlclty studies have been conducted by the NTP for
several PAEs Including BBP and DEHP (Kluwe et al.. 1982a.b; Huff and Kluwe.
1984). The tested PAEs discussed In this document are DEHP and BBP.
Essentially the same protocols were used for each compound. The compounds
(administered In the diet) were tested for 2 years In both Fischer 344 rats
and B6C3F1 mice using an untreated control group, a low-dose group and a
high-dose group. The high-dose used In testing was the estimated maximum
tolerated dose (HTD) determined by preceedlng 90-day subchronlc feeding
studies prior to the chronic exposure bloassay. The low-dose was one-half
of the estimated HTD. For each dose group, 50 animals of each sex and
species were tested. Animals that died during the study and animals
sacrificed at the end of the study were subjected to a gross necropsy and a
complete mlcropathologlc examination. Statistical comparisons of Incidences
of animals with systemic pathology, especially tumors at specific anatomical
sites and of survival and body weight gain, were made using both palrwlse
comparisons (Fisher's exact test) and trend tests (Cochran ArmHage trend
test).
DEHP was administered In the diet for 103 weeks at levels of 0, 6000 and
12.000 ppm for male and female F344 rats and 0, 3000 and 6000 ppm for male
and female B6C3F1 mice (NTP, 1982a). In this study procedure 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
04750 V-87 07/03/91
-------
rat body weight was used In 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). No clinical signs of toxlclty were observed
In either rats or mice. Survival was not significantly decreased In any
group except Ihe female mice receiving the low-dose level. The Increased
number of deaths In this group were not attributed to DEHP administration,
since pathologic changes In tissues were not observed and excessive deaths
did not occur at the higher dose. The nonneoplastlc lesions observed In
this study were discussed previously under chronic toxlclty.
Specific Incidences of neoplastlc lesions for the various treatment
groups are presented In Table V-ll. The major neoplastlc effect observed
among animals treated with DEHP was development of liver tumors. The Inci-
dence of animals with hepatocellular carcinomas was significantly Increased
among female rats fed DEHP at either 300 or 600 mg/kg bw when compared with
controls. Hale rats experienced a significant Increase of the incidence of
hepatocellular carcinomas and neoplastlc nodules only In the 600 mg/kg bw
DEHP group. Significant dose-related trends for Increased numbers of
animals bearing hepatocellular carcinomas and for Increased numbers of
animals bearing either hepatocellular carcinomas or neoplastlc nodules were
found In both male and female groups. Among male rats, Incidences of
animals with pituitary tumors, thyroid C-cell tumors or testlcular Inter-
stitial-cell tumors were all significantly reduced among treated groups by
both palrwlse comparison and trend tests (see Table V-ll). Comparison of
occurrence of non-neoplastlc effects In rats with occurrence of neoplastlc
effects showed that the presence of testlcular Interstitial cell tumors was
correlated (p<0.005) with either the presence of pituitary
04750 V-88 07/03/91
-------
en
o
TABLE V-11
Incidences of Animals with Neoplastlc Lesions In the NTP Carctnogentctty Btoassay of DEHP3
i
oo
CD
CO
•s.
to
Species
Rat
Rat
House
House
Sex Neoplastlc Lesions
H Liver, hepatocellular carcinoma0
Liver, neoplastlc nodule
Liver, hepatocellular carcinoma or neoplastlc noduled
Pituitary carcinoma'
Pituitary carcinoma or adenoma'
Thyroid. C-cell carcinoma'
Thyroid. C-cell carcinoma or adenoma'
Testls, Interstitial cell tumor"
f Liver, hepatocellular carcinoma1*
Liver, neoplastlc nodule0
Liver, hepatocellular carcinoma or neoplastlc nodule11
H Liver, hepatocellular carcinoma0
Liver, hepatocellular adenoma
Liver, hepatocellular carcinoma or adenoma"
F Liver, hepatocellular carcinoma11
Liver, hepatocellular adenoma
Liver, hepatocellular carcinoma or adenomad
Control
I/SO (2X)
2/50 (4X)
3/50 (6X)
4/46 (9X)
8/46 (17X)
4/48 (BX)
5/48 (10X)
47/49 (96X)
0/50 (OX)
0/50 (OX)
0/50 (OX)
9/50 (18X)
6/50 (12X)
14/50 (28X)
0/50 (OX)
1/50 (2X)
I/SO (2X)
Incidence1*
Low-Dose
1/49 (2X)
5/49 (10X)
6/49 (12X)
1/43 (2X)
6/43 (I4X)
1/47 (2X)
2/47 (4X)
42/44 (95X)
2/49 (4X)
4/49 (BX)
6/49 (12X)e
14/48 (29X)
11/48 (23X)
25/48 (52X)e
7/50 (14X)e
5/50 (10X)
12/50 (24X)e
High-Dose
5/49 (10X)
7/49 (14X)
12/49 (24X)e
0/49 (OX)
1/49 (2X)9
0/46 (OX)
0/46 (OX)5
11/48 (23X)1
8/50 (16X)J
5/50 (IOX)1
13/50 (26X)J
19/50 (38X)e
10/50 (20X)
29/50 (58X))
17/50 (34X)J
1/50 (2X)
18/50 (36X)1
'Source: Kluwe et al.. 1982a.b; Huff and Kluwe, 1984. Fischer 344 rats and B6C3F1 mice were fed diets containing O(control). 3000 (lower
dose, mice), 6000 (higher dose, mice; lower dose, rats) or 12.000 ppre (higher dose, rats) of OEHP for -2 years.
DInc1dences are expressed as the number of animals exhibiting the lesion over the number of animals examined microscopically; percentages
are given In parentheses.
(S1gn1f1cant dose-related Increased trend (p<0.05)
Significant dose-related Increased trend (p<0.01)
eSlgn1flcantly greater than controls (p<0.05)
'Significant dose-related decreased trend (p<0.05)
QSIgntflcantly less than controls (p<0.05)
"Significant dose-related decreased trend (p<0.0001)
'Significantly less than controls (p<0.0005)
If leant ly greater than controls (p<0.01)
-------
hypertrophy or the presence of testlcular degeneration. In addition to the
tumors presented In Table V-ll, myelomonocytlc leukemia, mammary flbro-
adenoma, clltoral gland carcinoma and uterine endometrlal stromal polyps
were observed In one or more rats, but their Incidences In treated animals
did not differ significantly from those found In controls.
The number of mice bearing hepatocellular carcinomas was significantly
Increased In both male and female groups receiving the high dose (780 mg/kg
bw) of DEHP and In female mice receiving the low dose (390 mg/kg bw) (see
Table V-11J. Trend tests showed significant dose-related effects for both
sexes. Metastases of the hepatocelTular tumors to the lungs were found In
12 male and 8 female DEHP treated mice bearing hepatocellular carcinomas.
Pulmonary metastases were not found In 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 In both sexes, and significant dose-related trends were present.
Lymphomas, hemanglomas, mammary gland adenocarclnomas and alveolar or
bronchlolar carcinomas or adenomas were also found 1n one or more treated
mice, but Incidences did not differ significantly from those observed In
controls. In conclusion, the DEHP feeding studies In rats and mice Indicate
that statistically significant Increases In hepatocellular carcinomas,
neoplastlc nodules and adenomas occurred. These tumors were found In both
species and both sexes. There were metastases of hepatocellular tumors to
the lungs of treated mice.
04750 V-90 07/03/91
-------
A summary of the Interpretation of the bloassay results by NTP Is shown
1n Table V-12. The only compound for which there was clear evidence of car-
clnogenlcUy 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 (MTD) had
been exceeded (based on differences 1n body weight gain) In both rats and
mVce 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 In 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 OEHP. The authors postulated that the effects of
DEHP were attributable to eplgenetlc mechanisms of cardnogenldty 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 Is metabolized
differently In rats than In humans, effects In these rodents cannot be
extrapolated to Indicate human risk.
04750 V-91 07/03/91
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TABLE V-12
Summary of the Carcinogenic Effects of DEHP on the NTP Bloassays
and Interpretation of These Findings*
Test Species Sex Neoplasms Interpretat1onb
Chemical
DEHP rats M Liver neoplastlc Some evidence
nodules/carcinomas
rats
mice
F
M&F
Liver carcinomas
Liver carcinomas
Clear evidence
Clear evidence
aSource: Huff and Kluwe, 1984
bEv1dence of Cardnogenldty-- Five categories of Interpretative conclu-
sions have been adopted for use In the NTP Technical Reports series to
specifically emphasize consistency and the concept of actual evidence of
carclnogenlclty. 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 either potency or mechanism (Huff and Kluwe, 1984).
04750 V-92 07/03/91
-------
On the other hand, Kluwe et al. (1983) defended the conclusions reached
In the NTP study on DEHP (Kluwe et al., 1982a) by noting that the MTD was
estimated based on prechronlc oral studies, and that the HTD was not tech-
nically exceeded since survival of animals was not adversely affected. In
response to the other criticisms, It was noted that the liver 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 (IARC, 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 In the NTP bloassay, Is Involved In a secondary
mechanism of cancer Induction (Reddy et al., 1986). Peroxlsomal prolifera-
tion Is discussed In detail In Chapter VII, Mechanisms of Toxldty.
Similar results were reported In cynomolgus monkeys (Short et al.,
1987). 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 V-93 07/03/91
-------
Ward et al. (1983) studied the patterns of promotion of hepatocellular
neoplasla by DEHP and phenobarbltol (PB) following Initiation by l.p.
dlethylnUrosamlne (DLN). 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 1n drinking water at 500 ppm. few foci of hyperplasla
were found In the liver at 2, 4 or 6 months 1n animals exposed only to DEN,
PB or DEHP, while numerous foci and hepatocellular neoplasms were found In
mice treated 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 of foci did not Increase between 4 and 6 months as they did for PB,
but the foci did Increase In mean diameter and volume as the study pro-
gressed. Foci and tumors appeared earlier In the higher dose group of DEHP
and, although the number of foci per unit volume of liver was similar for
all DEHP dose groups, the volumes of the fod were dose-related. The type
of hepatocytes found In the foci and neoplasms differed for PB and DFHP;
those for PB were predominantly eoslnophlUc hepatocytes while those In
DEHP-treated mice were predominantly basophlllc and were more malignant 1n
appearance. After 6 months exposure, the neoplasms In the high-dose DEHP
and DEN mice were significantly larger (p<0.02) than those for PB and DEN,
although hlstochemlstry revealed similarities In the lesions. DEHP did not
exhibit Initiating 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 F344/NCr rat livers. Rats were Initially Injected with 282 mg/kg DEN
04750 V-94 07/03/91
-------
and then fed diets containing 12,000 ppm DEKP 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 OEN-DEHP treated rats. Based on the above studies (Ward et al.. 1983,
1986) the Investigators suggest that liver cell replication Is 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 PB 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
promoting activity (Williams et al., 1987).
BBP. 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 1560
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 V-95 07/03/91
-------
evaluation of the animals for tumoMgenic responses. Incidences of tumors
at specific anatomical sites In BBP-treated male or female mice did not
differ significantly 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 rfwas probably carcinogenic In F344 female rats". A
summary of the Interpretive conclusions drawn from the NTP carclnogenesls
testing of BBP 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 significantly reduced total bone marrow cell counts at the 0.03 and
2.5X dose groups, but not at the 0.09, 0.28 or 0.83X dose groups when
compared with controls. This change was comprised primarily of decreases In
neutrophll metamyelocytes, bands, segmenters, lymphocytes, and basophlllc
rubMcytes.
The NTP Is currently repeating the rat portion of the cacner bloassay
for BBP. Testing began In June, 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 carclnogenlsls bloassays, Kluwe (1986)
compared the carcinogenic effects of D£HP and BBP and related compounds to
determine the structure-activity relationships. Among the PAEs shown to be
04750 V-96 08/08/91
-------
TABLE V-13
Incidences of female Rats with Tumors of the Hematopoletlc System
1n the NTP Carclnogenldty Bloasssay of BBPa'D
Incidence
HematopoletU System Tumor
Control
Low-Dose
Hlgh-Oose
Myelomonocytlc leukemia 7/49
Lymphoma 0/49
Myelomonocytlc leukemia or lymphoma 7/49
7/49
0/49
7/49
18/50C
1/50
19/50C
aSource: Kluwe at al., 1982b; NTP, 1982b
bFemale 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.
cS1gnUIcantly greater than controls, p<0.05
04750
V-97
07/03/91
-------
TABLE V-14
Summary of the Carcinogenic Effects of BBP 1n the NTP Bloassays
and Interpretation of These Findings*
, Species Sex Neoplasms Interpretation11
Chemical
rats M — Inadequate study
rats F Leukemia Some evidence
mice H&F — NO evidence
aSource: Huff and Kluwe, 1984
bEv1dence of Carclnogenlclty— Five categories of Interpretative conclu-
sions have been adopted for use In the NTP Technical Reports series to
specifically emphasize consistency and the concept of actual evidence of
carclnogenlclty. 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 either potency or mechanism (Huff and Kluwe, 1984).
04750 V_98 07/03/91
-------
potentially carcinogenic, the target sites of carcinogenic action varied.
For example, OEHP Induced hepatocellular carcinoma, while BBP was associated
with effects of the hematopoletlc system. It was concluded, therefore, that
the cardnogenlclty of PAEs may not be due to the acltlvHy of the phthalate
moiety but rather determined by the moiety attached to the phthalate, or to
a metabolic byproduct. Support for such an argument Is given by studies of
compounds containing the 2-ethylhexyl moiety. Comparison of results
obtained for DEHP, DEHA and two other compounds [d1(2-ethylhexyl)phosphate
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 hepatocarclnogenldty 1n female mice.
DBP. Data regarding the carclnogenldty of DBP could not be located
In the available literature.
PEP. Data regarding the cardnogenlclty of OEP could not be located
In the available literature.
PHP. Data regarding the cardnogenlclty of DMP could not be located
In the available literature.
Summary
The acute toxldty of PAEs tends to be Inversely related to the molecu-
lar weight of the compound. Signs of long-term toxldty 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 V-99 08/08/91
-------
The hepatotoxlc effects of PAEs have been studied by numerous Investi-
gators In a variety of species. Seth (1982) reviewed the hepatic effects of
PAEs and described both the morphologic and biochemical alterations
attributable to PAE exposure. Most Investigators have used DEHP as the
representative PAE 1n testing. Generally, enlargement of the liver has been
observed following oral or l.p. administration of PAEs. Examination of
tissue from enlarged mouse, rat, hamster and monkey livers has revealed
changes In morphology and biochemical constituents. Oral administration of
DEHP for 21 days was reported to cause dilation of smooth and rough endo-
plasmlc retlculum, mitochondria! swelling and Increase.In mlcrobodles In rat
liver (Lake et al., 1975). Oral administration of DEHP produced enlargement
of cells, dilation of smooth endoplasmlc retlculum and changes In hepatic
lysosomes of ferrets (Lake et al., 1977) and decreased glucose-6-phosphatase
In female rats (Mitchell et al., 1985). Information on the effects of
phthalates In primates Is limited to a study In which rhesus monkeys were
given plasma-solublUzed DEHP Intravenously (Jacobson et al., 1977). The
effects observed Included liver necrosis, Inflammatory cell Infiltration and
subtle changes 1n the clearance time of sulfobromophthaleln.
Reproductive and developmental effects have been reported for several
PAEs. Studies with DEHP have shown that this compound affects the fertility
and reproductive performance In both male and female mice. In males the
effects were associated with degenerative changes In the reproductive system
and adverse effects on sperm. Testlcular atrophy has been shown to occur
following exposure to DEHP, BBP and DBP. Long-term oral exposure to DEHP at
dietary levels as low as 150 mg/kg/day has Induced testlcular atrophy 1n
rats.
04750
V-100 08/08/91
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Testlcular 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 DEHP and DBF while the hamster appeared to be resistant to
the gonadal effects of these compounds and the corresponding monoesters at
the dose levels and durations tested (Gangolll, 1982).
Studies on the embryotoxlclty of PAEs seem consistent with other data
obtained using different toxlcologlc endpolnts (Tyl, 1988; NTP 1984a,b,
1985; Mitchell et al.. 1985; Dostal et al., 1987a). that 1s, there Is a
range of tox1c1t1es that varies as a function of the PAE being tested and.
In -general, high concentrations of this chemical are required to produce a
teratogenlc response. Host 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 Mshlmura, 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 Is
difficult at this time to define clearly the risk for the human population.
PAEs are generally regarded as nonmutagenU although mutagenlc responses
have been shown for some PAEs 1n some tests. DEHP 1s apparently metabolized
to a nongenotoxlc form In Intact animals but not by tissue preparations.
DEHP and BBP have been tested for carclnogenlclty In 2-year NCI/NTP car-
clnogenlclty bloassays. DEHP was found to Induce hepatocellular carcinomas
In 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 In the available literature.
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VI. HEALTH EFFECTS IN HUMANS
Introduction
Although PAEs are considered to have a low order of toxlclty, 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.t 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 have been limited by the Inability to discern doses and responses In
light of the ubiquity of phthalates 1n the environment. DEHP has been
detected In both transfused and nontransfused patients (WalUn 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 baby pants and vinyl fabrics covering playpen pads. A report by the
Consumer Products Safety Commission estimated possible Increased cancer risk
to children exposed to the above products (CPSC, 1983). The widespread
presence of PAEs In air, water, food and stored blood Indicates that humans
are subject to environmental and Industrial exposures to PAEs.
Clinical and Case Studies
DEHP. One of the earliest studies Involving an assessment of PAEs In
humans was performed by Shaffer et al. (1945). Two adult males were admin-
istered single oral doses of 5 or 10 g of DEHP In order to estimate Its con-
tent In urine. In each case 4.5% of the dose was recovered from the urine
In 24 hours. These experiments comprise the only controlled Ingestlon of
PAEs cited 1n the literature. The subject who received the larger dose
experienced mild gastric disturbances and moderate catharsis. No other
effects were reported at either dose.
04760 VI-1 08/15/88
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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 reapplled on the same spots after 10 days. These exposures did not
result 1n 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 human dlplold flbroblasts established from skin biopsies. DEHP was
solublllzed In sera collected and stored In polyvlnyl chloride (PVP 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 DEHP. cell growth was Inhibited by 20% and 50%, respectively.
These DEHP levels were comparable with concentrations detected In 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 In platelet concentrations stored at 22°C for
48 hours.
Chromosomal effects of DEHP (Stenchever etal., 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.
Allquots were diluted to final concentrations of 0.06, 0.6, 6.0 and 60.0
yg DEHP/mi of blood for the leukocyte Incubations. Incubations with
04760 VI-2 07/28/88
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DEHP were for 4 hours at 37°C. Phytohemaglutlnln was then added for 30
minutes to Initiate cell division and cells were cultured for 72 hours.
Mitosis was Inhibited by addition of democolclne 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 cultures. Fetal lung cells were Incubated with 6.0 yg DEHP (In
Polysorbate 80)/ma medium for 5 days. No significant difference In
aneuploldy between study and control cultures was seen.
Ishlkawa et al. (1983) determined that platelet function decreased as
DEHP concentrations Increased In PVC blood storage bags. Platelets demon-
strated a decrease 1n ADP-lnduced aggregation after at least 2 hours of
exposure to 100, 300 or 500 vq DEHP/ml. Maximum aggregation gradually
decreased with Incubation time, depending on the DEHP dose. Platelets
renewed with fresh plasma showed a restoration of aggregablllty. The degree
of restoration was decreased with Increasing DEHP dose.
The effects 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 compared with control cultures. As Indicated by figures these
decreases were dose dependent. Cells treated with 160 yM 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 (ID5Q), was cal-
culated to be 70 yH. These toxic effects were greater In replicating cell
04760 VI-3 08/15/88
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populations than In those treated after reaching confluency. Cells grown In
160 viH for 3 days and subsequently subcultured Into control medium showed
only 60% of control growth after 5 days In 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 sensHI-
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.
D8P. Atmospheric exposures to DBP were studied by Men'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 Ingestlon 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 VI-4 07/28/86
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white corpuscles In the urine). Unspecified treatment Initiated Immediately
allowed the subject to leave the hospital after 2 weeks without any after-
effects.
Ep1dem1olog1c Studies
Mllkov et al. (1973) performed a cross-sectional Investigation of
workers exposed 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 adlpate (DOA)]. Trlcresyl phosphate (TCP) was a component of the
Incombustible materials produced In 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 operators, 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) In 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 mill and calender
operators. The plastldzer level 1n the mixture preparation section was
found to be 1.7-40 mg/m3. Other contaminants (vinyl chloride, carbon
monoxide and hydrochloric add) around the calenders and rollers were either
below their maximum allowable concentrations or not detected.
The test procedures Included algeslmetry, olfactometry, audlometry,
vibration sensitivity and vestlbular function by the caloric method with
cold water (60 ms. 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 In 51.7% of the
subjects with a length of service 6-10 years and In 81.6% In those with >10
years. Polyneurltls was found 1n 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 nonoccuoa-
tlonal character was noted In the nervous system. An elevation In 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 1n 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 excit-
ability). This depression began with the first years of this occupational
contact, often 1n the absence of any health status complaints. The majority
of subjects showed an elevated threshold of excitability when tested by
04760 VI-6 07/28/88
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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 service. Audlometry did not reveal any pathology In auditory sensitiv-
ity. Blood 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.
Thless et al. (1978a) performed a morbidity study on 101 workers (97
males, 4 females) employed In a DEHP production plant. The age range of the
workers was 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 0.0006 and 0.01 ppm (detection limit not
stated). A clinical and occupational history was taken and the clinical
examination Included vital statistics, EKG, lung X-ray, and a complete
urinary status with uric acid and creatlnlne clearance. The blood analyses
Included a differential count and sedimentation rate, and thymol, total
protein, SGOT, SGPT, t-GT, LDH, alkaline phosphatase, cholesterol and
trlglycerldes determinations.
04760 VI-7 09/07/88
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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 epldemlologlc
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
compared with the general population. The study considered data prior to
1976. 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 In the exposed population. Eight cases of death were due to
04760 VI-8 08/15/88
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cancer. Thless 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
lymphocytes from a subset of this same study population. Lymphocytes were
cultured from 10 exposed production workers according -to a modified method
of Moorhead et al. (I960). 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 Upld soluble DEHP, from plastic
containers or catheter tubing (Marcel and Noel, 1970; Jaeger and Rubin,
1970. 1972, 1973).
04760 VI-9 09/07/88
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Hmman et al. (1975) studied DEHP levels In neonatal heart and GI tis-
sues. The study tissues were obtained from three Infants who previously had
umbilical 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 llveborn 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.+1.1 mg of DEHP/5 cm of catheter.
based upon the mean £ S.E. of the extraction of four'No. 5 French cath-
eters. The maximal amount contributed by blood products was estimated at 4
yg/ma, based upon a reference to Marcel (1973). The potential dosage
thus ranged from 0.04 mg 1n Infants receiving only 10 ma of blood to 1.4
mg 1n 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 DEHP 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
yg/g. respectively, were significantly higher than the corresponding
control levels, <0.07+0.03 and <0.07i0.04 yg/g. Three Infants who died of
necrotlzlng enterocolltls 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 VI-10 09/07/88
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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 OEHP and
the development of necrotlzlng enterocolltls. However, the study demon-
strated that DEHP accumulated In the tissues of critically 111 Infants.
Components of the catheters, Including DEHP, should be further Investigated
as potential vascular or 61 toxins, according to the authors of this
Investigation.
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 to quantify the levels of OEHP 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 Immediately after dialysis unless dialysis was terminated earlier. The
metabolic fate and toxlclty of these DEHP levels were not determined. Esti-
mates of the total amount of DEHP delivered to a patient during hemodlaly-
sls ranged 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
associated with the development of abnormal liver function tests In three
patients (two men, aged 25 and 40 years, and one 25-year-old woman), follow-
ing the use 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 10-20 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 In one case, jaundice. Liver biopsies In one patient
revealed changes 1n accordance with so-called nonspecific reactive
hepatitis; and In another patient, a hlstologlc picture compatible with a
diagnosis of viral hepatitis. Upon removal from the new dialysis machines
to dialysis systems In which DEHP 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
DEHP and Its desterlfled phthallc acid products, mono(a-ethylhexyl)
phthalate (MEHP) and phthallc add, were quantltated (HPLC/UV monitor) In 11
patients. These patients were undergoing maintenance hemodlalysls for
treatment of renal failure. The patients underwent hemodlalysls 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 VI-12 07/02/91
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Influence on the extraction of OEHP Into blood. Circulating levels of DEHP
and MEHP (1.91+2.11 w/mi and 1.33*0.58 pg/mi, respectively) during
dialysis did not correlate with the length of time the patients had been
undergoing dialysis. This, together with the observation that blood concen-
trations of OEHP during Interdlalysls were similar to those In 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 Upophlllc phthalate esters Into fatty tis-
sues. There was a strong correlation, however, between phthallc acid con-
centrations (5.22^3.94 jjg/mi) and the length 1n years of previous dialy-
sis treatments (r=+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 tr1-
glycerlde concentrations correlated most closely with the Teachability of
DEHP, although 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, Individuals who receive exposures above background or environmental
levels, such 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 VI-13 07/28/88
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Summary
Dlsplte widespread occurrence of PAEs, Information concerning the
effects of human exposure Is 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 DBP.
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 In human leukocytes and fetal lung cells.
In epidemlologic 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 1n 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
nerve damage was Indicated. Although the study was comprehensive In scope
H 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. Hematologlc 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.
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Parenteral administration of PAEs may Involve the greatest risk for
toxic effects, especially In Individuals requiring blood transfusions or
hemodlalysls. Despite the fact that PAEs may leach Into the contents of
plastic blood bags or plastic tubes, reports of hepatitis In hemodlalysls
patients and necrotlzlng enterocolltls In Infants given blood transfusions
or umbilical catheters could not be conclusively attributed to PAE exposure.
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VII. MECHANISMS OF TOXICITY
Introduction
The relationship between the toxlcoklnetlcs 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 DBP have been found to Interact with the toxlclty
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 al., 1979).
DEHP significantly (p<0.05) Increased barbiturate-Induced sleeping time 1n
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 toxlclty of organophosphate
Insecticides to house flies (Al-Badry and Knowles, 1980). Antagonism was
noted between the effects of DBP and zinc-Induced testlcular atrophy (Cater
et al., 1977). Methylenedloxyphenol compounds and paraoxon Inhibited DEHP
hydrolysis by rainbow trout liver \n_ 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 VII-1 07/02/91
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antlpyrlne metabolism as a model for metabolic clearance of drugs. Antl-
pyrlne's metabolism was Increased 1n normal and renal failure rats (Sprague-
Dawley rats In which renal failure was Induced by a two-step nephrectomy)
after treatment with OEHP. The plasma clearance was Increased and elimina-
tion half-life of antlpyrlne decreased upon OEHP 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 In antlpyrlne clearance than
did control animals after DEHP treatment.
Changes In hepatic enzyme activities are associated with liver enlarge-
ment and occur 1n animals exposed to PAEs (Seth, 1982). One change that has
been observed consistently following oral or l.p. administration of DEHP Is
a decrease In hepatic sucdnate dehydrogenase (SDH) activity occurring
specifically In the peMportal zones (Seth, 1982).
One target site for PAE effects on the liver Is mitochondria. Results
of In vitro studies have Indicated that several PAEs produce Inhibition of
mltochondrlal 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) In 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.
04770 VII-3 09/07/88
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DEHP has also been shown to affect carbohydrate metabolism. Decreased
levels of glycogen were reported In the livers of mice, rats and ferrets
receiving DEHP (Seth, 1982). Marked depression of glucose and glycogen
levels was found In the livers of rats fed diets containing 2 or 4% DEHP
(Sakural et a!., 1978). Glucogenesls and glycogenolysls are also Inhibited
by DEHP. However, no quantitative conclusion on the Inhibition of the
reaction could be reached In this experiment.
Agarwal et al. (1982a) also examined the effect of DEHP administration
(oral, 1.p.) upon hepatic enzymes, llpld peroxldatlon and hepatic sulfhydryl
content In rats. The authors concluded that the PAEs Interfered with bio-
transformation mechanisms of hepatic mlcrosomal drug-metabolizing enzymes.
After a single oral or 1.p. treatment of DEHP was administered to rats, the
activity of amlnopyrlne-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 were 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 In 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 amlnopyrlne-N-demethylase and aniline hydroxylase -were 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 pharmacologU agents.
Walseth et al. (1982) demonstrated contrasting results of PAE treatment
on rat liver and lung. DBF administered l.p. resulted In 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 (8[a]P) metabolism 1n 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 In the activity of hepatic
mlcrosomal enzymes, Khawaja and Dallner (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 BBP and DEP.
Cellular Effects
Ekwall et al. (1982) assayed 29 plastldzers Including DMP, DEP. DBP,
DEHP, BBP and three other PAEs, for cytotoxlclty of HeLa cells. Cyto-
toxlclty was measured by pH changes of the medium using phenol red as the
04770 VII-5 07/28/88
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Indicator and by microscopic Inspection of the cultures (the MIT-24 system).
A comparison of the results of this Vn vitro cytotoxldty test to other
cytotoxldty tests demonstrated that as the chain lengths of PAEs Increase,
llpophlllclty Increases. A comparison of these hi vitro cytotoxldty test
results with Iji vivo test results In mice suggest that a basal cytotoxlc
action to mouse tissues 1s responsible for the lethal action of plastlclzers
to mice.
The cytotoxlc mechanisms of PAEs may be better elucidated by studies of
subcellular distribution and activity as opposed to assays of tissue distri-
bution (Bell, 1982). Bell (1982) discussed a series of experiments con-
ducted In rats, rabbits and pigs that were directed at the Investigation of
PAE effects on Upld metabolism. In studies of rats and rabbits that were
fed DEHP, the dlester Impeded cholesterol synthesis by Inhibition of
3-hydroxy-3-methylglutaryl CoA reductase, which catalyzes the second step of
cholesterol 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 1n
these tissues was thought possibly to account for fetal abnormalities found
In the offspring of phthalate-treated dams, and testlcular atrophy In other
animals. Plasma and liver cholesterol levels were decreased In rats fed
either 08P or DEHP. Inhibition of cholesterol synthesis by these esters may
have been the underlying cause for this effect. Experiments with jji vitro
tissue slices of rats fed DEHP demonstrated that de novo fatty add
synthesis and esterlflcatlon are Inhibited In certain -tissues after PAE
administration. Phosphollpld synthesis may also be selectively affected
(Bell, 1982).
04770 VII-6 08/05/88
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Upon further Investigation Bell and Buthala (1983) discovered that DEHP
Inhibits mlcrosomal acylCoA:cholesterol acryltransferase (ACAT) 1n rats that
received this compound 1n the diet. The biosynthesis of cholesterol from
14C-mevalonate was also Inhibited In treated animals Indicating that other
mlcrosomal enzymes are Influenced by DEHP administration. The post-
mevalonate segment of the blosynthetlc pathway requires the Involvement of
numerous mlcrosomal enzymes, while cholesterol esterlfIcatlon Is largely
associated with ACAT.
Bell (1982) also described experiments performed on the effects of DEHP
on mltochondrlal 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 1n 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 OEHP directly added to an Isolated suspension will Inhibit aden'.ne
nucleotlde translocase. Thus, exchange of extramltochondrlal ADP for
Intramltochondrlal 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 In 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
(1982) noted that the biochemical transformations observed In these
experiments Indicated that the effects of PAEs may result from
04770 VII-7 09/07/88
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alterations of membrane fluidity. The UpophlUc properties of the PAEs
may, therefore, change the membrane environment sufficiently to modify
enzyme responses.
Melnlck and Schiller (1982) studied the effects of DMP, DBP and DEHP on
liver mitochondria Isolated from rats. Active transport of potassium Ions
(K*), respiration rates and succlnate cytochrome c reductase activities
were monitored. DBP was the most effective energy uncoupler as measured by
Interference with K* uptake Induced by three energy sources. It also led
to a nearly total loss of respiratory control. DMP was less effective In
th'ls regard; MEHP, but not the parent DEHP, was an effective uncoupler of
energy-linked reactions. The authors Indicated that PAEs may affect
mitochondrlal function by changing the Inner membrane permeability and
Inhibiting succlnate dehydrogenase activity.
Several studies evaluating the hepatotoxlc effects of PAEs have been
performed. Lists of effects upon hepatocytes and enzymatic processes can be
found In Tables VII-1 and VII-2. PAEs cause liver hypertrophy by Increasing
the number of hepatocytes (Canning et al., 1983). PAEs may change the
structure and function of the liver by Inducing peroxlsomes, greater numbers
of mitochondria and higher levels of enzymes (Gannlng et al., 1984). It was
noted that the capacity to Increase mitochondria synthesis Is a unique
characteristic of OEHP. Mitochondria, by contrast to other Intracellular
membranes, are seldom Increased (Canning et al., 1984).
The Induction pattern of DEHP In the liver has unique properties.
Gannlng et al. (1981) reported that after treatment of rats with DEHP,
hepatic changes Included Increases In the number of peroxlsomes. In the
04770 VII-8 07/28/88
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TABLE VII-1
Cellular Changes In Rat Hepatocytes Induced by
DEHP Administration3
Organelle
Change"
PeroxAsomes
Protein and phosphollpld
Beta-oxidation enzymes of fatty acids
Carnltlne-acetyl transferase
Catalase
Urate oxldase
Mitochondria
Protein and phosphollpld
B.eta-oxidation of fatty acids
Carnltlne-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
bSpec1f1c activities or amounts on protein basis compared with the control
04770
VII-9
09/07/88
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TABLE VII-2
Synthesis and Breakdown of Protein and Llpld 1n DEHP-Treated Rats3
Protein
Peroxlsomes
Catalase
Beta-oxidation enzymes
Mitochondria
Membrane
Beta-oxidation enzymes
Mlcrosomal membranes
Total cytoplasmU
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
Mlcrosomal phosphollplds Unchanged
Blood cholesterol
Total Unchanged
HDLb Decrease
LDLC Increase
VLDLd Unchanged
Increase
aSource: Canning et al., 1984
bHDL, high-density Upoproteln
CLDL, low-density Upoproteln
dVLDL, very low-density Upoproteln
04770
VII-10
07/28/88
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oxidation of CoA-Unked fatty acids, 1n mlcrosomal NADPH-cytochrome c reduc-
tase and cytochrome P-450 levels. 1n the number of mitochondria and In the
activity of carnltlne-acetyl transferase. Induction of the transferase was
attributed to an Increase In peroxlsomal Q-ox1dat1on. Gannlng et al. (1983)
demonstrated that although peroxlsomal and mHochondMal membranes were
Increased, the endoplasmlc retlculum was not changed In amount or appearance.
Increases In the activity of enzymes In rat hepatic cytosol have been
found upon application of various peroxlsome prollferators Including DEHP,
DAP and 2,4,5-tr1phenoxyacet1c acid among others (Katoh et al., 1984).
Administration of DEHP resulted 1n the Induction of catalase and two long-
chain acyl-CoA hydrolases. An Increase In peroxlsomal B-ox1dat1on was also
signaled by a marked Increase In palmHoyl-CoA oxidation after Ingestlon of
DEHP In the diet.
Primary rat hepatocyte cultures were used to ascertain effects of
various alkyl phthalate esters on peroxlsomal enzyme activities (Gray et
al., 1983). The authors concluded that straight-chain phthalates produce
few effects upon rat hepatic peroxlsomes. The 2-ethylhexyl ester, e.g.,
MEHP, Increased carnltlne acetyltransferase activity and palmltoyl CoA
oxidation, and produced Increased numbers of peroxlsomes.
The effects of different PAEs upon liver cells have been compared with
those of cloflbrate. another peroxlsome prollferator (Lake et al., 1984b).
Lake et al. (1984a) had previously determined that DEHP 1s a potent Inducer
of rat hepatic peroxlsomal enzyme activities. In the more recent study
(Lake et al., 1984b), rats were orally administered DEHP, d1-n-octyl
phthalate (OOP), mono-n-octyl phthalate (MOP) or cloflbrate for 14 days.
04770 VII-11 09/07/88
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This resulted 1n liver enlargement. Liver sections from DEHP and clofibrate
treated animals showed an increased number of peroxisomes. Both DEHP and
clofibrate stimulated the activities of peroxisomal marker enzymes,
increased microsomal cytochrome P-450 content and stimulated microsomal
laurlc add hydroxylation activity. The compounds, OOP and MOP, did not
produce such effects. The branched chain ester DEHP was thus determined to
exert effects that differed markedly from the straight chain analogue, OOP
and Us metabolite MOP. In addition, DEHP was shown to Induce forms of
cytochrome P-450 similar to those induced by clofibrate. Oklta and Chance
(1984) also demonstrated that DEHP, like clofibrate, -Increased microsomal
laurate hydroxylation activities. Potent Induction of the cytochrome P-450
mediated fatty acid w-hydroxylat1on reaction occurred in rats that were
fed a diet containing DEHP.
There may be some species variation In the biochemical actions of PAEs.
Lake et al. (1984a) compared DEHP, MEHP and cloMbrate-lnduced hepatic
peroxlsome proliferation in 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 in liver weight
and hepatic peroxlsome numbers, the response was more marked in rats. For
each of the three treatments, dose dependent Increases in the peroxisomal
marker, cyanide-Insensitive palmltoyl-CoA, and In carnltlne acetyltransfer-
ase were noted 1n the rats. Only small changes In these parameters were
found In the hamsters. The species variation in the effects of DEHP may
have been attributable to differences 1n peroxlsome proliferation or in the
metabolism of DEHP.
04770 VII-12 09/07/88
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The mechanism of carclnogenicHy for OEHP Is not well understood; how-
ever, it has been suggested that OEHP may fall Into the peroxlsome-prollfer-
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 1n relative liver weight and hepatic
peroxlsomal enzyme activities were similar In age groups showing markedly
different changes In body weight and survival rates. Similar Increases In
activities of both palmltoyl CoA oxldase and carnltlne acetyltransferase
were noted between suckling and adult rats Indicating that suckling rats are
equally 1f not more sensitive to the peroxlsomal proliferating effects of
OEHP {Dostal et al., 1987a) (see Table V-4). The Induction of perloxlsomes
and peroxlsomal enzyme activity as well as hypollpldemlc effects was not
detected In marmoset monkeys exposed either 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
toxlcologlc significance with regard to hepatocellular carcinoma.
Possible mechanisms for the hepatocarclnogenU effects of phthlates and
other peroxlsome prollferators have Included the generation of free radicals
from Increased hydrogen perloxlde (H?0?' production and decreased
catalase activity, and that peroxlsome Inducing chemicals and/or their
metabolites may act as promoters (Canning et al., 1984). The production of
the enzyme catalase by peroxlsomes catalyzes the breakdown of hydrogen
04770 VII-13 07/02/91
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peroxide to water. Hydrogen peroxide Hself or the hydroxyl Ion, that Is
formed from hydrogen peroxide, causes damage to DNA and chromosomes
(Turnbull and RodMcks, 1985). PAEs, such as OEHP, exhibit hypollpldemlc
activities common to several peroxlsome prollferators that Include liver
enlargement that Is not accompanied by frank hlstologlc liver damage,
proliferation of smooth endoplasmlc retlculum and an Increase In the number
of hepatic peroxlsomes {Cohen and Grasso, 1981). Warren et al. (1982)
hypothesized that 1f 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 that fatty add B-oxIdatlon, H202' Perox
-------
o
4fc
-J
Cntiri Intermediary HetaboIlM
PEHf
2-cthyJnei*nol
Oxidation ol
NCHP *nd
Kxcrction ol
MCHP .
Hetabollte*
Liver
ratty Acid
I ft -oiid.)
Oxidation
I-OH |
DHA
inactlvatioa fStrand Breakc)
I
Mutation
Reactive
Modular
•CarclnoMa*
02 * «jO
CO
C3
00
FIGURE V1I-1
Schematic of the peroxlsome proliferation hypothesis. At higher doses of OEHP. It Is proposed that
excess H2°2 °r o^er oxygen species are produced In excessive amounts because caUlase producllon does
nol Increase as rapidly as peroxide production.
Source: Turnbull and Rodrtcks. 1985
-------
In a more recent report Rodrlcks and Turnbull (1987) compared and
summarized the differences between peroxlsomes found In various mammalian
species. The most extensive studies on proliferation of peroxlsomes and
Induction of peroxlsomal enzymes have been In 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
crystalloid core 1s absent from the peroxlsome. When comparing peroxlsomal
data, there 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 chemically Induced peroxlsomal proliferation. Quantitative measurement
of the species differences Is not available. However, the authors speculate
that It may be due to differences In absorption, metabolism or Inherent
differences In hepatic susceptibility (Rodrlcks and Turnbull, 1987).
The possible DNA-b1nd1ng activity of OEHP has been Investigated by Albro
et al. (1983a). Ethylhexyl-labeled OEHP, but not ring-labeled DEHP, was
found to be associated with the ONA 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 In the labeled DNA, or
artlfactual binding from the sample preparation. The source of the 14C
may have been carbonyl phosphate, which Is a precursor for urea and pyrlmi-
dlne bases, von Oanlken et al. (1984) concluded that OEHP did not bind
covalently to hepatic DNA In 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 Toxldty
Gonadal toxIcHy In rats has been linked to the adverse effects of
phthalates upon testlcular zinc concentrations. Upon administration of DBP
or OEHP urinary excretion of zinc was enhanced and the testlcular zinc con-
tent decreased (Cater et al., 1977; Foster et al.t 1980; Thomas et al.
1982). Cater et al. (1977) concluded that after oral administration. DBP Is
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 1s, 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 1.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 In the GI tract (Gray and
Beamand, 1984). Thomas et al. (1982) provided s.c. and l.p. Injection data
that demonstrated the action of DEHP upon the depletion of endogenous
gonadal zinc was not a function of the Interference of the Intestinal
absorption of the divalent zinc Ion.
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 (GangolU, 1982). Oral administration of 14C-DBP (lactation
of 14C 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 gonadotropins.
04770 VII-17 07/02/91
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The D8P-1nduced testlcular Injury was not reversed by treatment with
testosterone or pregnant mare serum (Gangolli, 1982).
Summary
Research Into the mechanisms of PAE toxlclty In animal tissues has
Indicated 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-llplds 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,
mltrochondrla and enzymes of fatty acid oxidation. Studies primarily on
DEHP Indicate that llpld and protein metabolism are inhibited. These
effects on carbohydrate metabolism are also associated with depressions In
the energy coupling systems of the liver, Including the mitochondria.
Inhibition of cholesterol synthesis In various organs occurs when phthalates
Inhibit an enzyme necessary for the conversion of acetate to cholesterol.
Although PAEs may become associated with hepatic DNA. the data Indicate that
this does not occur through covalent binding, but rather as a result of the
blosynthetlc Incorporation of PAE metabolites Into the genetic material.
The gonadal toxldty of PAEs has been related to an effect of these
compounds 1n decreasing endogenous testlcular zinc. Researchers have
Investigated several possible mechanisms of PAE toxlclty; however, there is
no conclusive evidence on any one mechanism.
04770 VII-18 07/02/91
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VIII. QUANTIFICATION OF TOXICOLOGIC EFFECTS
Introduction
The quantification of toxlcologlc 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 which no adverse, noncarclnogenlc health effects occur,
while carcinogens are assumed to act without a threshold.
In the quantification of noncarclnogenlc effects, a Reference Dose
(RfO), [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 1s derived from a no-observed-
adverse-effect level (NOAEL). or lowest-observed-adverse-effect level
(LOAEL), Identified from a subchronlc or chronic study, and divided by an
uncertainty factor(s) times a modifying factor. The RfD Is calculated as
follows:
RfD . (NOAEL or LOAEL) mg/Rg bw/day
[Uncertainty Factor(s) x Modifying Factor]
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 toxlcologlc effects for the chemical. In order to ensure that
uncertainty factors are selected and applied In a consistent manner, the
04780 VII1-1 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)
Use a 10-fold factor when extrapolating from valid experimental
results from studies using prolonged exposure to average healthy
humans. This factor Is Intended to account for the variation
In sensitivity among the members of the human population. [10H]
Use 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.
[10A]
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 RfO from a
LOAEL Instead of a NOAEL. This factor Is Intended to account
for the uncertainty In extrapolating from LOAELs to NOAELs.
[10L]
Modifying Factor (MF)
Use professional Judgment to determine another uncertainty
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 Is 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 Interspecles differences. Additional
considerations not Incorporated In the NAS/ODW guidelines for selection of
an uncertainty 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
VHl-2 07/02/91
-------
From the RfD, a Drinking Water Equivalent Level (DUEL) 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 DUEL provides the noncarclnogenic health effects basis for establishing
a drinking water standard. For Ingestlon data, the DWEL Is derived as
follows:
DWEL = («fP) * (Body weight In kg) =
Drinking Water Volume In I/day
where:
Body weight = assumed to be 70 kg for an adult
Drinking water volume = assumed to be 2 I/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:
UA (NOAEL or LOAEL) x (bw) .a
HA = •* ' '—•• = mg/l
(UF) x ( l/day) y
Using the above equation, the following drinking water HAs are developed
for noncarclnogenic effects:
1. 1-day HA for a 10 kg child Ingesting 1 I water per day.
2. 10-day HA for a 10 kg child Ingesting 1 l water per day.
3. Longer-term HA for a 10 kg child Ingesting 1 i. water per day.
4. Longer-term HA for a 70 kg adult Ingesting 2 1 water per day.
04780 VI11-3 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 Is 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 derived 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 Is generally derived from a study of subchronlc duration
(exposure for 10% of animal's lifetime).
The U.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 B: Probable Human Carcinogen. Sufficient evidence of
carclnogenlclty In animals with limited (Group 81) or Inade-
quate (Group 82) evidence In humans.
Group C: Possible Human Carcinogen. Limited evidence of
carclnogenlclty In animals 1n the absence of human data.
Group 0: Not Classified as to Human Carclnogenlclty. Inade-
quate human and animal evidence of carclnogenlclty or for which
no data are available.
Group E: Evidence of Noncarclnogenldtv for Humans. No
evidence of carclnogenlclty In at least two adequate animal
tests In different species or In both adequate epldemlologlc
and animal studies.
If toxlcologlc 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. This conversion Includes correction
for noncontlnuous exposure, less than lifetime studies and for differences
In size. The factor that compensates for the size difference 1s the cube
root of the ratio of the animal and human body weights. It is assumed that
the average adult human body weight Is 70 kg and that the average water
consumption of an adult human Is 2 I 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
Ingestlon 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
likely 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. Weibull, logit and probit. 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 in scientific measurement. In most cases, only
studies using experimental animals have been performed. Thus, there is
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 water, the Impact of the experimental animal's age, sex and
species, the nature of the target organ system(s) examined and the actual
rate of exposure of the Internal targets In 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. When there Is exposure to more than one contaminant, additional
uncertainty results from a lack of Information about possible synerglstlc or
antagonistic effects.
Noncarclnogenlc Effects
PAEs appear to be ubiquitous based on their detection In a variety of
environmental media Including air, water, food and soil. They have also
been detected In human tissues. Despite widespread occurrence of PAEs,
Information concerning the effects of human exposure to these compounds Is
limited. The available studies on human effects are not suitable for use In
risk assessment because of the small numbers of subjects studied and the
lack of quantitative Information on levels and duration of exposure.
However, the human studies and case reports provide supplemental Information
for comparison with health effects reported In animal studies.
Information on the acute oral toxlclty of PAEs In humans 1s limited to
the effects observed In three Individuals exposed to DEHP. No effects were
observed In one adult male subject administered an oral dose of 5 g of
04780 VIII-6 08/16/88
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DEHP. A second adult male subject given an oral dose of 10 g of OEHP exper-
ienced mild gastric disturbances and moderate catharsis (Shaffer et al.,
1945). Accidental Ingestion 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 expected, 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 OBP In this group of workers. .In vitro studies of
human tissue and cell cultures revealed that PAEs Inhibited cellular growth
and decreased platelet function but did not produce chromosomal damage 1n
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 hemodlalysls due to extraction of PAEs from plastic blood bags or
plastic tubing used in these treatments. However, reports of hepatitis In
hemodlalysls patients and necrotlzlng enterocolHIs In Infants given blood
transfusion 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 OEHP with the exception
of rats (Tanaka et al., 1975; Williams and Blanchfleld, 1975; Albro et al.,
1982). The role that glucuronlde conjugation may play 1n the sensitivity
between species to toxic endpoints Is not known; therefore, studies with
rats will still be considered with other test species for quantification of
toxlcologlc effects.
04780 VIII-7 07/31/91
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There Is no common toxic effect that PAEs as a group of compounds have
been shown to produce. Testlcular atrophy and hepatic Involvement are two
effects observed for several, though not all, PAEs. There Is evidence that
some effects of PAEs, such as the suspected carcinogenic effects of DEHP,
are related to the moiety attached to the phthalate group rather than the
phthalate group Itself {Kluwe, 1986). Thus, different PAEs may have differ-
ent patterns of toxldty. For example, DEHP Induces hepatocarclnomas 1n
rats and mice, whereas BBP appears to affect the hematopoletlc system and
may Induce leukemia In female rats. Because a single toxlcologlc pattern
has not been Identified for PAEs, the HAs, DWELs and cancer risk levels are
calculated for Individual PAEs rather than for the group of compounds
generlcally.
Studies Considered for Noncarclnoqenlc Quantifications — DEHP. DEHP
has been studied more extensively than any other PAE, In part because It Is
the most widely used plastlclzer. Enlarged liver and testlcular atrophy are
the two most commonly observed effects of DEHP In rats. Mangham et al.
(1981) conducted a short-term test to examine the testlcular and hepatic
effects of 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 In males. Also, body weight
gain was significantly decreased In males. The testlcular effects observed
are consistent with results of an earlier study by Gray et al. (1977) In
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, Mitchell 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. Hlstopathologlc 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
1000 mg/kg/day DEHP. After 14 days significant liver enlargement was noted
at the 50 and 200 mg/kg/day doses In male rats. There were no significant
differences 1n 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 endoplasmlc 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.
Subchronlc 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 VIII-9 07/02/91
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dogs (Harris et al.. 1956). The study In 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 Wlstar albino rats diets
containing 50, 200 and 1000 mg/kg/day DEHP for 9 months. Necropsy of the
thoracic, abdominal and other regions was carried out. The livers were
subjected to extensive hlstologlc, electron microscopic and biochemical
examination. Significant liver enlargement was observed In male rats at all
dose levels. 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
Tysosomes at 200 and 1000 mg/kg/day.
In the study by Gray et al. (1977), male and female CO rats were fed
dietary levels of 0, 0.2, 1 and 2% DEHP In the diet for 17 weeks. Dally
doses calculated from food consumption data corresponded to 143, 737 and
1440 mg/kg/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 exposed 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% level, liver weight was Increased in both sexes and spermatogenesls was
decreased In males.
Carpenter et al. (1953) conducted chronic toxlclty testing In rats,
guinea pigs 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 1n 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 al., 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 In this study. A group of four dogs given capsules 5 times/week
containing 0.03 ml/kg/day for 19 doses, then 0.06 mil/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%
DEHP. 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, food 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 mg/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 oral dose of 0.05 mi/kg administered by gavage to mice on day 7 of
gestation was associated with a decrease 1n body weight of viable fetuses;
however, no abnormal fetuses were observed. Since the density of OEHP Is
0.985 g/ml, the 0.05 ml/kg dose 1s equivalent to 49 mg/kg. Using the
dose-response curve for resorpMons and deaths, the authors calculated the
NOEL for fetal lethality to be 64 mg/kg. Shlota and Nlshlmura (1982)
administered OEHP In the diets of ICR-ICL mice on days 0-18 of gestation.
At the 0.05% level (70 mg/kg/day). 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 anomolles were observed. At a dietary level of 0.1% (190 mg/kg/
day) the number of resorptlons and dead fetuses were Increased, although the
statistical significance of this Increase was marginal (p=0.05). At 0.25%
(410 mg/kg/day). an Increased number of malformations were observed 1n
addition to Increased resorptlons and dead fetuses, decreased maternal and
fetal weights, 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 toxlclty In Fischer
344 rats and CD-I 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 In rats and mice, respectively. Tyl et al. (1988) concluded that
OEHP was not teratogenlc at any dose tested 1n Fischer 344 rats. However,
treatment did produce maternal and other embryofetal toxlclty 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 toxlclty also Increased Incidence of malformations. A dose of
0.05% (91 mg/kg/day) DEHP produced Increased Incidence of malformations
without maternal or embryofetal toxlclty. An embryofetal NOEL In mice was
determined to be 0.025% (44 mg/kg/day) DEHP.
A study by NTP (1984a) tested CD-I mice using a newly developed testing
scheme designated "Fertility Assessment by Continuous Breeding". Result-; of
this study were similar to those of Shlota and Nishlmura (1982); however,
the NTP study focused on fertility effects rather than teratogenlclty. 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. Dally Intakes of DEHP were not calculated by the authors.
However. 1f one assumes the same dally Intake rate as that calculated for
the low-dose CD-I mice In the carclnogenlclty bloassay by Kluwe et al.
(1982a) of 735 mg/kg/day (averaged for males and females) for a 0.3% dietary
level, dally DEHP Intakes for the lower dietary levels of 0.01 and 0.1%
04780 VI11-13 07/02/91
-------
would be -24 and 243 mg/kg/day, respectively. It should be noted that these
calculations do not account for differences In food consumption due to preg-
nancy, age of mice or any additional differences between the two studies.
At the 0.3°/o level, complete suppression of fertility was observed. At the
0.1% level, fertility was decreased and various reproductive parameters were
significantly decreased. These parameters Included number of Utters per
pair, and number of live pups per litter, 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 males were mated to the
0.3%-treated females and control females were mated to 0.3°/.-treated males.
In addition, control males were bred to control females to serve as the con-
trol group for the second phase. Results of this phase of testing revealed
that the decreased fertility was attributable to effects of DEHP 1n both
males and females.
Quantification of Noncarclnoqenlc Effects — DEHP.
Assessment of Acute Exposure Data and Derivation of 1-day HA — Liver
enlargement, testlcular atrophy 1n males, depressed weight gain and death
have all been observed after oral administration of single doses of DEHP to
rats. LD 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 OEHP than are
adults. Dostal 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/group) 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
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1n all groups and Increased mortality In 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 llver-to-body weight ratios.
Effects of acute oral exposure to DEHP on the liver have been studied by
Mitchell et al. (1985) and Hangham el al. (1981). Mitchell et al. (1985)
administered DEHP In the diet of rats (4/sex/group) at nominal doses of 50,
200 and 1000 mg/kg/day. Hlstopathologlc, biochemical cytogenetlc analyses
were conducted on days 3, 7, 14 and 28, and at 9 months of dosing.
Indications of hepatotoxldty (Increased liver weight, decreased hepatic
glucose-6-phosphatase activity) were first observed In the 50 mg/kg/day dose
males at 14 days of treatment. Mangham et al. (1981) observed decreased
testlcular weight, microscopic changes In the seminiferous tubules, enlarged
liver, 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 observation* are supported by the work of TomUa et al.
(1982a). Mice were administered slmjle doses of 50 vl OEHP/kg (-49
mg/kg) or TOO nl 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 toxUUy. From the
studies described previously, H Is not possible to determine whether a dose
of 44 mg/kg/day, the NOAEL for developmental effects, would cause liver
effects In rats, observed at 100 but not 10 mg/kg/day. Therefore, the NOAEL
for liver 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 =10mq/kq/daY x 10 kq = ]
100 x 1 l/day
where:
10 mg/kg/day = NOAEL based on lack of liver enlargement (Dostal et
al., 1987a)
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 i/day = assumed water consumption by a child
Assessment of Short-Term Exposure Data and Derivation of 10-day HA —
Effects on the liver appear to be the most sensitive endpolnt of toxldty
for 10-day 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 1s consistent with the 1-day HA If U were adjusted for a 10-day
exposure period. However, the Mitchell et al. (1985) study examined other
organs 1n addition to the liver and provides a better estimate of a 10-day
04780
VIII-16 07/02/91
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exposure. The 10-day HA Is also protective of developmental toxklty
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_d HA s50mq/kq/daY x 10kg = Q 5
1000 x 1 I/day
where:
50 mg/kg/day = LOAEL based on liver enlargement {Mitchell et al..
1985)
10 kg = 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
1 l/day = assumed water consumption by a child
Derivation of Longer-term HA — Subcnronlc oral studies nave been
conducted with DEHP. however none of the studies Identify a NOAEL. Mitchell
et al. (1985) observed significant liver enlargement In male tats
administered 50, 200 or 1000 mg/kg/day for 9 months. No clear progression
of hepatotoxlc effects was observed from 3-, 7-, 14- or 28-day time points.
This Is supported by the subchronlc study by Gray et al. (1977) where liver
weights were Increased In both sexes of rats and spermatogenesls was
decreased In males (143 mg/kg/day dose level 1n males; 154 mg/kg/day dose
level in females).
Deriving the longer-term HA based on the LOAEL 0-f 50 mg/kg/day Is
protective of the reproductive (NTP, 1984a) and developmental toxlclty (Tyl
et al., 1988} observed 1n mice at doses of ?43 and 91 mg/kg/day, respec-
tively. The reproductive study by NTP (1984a) showed no effects on
04780 VI1I-17 07/02/91
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fertility 1n mice fed 0.01% (24 mg/kg/day) In the diet for 7 days prematlng
and 98 days continuous breeding. The next highest dietary level of 0.1%
(240 mg/kg/day) significantly reduced fertility. Tyl et al. (1988) observed
fetotoxlclty 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/kg/day x 10 kg
Longer-term HA = = 0.5 mg/l
y 1000 x 1 I/day
(child)
where:
50 mg/kg/day = LOAEL based on liver enlargement (Mitchell et al.,
1985)
10 kg = 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
1 l/day = assumed water consumption by a child
50 mq/kq/dav x 70 kg
Longer-term HA = = 1.75 mg/l
1000 x 2 i/day „ , „
(adult) (rounded to 2 mg/i)
where:
50 mg/kg/day = LOAEL based on liver enlargement (Mitchell et al.,
1985)
70 kg = 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
2 i/day = 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 DUEL — 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-
port these results. Carpenter et al. (1953) also reported that no effects
were observed In 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% OEHP (64 mg/kg/
day). No hlstologlc 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 Nlshlmura (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 In 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 DWEL (U.S. EPA. 1991).
04780 VIII-19 07/02/91
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Using this LOAEL, the DWEL would be derived as follows:
RFD . . 0.019 mg/kg/day
1000 (rounded to 0.02 mg/kg/day)
where:
19 mg/kg/day = LOAEL derived from oral exposure to guinea pigs
(Carpenter et al., 1953)
1000 = uncertainty factor, according to U.S. EPA and
ODW/NAS guidelines for use with a LOAEL from a
subchronlc animal study
0.02 ^/kg/day « 70 kg
2 I/day
where:
0.02 mg/kg/day = RfD
70 kg = assumed weight of an adult
2 l/day * assumed water consumption by an adult
The 1-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 Noncarclnoqenlc Quantification — BBP.
Toxlclty of BBP 1s limited to a few studies. The most commonly observed
effects are liver and kidney enlargement and testlcular atrophy. Agarwal et
al. (1985a) conducted a 14-day dietary study of male Fischer 344 rats fed
levels of 0.625. 1.25, 2.5 and 5.0% BBP. At the 0.625% and 1.25% levels.
04780
VIII-20 07/02/91
-------
TABLE VIII-1
-J
00
0
»— «
•-^
0
CJ
ID
juuuiai j vi
Compound Criteria
Level
DEHP 1-day HA
10-day HA
Longer-term
HA
DWEL
BBP 1-day HA*
10-day HA
Longer-term
HA
DUEL
DBP 1-day HA
10-dav HA
uaia ujtru lu u
Dose
(mg/kg/day)
10
50
50
19
160
160
159
159
500
125
ci ivc nn aim
Study
Duration
5 days
14 days
9 months
1 year
14 days
14 days
26 weeks
26 weeks
14 days
1 year
unLL »a iuci i ui i
Species/
Effect Level
rat/NOAEL
rat/LOAEL
rat/LOAEL
guinea pig/
LOAEL
rat/NOAEL
rat/NOAEL
rat/NOAEL
rat/NOAEL
rat/NOAEL
rat/NOAEI.
jLiir, uur, utr, unr anu
Value of HA or DUEL
Adult Child
(mg/l) (mg/l)
1
0.5
2 0.5
0.7
20
20
60 20
7
50
10
uur
Reference
Dostal
et al., 1987a
Mitchell
et al., 1965
Mitchell
et al.. 1985
Carpenter
et al., 1953
Lake et al..
1978
Lake et al..
1978
NTP. 1985
NTP, 1985
Cater et al..
1977
Smith. 1953
-------
1ABLE VIIl-1 (cent.)
oo
o
INJ
IVJ
O
•^J
^v
CO
>x
LD
Compound
DBP
OEP
DHP
Criteria Dose
Level (mg/kg/day)
Longer-term 125
HA
DUEL 125
1-day HA
10-day HA
Longer-term 750
HA
OWEL 750
1-day HA
10-day HA
Longer-term
OWEL
Value of HA or DUEL
Study Species/ Reference
Duration Effect Level Adult Child
(dig/a) (mg/1)
1 year rat/MOAEL 40 10 Smith, 1953
I year rat/NOAEL 4 Smith. 1953
NO
NO
16 weeks rat/NOAEL 300 BO Brown etal.,
1978
16 weeks rat/NOAEL 30 Brown et al.,
1978
ND
NO
ND ND
ND
'Adopted from the 10-day HA
NO - Not determined (Inadequate data for derivation)
-------
liver and kidney weights were significantly Increased. In addition, the
Incidence of proximal tubular regeneration of the kidney IncMised 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
0.625%, 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
dally 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 peroxisomes. Effects on testes weights
were not observed In 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-Oawley and Wistar Albino rats were treated with 480 and 1600
mg/kg/day BBP for 14 days. A significant depression In either absolute or
relative liver and testes weight was observed In both strains of rats at
1600 mg/kg/day BBP. Hlstologic examination revealed testlcular atrophy In
both strains (1600 mg/kg/day) with the extent of the lesions being more
severe In the Sprague-Dawley strain. At 480 mg/kg/day BBP. 1/6 had testlcu-
lar atrophy, whereas the Wlstar albino strain revealed no histologic changes.
There are few oral long-term BBP studies. In a final report, NTP (1985)
conducted toxldty and mating trial studies in F344 rats. The toxirity
portion was 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 in the diet for 26 weeks. Powdered BBP was
mixed In to standard rodent meal diet. Because of the manner in 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 5% 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
VHl-24 07/02/91
-------
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, I1ver-to-body weight, I1ver-to-bra1n 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%
group 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 1n any other
organs.
Hlstopathologlc changes were also seen at the 2.5% BBP level after 10
weeks of exposure In 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% In 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 V1II-25 07/02/91
-------
at levels of 0.25 (125 mg/kg/day) and 0.50% (250 mg/kg/day) For 90 days
showed no toxic effects. A dietary level of 1.0% (500 mg/kg/day) BBP
resulted In Increased liver weight. Levels of 1.5 (750 mg/kg/day) and 2.0%
{1000 mg/kg/day) BBP were associated with Increased liver weight and a
decrease In growth rate. No effects were observed In dogs administered BBP
In 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.
Quantification of Noncardnoqenlc 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 on acute oral toxklty 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/J. be adopted as a conservative
estimate for the 1-day HA.
Assessment 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 B8P to male F344 rats
In the diet 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 hlstopathologlc changes
(proximal tubular regeneration) were also noted In 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 calculated to be 375 mg/kg/day.
04780 VIIl-26 07/31/91
-------
In male^Sprague-Dawley rats administered 160, 480 or 1600 mg/kg/day BBP
for 14 days by gastric Intubation, biochemical or morphologic changes In the
liver as well as effects on testes weights were not observed In the 160
mg/kg/day dose group (Lake et al., 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 In 1/6
Sprague-Dawley rats, whereas the Wlstar albino strain revealed no such
effects (Lake et al., >978).
When comparing the two studies Lake et al. (1978) Identifies a NOAEL of
160 mg/kg/day. It Is questionable whether 480 rng/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 In deriving the 10-day
HA. Although the method of treatment was gavage in the study by Lake et al.
(1978) and diet in the study by Agarwal et al. (1985a), treatment-related
effects across similar dose ranges. Including liver effects in both studies
In two sensitive strains of rats, support use of 160 mg/kg/day as NOAEL in
rats given BBP orally for 14 days.
The 10-day HA is calculated as follows:
10-day -A J60 mq/kq/day x 10 kq = ^ (rounded to 20 mg/i)
100 x 1 i/day
04780 VIII-27 07/31/91
-------
where:
160 mg/kg/day = NOAEL based on the absence of liver and testlcular
effects from animal data (Lake et al., 1978)
10 kg = assumed weight of a child
100 = uncertainty factor, according to U.S. EPA and
OOH/NAS guidelines for use with a NOAEL from an
animal study
1 l/day = assumed water consumption by a child
Assessment of Longer-term HA — Long-term exposure to BBP causes
adverse effects to the testes of male rats. The only study available for
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
testlcular 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 1n the brain, right
kidney, right testes and liver. Rats given dietary levels of 0, 0.03, 0.09,
0.28 and 0.83% BBP for 26 weeks exhibited no grossly observable effects on
male reproductive organs. Corresponding doses assuming -300 g bw and -17 g
of food consumption/day from data presented In 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
llver-to-body weight and llver-to-braln weight ratios and Increases In mean
corpuscular hemoglobin. Llver-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
04780 V1I1-28 07/02/91
-------
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 . 159 = 15'9
(child) IWW A ' "uaj (rounded to 20 mg/i)
where:
159 mg/kg/day = NOAEL based on the absence of Increased liver
weights In rats (NTP, 1985)
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 . 159 mq/kq/daY x 70 kq = ^
(adult) IUU x * a/aay (rounded to 60 mg/i)
where:
159 mg/kg/day = NOAEL based on the absence of Increased liver
weight In rats (NTP, 1985)
70 kg = assumed weight of an adult
100 = uncertainty factor, according to U.S. EPA and
ODW/NAS guidelines for use with a NOAEL from an
animal study
2 i/day = assumed water consumption by an adult
04780 VIII-29 07/31/91
-------
Assessment of Long-Term Exposure Data and Derivation of a DUEL -- NTP
(1985) 1s also the only available sludy for the derivation of the DWEL (U.S.
EPA, 1991). The OWEL Is derived as follows:
Step 1 - RfD Derivation
Rf0 m = Q 159 mg/kg/day
uuu (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
ODW/NAS guidelines for use with a NOAEL from animal
data, for less than lifetime exposure and to protect
sensitive members of the human population
Step 2 - DUEL Derivation
OWEL = 0.2 mq/kq/day x 70 kq = ?
2 I/day
where:
0.2 mg/kg/day = RfD
70 kg = assumed weight of an adult
2 l/day = assumed water consumption by an adult
The 1-day HA. 10-day HA and DWEL values calculated for B8P and the
effects levels used In calculation are summarized In Table VIII-1.
Studies Considered for Noncardnoqenlc Quantification — DBP. No
Information was found In the available literature on the effects of DBP in
humans and Information on effects In animals Is limited. The teratogenlc
effects of PAEs following oral administration were studied by Nlkonorow et
04780 VIII-30 07/31/91
-------
al. (1973). In this study female Wlstar rats were administered 120 and 600
mg/kg/day OBP 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,
paws on the front and hind legs, or Mb fusion 1n fetuses from treated rats
at either dose level when compared with the control animals.
Cater et al. (1977) found that DBP Induced testlcular atrophy In young
(3-4 weeks old) male Sprague-Dawley rats. DBP was dissolved In corn oil 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 mi/kg. Testes
weights were measured on days 4 and 6 for 500, 1000 and 2000 mg/kg/day doses
of DBP. 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 In 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 In 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 DBP
exposure revealed a diminution of both spermatocytes and spermatogonla.
04780 VIII-31 07/02/91
-------
In a dietary study OBP was fed to male and female Fischer 344 rats at 0,
0.6, 1.2 and 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
significantly Increased In both male and female rats at all treatment
levels. Male 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 laurlc
acid 11- and 12-hydroxylase Increased In males given 0.6, 1.2 and 2.5% and
In females given 2.5%. Cyanide-Insensitive palm-ltoyi CoA oxidation
Increased at 1.2 and 2.5% In males and 2.5% In females.
Smith (1953) studied the effects of feeding OBP to groups of 10 male
5-week-old Sprague-Oawley rats, weighing 55-65 g. Rats were fed dietary
levels of 0, 0.01. 0.05, 0.25 and 1.25% OBP for 1 year. The dietary Intakes
for DBP were 0, 5, 25, 125 and 600 mg/kg/day, respectively, estimated from a
figure depleting dally Intake In mg/kg 1n Smith (1953). Survival rates were
not reported for the three lowest dose groups. In the group fed 1.25% OBP.
half (presumably 5/10) of the animals died during the first week of the
experiment while the remaining animals gained weight comparable with
controls. It was not Indicated whether the deaths were thought to be
treatment-related. Necropsies were performed when rats showed marked weight
loss or signs of severe Infection. Animals alive at the end of 1 year were
sacrificed end examined for gross pathologic changes. While It was stated
that several organs were sectioned and stained, the results of hlstologlc
evaluation 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% DBP. The dally Intake of food and
plastlclzer {mg/kg bw/day) decreased as the rats Increased in size. No
changes in hematologlc parameters or gross pathology were observed at any
dose level.
Shlota and Nishimura (1982) found retarded ossification In mice fed
diets of 80. 180, 370, 660 and 2100 mg/kg/day DBP 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 OBP.
Quantification of Noncarcinoqenlc 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 toxicity
of DBP to humans. Cater et al. (1977) found that OBP Induced testicular
atrophy In young (3-4 weeks old) male Sprague-Dawley rats. OBP 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
04780
VHI-33 05/16/91
-------
significantly reduced testes weight and at 2000 mg/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, CMA (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 \n male rats. Liver weights Increased but were not statistically
significant. 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 s 500 mo/kq/daY x 10 kq = 50
100 x 1 a/day
where:
500 mg/kg/day = NOAEL based on the absence of decreased testes
weight from animal data (Cater et al., 1977)
10 kg = assumed weight of a child
100 = uncertainty factor, according to U.S. EPA and
OOW/NAS guidelines for use with a NOAEL from an
animal study
1 ft/day = 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/day 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 fur growth,
survival, gross pathology or hematology after 1 year exposure of DBP 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 1n addition to the longer-term HAs and OWE I.
The 10-day HA 1s calculated as follows:
A* 125 mq/kq/dav x 10 kg
-day = inn it i o/dav = 12'5 mg/l
100 x 1 i/day (rounded to 10 mg/a)
where:
125 mg/kg/day = NOAEL based on the absence of Increased mortality
and hematologlc 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
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 VI1I-35 07/31/91
-------
the 50% survival rate among the high-dose group, combined with the lack of
mlcropathologlc examination, must be noted 1n interpreting these results.
Longer-term HA . 1?5 "™ *q - 12-5 mg/l
{child)
' ' (rounded to 10 mg/i)
where:
125 ing/kg/day = NOAEL In male rats based on the absence of
Increased mortality and hematologlc effects (Smith,
1953)
10 kg = assumed weight of a child
f
100 = uncertainty factor, according to' U.S. EPA and
ODH/NAS guidelines for use with a NOAEL from an
animal study
1 I/day =• assumed water consumption by a child
, ftnn_r lorm u. 125 mg/kg/day x 70 kg .„ _.
Longer-term HA * - IQQ x 2 = 43'75
10° X Z
(adult) (rounded to 40 mg/t)
where:
125 mg/kg/day = NOAEL in male rats based on the absence of
Increased mortality and hematologlcal effects
(Smith, 1S53)
70 kg = assumed weight of an adult
100 = uncertainty factor, according to U.S. EPA and
ODW/NAS guidelines for use with a NOAEL from an
animal study
2 l/day = assumed water consumption by an adult
Assessment of Long-Term Exposure Data and Derivation of a DUEL —
Smith (1953) is also the only available study for the derivation of the OWEl
(U.S. EPA, 1991). The DUEL is derived as follows:
04780 Vlll-36 07/31/91
-------
RfD . = 0.125 nig/kg/day
(rounded to 0.1 mg/kg/day)
where:
125 mg/kg/day = NOAEL In male rats based on the absence of
Increased mortality and hematologic effects (Smith,
1953)
1000 = uncertainty factor, according to U.S. EPA and
ODU/NAS guidelines for use with a NOAEL from a
subchronlc animal study
nuFi 0.1 mq/kg/dav x 70 kg o c mn/a
DUEL = 2 o/dav = 3'5 mg/l
c y {rounded to 4 mg/l)
where:
0.1 mg/kg/day = RfD
70 kg = assumed weight of an adult
2 l/day = assumed water consumption by an adult
Studies Considered for Noncarclnoqenlc Quantification — PEP. No
information was available on the effects of DEP 1n humans. Information on
DEP toxlclty in animals Is 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%. DEP 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 hlstopathologic examination was
performed only on the 5.0% dose group. Statistical analysis was only
04780 VIII-37 07/31/91
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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 1n the diet at levels of 0, 0.5. 1.5, 2.0 and 2.5% for 1
year. Problems were encountered with palatablllty of DEP In the diet. As a
result, the dogs received varying exposures to OEP before each dog atlalned
stabilization 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. No effects were noted In dogs as a result of DEP exposures.
Brown et al. (1978) also studied the long-term oral toxlclty of DEP In
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/kg/day In females, respectively. Autopsies and hlstologlc exami-
nations were conducted at the end of 16 weeks. No changes 1n behavioral
patterns or clinical signs of toxlclty were observed. Female rats fed diets
containing 1% DEP and both sexes fed diets of 5% DEP gained significantly
less weight 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 palatablllty was the possible cause
In decreased weight gain, a paired-feeding study was conducted. Test rats
fed 5% DEP consumed more food (total) and gained less weight than controls.
Weights of the brain, heart, spleen and kidney were significantly lower In
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 VI11-38 07/02/91
-------
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 1n gross or microscopic pathology were not observed. No other
effects were observed.
Quantification of Noncardnogenlc 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.OX DEP (150, 770 and 3160 mg/kg/day.
male; ISO. 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
palatablllty. 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 VII1-39 05/16/91
-------
(without correction) and the small magnitude of the changes Indicates that
the 1% feeding level (750 mg/kg/day) represents a NOAEL In 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:
Longer-term HA = 75° «*'W**1 * ™ ** . 75 mg/i
(child) 10° x } l/day (rounded to 80 mg/i)
where:
750 mg/kg/day = NOAEL, based on the lack of kidney and body weight
effects. The dose level of 750 mg/kg/day was
chosen since the females were considered more
sensitive than the males (Brown et al., 1978).
10 kg = assumed weight of a child
1 l/day = assumed water consumption by a child
100 = uncertainty factor, according to U.S. EPA and
OOU/NAS guidelines for use with a NOAEL from an
animal study
for a 70 kg adult:
Longer-term HA . 7S° - 262.5 mg/l
(adu1t) IWU * * x/uay (rounded to 300 mq/l]
where:
750 mg/kg/day = NOAEL, based on the lack of kidney and body weight
effects. The dose level of 750 mg/kg/day was
chosen since the females were considered more
sensitive than the males (Brown et al., 1978).
2 l/day = assumed water consumption by an adult
100 = uncertainty factor, according to U.S. EPA and
OOW/NAS guidelines for use with a NOAEL from an
animal study
The longer-term HA values for a 10 kg child and a 70 kg adult are 80
mg/i and 300 mg/i, respectively.
04780 VI[1-40 08/08/91
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Assessment of Long-term Exposure Data and Derivation of a DUEL —
There are two possible long-term studies for derivation of a lifetime DUEL.
The Brown et al. (1978) 16-week study as described for the longer-term HA Is
also considered In deriving the DUEL. In a 2-year dietary study, Food
Research Laboratories. Inc. (1955) observed similar results at 5.0% DEP as
In the Brown et al. (1978) study. They reported a NOEL at 2.5X or 1250
mg/kg/day. Deficiencies In the reporting of the study reduce confidence In
the use of this data, since complete hlstopathologles 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 m 750 "q/kq/day . n.75 mg/kg/day
IUUU (rounded to 0.8 mg/kg/day)
where:
750 mg/kg/day = NOAEL 1n 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.r, 0.8 mq/kq/day x 70 kg ._ ... . . . __
DWEL = a—a ' * = 28 mg/l (rounded to 30 mg/l
2 l/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 VIII-41 08/08/91
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Studies Considered for Noncardnoqenlc Quantification — PHP. No
Information was available on the effects of DMP In humans. The only studies
available on acute oral toxlclty In animals used lethality as the toxic end-
point. The only long-term oral data was from an unpublished review article
(Lehman, 1955).
Quantification of Noncarclnoqenlc Effects — PHP. The 1-day, 10-day
or longer-term HAs and a lifetime DUEL for PHP 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 numbers of subjects studied and the lack of quantitative Information
on levels and duration of exposure. The human studies were designed lo
assess toxic effects caused by PAEs. However, there Is adequate data to
consider DEHP to be a Group B2 compound (I.e., probable human carcinogen)
based on significant Increases In liver tumor responses In rats and mice of
both sexes. BBP has been classified as a Group C compound (I.e., possible
human carcinogen) based on mononuclear cell leukemia In female rats. OBP.
DEP and DMP are classified as Group P (I.e., not classifiable) since
pertinent data regarding carclnogenlclty was not located In the available
literature (U.S. EPA. 1986; These classifications have all been verified
by the CRAVE Work Group.
Studies Considered for Carcinogenic Quantification — OEHP. In an NTP
study (1982a). 50 male and 50 female Fisher 344 rats per group were fed 6000
04780 VIII-42 08/08/91
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or 12,000 ppm DEHP \n the diet for 103 weeks. Similarly, groups of 50 male
and 50 female B6C3F1 mice were given 3000 or 6000 ppm OEHP 1n 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 In 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
In 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 hlstologlc examination of tissues was made.
Treated 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 In rats and hepatocellular carcinomas and
hepatocellular adenomas In mice. Metastasis of liver carcinoma to the lung
In mice was found In 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 DEHP In
Sherman rats. The untreated control group and each treatment group con-
04780 VIII-43 08/08/91
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00
o
TABLE VIM-2
Results of a 2-Year Carclnogentctty Btoassay of DEHP In Rats and Hlcea
o
vO
OD
oo
Species Sex Types of Liver Tumor
Rat H hepatocellular carcinomas
hepatocellular carcinomas
and neoplasltc nodules
F hepatocellular carcinomas
hepatocellular carcinomas
and neoplasttc nodules
Mouse N hepatocellular carcinomas
hepatocellular carcinomas
and hepatocelluar adenomas
F hepatocellular carcinomas
hepatocellular carcinomas
and hepatocellular adenomas
Lifetime Dose
-------
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 reducpt! 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 litter was reduced to two males and two females when the
pups had reached 15 days of age, and 32 males and 32 females 1n 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 F, 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 OEHP was not evident In this study. However,
this study Is weakened by the fact that of the 32 animals of each sex In
each group of the study (excluding the F, animals, all of which were
allowed to survive 1 year), only eight were allowed to survive beyond 1 year
04780 V1II-45 08/08/91
-------
of treatment. Furthermore, mortality was high with respect to all groups.
Hence, an Insufficient number of animals were available for a lifetime
feeding study of DEHP carclnogenldty 1n rats.
Carpenter et al. (1953) also Investigated the toxUHy of DEHP 1n long-
term studies In guinea pigs and dogs. Groups of 23 or 24- guinea pigs of
each sex were fed 1300 or 4000 ppm DEHP In 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 mi/kg for the Initial 19
doses followed by 240 doses at 0.06 ml/kg. The dogs were sacrificed at
the end of 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 Is considered by U.S. EPA to be sufficient and
there Is no human data, according to the U.S. EPA Guidelines for Carcinogen
Risk Assessment, DEHP Is classified as a 82 carcinogen (U.S. EPA, 1986).
This classification was verified (10/07/87) by the CRAVE Work Group (U.S.
EPA, 1991).
Quantification of Carcinogenic Effects — DEHP. The risk calculation
Is based on the liver tumor data from the NTP study (1982a) on DEHP.
Hepatocellular carcinoma and hepatocellular adenoma Incidence were reported
In both male and female rats and male and female mice. -However, male mice
were the most sensitive group. The combined Incidence of hepatocellular
carcinomas and adenomas In male mice (see Table V1II-2) was 14/50 for con-
04780 VI11-46 08/08/91
-------
trol animals. 25/48 for 390 mg/kg/day animals and 29/50 for 780 mg/kg/day
animals.
NTP (1982a), Kluwe et al. (1982a), U.S. EPA (19870) and IARC (1982)
concluded that these results provide sufficient evidence of d1(2-ethyl-
hexyl) phthalate-lnduced carclnogenldty In rats and mice. This conclusion.
however. Is disputed. Northrup et al. (1982) claim that the NTP (1982a)
results are equivocal since the MTD was exceeded In 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 Is metabolized differently
In rats than 1n 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 liver tumors was Increased In DEHP-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 Rodrlcks (1985) concluded that using
NTP (1982a) data to estimate DEHP-lnduced carcinogenic risk to humans will
probably overestimate actual risk. This conclusion was based on the
differences between rodents and primates In 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 Is 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 In general.
04780 VIII-47 08/08/91
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The dose-response data used In the potency calculations Included rats
wHh either hepatocellular carcinomas or neoplastlc nodules and mice with
either hepatocellular carcinomas or adenomas In the NTP (1982a) bloassay.
Male and female response data from the rat and mouse were used to calculate
a qj* value (Table VIII-3). The oral slope factors were 3.1BxlO'» and
4.52xlO~" (mg/kg/day)"* for male and female rats, and 1.41xlO'2 and
1.03xlO~* (mg/kg/day)'1 for male and female mice. The value of
1.41xlO~a represents the most sensitive response and hence Is 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
metabolism and pharmacoklnetlc considerations should be accounted for 1n the
dose response analysis. The examination of these factors has been done by
Turnbull and RodMcks (1985). but has not been further evaluated 1n this
document. 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 l of water for a 70-year lifetime with a
concentration of contaminant Is 4.0xlO"T (vg/lp1. Since risk 1s
assumed to be linear with dose In this range, risk factors of 10"*, 10"s
and 10"6 correspond to 300. 30 and 3 wg/i, respectively.
Studies Considered for Carcinogenic Quantification — BBP. A bloassay
was performed to evaluate the carcinogenic potential of BBP In rats and mice
(Kluwe et al., 1982b; NTP, I982b). 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 mice. Untreated groups of
SO males and 50 females of each species were used as controls. The female
04780 VIII-4B 09/05/91
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TABLE VIIJ-3
Cancer Risk Calculations
Animal
Dose
(mg/kq/day)
Animal/Sex
Rat/male
Rat/female
Mouse/male
Mouse/female
Low
300
300
390
390
High
600
600
780
780
Human
(Dose
Low
52
46
32
31
Equivalent
mq/kq/day)
High
100
89
65
59
Human Potency
Oral Slope
Factor
(mg/kg/day)"1
3.18xlO'3
4.52xlO'3
1.41x10"*
1.03xlO"2
04780
VIII-49
08/08/S1
-------
rats and both sexes of mice were maintained on these diets For 103 weeks;
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
terminated. No chronic or carcinogenic effects were observed In male or
female mice. Among female rats, however, an Increase In mononuclear cell
leukemia was observed at the higher dose level.
Quantification of Carcinogenic Effects -- BBP. The available data
meets the criteria for limited animal evidence based on mononuclear cell
leukemia In female rats. Hence BBP is 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 Group (U.S. EPA, 1991). A bloassay was performed by the NTP (1982b) to
evaluate the carclnogenldty of BBP 1n 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 both concurrent controls and historical controls. The conclusions
reached by the peer review group of this study Indicate that B8P was
"probably" carcinogenic In female rats. Although the Increase In leukemia
was statistically 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 VI11-50 08/08/91
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Studies Considered for Carcinogenic Quantification — DBP. Pertinent
data regarding the carclnogenlclty 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 — PEP. Pertinent
data regarding the carclnogenlcUy of DEP could not be located 1n the
available literature. According to U.S. EPA guidelines DEP Is 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 carclnogenlcUy of DMP could not be located In 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 Hyg1en1sts has set a TLV of 5
mg/m3, 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 VI11-51 08/08/91
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Uons: Group B2 for DEHP (10/07/67); Group C for BBP (08/26/87); and Group D
for OBP, DEP and OMP (08/26/87). The oral slope factor for DEHP, the only
one of these five phthalates to have a quantitative cancer risk assessment,
Is 4xlO~7 tug/a)"1. These assessments are also available on IRIS
{U.S. EPA, 1991).
Interactions with Other Chemicals
PAEs have been shown to Interact with other compounds In a synerglstlc
or antagonistic manner. Carbon tetrachlorlde. barbiturates and
organophosphate 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).
OEHP has been shown to Increase antlpyrlne metabolism In rats, possibly by
Inducing hepatic mlcrosomal enzymes (Pollack and Shen, 1984). Interaction
between DEHP and ethanol In rats has been studied by Agarwal et al.
(1982a). OEHP produces changes In the pharmacologlc response to ethanol by
altering the activities of alcohol dehydrogenase and aldehyde dehydrogenase.
Agarwal et al. (1982b) examined the effects of DEHP administration on
phenobarbltal-lnduced sleeping time In rats. The authors concluded that
PAEs Interfere with blotransformatlon mechanisms of hepatic mlcrosomal drug-
metabolizing enzymes. The effects of OEHP 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 Risk
Patients receiving blood transfusions or hemodlalysls constitute a
high-risk subpopulatlon for PAE exposure. This group ir.ay receive excessive
quantities of PAEs during transfusion or hemodlalysls due to leaching of PAE
plastldzers from plastic blood bags or plastic tubing.
Hlllman et al. (1975) studied the occurrence of necrotlzlng enterocoll-
tls and DEHP tissue concentrations In Infants who had received treatment
using arterial catheters containing DEHP. Higher DEHP content was found 1n
catheterlzed Infants with necrotlzlng enterocolHIs than In Infants that had
been catheterlzed but did not develop this disease. While the study did not
show a causal relationship. 1t 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 1n 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
study looked at workers exposed to DEHP 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 VIII-53 08/08/91
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Abe. S. and M. Sasaki. 1977. Chromosome aberrations and sister chromatld
exchanges In Chinese hamster cells exposed to various chemicals. J. Natl.
Cancer Inst. 58: 1635-1641.
ACGIH (American Conference of Governmental Industrial Hyglenlsts). 1985.
Documentation of the Threshold Limit Values and Biological Exposure Indices,
2nd ed. Cincinnati, OH.
Agarwal, O.K. 1986. Letters to the Editor. Toxlcol. Appl. Pharmacol. 82:
383-385.
Agarwal, O.K., S. Agarwal and P.K. Seth. 1982a. Effect of dl-{2-ethyl-
hexyl) phthalate on drug metabolism, llpld peroxldatlon, and sulfhydryl con-
tent of rat liver. Drug Metab. Dlspos. 10(1): 77-80.
Agarwal, O.K., S. Agarwal and P.K. Seth. 1982b. Interaction of dl-(2-
ethylhexyl) phthalate with the pharmacological response and metabolic
aspects of ethanol In mice. Blochem. Pharmacol. 311(21): 3419-3423.
Agarwal, O.K., R.R. Maronpot, 3.C. Lamb, IV and W.H. Kluwe. 1985a. Adverse
effects of butylbenzyl phthalate on the reproductive and hematopoletlc
systems of male rats. Toxicology. 35: 189-206.
Agarwal, O.K., W.H. Lawrence and 0. Autlan. 1985b. AntlfertllUy and muta-
genlc effects In mice from parenteral administration of d1-2-ethylhexyl
phthalate (DEHPJ. J. Toxlcol. Environ. Health. 16: 71-84.
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Agarwal. O.K.. W.H. Lawrence. L. J. Nunez and J. Autlan. 1985c.
MutagenlcUy evaluation of phthallc add esters and metabolites In
Salmonella typhlmurlum cultures. J. Environ. Health. 16: 61-69.
Agarwal. O.K.. S. Eustls. J.C. Lamb IV. J.R. Reel and W.M. Kluwe. 1986.
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Al-Badry, M.S. and C.JD. Knowles. 1980. Phthalate-organophosphate Inter-
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Environ. Contam. Toxlcol. 9: 147-161.
Albro. P.W. 1986. Absorption, metabolism and excretion of dl(2-ethylhexyl)
phthalate by rats and mice. Environ. Health Perspect. 65: 293-298.
Albro. P.W. and B. Moore. 1974, Identification of the metabolites of
simple phthalate dlesters In rat urine. J. Chromatogr. 94: 209-218.
Albro. P.W.. R. Thomas and L. Flshbeln. 1973. Metabolism of dlethylhexyl
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Albro. P.M., J.T. Corbett, J.L. Schroeder, S. Jordan and H.B. Matthews.
1982. Pharmacolclnetlcs. Interactions with macromolecules and species
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Albro, P.W., J.T. Corbett, J.L. Schroeder and S.T. Jordan. 1983a. Incorpor-
ation of radioactivity from labeled d!-(2-ethylhexyl) phthalate Into DNA of
rat liver \jn vivo. Chem.-B1ol. Interact. 44: 1-16.
Albro, P.W., I. Tondeur, 0. Marbury, S. Jordan, J. Schroeder and J.T.
Corbett. 1983b. Polar metabolites of dl-12-ethylhexyl) ph.thalate In the
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