0053
January 1992
FINAL
DRINKING WATER CRITERIA DOCUMENT
FOR
DI{2-ETHYLHEXYL)ADIPATE
Health and Ecological Criteria Division
Office of Science and Technology
Office of Water
U.S. Environmental Protection Agency
Washington, DC 20460
CT3
CV'
HEADQUARTERS LIBRARY
ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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TABLE OF CONTENTS
Pace
LIST OF FIGURES ............. . ....... v
LIST OF TABLES ...................... v
FOREWORD ........................ vii
PREFACE ............ . ....... ..... viii
I. SUMMARY ..................... . . . . M
II. PHYSICAL AND CHEMICAL PROPERTIES ........ ..... II-l
A. General Properties ............... . . II-l
B. Applications and Uses ................ II-l
C. .Production .................. ... II-4
D. Summary ............. . ......... II-4
III. TOXICOKINETICS ...................... III-l
A. Absorption ................ . ..... III-l
8. Distribution .................... III-2
C. Metabolism ..................... III-4
D. Excretion ...................... III-6
E. Bioaccumulation and Retention ............ . III-7
F. Structure-Activity Relationships .......... III-7
G. Summary ....................... III-8
IV. HUMAN EXPOSURE .................. .... IV-1
- V. HEALTH EFFECTS IN ANIMALS ................ V-l
A. Short-Term Exposure .................. V-l
1. Acute Toxicity ................ . V-l
2. Subacute Toxicity ................ V-l
3. Studies on Enzyme Induction ........... V-4
B. Long-Term Exposure ................. V-ll
C. Reproductive/Teratogenic Effects .......... V-13
D. Mutagenicity . . ................ . . V-24
E. Carcinogenicity ...... ............. V-27
F. Summary ....................... V-32
VI. HEALTH EFFECTS IN HUMANS ................. VI-1
iii
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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 anticipated 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 ptiarmacokinetics,
human exposure, acute and chronic toxicity to animals and humans, epidemiology,
and mechanisms of toxicity were 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 was comprehensive,
only the reports considered most pertinent in the derivation of the MCLG are
cited in the document. The comprehensive literature data base in support of this
document includes information published up to April 1987; however, more recent
data have been added during the review process and in response to public
comments.
When adequate health effects data exist, Health Advisory values for less-
than-lifetime exposures (One-day, Ten-day, and Longer-term, approximately 10% of
an individual'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.
James R. Elder
Director
Office of Ground Water and Drinking Water
Tudor T. Davies
Director
Office of Science of Technology
vii
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PREFACE
Of a total of 125,186,000 pounds of adipate ester plasticizers produced in
the United States In 1985, 36,991,000 pounds (29.5%) consisted of di(2-
ethylhexyljadipate (DEHA). No other single adipate constituted more than 7.1%
of the total production, although 57% of total production was attributed to "all
other adipic acid esters" (U.S. International Trade Commission, 1986).
Little information was found in the available literature on the toxic
effects of adipic acid ester plasticizers other than DEHA. Owing to the
importance of DEHA in terms of production volume, however, and to the scarcity
of data on other adipates, this document considers, in most instances, only DEHA.
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TABLE OF CONTENTS (continued)
Paoe
VII. MECHANISMS OF TOXICITY VII-1
A. Peroxisome Proliferation VII-1
8. Activity of Hydrolysis Products . . . VII-1
C. Synergism and Antagonism VII-3
D. Summary ..... VII-3
VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS VIIM
A. Procedures for Quantification of Toxicological
Effect .-.•••• VIII-1
1. Noncarcinogenic Effects ... VIII-1
2. Carcinogenic Effects VIII-3
B. Quantification of Noncarcinogenic Effects for
Adipates . V VIII-5
1. One-day Health Advisory for a Child VIII-6
2. Ten-day Health Advisory for Children and Adult . VIII-6
3. Longer-term Health Advisory for Children and
Adults VIII-8
4. Reference Dose and Drinking Water Equivalent
Level VIII-10
C. Quantification of Carcinogenic Effects for Adipates . VIII-11
1. Categorization of Carcinogenic Potential .... VIII-11
2. Quantification of Carcinogenic Effects VIII-13
D. Existing Guidelines and Standards VIII-13
E. Summary ...... VIII-13
IX. REFERENCES IX-1
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LIST OF FIGURES
Figure No.
Page
V-l Effect of Di(2-ethylhexyl)adipate (DEHA) Feeding on i
Body Weight in F344 Rats ,.,.; V ...-...= V-16
V-2 Effect of Di(2-ethylhexyl)adipate (DEHA) Feeding on Body . ' '
Weight in B6C3F, Mice ' V-17
•>
•' - . !
LIST OF TABLES
Table No.
II-l. Physical and Chemical Properties of Di{2)-ethy1hexy1)-
adipate (DEHA) II-2
III-l Distribution of Radioactivity in Rats After Oral
Administration of ["C]Di (2-ethylhexylJadipate (DEHA) . . III-3
III-2 Appearance of Metabolites After Oral Administration of
• Di(2-ethylhexyl)adipate (DEHA) to Rats III-5
V-l Body Weight Gain in Rats and Mice Fed Di(2-ethylhexyl)-
adipate (DEHA) For 14 Days V-2 ,,
V-2 Calculation of Average Daily Doses in Rats and Mice Fed !
Di(2-ethylhexyl)adipate (DEHA) for 14 Days ........ V-3 '
V-3 The Effect of Dietary Ingestion of Di(2-ethy1hexyl)adipate •
(DEHA) on Liver Catalase and Peroxis'omal beta-Oxidation
Activities . . . V-5 ;
V-4 Effect of Dietary Administration of Plasticizers and i
Related Compounds on Weight Gain, Liver Weight,
Peroxisome-Associated Enzymes, and Hepatic Peroxisome ',
Proliferation in Male F344 Rats V-8 ;
V-5 Effect of Dietary Di(2-ethylhexyl)adipate (DEHA) on . ;
Hepatic Synthesis of Squalene and Cholesterol in Rats . . V-10
V-6 Body Weight Gain in Rats and Mice Fed Di(2-ethylhexyl)- ' ;
adipate (DEHA) for 91 Days V-12 :
V-7 Daily di(2-ethylhexylJadipate (DEHA) Consumption in Rats •
and Mice Fed Diets Containing DEHA for 91 Days .V-14
V-8 Estimated Average Daily Consumption of di(2-ethylhexyl)-
adipate (DEHA) in Rats and Mice in a 2-Year Study ... V-15
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LIST OF TABLES (contined)
Table No. Page
V-9 Embryonic and Fetal Toxicity and Teratogenicity of
Adipate Esters in Rats V-18
V-10 Litter Data for Rats Fed Di(2-ethylhexyl)adipate (DEHA)
in a Developmental Toxicity Study V-21
V-ll Minor Abnormalities and Variants in Litters and Fetuses
in a Developmental Study in Rats Fed Di(2-ethylhexyl)-
adipate (DEHA) V-23
V-12 Mean Liver Weights in F0 Males and Females Fed Di(2-ethyl-
hexyl) adipate (DEHA) in a Developmental Toxicity Study . V-25
V-13 Cumulative Mean Weight Gain in Pregnant Females and
Their Pups in a Reproductive Toxicity Study in Rats Fed
Di(2-ethylhexyl)adipate (DEHA) . V-26
V-14 Incidence of Hepatocellular Tumors in Mice Fed Diets
Containing di(2-ethylhexyl)adipate {DEHA) for 2 Years . . V-29
V-15 Comparative Effects of Compounds With a 2-Ethylhexyl
Moiety on the Occurrence of Hepatocellular Tumors in
Rats and Mice - V-31
VII-1 Effect of Feeding Di(2-ethylhexyl)adipate (DEHA) or Its
Metabolites on Liver Function in Rats VII-2
VIII-1 Summary of Candidate Studies for Derivation of the
Ten-day Health Advisory for Di(2-ethylhexyl)adipate (DEHA) VIII-7
VIII-2 Summary of Candidate Studies for Derivation of the
Longer-term Health Advisory for Di(2-ethylhexyl)-
adipate (DEHA) VIII-9
VIII-3 Summary of Quantification of Toxicological Effects for
Di(2-ethylhexylJadipate (DEHA) VIII-14
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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 anticipated 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 pharmacokinetics,
human exposure, acute and chronic toxicity to animals and humans, epidemiology,
and mechanisms of toxicity were 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 was comprehensive,
only the reports considered most pertinent in the derivation of the MCLG are
cited in the document. The comprehensive literature data base in support of this
document includes information published up to April 1987; however, more recent
data have been added during the review process and in response to public
comments.
When adequate health effects data exist, Health Advisory values for less-
than-lifetime exposures {One-day, Ten-day, and Longer-term, approximately 10% of
an individual'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.
James R. Elder
Director
Office of Ground Water and Drinking Water
Tudor T. Davies
Director
Office of Science of Technology
vn
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PREFACE
Of a total of 125,186,000 pounds of adipate ester plasticizers produced in
the United States in 1985, 36,991,000 pounds (29.5%) consisted of di(2-
ethylhexyljadipate (DEHA). No other single adipate constituted more than 7.1%
of the total production, although 57% of total production was attributed to "all
other adipic acid esters" (U.S. International Trade Commission, 1986).
Little information was found in the available literature on the toxic
effects of adipic acid ester plasticizers other than DEHA. Owing to the
importance of DEHA in terms of production volume, however, and to the scarcity
of data on other adipates, this document considers, in most instances, only DEHA.
This document was prepared under a contract to Environmental Management
Support, Inc., with the Health and Ecological Criteria Division, Office of
Science and Technology, Office of Water, U.S. Environmental Protection Agency
(U.S. EPA), Washington, DC .{Robert Cantilli, Lead Scientist and Contract
Manager).
VII 1
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I. SUMMARY
Di(2-ethylhexyl)adipate (DEHA) is a light-colored, oily liquid. It has
low solubility in water (0.78 mg/L at 22°C) and low vapor pressure {0.01 mmHg
at 20"C). DEHA and other esters of adipic acid (AA) are widely used as
plasticizers in a variety of common products, including synthetic rubber, food
packaging materials, and cosmetics.
-Studies with Wistar and F334 rats, B6C3F, mice, and cynomolgus monkeys
indicate that DEHA is readily absorbed and extensively metabolized following
oral administration of single doses as high as 5,000 mg/kg. In mice, absorp-
tion via the gastrointestinal (GI) tract is apparently faster in females than
in males. In the GI tract, DEHA is hydrolyzed to monoethylhexyladipate (MEHA)
and AA with the release of 2-ethylhexanol (2-EH). Radiolabeled residues
distribute throughout the body, primarily in the form of AA and 2-EH, although
other more polar residues are also found. In rats administered a single oral
dose, highest radioactive label initially occurs in the stomach, intestine,
and adipose tissues, reaching a maximum at 6 to 12 hours, and then falling to
low levels by 48 to 96 hours with no preferential retention in any tissue. In
mice administered radioactive DEHA, almost all the radioactivity is eliminated
from the body via the urine and expired air, with very little being eliminated
in the feces or retained in the body. However, in F344 rats and monkeys, the
feces were found to contain up to 20 and 40%, respectively, of the
radioactivity.
Metabolites found in urine include AA and oxidation products of 2-EH
and/or their glucuronides. DEHA, MEHA, 2-EH, or oxidized metabolites of MEHA
were not detected in the urine of rats or mice, although small amounts of
2-EH, DEHA, and MEHA (but not oxidized metabolites of MEHA} were found in the
urine of cynomolgus monkeys.
The acute oral toxicity of DEHA is low. The LDW estimates in rats and
mice range from 9 to 45 g/kg. Short-term feeding studies (<30 days) indicate
that rats and mice gain weight normally when given diets containing up to 1.2%
DEHA (average daily dose of 1 to 2 g/kg/day). Diets with higher levels of
DEHA may cause decreased weight gain or weight loss. Long-term feeding
M
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studies (>30 days) suggest that diets containing 1.2% DEHA (average daily dose
about 0.6 to 1.2 g/kg/day) lead to decreased weight gain in rats and mice.
DEHA feeding has also resulted in several changes in the liver. Doses of
0.25 or 0.5 g/kg/day for 14 days produced a decrease in cholesterogenesis and
altered phospholipid synthesis in rat liver accompanied by decreased serum
cholesterol levels. Doses of about 1.5 g/kg/day for 7 to 21 days caused
peroxisome proliferation and hepatic enlargement in rats. Structure-activity
relationships indicate that these effects are strongly associated with the 2-
ethylhexyl moiety.
In a developmental toxicity feeding study in Wistar-derived rats,
dietary administration of 12,000 ppm {corresponding to a daily intake of
1,080 mg/kg body weight) in pregnant females caused slight maternal toxicity
evidenced by significantly (p <0.01) decreased gestational weight gain and
fetal toxicity seen as slightly reduced ossification and variations in ureter
development. Since there were no fetal effects without maternal toxicity,
DEHA is not a selective developmental toxicant; the NOAEL for maternal and
fetal effects was 170 mg/kg/day.. DEHA did not affect fertility or any
reproductive parameters in a one-generation reproductive study at the same
dietary levels (0, 300, 1,800, or 12,000 ppm). Reduced maternal weight gain
during pregnancy and reduced offspring weight gain during lactation was
observed in the high-dose (1,080 mg/kg). However, there were no effects on
embryo/fetal development or offspring survival. The NOAEL of 170 mg/kg/day
supports that in the developmental study.
Di(2-ethylhexyl )adipate was not rautageni.c in the Ames test. In a 2-year
feeding study, average daily doses of 0.7 or 1.7 g/kg/day were not
carcinogenic in male or female rats, but in female mice, doses of 2.9 or
8-2 g/kg/day caused an increased incidence of hepatocellular carcinomas.
Structure-activity studies have indicated that 2-ethylhexyl-containing
compounds elicit the same type of tumorigenic response, namely hepato-
carcinogenicity, to varying degrees in mice, particularly in females,
suggesting that this moiety may have a propensity for causing
hepatocarcinogenicity. In contrast, the toxic manifestations of phthalic acid
1-2
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esters (PAEs), including di{2-ethylhexyl}phthalate, are closely correlated
with their ester substituents. Although many PAEs possess some carcinogenic
activity, target sites are dissimilar, suggesting the absence of a common mode
of action.
No data were found in the available literature on the toxic effects of
DEHA in humans.
The mechanism of DEHA toxicity is not entirely known. DEHA belongs to a
diverse group of chemicals that induce the proliferation of hepatic
peroxisomes and are associated with hepatic cancer in female mice. In recent
years, peroxisome production and induction of peroxisome-associated enzymes in
the livers of rodents exposed to di(2-ethy1hexyl)adipate and related compounds
have been extensively studied. The induction of peroxisomes in OEHA-treated
rodents is associated with a severalfold increase in the activity of the
peroxisomal fatty acid beta-oxidation system and with a two-fold increase in
catalase activity. In addition, long-term exposure to these peroxisome
proliferators results in the induction of hepatocellular carcinomas in mice.
The lack of mutagenicity of these agents, combined with consistent findings of
proliferation of hydrogen peroxide-generating peroxisomes, indicates that
persistent proliferation of pt :xisomes serves as an endogenous initiator of
neoplastic transformation by enhancing oxidative stress.
. No suitable data were found for calculating One-day and Ten-day HA values
for DEHA; therefore, the Longer-term HA value of 20,000 ng/L is taken as a
conservative estimate of both HA values. 'The Longer-term Health Advisory is
based on a one-generation reproduction study in rats where the NOAEL was
170 mg/kg/day, and the LOAEL was 1,080 mg/kg/day. The LOAEL was based on
increased liver weight gain in dams and decreased litter size and weight gain
in pups. Using the NOAEL, the Longer-term Health Advisory values of 20,000
and 60,000 pg/L were calculated for children and adults, respectively. Based
on a developmental toxicity study in rats, a NOAEL of 170 mg/kg/day was
identified, which was supported by the one-generation reproductive study.
Using this NOAEL, a Reference Dose (RfD) of 0.6 mg/kg/day was established, and
a Drinking Water Equivalent Level (DWEL) of 20,000 pg/L was calculated. Based
on a carcinogenicity assessment for oral exposure to DEHA (NTP, 1982) which
1-3
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provides evidence for an increase in hepatocellular adenomas and carcinomas
{combined}, the Oral Slope Factor (q,*) was 1.2 x 10° (mg/kg/day)*', and the
Drinking Water Unit Risk was 3.4 x 10" ([ig/l}"; a risk level of 1(T*
corresponds to a drinking water concentration of 30 jig/L.
1-4
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II. PHYSICAL AND CHEMICAL PROPERTIES
A. GENERAL PROPERTIES
Di(2-ethylhexyl)adipate (DEHA) is a light-colored, oily liquid with the
formula CMH«,Q.. The compound is slightly soluble in water (less than
0.78 mg/L) (Felder et a!., 1986} but is soluble in acetic acid, acetone,
diethyl ether, and ethanol (Weast, 1979). Table II-l summarizes the important
physical and chemical properties of DEHA.
B. APPLICATIONS.AND USES . •
DEHA and other adipate esters are used primarily as plasticizers.
Plasticizers are organic chemicals that are added to synthetic plastics and
resin materials to improve workability during fabrication, to extend or modify
the natural properties of these materials, or to achieve new properties not
present in the original material. DEHA imparts low-temperature flexibility
and is frequently used in products intended for low-temperature uses (IARC,
1982). A large quantity of di(2-ethylhexyl)adipate is used in the manufacture
of polymeric substances such as polyvinyl chloride (PVC). The U.S. Food and
Drug Administration (U.S. FDA, 1980) currently allows up to 50% DEHA in
polymeric products that have contact with food, including adhesives,
cellophane, closures with sealing gaskets for food containers, water-soluble
hydroxyethyl cellulose film, and rubber articles intended for repeated use.
Polyvinyl chloride film used for wrapping meat contains levels up to 30%
plasticizer, including DEHA (Boettner and Ball, 1980).
Production of synthetic rubber, nitrocellulose, ethyl cellulose, and
vinyl copolymers also involves the use of DEHA. Consumer products that
contain DEHA include bath oils, eye shadow, cologne, cosmetic foundations,
rouge, blusher, nail polish remover, moisturizers, and indoor tanning
preparations (IARC, 1982).
II-l
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Table II-l. Physical and Chemical Properties of Di(2-ethylhexyl)adipate
Property
Value
CAS Registry No.
Chemical abstract name
IUPAC systematic name
Registry of Toxic Effects of
Chemical Substances (RTECS) No.
Synonyms
Chemical formula
Structural formula
Molecular weight
Trade names
Boiling point
Melting point
103-23-1
Hexanedioic acid, bis(Z-ethylhexyl)
ester
Adi pic acid, bis(2-ethylhexyl)
AU9700000
BEHA; bis{2-ethylhexyl)adipate;
DEHA; di-(2-ethylhexyl)adipate;
DOA; dioctyl adipate; hexanedioic
acid; dioctyl ester; octyl adipate
I-
0
370.64
Adipol 2EH; Bisoflex DOA; Effemoll
DOA, Effemoll Flexol Plasticizer
10-A; Ergoplast AdDO; Flexol A26;
Flexol Plasticizer A-26; Kemsler
5652; Kodeflex DOA; Moll an S;
Mcrcplcx DOA; NCI C54386; Plastomol
DDA; PX-238; Reomol OOA; Rucoflex
Plasticizer DOA; Sicol 250; Staflex
DOA; Truflex DOA; Uniflex DOA;
Vestinol OA; Wickenol 158; Witamol
320
214eC (5 mmHg)
-67.8°C
(continued)
H-2
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Table II-l. (continued)
Property
Value
Specific gravity (25/4°C)
Flashpoint
Refractive Index (20°C)
0.922
384° C
1.4474
Vapor pressure
Conversion factor: air (ppm to
mg/mj)
Solubility
Hater (delom'zed) at 22°C
Acetic acid
Acetone
D1ethyl ether
Ethanol
Octanol/water partition
coefficient
Measured bioconcentration factor
<0.01 mmHg at 20°C
2.60 mmHg at 200°C
0.0659
0.78 ± 0.16 mg/L
Soluble
Soluble
Soluble
Soluble
>1.3 x 10*
27
SOURCE: Adapted from Felder et al. (1986); Weast and Astle (1986); Sandmeyer
and Kirkwin (1981); Weast (1985-86); IARC (1982).
II-3
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C. PRODUCTION
DEHA is produced commercially by reacting excess 2-ethylhexanol with
adipic acid (AA) in the presence of an acid catalyst such as sulfuric acid or
para-toluene sulfonic acid (IARC, 1982). Of a total of 125,186,000 pounds of
adipate ester plasticizers produced in the United States in 1985, 36,991,000
pounds (29.5%) consisted of DEHA (U.S. ITC, 1986).
The following information on adipic acid ester production in the United
States was reported by the U.S. International Trade Commission (U.S. ITC,
1986):
Total U.S. production
Adi pic acid ester fx 1.000 pounds) % of total
Di(2-ethylhexyl)adipate (DEHA) 36,991 29.5
DHridecyl adipate 8,952 7.1
m-Octyl-rn-decyl adipate 5,331 4.3
Diisooctyladipate 1,496 1.2
Diisodecyladipate 1,400 1.1
Diisobutyladipate 212 0.2
All other adipic acid esters 70,804 56.6
Total produced 125,186 100.0
D. SUMMARY
DEHA is the di(ethylhexyl) ester of adipic acid (AA). It is a clear,
oily liquid, with low water solubility (less than 0.78 mg/L at 22°C) and low
volatility (vapor pressure <0.01 mmHg at 20°C). The esters of AA, including
DEHA, are used as plasticizers in a variety of products such as PVC and other
plastics, cellophane, rubber, and cosmetics.
II-4
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III. TOXICOKINETICS
A. ABSORPTION
D1(2-ethylhexyl)adipate (DEHA) is readily absorbed from the
gastrointestinal (GI) tracts of mice, rats, and cynomolgus monkeys. Takahashi
et al. (1981) studied the metabolism of DEHA in two male Mistar rats (150 to
270 g) given a single oral dose of 500 mg/kg of [14C-carbonyl]DEHA
(1.26 (iCi/rat) as a saturated solution in dimethyl sulfoxide (DMSO). • Within
48 hours, 90.0 and 93.9% of the radioactivity were excreted in expired air and
urine, while only 1.4 and 5.0% of the dose, respectively, were, detected in the
feces of each animal. This indicates that DEHA is rapidly absorbed and
excreted in urine and expired breath following oral ingestion. It should be
noted that the use of DMSO may have increased the degree of absorption of DEHA
as compared with the absorption of DEHA administered in water.
The absorption, disposition, and metabolism of ["C-2-hexyl]DEHA was
studied by Guest et al. (1985) in male and female B6C3F, mice receiving single
oral doses of 50, 500, or 5,000 mg/kg DEHA (dosing vehicle not indicated). At
6 hours after dosing, the GI tract of males and females contained 56 to 70%
and 30 to 38% of the total radioactivity, respectively. The levels of the
radioactivity found indicated faster absorption via the GI tract in females
than in males. Mice receiving either the 50- or 500-mg/kg DEHA dose excreted
91, 7, and 1 to 2% of the radioactive dose in the urine, feces, and expired
air at 24 hours, respectively. Mice receiving 5,000 mg/kg excreted 65 to 70%
and 3% of the radioactive dose in the urine and feces, respectively, in
24 hours; 20 and 0.3 to 1.3% of the total radioactivity remained in the GI
tract and tissues, respectively.
Differences in the disposition and metabolism of ["C-2-hexyl]DEHA at
500 mg/kg dose were studied in male and female B6C3F, mice, F344 rats, and
cynomolgus monkeys by El-hawari et al. (1985). The distribution of
radioactivities at 24 hours in urine, feces, expired air, and tissues was 91,
6 to 8, 1 to 2, and <1% of the dose, respectively, for mice and 74 to 78, 15
to 20, 1 to 2, and about 6%, respectively, for rats. The monkeys excreted 49
III-l
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to 69% and 23 to 40% of the radioactivity in the urine and feces,
respectively, in 48 hours, Only traces were recovered in tissues.
8. DISTRIBUTION
Five groups of male Wistar rats (three rats/group) were dosed (as
previously described in Section III.A, Absorption) and sacrificed at 6, 12,
24, 48, or 96 hours (Takahashi et al., 1981). Tissue samples (blood, brain,
heart, lung, liver, spleen, kidney, stomach, intestine, testicle, thymus,
muscle, and adipose) were removed, and their radioactive contents were
measured. The results are shown in Table III-l. At 6 hours, the largest
amount of radioactivity was found in the stomach (29.10 ± 9.40% of dose),
adipose tissue (8.21 ± 8.87% of dose), and intestine (5.53 ± 4.03% of dose).
(It is not clear whether the measurements of stomach and intestinal
radioactivity were of tissue content only or whether gastric or intestinal
contents were also included.) No other tissue examined contained more than
the 1.21 ± 1.00% of the dosed radioactivity that was found in the muscle. In
liver, kidney, testicle, and muscle, the amount of residual radioactivity
reached a maximum at 12 hours, while radioactivity in all other tissues
reached a maximum at 6 hours. The significance of these data should be
interpreted cautiously, since the standard deviations are relatively large.
Analysis of distribution in terms of percent-of dose/tissue is affected
by the relative contribution to total body weight of different tissues. It is
also of interest, therefore, to examine specific activity/tissue as a measure
of affinity of dosed radioactivity for different tissues. Aside from stomach,
tbe highest specific activity at 6 hours was found in adipose tissue
(33,711 ± 42,658 dpm/g tissue). Specific activities at 6 hours in. other
tissues (excluding intestine, 9,619 ± 7,050 dpm/g) ranged .from
5,853 ± 1,553 dpm/g (kidney) to 537 ± 476 dpm/g (muscle). In addition to
stomach and intestine, relatively high specific activities were noted for
liver, kidney, and adipose tissues. There was a gradual decline in
radioactivity within 6 to 12 hours in all tissues. At the end of 96 hours,
less than 1% remained in tissues. It is possible that stomach and intestinal
contents may have contributed to the radioactivity measured in those organs.
III-2
-------
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III-3
-------
Tht significance of these data also should be interpreted cautiously, because
the standard deviations are relatively large.
In mice administered [I4C]DEHA {5,000 mg/kg), about 20% and 0.3 to 1.3%
of the radioactive dose remained in the GI tract and tissues, respectively
(Guest et al., 1985). Little, if any, radioactivity was found in tissues of
animals receiving either 50 or 500 mg/kg DEHA. Similarly, El-hawari (1985)
found about 1%, 6%, and traces of the radioactive dose (500 mg/kg) in tissues
of mice, rats, and cynomolgus monkeys, respectively.
C. METABOLISM
Takahashi et al. (1981) studied DEHA metabolism both in vivo and in vitro
in rats. For the in vivo studies, 100 mg nonlabeled OEHA (as a 5% solution in
DMSO) was administered by gavage to five male Wistar rats (500 mg/kg, assuming
body weights of 0.200 kg). Urine, blood, stomach, small intestine, and liver
were extracted with ether at 1, 3, or 6 hours after dosing, and the extracts
analyzed by gas chromatography. The results (shown in Table III-2) indicate
that ingested DEHA is hydrolyzed to monoethylhexyladipate (MEHA) and to adipic
acid (AA) in the stomach. The concentration of DEHA in the stomach declined
with time, accompanied by the appearance of MEHA and AA. Peak values of these
hydrolysis products occurred at 3 hours. In the intestine, the amount of AA
Increased with time, but neither DEHA nor MEHA was found. Similarly, only AA
could be detected in the blood, liver, and urine. Adipic acid appeared in the
urine 1 hour after DEHA administration, and its excretion reached 20 to 30% of
the administered dose of radioactivity within 6 hours.
Guest et al. (1985) found that the "C activity in the GI tract of mice .
dosed with ["C]DEHA consisted largely of DEHA, MEHA, and 2-ethylhexanol
(2-EH), whereas the liver contained more polar components of 2-EH (not
specified). The urine contained metabolites of 2-EH including ethylhexanoic
acid (2-EHA), 2-EHA glucuronide, 5-hydroxy-EHA, and 2-ethyl-l,6-hexanedioic
acid (2-EHDA). The proportion .of highly oxidized metabolites (5-hydroxy-EHA
III-4
-------
Table III-2. Appearance of Metabolites After Oral Administration
of Di(2-ethylhexy1)adipate (OEHA) to Rats
Rat
1
2
3
4
5
PC]
Time
(hr)
1
3
3
6
6
Metabolites exoressed as
Urine
AA*
5.7
5.6
1.5 .
23.0
29.5
Blood
AA
0.5
0.5
0.7
0.6
AA
3.9
8.9
9.8
3.3
3.6
percentage of administered
Stomach
MEHA'
8.7
6.3
11.6
4.5
1.0
DEHAC
57.4
43.2
45.6
5.6
2.9
Intestine
AA
2.0
4.4
13.8
19.7
19.1
dose
Liver
AA
0.2
1.7
0.5
2.1
3.3
'Adipic acid.
'Monoethylhexyladipate.
*Di(2-ethylhexyl)adipate.
SOURCE: Adapted from Takahashi et'al. (1981).
III-5
-------
and 2-EHDA) increased with dose. No 2-EH, DEHA, MEHA, or oxidized metabolites
of MEHA were detected in the urine.
El-hawari et al. (1985) identified the urinary metabolites following
administration of 500 mg/kg [u-2-hexyl]DEHA to mice, rats, and cynomolgus
monkeys. In mice, the urine contained metabolites of 2-EH, including 2-EHA,
its glucuronide, 5-hydroxy-EHA, and 2-EHDA. No sulfate conjugates were
detected. Rat urine had more oxidized metabolites and less 2-EHA glucuronide.
Monkey urine contained MEHA, its glucuronide, and small amounts of 2-EH and
DEHA. No oxidized metabolites of MEHA were detected.
In vitro hydrolysis of DEHA and MEHA was investigated in tissue
preparations from liver, pancreas, and small intestine. Extracts from each
of these tissues hydrolyzed DEHA to MEHA and AA, and MEHA to AA, with the
highest rate of hydrolysis found in intestinal extracts (Takahashi et al.,
1981).' • '
D. EXCRETION
Takahashi et al. (1981) studied the excretion of [14C-carbonyl]DEHA in two
male Wistar rats given single oral doses of 500 mg/kg [UC]DEHA (as a
saturated solution in DMSO). Most of the administered label was eliminated in
the urine (individual values of 38 and 54%) or expired as carbon dioxide
(individual values of 40 and 59%) within 48 hours. There was little excretion
in the feces (individual values of 1 and 5%).
Similarly, Guest et al. (1985) found that B6C3F, mice receiving oral
doses of 50 or 500 mg/kg ["C-2-hexyl]DEHA excreted within 24 hours 91, 7, and
1 to 2% of the radioactive dose in urine, feces, and expired air,
respectively. Mice receiving 5,000 mg/kg excreted 65 to 75% in the urine and
3% in the feces within 24 hours. A total of 20% of the radioactivity was
found in the GI tract 24 hours after dosing.
i
The elimination of UC in the urine, feces, and expired air of B6C3F,
mice, F344 rats, and cynomolgus monkeys following oral administration of [UC-
2-hexyl]DEHA exhibited similar patterns. The distribution of radioactivity at
III-6
-------
24 hours in the urine, feces, and expired air was 91, .6 to 8, and 1 to 2% of
the radioactivity, respectively, in mice; and 74 to 78, 15 to 20, and 1 to 2%,
respectively, in rats. The monkeys excreted 49 to 69% of the radioactivity in
urine and 23 to 40% in the feces in 48 hours.
E. BIOACCUMULATION AND RETENTION • \ . -
The data in Table III-l show that DEHA is cleared from a 1*1 tissues of the
body within 48 to 96 hours. The results of Takahashi et al. (1981), Guest
et al. (1985), and El-hawari et al. (1985) indicate that elimination Is rapid,
and there is no organ-specific retention.
F. STRUCTURE-ACTIVITY RELATIONSHIPS
Fox et al. (1984) studied the in vitro hydrolysis of selected
plasticizers by gut homogenates from Sprague-Dawley rats. The kinetics of.
2-EH formation and disappearance of the parent compound were followed for
30 minutes. The study showed that formation of 2-EH increased with time for
DEHA, di{2-ethylhexyl)phthalate (DEHP), and di(2-ethylhexyl)terephthalate
(DEHT), but not for tri(2-ethylhexyl)trimillitate (TEHT). The stoichiometry
\
of the hydrolysis reaction indicated that DEHA and DEHT are converted to 2-EH
and their respective diacids, whereas DEHP is largely converted to 2-EH and
mono(2-ethylhexyl)phthalate.. The disappearance half-lives for DEHA, DEHP, and
DEHT were 6, 12.6, and 53.3 minutes, respectively. There was no evidence for
the hydrolysis of TEHT. The authors concluded that the rates of hydrolysis .
may have a bearing both on the extent of absorption and on the specific
chemical species absorbed.
In a comparative pharmacokinetic study of DEHP in rats and marmosets, the
latter exhibited a lower excretion profile and tissue levels after ip or iv
administration. The urinary metabolite pattern in the marmoset was, in many
respects, qualitatively similar to but quantitatively different from that of
the rat. Marmosets excreted principally conjugated metabolites from omega-1
oxidation, whereas rats excreted nonconjugated metabolites produced by omega,
omega-1, and beta-oxidation routes (Rhodes et al., 1986).
III-7
-------
Albro (1986) conducted a detailed study on the absorption, metabolism,
and excretion of DEHP in CD-I rats and B6C3F, mice. The metabolic pathways of
phthalates with saturated alkyl groups were postulated based on in vivo and
jn vitro data. The pathways involve hydrolysis (nonspecific lipase,
microsomal esterase), oxidation (microsomal monooxygenase, alcohol
dehydrogenase, aldehyde dehydrogenase), and conjugation (glucuronide
formation). The mouse urine contained all the metabolites found in the rat
urine plus glucuronide ester conjugates. However, after hydrolysis of the
conjugates, the proportion of metabolites was quite different for the two
species. Thus, the 2-ethyl-5carboxypentyl phthalate metabolite was by far the
most abundant in rat urine, whereas its presumed oxidation product 2-ethyl-3-
carboxypropyl phthalate predominated in the mouse urine.
G. SUMMARY
Studies with Wistar and F334 rats, B6C3F, mice, and cynomolgus monkeys
indicate that DEHA is readily absorbed and extensively metabolized following
oral administration of single doses as high as 5,000 mg/kg. Absorption via
the GI tract of mice is apparently faster in females than in males. In the GI
tract, DEHA is hydrolyzed to monoethyhexyl adipate (MEHA) and adipic acid (AA)
with the release of 2-ethoxyethanol (2-EH). Radiolabeled residues distribute
throughout the body, primarily in the form of AA and 2-EH, although other more
polar residues are also found. Following a single oral dose to Wistar rats of
500 mg/kg, tissue levels reach a maximum within 6 to 12 hours, with the
highest levels of radioactivity occurring in stomach, intestine, and adipose
tissue. Radiolabeled residues in tissue fall to low levels by 48 to 96 hours,
with no preferential retention in any tissue.
In mice, greater than 90% of the radioactivity administered is eliminated
from the body into the urine and expired air; very little is eliminated in the
feces or retained in the body. However, in F344 rats and monkeys, up to 20
and 40%, respectively, was found in the feces. Approximately 40 to 59% of the
AA is further metabolized, and the ["C]carbonyl moiety is eliminated in
expired air as carbon dioxide. Similarly, approximately 1 to 2% of the [I4C-
2-hexyl]DEHA is eliminated as carbon dioxide via expired air in mice and rats.
No 2-EH, 2-DEHA, MEHA, or oxidized metabolites of MEHA were detected in the
III-8
-------
urine of rats or mice, although small amounts (not specified) of 2-EH, OEHA,
and MEHA (but not the oxidation metabolites of MEHA) were found in the urine
of cynomolgus monkeys. Metabolites found in urine include AA and oxidation
products of 2-EH and/or their glucuronides.
The in vitro hydrolysis of selected plasticizers by rat gut homogenates
shows that DEHA and DEHT are largely converted to 2-EH and their diacids,
whereas OEHP is largely converted to 2-EH and mono(2-ethylhexyl)phthalate.
The half-lives for DEHA, DEHP, and DEHT disappearance, were 6, 12.6, and
53.2 minutes, respectively. The rates of hydrolysis of plasticizers in the
gut appear to affect the absorption of chemicals, qualitatively as well as
quantitatively.
III-9
-------
IV. HUMAN EXPOSURE
To be provided by the Science and Technology Branch, Office of Drinking
Water.
IV-1
-------
V. HEALTH EFFECTS IN ANIMALS
The health effects of di(2-ethy1hexyl)adipate (DEHA) and related
compounds have been reviewed by several Investigators (e.g., CTFA, 1984;
Felder et al., 1986; IARC, 1982; Jackh et al., 1984; Kluwe, 1986; Reddy
et al., 1986). Although the majority of data are on DEHA, limited studies on
diisopropyladipate (CTFA, 1984) and diisononyladipate (McKee et al., 1986)
have also been reported.
A. SHORT-TERM EXPOSURE
1. Acute Toxlcitv
Di(2-ethylhexyl)adipate is only slightly toxic, with LDMs reported in
the range of 9 to 46 g/kg. Smyth et al. (1951) reported the single oral LDSO
dose of DEHA in male Carworth-Wistar rats as 9.11 g/kg. The National
Toxicology Program (NTP, 1982) estimated the acute oral LDM in male and
female F344 rats to be 45 and 24.6 g/kg, respectively, and in male and female
B6C3F, mice to be 15 and 24.6 g/kg, respectively. Intraperitoneal
administration of DEHA at dose levels as high as 92 g/kg in male ICR/Harlan
albino mice and 46 g/kg in male Sprague-Dawley rats were reported as nonlethal
(Singh et al., 1973, 1975).
2. Subacute Toxicitv
The National Toxicology Program (NTP, 1982) reported the effects of
feeding DEHA for 14 days on body weight gain in rats and mice. Groups of five
male and five female F344 rats (137.5 and 103.5 g average initial weight,
respectively) or B6C3F, mice (23 and 18 g average initial body weight,
respectively) were fed diets containing various DEHA concentrations (3.1 to
100 g DEHA/kg food). Body weight gain was measured after 14 days. The
results are shown in Table V-l, and the estimated average daily doses supplied
by these diets are shown in Table V-2. Ingestion of diets containing 12.5 g
DEHA/kg food (estimated average daily intake of about 1.4 to 2.8 g/kg/day) or
less did not result in a significant decrease in body weight gain in either
species relative to control. However, ingestion of diets containing 25 g
V-l
-------
Table V-l. Body Weight Gain in Rats and Mice Fed Di(2-ethy1hexyl)adipate (DEHA) for
14 Days
OEHA in diet [q/kal*
Species
(sex)
Rat
(M)
Rat
(F)
House
(H)
House
m
Parameter
Initial wt (g)
Final »t (gj
Final wt (XC)e
Initial wt (g)
Final wt (g)
final Mt (XC)
Initial wt (g)
Final wt (g)
Final wt (XC)
Initial «t (g)
Final wt (g)
Final wt (XC)
0
132.5
150.0
[100]
103. 5
124.4
[100]
23.0
25. 2
[100]
18.0
18.0
[100]
3.1
132.5
190.0
127
—
--
23.0
25.2
100
,.
.-
6.3
132.5
182.6
122
103.5
121.4
98
23.0
25.6
102
18.0
20.0
106
12.5
132.5
177.8
, 119 •'
103.5
125.0
100
23.0
24.8
98
18.0
18.2
97
25
132.5
159.6
106
103.5
119.0
96
23.0
23.6
94
18.0
17.4
93
50
132.5
145.6*
97
103.5
109.6"
88
23.0
20.6 '
32
18.0
15.2
81
100
*•
—
103. S
82. Q6
66
..
--
18.0
11. 5"
61
'Data are the means for five animals. No estimate of variance was provided.
"Reduced food consumption was noted in these cases.
'Final body weight, expressed as the percent of the final body weight of control (untreated) animals.
SOURCE: Adapted from NTP (1982).
V-2
-------
Table V-2. Calculation of Average Daily Doses in Rats and Mice Fed
Di(2-ethylhexyl)adipate ;0£HA) for H Days
Species
(sex)
Rat (M)
Rat (F)
Mouse (M)
Mouse (F}
DEHA in
diet (g/kg)
3.1
6.3
12.5
25
50
6.3
12.5
25
50
100
3.1
6.3
12.5
25
50
6.3
12.5
25
50
100
Estimate
mean body
weight (kg)'
0.161
0.158
0.155
0.146
0.139
0.112
0.114
0.111
0.107
0.093
0.024
0.024
0.024
0.023
0.022
0.019
0.018
0.018
0.017
0.015
Assumed food
intake (g/day)'
.18
18
18
18
15e
13
13
13
12C
ir
4
4
4
4
4
4
4
4
4
3C
Calculated
dose
(mg/kg/day)
• 350
720
1,450
3,080
5,400
730
1,430
2,930
5,610
11,830
520
1,050
2,080
4,350
9,090
1,330
2,780
5,560
11,760
20,000
"Calculated as the mean of the initial and final body weights (see Table V-l)
"Food consumption for the rat from Altman and Dittmer (1974). Food
consumption for the mouse from Arrington (1972).
'Reduced food intake was observed in the animals as compared with control.
SOURCE: Adapted from NTP (1982).
V-3
-------
DEHA/kg food (estimated average daily intake of about 2.9 to 5.6 g/kg/day) and
above resulted in weight gain depression in male and female mice and in female
rats. No other observations were reported'in this study. NOAELs of 1.4 and
2.8 g/kg/day for rats and mice, respectively, were identified from this study.
Fassett (1963) described two unpublished studies conducted by Hodge at
the University of Rochester. In one study, DEHA was fed to rats at levels of
0.5, 2, or 5% of the diet for 1 month, which corresponds-to doses of 0.25, 1,
or 2.5 g OEHA/kg/day, assuming a body weight of 0.3 kg and a daily food
consumption of 15 g (Arrington, 1978). The only abnormality observed was a
retardation of growth at the 5% level (2.5 g DEHA/kg/day). No effects were
found at any dose-level in the blood or urine, and no histopathological
changes (tissues examined not specified) were observed. A NOAEL of 1 g/kg/day
has been identified from this study. In the second study, dogs were
administered 2 g DEHA/kg/day in the diet for 2 months. The only effect
observed was a transient loss of appetite. No changes were observed in the
blood or urine, and no histopathological changes (tissues examined not
specified) were noted.
3. Studieson Enzvme Induction
Reddy et al. (1986) compared the potency of DEHA,
di(2-ethylhexyl)phthalate (DEHP), and ciprofibrate to induce hepatic
peroxisomal proliferation when fed in the diet to groups of F344 male rats for
3 days. Dietary levels of DEHA and DEHP were 0.25, 0.5, 1, and 2%, which
correspond to 125, 250, 500, or 1,000 mg/kg/day assuming a body weight of
0.3 kg and a daily food consumption of 15 g (Arrington, 1978). Table V-3
presents the.results of changes in the catal.ase and peroxisomal
["C]palmitoyl-CoA oxidation system. The changes in the beta-oxidation system
appeared to parallel the alterations in peroxisome volume density. The
increase in specific activity of catalase was about twofold at a maximum level
of peroxisome proliferation. The increase in peroxisome proliferation was
associated with specific changes in the composition of hepatocyte proteins. Of
particular interest was the marked increase in the peroxisome proliferation
V-4
-------
Table V-3. The Effect of Dietary Ingestion of Di{2-ethy1hexyl)adipate
(DEHA) on Liver Catalase and Peroxisomal beta-Oxidation
Activities
Group
Dose
(% in diet)'
Catalase
(units/mg protein}8
[l-"C]Palmitoyl-CoA
oxidation
liver)"
Normal
42 ± 3
1.1 ± 0.01
Ciprofibrate
DEHP
DEHA
0.001
0.01
0.02
0.25
-0.50
1.0
2.0
0.25
0.50
1.0
2.0
52 + 2
76 + 3
98 + 3 .
48 + 3
55 + 2
. 58 + 3
70 + 3
46 + 3
49 + 2
57 + 4 -
63 T 3
5.8 + 2.5
12.9 + 0.5
14.6 + 0.2
4.4 +0.1
6:1 + 0.3
5.9 + 0.9
10.7 + 1.0
2.9 + 0.9
2.8 + 0.1
3.7 + 0.1
6.8 + 0.4
'DEHA was mixed in the powdered rat chow at the level (% w/w) indicated.
Male F344 rats were maintained on these diets for 3 days before sacrifice.
"The values are mean i SD of three to four animals in each group.
SOURCE: Adapted from Reddy et al. (1986).
V-5
-------
peroxisome proliferation which associates with an Increased amount of
polypeptide PPA-80 (molecular weight 80,000), a bifunctional protein of the •
peroxisomal fatty acid beta-oxidation system (Reddy et al., 1986). A dose-
dependent increase in the amount of PPA-80 was evident with ciprofibrate,
DEHP, and OEHA. Ciprofibrate was the most potent and OEHA the weakest inducer
among these. Peroxisome proliferation appeared to be tissue specific, limited
largely to hepatocytes. A correlation between the hepatocarcinogenicity
potency and the peroxisome proliferation, peroxisomal beta oxidation,.and
PPA-80 content was apparent. A LOAEL of 125 mg OEHA/kg/day has been
identified from this study, but a NOAEL was not established.
Kawashima et al. (1983a) fed a diet containing 2% DEHA to four male
Wistar rats (150 to 200 g) for 7 days. This corresponds to a dose of about
1.4 g/kg/day, assuming a mean body weight of 0.175 kg and daily food
consumption of 12 g (Arrington, 1978). The rate of stearoyl-coenzyme A (CoA)
desaturation in rat liver microsomes was increased (twice the control value),
in association with an increase in the activity of peroxisomal beta-oxidation
(five times the control value) and a slight increase in catalase activity (no
p values given). In a similar study (Kawashima et al., 1983b), male Wistar
rats (160 to 180 g) were fed a diet containing 2%.OEHA for 7 days. This
corresponds to a dose of 1.4 g/kg/day, assuming a mean body weight of 0.175 kg
and daily foo'd intake of 12 g (Arrington, 1978). Both the oleic acid-binding
capacity and fatty acid-binding protein content in the liver were increased
(twice the control value) in association with an increased peroxisomal beta-
oxidation activity (six times the control value; no p values given).
Increased levels-'of fatty acid-binding protein resulted in an increased uptake^
of fatty acids into the liver.
The effect of administering DEHA by gavage for 14 days (2 g/kg/day in
corn oil) on cellular hydrogen, peroxide levels in male Fischer 344 rats (250
to 350 g) and female 66C3F, mice (18 to 28 g) was studied by Tomaszewski
et al. (1986). The activity of enzymes responsible for the production
(peroxisomal palmitoyl CoA oxidase, PCO) and degradation (catalase, CAT;
glutathione peroxidase, GSHPx) of hydrogen peroxide was assayed in liver
homogenates prepared from the animals. The activities of the rat peroxisomal
enzymes PCO and CAT were enhanced fivefold and twofold (p <0.01),
V-6
-------
respectively, and the activity of GSHPx was decreased by 40% (not
statistically significant). In mice, OEHA enhanced the activities of the PCO
and CAT enzymes fourfold and twofold (p <0.01), respectively; the GSHPx
activity was not affected. The rates of formation of hydrogen peroxide by PCO
and degradation by CAT were used to estimate the steady-state hydrogen
peroxide concentration during peroxisomal oxidation of palnritoyl CoA. The
authors concluded that increases in peroxisomal steady-state hydrogen peroxide
concentration for F344 rat liver homogenate correlated well with the.
carcinogenic potential of the chemicals.
Moody and Reddy (1978) : jdied in the effects of five structurally
related plasticizers on hecanc enzyme induction. The plasticizers were
di(2-ethylhexylJphthalate (DEHP), di(2-ethylhexyl)adipate (DEHA),
di(2-ethylhexylJsebacate (DEHS), adipic acid, and diethylphthalate. They were
fed to male F344 rats (150 to 180 g) for 3 weeks at a dietary concentration of
2%. This corresponds to a dose of about 1.5 g/kg/day assuming an average body
weight of 0.165 kg and food consumption of 12 g/day (Arrington, 1978)..
Hepatic peroxisome proliferation was observed in three plasticizers: DEHP,
DEHA, and DEHS (Table V-4). this accompanied an increase in liver weight and
enhancement of hepatic activities of catalase and carnitine acetyltransferase.
The body weights were not affected. No change in peroxisome proliferation and
activity of catalase, an enzyme found in peroxisomes, occurred in the other .
two plasticizers (adipic acid and diethyl phthalate). Structurally DEHP,
DEHA, and DEHS contain an ester linkage to 2-ethylhexyl alcohol. It therefore
appears that the active moiety, in these plasticizers, that induces hepatic
peroxisome proliferation may be in part the metabolite 2-ethylhexyl alcohol. ^
This observation was confirmed in the feeding studies with 2-ethylhexyl
alcohol and 2 ethyl-hexanoic acid (Table V-4), which induced changes
comparable to those induced by DEHP, DEHA, and DEHS. From this study, a LOAEL
of 1.5 g/kg/day is identified for DEHA and is based on increased liver weight
(p - 0.001), and enhancement of catalase (p <0.001) and .carnitine acetyl
transferase activities (p <0.001).
Bell (1983) studied the effect of DEHA on hepatic synthesis of
cholesterol and phospholipids. Male rats (Upjohn:TUC(SD)spf) weighing 150 to
160 g were fed a diet containing either 0.5 or 1% DEHA for 14 days. This
V-7
-------
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corresponds to a dose of about 0.39 or 0.77 g/kg/day, respectively, assuming
an average body weight of 0.155 kg and daily food consumption of 12 g
{Arrington, 1978). After 2 weeks, the livers of the animals were removed and
minced, and the rates of incorporation of ["C]acetate or ["CJmevalonate into
squalene and cholesterol were measured. The results are shown in Table V-5.
Synthesis of squalene from either acetate or mevalonate was significantly
reduced in liver minces from animals receiving either the.low (0.5%) or high
(1%) dose. Cholesterol synthesis from acetate was reduced by 41% (not
statistically significant) at the low dose, and by 62% (p <0.001) at the high
dose. Cholesterol synthesis from mevalonate was not significantly affected at
either dose. The author speculated that OEHA affected sterol synthesis at a
site prior to mevalonate formation. The partial inhibition of mevalonate
incorporation may be explained if one or more of the enzymes between
mevalonate and squalene is substrate induced (and, hence, decreased in
activity as endogenous mevalonate synthesis is.reduced). Serum cholesterol
levels were decreased about 13% (p <0.02) in animals fed 1% DEHA and about 7%
(not statistically significant) in animals fed 0.5% DEHA diets. Serum x
triglyceride levels were not statistically different in animals fed either
total 0.5 or 1% DEHA in their diets. In rats fed 1% DEHA, incorporation of
["C]oleate or ["C]acetate into total liver phospholipids, triglycerides, and
steryl esters was not significantly affected, although there was a
redistribution of [14C]acetate incorporation into individual phospholipid
classes (a decrease in sphingomyelin, p <0.05; a decrease in phosphatidyl
choline, p <0.05; and an increase in phosphatidyl ethanolamine, p <0.01). A
NOAEL of 0.39 g/kg/day has been identified from this study.
Oi(2-ethylhexylJadipate has also been found to cause a decrease in serum '
cholesterol and triglycerides in rats. Moody and Reddy (1982) fed nine male
F344 rats (body weights not given) diets containing 2% DEHA for 3 weeks. This
corresponds to a dose of about 1.5 g/kg/day, assuming a mean body weight of
0.165 kg and food consumption of 12 g per day (Arrington, 1978). After
3 weeks, serum cholesterol levels in treated animals decreased by about 15%
(p <0.05) compared with the control, and serum triglyceride levels decreased
by >66% (p
-------
Table V-5. Effect of Dietary Di(2-ethylhexy1)adipate (DEHA) on Hepatic
Synthesis of Squalene and Cholesterol in Rats
DEHA
in diet (%}
0.5
1.0
Precursor
Acetate
Mevalonate
Acetate
Mevalonate
Amount
Squalene
20*
.55'
68'
of oroduct (% control )
Cholesterol
59
78
38C
98
'p
-------
Bell (1984) studied the effect of DEHA on plasma lipids and hepatic
cholesterogenesis in male rats (Upjohn.-TUC(SD)spf, 200 to 225 g) fed a diet
containing 1% DEHA for 7 weeks. This corresponds to a dose of 0.7 g/kg/day,
assuming an average body weight of 212.5 g and daily consumption of 15 g of
food (Arrington, 1978). The cholesterol level was lowered by 15% at weeks 2
and 5 (p <0.002), but normalized by week 7. Plasma triglycerides were within
normal range at week 2 but fell 40% by week 5 (p <0.05). By week 7, the
triglyceride levels were below the control, but this was not statistically
significant. Feeding DEHA in the diet significantly (p <0.05) reduced the
free plasma cholesterol; however, esterification by lecithin-cholesterol
acetyltransferase was not affected. The hepatic in vitro incorporation of
["C]octanoate into cholesterol was reduced about 66% (p <0.05) with DEHA
feeding, whereas incorporation into phospholipids, triglycerides, and free
fatty acids was not significantly affected. The author concluded that the
plasma cholesterol-lowering effect of DEHA seen at weeks 2 and 5 was not a
simple reflection of an inhibition of hepatic cholesterogenesis, since the
plasma cholesterol level normalized by week 7 despite the fact that sterol
synthesis was reduced by 50%. The author suggested that plasma excretion and
plasma lipoprotein turnover may be the factors involved.
B. LONG-TERM EXPOSURE
In a study by Smyth et al. (1951), Sherman rats (five/sex/group) were
administered DEHA in the diet at doses ranging from 0.16 to 4.74 g/kg for
90 days. At 4.74 g DEHA/kg/day, increased mortality (no p value given) was
observed. Animals receiving 2.92 g OEHA/kg/day exhibited reduced growth,
reduced appetite, altered liver or kidney weights, and microscopic lesions (no
details given) "in the liver, kidneys, spleen, or testes (no p value given for
any parameter).
DEHA/kg/day.
No effects were observed at or below doses^of 0.61 g
The National Toxicology Program (Mason Research Institute, 1977; NTP,
1982) reported the effect of feeding DEHA for 91 days on body weight gain in
rats and mice. Groups of 10 male and 10 female F344 rats (average initial
body weights of 80 and 71 g, respectively) or B6C3F, mice (average initial
body weights of 20.7 and 16.8 g, respectively) were fed various amounts of
DEHA (1.6 to 25 g/kg feed). The effect of this feeding regimen on body weight
gain is summarized in Table V-6, and the daily OEHA consumption is shown in
V-ll
-------
Table V-6. Body Weight Gain in Rats and Mice Fed
Di(2-ethylhexyl)adipate (DEHA) for 91 Days
Species
(sex) Parameter
DEHA in diet
1.6
3.1
6.3
12.5
25
Rat
(M)
Rat
(F-)
Mouse
(M)
Mouse
(H ,
^•••••••••^•••i
Initial wt (g)
Final wt (g)
Final wt (%C)b
Initial wt (g)
Final wt (g)
Final wt (%C)
Initial wt (g)
Final wt (g)
Final wt (%C)
Initial wt (g)
Final wt (g)
Final wt (%C)
80
342
[100]
71
193
[100]
20.2
33.3
[100]
16.8
25.3
[100].
™.^^^— «^— -
80
320
94
71
197
102
20.7
33.5
101
16.8
25.0
99
80
330
96
71
191
99
20.7
30.7
92
16.8
25.6
101
80
325
95
71
195
101
20.7
32.4
103'
16.8
21.5
85
80
312
91
71
186
96
20.7
31.8
95
16.8
25.8
102
80
296
87
71
183
95
20.7
30.5
92
16.8
24.2
96
'Data are the means for 10 animals; no estimate of variance was Prided.
'Final body weight, expressed as the percentage of the final body weight of
control (untreated) animals.
SOURCE: Adapted from NTP (1982).
V-12
-------
Table V-6. Body Weight Gain in Rats and Mice Fed
Di(2-ethylhexyl)adipate (DEHA) for 91 Days
Species
(sex) Parameter
DEHA in diet (g/kol1
1.6
3.1
6.3
12.5
25
Rat
(M)
Rat
(F)
Mouse
(M)
Mouse
(F)
Initial wt (g)
Final wt (g)
Final wt (%C)5
Initial wt (g)
Final wt (g)
Final wt (%C)
Initial wt (g)
Final wt (g)
Final wt (%C)
Initial wt (g)
Final wt (g)
Final wt (%C)
80
342
[100]
71
193
[100]
20.2
33.3
[100]
16.8
25.3
[100]
80
320
94
71
197
102
20.7
33,5
101
16.8
25.0
99
80
330
96
71
191
99
20.7
30.7
92
16.8
25.6
101
80
325
95
71
195
101
20.7
32.4
103
16.8
21.5
85
80
312
91
71
186
96
20.7
31.8
95
16.8
25.8
102
on
296
87
71
183
95
20.7
30.5
92
16.8
24.2
96
'Data are the means for 10 animals; no estimate of variance was provided.
"Final body weight, expressed as the percentage of the final body weight of
control (untreated) animals.
SOURCE: Adapted from NTP (1982).
V-12
-------
Table V-7. At 25 g DEHA/kg food, decreased weight gain was observed in rats
and mice of both sexes. At 12.5 g OEHA/kg diet, .a slight decrease in weight
gain was also seen in male and female rats and male mice. At 6.3 g DEHA/kg
diet, a slight decrease in weight gain was seen in male rats and female mice.
After 91 days, the animals were sacrificed and necropsied, and tissues were
preserved for histopathologic examination. No compound-related histopathology
was observed. Based on decreased weight gain, a NOAEL of about 400 mg DEHA/kg
body weight has been identified for rats and 1,300 mg DEHA/kg body, weight for
mice. These values are average values for both sexes and are based on food
consumption data.
The only chronic study of DEHA toxicity found in the literature was a
carcinogenicity study conducted by the National Toxicology Program (NTP,
1982). Groups of 50 male and 50 female F344 rats (5 weeks old) or B6C3F, mice
(6 weeks old) were fed diets containing 0, 12, or 25 g OEHA/kg food for
2 years. The estimated average daily DEHA consumption is summarized in
Table V-8. There was no dose-related effect of DEHA on longevity in any of
the four test groups. The effect of DEHA on body weight gain is shown in
Figures V-l and V-2. The high dose (25 g DEHA/kg food) produced a marked
decrease in body weight gain in both rats and mice, ranging from about 18 to
36% compared to control. The effect of the low dose (12 g OEHA/kg food) was
less dramatic, but an inhibition of body weight gain (about 7 to 18% compared
to control) was seen in both rats and mice. The daily intake at the low dose
was 0.82 g OEHA/kg body weight/day for rats and 2.65 g/kg/day for mice (both
sexes combined).. The LOAEL for rats was 0.82 g/kg/day.
£. REPROOUCTIVE/TERATOGENIC EFFECTS
Singh et al. (1973) administered DEHA and other adipate esters by ip
injection to groups of five Sprague-Dawley rats on days 5, 10, and 15 of
gestation; DEHA was given at dose levels of 1, 5, or 10 ml/kg (0.9, 4.6, or
9.2 g/kg), which were about 2, 5, or 10% of the acute oral LDSD values,
respectively. The results are summarized in Table V-9. No increase in
embryolethality occurred, but reduced fetal weight (p <0.05) was seen at the
two highest dose levels (4.6 and 9.2 g/kg). Except for one skeletal
V-13
-------
Table v-7. Daily Di(2-ethylhexyl)adipate (DEHAJ Consumption in
Rats and Mice Fed Diets Containing DEHA for 91 Days
" "• " - - !l
DEHA consumption (mg/kg/day/animal) . j
at dietary levels (a/kal of: i
Species (sex)
Rat (M)
Rat (F)
Mouse (M)
Mouse (F)
Study week
Week 4
Week 8
Week 12
Average
Week 4
Week 8
Week 12
Average
Week 4
Week '8
Week 12
Average
Week 4
Week 8
Week 12
Average
1.6
128
95
79
101
105
112
Ml
109
347
371
293
337
429
565
362
452
3.1
228
164
165'
186
241
237.
235
238
780
732
649
720
808
1,046
782
612
6.3
528
364
360
417
437
374
394
402
821
1,340
1,063
1,075
1,435
1,759
1,432
1,542
12.5
900
634
603
712
814
795
700
770
1,725
2,246
1,937
1,970
2,751
4,029
4,163
3,648
il
25 |!
- i
1,774 ' :t
1,314
1,314
1,467 j
1,726 i
1,589 i
1,437 ||
t
i , 584 ;i
4,701 |
6,265 |
4,382 |
5,116 (
7,105 :]
13,451 !|
6, '358 •••
it
8,971 l>
i
SOURCE: Adapted from Mason Research Institute (1977).
V-14
-------
Table V-8. Estimated Average Daily Consumption of Qi(2-ethylhexyl)adipate
(DEHA) in Rats and Mice in a 2-Year Study
DEHA in diet (a/kcrt
Species (sex)
Rat (M)
Rat (F)
Mouse (M)
Mouse (F)
Parameter*
Body weight (g)
Daily food consumption (g)
Daily DEHA intake (g/kg/day}
Body weight (g)
Daily food consumption (g)
Daily OEHA intake (g/kg/day)
Body weight (g)
Daily food consumption (g)
Daily DEHA intake .(g/kg/day)
Body weight (g)
Daily food consumption (g)
Daily DEHA intake (g/kg/day)
12
363.0 .
21.1
0.697'
240.0
17.2
0.860
38.6
7.8
2,425
31.8
7.7
2.905
25
318.0
19.2
• 1.509"
206.0
13.8
1.674
33.3
8.1
6.08
26.4
8.7
8.239
''Values represent the mean body weight, food consumption, and DEHA consumption
for the entire study (103 weeks).
SOURCE: Adapted from Mason Research Institute (1979).
V-15
-------
f
• s
83
' 0
e go o
A A
8
9
A
MAUMTV
i
TIMI ON tTWOT (WIIRSt
HMAUMfl
A
TMMI ON fTVOT (WE.
Figure V-l. Effect of di(2-ethylhexyl)adipate (D£HA) feeding on body weight
in F344 rats.
SOURCE: Adapted from NTP (1982).
V-16
-------
I
5
B
a e
0'
2
s
O A
A A A
8
MALI MtCI
O MiOBH
TIME ON tTVIOT (WIIKt)
S
i
!•
a i
00
DO
O O
.00 O
oooo
00
o o
a e o
o e
o o
0? 2 A A
KMALE MfCI
TIM! ON STVDT (Wf IKSI
Figure V-2. Effect of di(2-ethylhexyl)adipate (DEHA) feeding on body weight
in B6C3F, mice.
SOURCE: Adapted from NTP (1982).
V-17
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abnormality, no gross or visceral abnormalities were produced at the 1-ml/kg
level. Controls ("blunt needle") show one skeletal abnormality. Singh et al.
(1973) concluded that adipates are less teratogenic than the comparable
phthalates when administered during gestation in equivalent fractions of the
respective L0,0 doses. A NOAEL of 0.9 g/kg/day has been identified from this
study.
ICI (1988a) conducted a developmental .toxicity study in which groups of
24 pregnant female AlpkrApfSD (Wistar derived) rats received diets containing
0, 300, 1,800, or 12,000 ppm di(2-ethylhexyl)adipate (DEHA) from days 1 to 22
of gestation. Dosing was initiated on day 1 of gestation when body weights of
successfully mated females ranged from 218 to 278 g. The compound intakes
were approximately 28, 170, or 1,080 mg DEHA/kg/day in the low-, mid-, and
high-dose groups. No compound-related signs of toxicity were observed, i
Maternal mean body weight gain in dams receiving 12,000 ppm was significantly
(p <0.01) depressed throughout gestation; from days 1 to 7, mean weight gain
in high-dose dams was 22.4 g compared to 32.9 g for controls, and from days 1
to 22, mean weight.gain was 129.3 g compared to 148 g for controls. A slight
decrease in food consumption (6%) was also observed from days 1 to 21.
Table V-10 summarizes litter data. No effect was reported at any dose for
fetal weight, litter weight, gravid uterus weight, or numbers of intrauterine
deaths. A minimal increase in pre-implantation loss with an associated
decrease in litter size was seen at 12,000 ppm.
Major skeletal defects in offspring occurred at a low incidence and were
considered unrelated to dosing with DEHA. Seven fetuses from one control
female had multiple skull defects. Excluding these defects, one fetus in the
1800-ppm group and one fetus in the 12,000-ppm group had major defects of the
vertebral column. Major visceral defects included the following: absence of
adrenal glands, kidneys, and ureter in a control fetus; one fetus in the 300-
ppm group had a liver cyst, and another fetus had a small kidney; a fetus in
the 12,000-ppm group displayed situs inversus total is.
Minor skeletal defects and variants in ureter development were observed
with a slightly increased incidence in the 12,000-ppm group when compared to
V-20
-------
controls. Table V-ll summarizes the incidence of the reported changes. The
laboratory's classification of specific findings as minor defects and skeletal
variants was not clarified but it was sufficiently broad so that 96% of the
control fetuses exhibited one or more skeletal findings. The highest dose
level was associated with slight fetal toxicity evidenced by reduced
ossification in this group. Effects on ossification at 1800 ppm were not
considered of toxicologic importance. Although the litter incidence of
bipartite 5th sternebrae was significantly increased in all dosage groups
compared to concurrent control, the incidence in any group di'd not exceed that
of the recent laboratory historical control range of 6 to 16% on a fetus
basis. It was reported that the incidence of unilateral kinked ureter and
unilateral dilated ureter were increased in the mid- and high-dose groups.
However, only the litter incidence of slightly dilated ureter was increased
significantly (p <0.05) in the high-dose groups. Since the differences
between groups was slight, the toxicologic importance of this variation is
questionable.
The study demonstrates that at a dose resulting in slight maternal
effects, slight fetal effects were observed. There is no indication that OEHA
is a selective developmental toxicant. The LOAEL for both maternal and fetal
toxicity is 1,080 mg/kg/day, and the NOAEL is 170 mg/kg/day.
ICI (1988b) conducted a fertility study in which groups of 15 male and
30 female A1pk:ApfSD (Wistar derived) rats were fed diets containing 0, 300,
1,800, or 12,000 ppm di(2-ethylhexyl) adipate (DEHA) from weaning. Compound
intake in dams corresponded to approximately 28, 170, or 1,080 mg DEHA/kg/day
in the low-, mid-, and high-dose groups. After 10 weeks, the animals were
mated to produce a single litter, which was reared to day 36. Dosing was
continued throughout the study.
No effects on body weight or food consumption were observed in the
premating phase; however, mean weight gain during gestation was significantly
depressed in F0 females receiving 12,000 ppm (114.8 g compared to 127.4 g for
controls). A decrease in liver weights was observed at the 12,000-ppm dose in
V-22
-------
Table V-ll. Minor Abnormalities and Variants in Litters and Fetuses
in a Developmental Study in Rats Fed Di(2-ethylhexyl)adipate
(DEHA)
Site/Abnormality
No. of litters (% fetuses) affected
at dietary level
300
1,800
12,000
Parietals partially ossified,
unilateral
7 (3.9) 3 (3.0)
8 (7.9*) 10 (9.9*)
Cervical vertebrae (M)*
5th - Centrum not ossified
W
7th - Transverse process,
partially ossified (V)
Sternebrae
5th - Bipartite (M)
Ventral tubercle, not ossified
(M)
Ureter
Kinked (V)
Dilated (slight, unilateral)
(V)
Dilated (moderate, unilateral)
(M)
3
19
6
7
12
12
2
(1.
1)
(23)
(5.
(3.
(7.
(20
(2.
7)
9)
8)
.2)
1)
4
19
14*
5
14
16
5
(1.
(22
5)
•4)
(10.6)
(2-
(9.
(21
(2.
3)
1)
.3)
3)
4
22
(1.
(31
8)
-7*)
15** (9.0)
7
13
17
6
(2.
(11
(21
(3-
9)
•9)
•2)
2)
5
22
15
12
16
19
2
(4.5*)
(38.7*)
** (15.6)
(10.7*)
(12.3)
* (26.3)
(0.8)
*V = variation; M » minor abnormality.
*Significantly different from control value, p <0.05.
**Significantly different from control value, p <0.01.
V-23
-------
male parents postmating and in female parents after weaning their litters
(Table V-12). No effects of dosing on male or female fertility were observed.
The.pre-coital interval was between 2.73 and 3.06 days in all groups and the
gestational length between 22.1 and 22.3 days.
The number of live pups was unaffected, ranging from 95.8 -to 99.3% in all
groups, and there was no effect on pup 21-day survival when whole litter
losses were excluded. However, the mean litter size 'in the 12,000-ppm group
(9.8) was slightly smaller than in the,control group (10.9) or the low- cr
mid-dose groups (10.8 and 10.3, respectively). Mean birth weight of pups did
not differ between groups (5.9 to 6.1 g in males and 5.5 to 5.7 g in females),
but pup weight gain from day 1 to 36 in the 12,000-ppm group was significantly
(p <0.01) lower than control gain. Table V-13 summarizes weight gain data for
dams and pups... The results of this study support the NOAEL of 170 mg/kg/day
and the LOAEL of 1,080 mg/kg/day in the ICI (1988a) developmental toxicity
study.
0. MUTAGEN1CITY
OEHA was negative in a battery of genetic toxicity assays including the
Ames, mouse lymphoma L5178Y, rat hepatocyte unscheduled DMA synthesis, mouse
micronucleus, and BALB 3T3 transformation assays (Barber et al., 1985; Simmon
et al., 1977; Zeiger et al., 1982). DEHA did not bind covalently to DNA in
the livers of female mice (Von Oaniken et al., 1984), and no evidence of
rnutagenic activity was found in the urine of rats after oral dosing with DEHA
(DiVincenzo et al.," 1985).
Singh et al. (1975) conducted a dominant lethal test in Harlan/ICR albino
Swiss mice. Groups of 10 males received single intraperitoneal injections of
0, 0.45, 0.9, 4.6, or 9.2 g DEHA/kg body weight, and each male was caged with
2 different female mice each week for 8 weeks. Pregnant females were
sacrificed on gestation days 15 to 17; and corpora lutea, total implantations,
pre-implantation losses, early and late fetal deaths, and viable fetuses were
scored. In the high dose, there was a significant increase in early fetal
deaths, indicating dominant lethal mutations. Mean incidences of early fetal
V-24
-------
Table V-12. Mean Liver Weights in F8 Males and Females Fed
Di(2-ethylhexyl)adipate(DEHA) in a Developmental
Toxicity Study
Sex
Mean liver weights fq) at dietary levels fppm)
0 300 1,800 12,000
Males
Females
19.1
13.2
19.7
13.2
19.5
13.3
22.5**
15.5**
**Significantly different from control value, p <0.01.
V-25
-------
Table V-13. Cumulative Mean Weight Gain in Pregnant.Females and Their Pups
in a Reproductive Toxicity Study in Rats Fed
Di(2-ethylhexyl)adipate. (DEHA)
0
Gestation Dav
8 24.6
15 59.1
22 127.4
Lactation Dav
11 .12.4
22 33.2
36 115.2
11 11.8
22 31.7
36 . 103.0
Mean weiaht(s)
300
26.5
60.7
131.1
11.9
33.7
113.6
11.7
32.4
100.5
aains at dietary
1,800
F. Females
26.5
59.1
123.3
F. Males
1
13.1
35.6
118.2
F. Females
12.6
34.2
104.5
levels (pom)
12,000
21.2
50.4**
114.8**
10.5**
29.3*
100.9**
. 10.5*
28.7
93.0**
*Significantly different from control value, p <0.05.
**Significantly different from control value, p <0,01.
V-26
-------
deaths/pregnancy were as follows: 0.29, 0.39, 0.48, 0.74, and 0.96 at doses
of 0, 0.45, 0.9, 4.6, or 9.,2 g DEHA/kg body weight. A dose-related decrease
in fertility was also reported (67% pregnancies at the high dose as compared
to 82% in controls).
The genotoxic potential of diisononyladipate was evaluated by McKee
et al. (1986) in a battery of in vitro tests. Diisononyladipate did not
exhibit any evidence of mutagenic or transforming potential in the •
Salmonella/ mammalian microsome mutagenicity assay, the mouse lymphoma.TK +/-
assay, and two tests of morphologic transforming ability (the BAL8 3T3 and the
Syrian hamster embryo in vitro assays).
Jones et al. (1975) studied the effect of DEHA on the human diploid cell
strain WI-38. The dose that causes 50% growth inhibition in tissue culture
(IDSO) was found to be 32 pM (11.9 mg/L). The authors concluded that DEHA has
a high intrinsic toxicity. Ekwall et al. (1982) studied the toxicity of DEHA
to HeLa cells using the Metabolic Inhibition Test supplemented by microscopy
of cells after a ?.*•• . ;r incubation period (known as the MIT-24 test system).
The 7-day IC50 (50% inhibitory concentration based on two data points only,
total and partial inhibition) was 310 mM (115 g/L), which is considered to be
highly nontoxic to these cells. Ekwall et al. suggested that this low
toxicity may be due to the insolubility of DEHA in water. The authors also
addressed the discrepancy between their results and those of Jones et al.
(1975). They suggested that the high intrinsic toxicity 'to human diploid
cells observed b;, r.-rs et a.l, may be due to the use of a supersaturated
solution, long stirring with serum and medium prior to the study (which may
have led to the production of more toxic metabolites), and a longer incubation
time (9 days).
E. CARCINOGENICITY
The National Toxicology Program (NTP,. 1982) evaluated the carcinogenic
potential of DEHA in mice. Two groups of 50 male and 50 female 86C3F, mice,
initially 6 weeks old, were fed diets containing 12 or 25.g DEHA/kg food for
103 weeks. Control groups of 50 male and 50 female.mice were included. Two
V-27
-------
batches of OEHA were used, one 98.1% and the other 99.7% pure. The former
contained at least seven unidentified organic Impurities, and the latter
contained five impurities identified by gas-liquid chromatography (GLC). The
estimated average dally consumption of DEHA is listed in Table V-8. Survival
rates at the end of the study (105 to 106 weeks) ranged from 64 to 84%.
Except for liver tumors, no tumors at other sites were statistically
increased from controls. The incidences of total hepatocellular tumors,
adenomas, and carcinomas as a function of dose are shown in Table V-14.
In male mice, a statistically significant increase in hepatocellular
adenomas, but not carcinomas, was observed at the high-dose level (p = 0.021),
and there was a postive dose-related trend (p » 0.013). The combined adenoma
or carcinoma incidences were also significantly greater than controls at the
high dose (p = 0.003), and there was a dose-related linear trend (p = 0.002).
Although the frequency of total hepatocellular tumors in male mice increased
from 26% (control) to 41% (12 g/kg dose) or 55% (25 g/kg dose), this increase
could not be clearly attributed to compound administration when compared with
the incidence in historical laboratory controls, (116/398, 29.1%). Time-to-
tumor analysis did not show a significant difference between control and dose
groups. Based on .the lack of an increase in hepatocellular carcinomas and the
lack of significance of the adenoma increase when compared with historical
controls, the results in male mice are considered equivocal.
In female mice, there was a significant increase (p <0.001) in hepatocel-
lular carcinomas'and in combined hepatocellular adenomas and carcinomas at *•
both dose levels. There was also an increase in hepatocellular adenoma
frequency, but this was not statistically significant at either dose.
Overall, the frequency of total hepatocellular tumors in female mice
increased from 6% (control) to 38% (12 g/kg dose) or 37% (25 g/kg dose).
Laboratory historical control incidence was 31/397 (7.8%). In females, times
to observation of the first hepatocellular tumors were 106, 85, and 79 weeks
in the control, low-, and high-dose groups, respectively.
V-28
-------
Table V-14. Incidence of Hepatocellular Tumors in Mice
Fed Diets Containing Di(2-ethylhexyl)adipate
(DEHA) for 2 Years
Sex
Males
Males
Males
Females
Females
Females
OEHA
(ppm in feed)
0
12,000
25,000
0
12,000
25,000
Total No.
of tumors1
13/50 (26)
p=0.002"
20/49 (41)
N.S.
27/49 (55)
p=0,003
3/50 (6)
p=0.001
19/50 (38)
p <0.001
18/49 (37)
p <0.001
' ' No. Of
adenomas
6/50 (12)
p-0.013
8/49 (16)
N.S.
15/49 (31)
p=0.021
2/50 (4)
N.S.
5/50 (10)
N.S.
6/49 (12)
N.S.
NO. Of
. carcinomas
7/50 (14)
N.S.
12/49 (24)
N.S.
12/49 (24)
N.S.
1/50 (2)
p=0.003
14/50 (28)
p cO.OOl
12/49 (24)
p=0.001
'Number of hepatocellular tumor-bearing animals/number of animals examined at
site (%}.
"Beneath the incidence of tumors in the control group is the probability level
for the Cochran-Armitage test when the p value is less than 0.05; otherwise,
"not significant" (N.S.) is indicated. Beneath the incidence of tumors in a
dosed group is the probability level for the Fisher exact test for the
comparison of that dosed group with the control group when the p value is
less than 0.05; otherwise, N.S. is indicated.
-SOURCE: Adapted from NTP (1982).
V-29
-------
No evidence of carcinogenicity was observed in a companion study with
rats (NTP, 1982). Three groups of 5-week-old F344 rats, 50 males and
50 females per group, were fed diets containing 0, 12, or 25 g DEHA/kg food
(the same purity as in the mouse studies described in the preceding paragraph)
for 103 weeks. Estimated average daily DEHA consumption is shown in
Table V-8. Survival at 105 to 107 weeks was 68% in male controls and low-dose
males, and 80% in high-dose males; and 58% in control females, 78% in low-dose
females, and 88% in high-dose females. There was no difference in the
incidence or type of tumors between treated and control animals that could be
attributed to DEHA administration. The most common tumors were myelomonocytic
leukemia, lymphoma, pituitary tumors, and interstitial cell tumors of the
testes (NTP, 1982),
Four compounds containing the 2-ethylhexyl moiety (DEHP, OEHA,
tris(2-ethyl-hexyl)phthalate (TEHP), and 2-ethylhexyl sulfate (EHS) have
exhibited evidence of liver carcinogenicity in rodents (Kluwe, 1986; Kluwe
et al., 1985). The studies were performed with F344 rats and B6C3F, mice and
include those conducted with DEHA (NTP, 1982). - All four chemicals exhibited
evidence of hepatocarcinogenicity ranging from very strong (DEHP) to equivocal
(EHS). The data in Table V-15 indicate a greater, relative susceptibility in
mice than in rats and of females than of males to the hepatocarcinogenic
activity of the 2-ethylhexyl-containing chemicals. Those compounds exhibiting
the strongest carcinogenic activity in female mice (DEHP, DEHA) were also
carcinogenic in male mice. The strongest carcinogen in mice (DEHP) was also
carcinogenic in rats. Based on these sex and species differences, the authors
concluded that the carcinogenic activity of this group of compounds is of a
quantitative rather than of a qualitative nature; i.e., the compounds elicit
the same type of tumorigenic response but to varying degrees.
In contrast, when the toxic manifestations and/or carcinogenic activity
of several phthalic acid esters (e.g., phthalic anhydride, di(2-ethylhexyl)-
phthalate, butyl benzylphthalate, diallylphthalate) in the same species were
analyzed, the target sites for such effects were dissimilar (Kluwe, 1986).
The author reported that these findings suggest the absence of a common mode
of action among these phthalic acid esters. Kluwe (1986) and Kluwe et al.
V-30
-------
Table V-15. Comparative Effects of Feeding of Compounds With a 2-Ethylhexyl
Moiety on the Occurrence of Hepatocellular Tumors in Rats
and Mice1
Rats
Type of
hepatocellular
tumor
All (combined)'
Carcinomas
Males
Test
compound"
DEHP
DEHA
TEHP
2-EHS
DEHP
DEHA
TEHP
2-EHS
Low
dose
m
-
.
-
.
-
-
•
High
dose
+
-
-
-
+
-
-
~
Females
Low
dose
+
-
-
-
.
-
-
•
High
dose
+-H-
-
-
-
++
-
-
•
Mice
Males
Low High
dose dose
•*• ++
++
.
-
+
-
-
-
Females
Low High
dose dose
•H-+ ++++
+++ +++
+
+
•H- ++++
•»•++ ++
+
i
*The level of statistical significance is as follows: -, p >0-05; +, p <0.05; +,
0.01
-------
(1985) concluded that the 2-ethylhexyl mo.iety may have a propensity for
causing hepatocarcinogenesis in female mice.
F. SUMMARY
The acute oral toxicity of DEHA is low, with LD50 estimates in rats and
mice ranging from 9 to 45 g/kg. Short-term feeding studies (<30 days)
indicate that rats and mice gain weight normally when given diets containing
1.2% DEHA or less (average daily dose of about 1 to 2 g/kg/day). Diets with
higher levels may cause decreased weight gain or weight loss. Long-term
feeding studies (>30 days) suggest that diets containing 1.2% (12 g/kg food)
DEHA (average dose of about 0.6 to 1.2 g/kg/day) lead to decreased weight gain
in rats and mice. The low toxic potency of the compound allowed for relatively
high doses to be given during the chronic studies.
In a developmental toxicity feeding study in pregnant Wistar rats,
dietary administration of 12,000 ppm DEHA (corresponding to a daily dose of
1,080 mg/kg) during gestation resulted in slight reductions in maternal weight
gain. Fetal toxicity was also seen at this dose as well as reduced
ossification and variations in ureter development. The LOAEL for maternal and
fetal toxicity corresponded to a DEHA intake of 1,080 mg/kg/day, and the NOAEL
corresponded to 170 mg/kg/day. DEHA did not affect fertility in a one-
generation reproductive toxicity study at the same dietary levels as in the
developmental study or did it alter any reproductive parameters, but it caused
reduced maternal and offspring weight gain at 1,080 mg/kg/day, supporting the
developmental toxicity NOAEL of 170 mg/kg/day.
DEHA feeding results in several changes in the liver. Doses of 0.25 or
0.5 g/kg/day for 14 days produced a decrease in cholesterogenesis and altered
phospholipid synthesis in rat liver, accompanied by decreased serum
cholesterol levels. Doses of about 1.5 g/kg/day for 7 to 21 days caused
peroxisome proliferation and hepatic enlargement in rats. Structure-activity
relationships indicate that these effects are strongly associated with the
2-ethylhexyl moiety.
V-32
-------
In recent years, peroxisome production and induction of peroxisome-
associated enzymes in the livers of rodents exposed to OEHA and related
compounds have been extensively studied. The induction of peroxisomes is
associated with a several-fold increase in the activity of the peroxisomal
fatty acid beta-oxidation system and a twofold increase in catalase activity.
In addition, long-term exposure to peroxisome proliferators may result in the
induction of hepatocellular carcinomas in rats and mice. The lack of
mutagenicity of these agents, combined with consistent findings of
proliferation of hydrogen peroxide-generating peroxisomes, indicates that
persistent proliferation of peroxisomes serves as an endogenous initiator of
neoplastic transformation by enhancing oxidative stress.
OEHA was negative in a battery of genetic toxicity assays, including the
Ames test, mouse lymphoma test, rat unscheduled DNA synthesis, the mouse
micronucleus test, and BALB 3T3 transformation assays. It was positive in a
dominant lethal test in mice. In a 2-year carcinogenicity feeding study in
B6C3F1 mice, doses of 2.9 or 8.2 g DEHA/kg body weight caused an increase in
hepatocellular adenomas and carcinomas (combined) in females, but no
significant corresponding effects were found in male mice. In a similar study
in F344 rats, no carcinogenic response was observed.
V-33
-------
VI. HEALTH EFFECTS IN HUMANS
No studies were found in the available literature on the effects of oral
ingestion of DEHA in humans. Malette et al. (1952) reported that pure DEHA
did not cause skin Irritation or sensitization in 15 to 30 subjects (the exact
number is unclear from the study).
VI-1
-------
VII. MECHANISMS OF TOXICITY
A. PEROXISOME PROLIFERATION
Di(2-ethy1hexyl)adipate (DEHA) induces hepatic peroxisomal proliferation
(Moody and Reddy, 1978; Kawashima et al. 1983a,b; Reddy et al., 1986;
Tomaszewski et al., 1986). Hepatic peroxisomal proliferation -induced by a
variety of compounds has been associated with hepatic cancer in several
studies (e.g., Ames, 1983; Reddy et al., 1986). No definitive mechanistic
link has been proven between peroxisome proliferation and cancer or any other
form of toxicity, but it has been proposed that peroxisome proliferation is
associated with formation of increased amounts of reactive oxygen species that
could damage critical macromolecules such as ONA (Ames, 1983; Tomaszewski
et al., 1986).
8. ACTIVITY OF HYDROLYSIS PRODUCTS
The toxic effects of DEHA may be related to the two main metabolites,
adipic acid (AA) and 2-ethylhexanol (2-EH), which are formed during
hydrolysis. Di(2-ethylhexy1)adipate is rapidly hydrolyzed in the stomach to
monoethylhexyladipate (MEHA) and to AA with the release of two molecules of 2-
ethylhexanol (Takahashi et al., 1981). Moody and Reddy (1978, 1982) compared
the effects of DEHA, AA, and 2-ethylhexyl alcohol on a number of parameters
related to liver function. Male F344 rats (150 to 180 g) were fed diets
containing 2% DEHA,. AA, or 2-ethylhexyl alcohol for 3 weeks. These diets did
not cause any significant change in weight gain over the 3-week period. A
number of parameters related to liver function were measured, and the results
are shown in Table VII-1. As discussed in Chapter V, DEHA feeding results in
proliferation of hepatic peroxisomes accompanied by increased liver weight,
increased peroxisomal enzymes, and decreased serum levels of cholesterol and
triglyceride. This pattern of effects is closely mimicked by 2-ethylhexyl
alcohol feeding, but it is not mimicked by AA feeding. Moreover,
di(2-ethylhexyl)phthalate and di(2ethylhexyl)sebacate also produced a pattern
of change similar to DEHA. The authors concluded that 2-ethylhexyl alcohol is
the active part of the molecule responsible for peroxisome proliferation. .
VII-1
-------
fable VIM. Effect of Feeding Di(2-ethylhexyl)adipate (DEHA) or Its
Metabolites on Liver Function in Rats
Parameter
Liver weight
(% body weight)- -
Liver catalase
(units/mg protein)
Liver carnitine
acetyltransferase
(units/mg protein) .
Hepatic peroxisome
prol iferation
Serum cholesterol
(mg/100 mL)
Serum triglyceride
(mg/100 mL)
None
3;8+0.05
(13)
44.0+2.7
(13).
2.7+0.5
(13) '
_
(13)
46.1+4.8
(13)
114.8+17.8
(13)
Additions
2% DEHA
5.6+0.08'
(8)
86.0+6.1"
(8)
30.7+3.0*
18)
++++
(8)
39.4+3.3'
(9)
39.0+6.9*
(9)
to the diet
2% Ethyl/
2% 2-Adipic hexyl
acid alcohol
3.9+0.02
"(4)
41.0+1.9
(4)
2.0+0.0
"(4)
•
(4)
52.8+3.3*
(4)
119.6+24.4
(4)
' 4.9+0.1"
(5)
63.0+5.2'
(5)
40.1+3.1"
(5)
++++
(5)
40.0+4.6*
(5)
59.2+23.9'
("5)
''All values shown are the mean + SE, with the number of experimental animals
shown in parentheses.
V<0.001.
cp <0.01.
"The ratio between mitochondria and peroxisomes in normal rat liver cells is
approximately 5:.l. The peroxisome proliferation was assessed semiquantita-
tively by determining the mitochondria!-peroxisome ratio: -, 5:1 (normal);
++++, 1:1 or more.
-------
c.
:GISM AND ANTAGONISM
. No information was found in the available literature on the interaction
of OEHA with other chemicals.
0. SUMMARY
The mechanism of OEHA action in biological systems is not entirely-known.
It has been proposed that OEHA may act by inducing peroxisome proliferation in
the liver. It appears that 2-ethylhexyl alcohol is the active moiety with • .
regard to this effect. It has been suggested that peroxisome proliferation -
leads to . ~:uction of excess amounts of reactive oxygen species, which may.
damage critical macromolecules such as ONA. .
In recent years, peroxisome production and induction of peroxisome- .
associated enzymes in the livers of rodents exposed to OEHA and related .
compounds have been extensively studied. The induction of peroxisomes is
associated with a several-fold increase in the activity of the peroxisomal
fatty acid beta-oxidation system and a twofold increase in catalase activity.
In addition, long-term exposure to these peroxisome proliferators results in
the induction of hepatocellular carcinomas in rats and mice. The lack of
mutagenicity of these agents, combined with consistent findings of
proliferation of hydrogen peroxide-generating peroxisomes, -indicates that
persistent proliferation of peroxisomes serves as an endogenous initiator of
neoplastic transformation by enhancing oxidative stress.
VII-3 .
-------
VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS
The quantification of lexicological effects of a chemical consists of an
assessment of noncarcinogenic and carcinogenic effects. Chemicals that do not
produce carcinogenic effects are believed to have a threshold dose below which
no adverse, noncarcinogenic health effects occur, whereas carcinogens are
assumed to act without a threshold.
A. PROCEDURES FOR QUANTIFICATION OF TOXICOLOGICAL EFFECTS
1. Noncarcinogenic Effects
In the quantification of noncarcinogenic effects, a Reference Dose (RfD},
formerly called the Acceptable Daily Intake (ADI), is calculated. The RfD is
an estimate of a daily exposure of the human population that is likely to be
without appreciable risk of deleterious health effects, even if exposure
occurs over a lifetime. The RfD is derived from a No-Observed-Adverse-Effect
LeveT (NOAEL)/ or Lowest-Observed-Adverse-Effect Level (LOAEL), identified
from a subchronic or chronic study, and divided by an uncertainty factor (UF).
The RfD is calculated as follows:
RfD - (NOAEL or LQAEL) « mg/kg bw/day
Uncertainty factor
Selection of the uncertainty factor to be employed in the calculation of
the RfD is based.'on.professional judgment while considering the entire data
base of toxicological effects for the chemical. To ensure that uncertainty
factors are selected and applied in a consistent manner, the Office of Water
(OW) employs a modification to the guidelines proposed by the National Academy
of Sciences (NAS, 1977, 1980) as follows:
• An uncertainty factor of 10 is generally used when good chronic or
subchronic human exposure data identifying a NOAEL are available and
are supported by good chronic or subchronic toxicity data in other
species. '
VIII-1
-------
• An uncertainty factor of 100 is generally used when good chronic
toxicity data identifying a NOAEL are available for one or more
animal species (and human data are not available), or when good .
chronic or subchronic toxicity data identifying a LOAEL in humans are
available.
• An uncertainty factor of 1,000 is generally used when limited or
incomplete chronic or subchronic toxicity data are available, or when
good chronic or subchronic toxicity data identifying a LOAEL, but not
a NOAEL, for one or more animal species are available..
The uncertainty factor used for a specific risk assessment is based
principally on scientific judgment; rather than scientific fact, and accounts
for possible intra- and interspecies differences. Additional considerations
v
not incorporated in the NAS/OW 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 effect, and the counterbalancing of
beneficial effects.
From the RfD, a Drinking Water Equivalent Level (DWEL) can be calculated.
The DWEL represents.a medium-specific (i.e., drinking water) lifetime exposure
at which adverse, noncarcinogenic health effects are not expected to occur.
The DWEL assumes 100% exposure from drinking water. The DWEL provides the
noncarcinogenic health effects basis for establishing a drinking water
standard. From i.ngestion data, the DWEL is derived as follows:
DWEL = RfD x (body weight in ko.)
Drinking water volume in L/day
mg/L (.
where: ' ~
Body weight » assumed to be 70 kg for an adult.
Drinking water volume - assumed to be 2 L per day for an adult.
;t
i
i
In addition to the RfD.and the DWEL, Health Advisories (HAs) for
exposures of shorter duration (One-day, Ten-day, and Longer-term HAs) are
determined. The HA values are used as informal guidance to municipalities and
VIII-2
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other organizations when emergency spills or contamination situations occur.
The HAs are calculated using a similar equation to the RfD and DUEL; however,
the NOAELs or LOAELs are identified from acute or subchronic studies. The HAs
are derived as follows:
HA - (NOAEL or LOAEL1 x fbw) = rag/L ( jig/L)
( L/day) x (UF)
"Using the above equation, the following drinking water HAs are'developed'
for noncarcinogenic effects:
1. One-day HA for a 10-kg child ingesting 1 L water per day.
2. Ten-day HA for a 10-kg child ingesting 1 L water per day.
3. Longer-term HA for a 10-kg child ingesting 1 L water per day.
4. Longer-term HA for a 70-kg adult ingesting 2 L water per day.
The One-day HA calculated for a 10-kg child assumes a single acute expo-
sure to the chemical and is generally derived from a study of less than
7 days' duration. The Ten-day HA assumes a limited exposure period of 1 to
2 weeks and is generally derived from a study of less than 30 days' duration.
A Longer-term HA is derived for both a 10-kg child and a 70-kg adult and
assumes an exposure period of approximately 7 years (or 10% of an individual's
lifetime.). A Longer-term HA is generally derived from a study of subchronic
duration (exposure for 10% of an animal's lifetime).
•*
2. Carcinogenic Effects *
The EPA categorizes the carcinogenic potential of a chemical, based on
the overall weight of evidence, according to the following scheme:
• Group A: Known Human Carcinogen. Sufficient evidence exists from
epidemiology studies-to support a causal association
between exposure to the chemical and human cancer.
VIII-3
-------
t Group B: Probable Human Carcinogen. Sufficient evidence of
carcinogenicity in animals with limited (Group Bl) or
inadequate (Group 62} evidence in humans.
• Group C: Possible Human Carcinogen. Limited evidence of
carcinogenicity in animals in the absence of human data.
• Group D: Not Classified as to Human Carcinogenicitv. Inadequate
human and animal evidence of carcinogenicity or for which
no data are available.
• Group £: Evidence of Noncarcinogenicitv for Humans. No evidence of
carcinogenicity in at least two adequate animal tests in
different species or in both adequate epidemiologic and
animal studies.
If toxicological evidence leads to the classification of the contaminant
as a known, probable, or possible human carcinogen, mathematical models are
used to calculate the estimate of excess cancer risk associated with the
ingestion of the contaminant in drinking water. The data used in these
estimates usually come from lifetime exposure studies in animals. To predict
the risk for humans from animal data, animal doses must be converted to
equivalent human doses. This conversion includes correction for noncontiguous
exposure, less-than^lifetime studies, and for differences in size. The factor
that compensates, for the size difference is the cube root of the ratio of the
animal and human body weights. It is assumed that the average adult human
"body weight is 70 kg and that the average water consumption of an adult human
is 2 liters 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 via
ingestion of water. The cancer unit risk is usually derived from a linearized
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
VIII-4
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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 biological mechanisms involved in cancer to suggest that
any one of these models is able to predict risk more accurately than any
others. Because each model is based on differing assumptions, the estimates
that are 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 due to the systematic and
random errors in scientific measurement. In most cases, only studies using
laboratory animals have been performed. Thus, there is 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 in drinking water; the impact of
the laboratory 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
laboratory animals or humans. Dose-response data usually are available only
for high levels of exposure, not for the lower levels of exposure closer to
where a standard may be set. When there is exposure to more than one contami-
nant, additional uncertainty results from a lack of information about possible
synergistic or antagonistic effects.
8. QUANTIFICATION OF NONCARCIMOGEN1C EFFECTS FOR ADIPATES
Identifying the most appropriate studies for calculation of the One-day,
Ten-day, and Longer-term Health Advisories (HAs) depends on which effect of
DEHA is selected as the most appropriate index of DEHA toxicity.
Although DEHA has been shown to decrease the rates of synthesis of
cholesterol and triglyceride in the liver (Bell, 1983, 1984; Moody and Reddy,
1982), this is not considered a primary toxic effect.
Several studies (e.g., Smyth et al., 1951; NTP, 1982) have shown that
DEHA administered in the diet results in decreased weight gain, although it
is not clear whether this is a primary toxic effect or is secondary to reduced
food intake.
VIII-5
-------
1. One-day Health Advisory for 'a Child
No studies of DEHA acute oral toxicity (other than lethality) were found
in the available literature. The studies by Singh et al. (1973, 1975} were
not selected because the exposure route was via ip injection. In the absence
of a Ten-day HA, the Longer-term HA value will be used as a conservative
estimate of the One-day HA.
2. Ten-dav Health Advisory for Children and Adults
The studies considered for calculation of the Ten-day HA values for OEHA
are summarized in Table VIII-1. None of the studies were suitable for cal-
culating the Ten-day HA. Kawashima et al. (1983a) fed a diet containing OEHA
(1,400 mg/kg) to rats for 7 days and measured the rate of stearoylcoenzyme A
desaturation in rat liver microsomes, peroxisomal beta-oxidation activity, and
catalase activity. This study was not selected for derivation of the Ten-day
HA value because the observations are based on a single dose. Moreover, the
toxicological significance of endpoints such as enzyme induction is uncertain.
Similarly, a 14-day feeding study in rats by Bell (1983) was not selected
because the endpoint studied was cholesterol synthesis, the toxicological
significance of which is uncertain.
NTP- (1982) conducted a 14-day dose-response study in rats and mice fed
DEHA at doses ranging from 2.1 to 2.8 g/kg/day in mice and from 1.4 to
1.5 g/kg/day in.rats. This study was not selected because the only endpoint •
studied was body weight. No histopathology was conducted.
The 1-month feeding study with rats described by Fassett (1963) was not
selected because very limited information was presented. The only effect
observed was growth retardation. No histopathological changes were found, but
tissues examined were not specified.
In a 3-week feeding study by Moody and Reddy (1978), DEHA was fed to male
rats at a dose of 1.5 g/kg/day for 3 weeks and its effects on the liver were
examined. After 3 weeks, significant increases in liver weight (p • 0.001),
and catalase (p = <6.001) and carnitine acetyltransferase activities
VIII-6
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{p <0.001) were observed. At this dose, no effects on body weight were
observed. The LOAEL of 1,500 mg/kg/day identified from this study is much
less than the NOAEL of 2,800 mg/kg/day identified in the NTP (1983) study
reported above. However, the study by Moody and Reddy (1978) is based on a
single dose and cannot be used to calculate the Ten-day HA. Consequently, the
Longer-term HA will be used as a conservative estimate of the Ten-day HA.
3. Longer-term Health Advisory for Children and Adults
Table VIII-2 summarizes studies that were considered for calculation of
the Longer-term HA values for DEHA. The studies conducted with rats (Smyth
et al., 1951; NTP, 1982) are in general agreement, identifying NOAEL values of
402 to 610 mg/kg/day and LOAEL values of 770 to 2,920 mg/kg/day. The
corresponding value in mice was somewhat higher (NOAEL values of
1,075 mg/kg/day and LOAEL values of 1,970 mg/kg/day). In a one-generation
reproduction study in rats (ICI, 1988b), the NOAEL was 170 mg/kg/day and the
LOAEL was 1,080 mg/kg/day based on increased liver weights in both sexes,
decreased gestational weight gain in dams, reduced weight gain in pups, and a
decreased total litter size. The reproduction study was selected as most
appropriate for derivation of the Longer-term HA values; the LOAEL and NOAEL
were also supported by a developmental toxicity in rats at the same dose
levels (ICI, 1988a).
\
The Longer-term HA for a 10-kg child is calculated as follows:
(170 ma/koydayHlO kg)
(1 L/day)(100)
17 mg/L (rounded to 20,000 ng/L)
where:
170 mg/kg/day
NOAEL, based on decreased gestational weight gain
in pregnant females, increased liver weights in both
sexes, and decreased litter size and pup weight gain.
10 kg ,= assumed weight of a child.
VIII-8
-------
Table VIII-2. Summary of Candidate Studies for Derivation of the Longer-term
Health Advisory for Di(2-ethylhexyl)adipate (DEHA)
Exposure
duration NOAEL LOAEL
Species Route-' (days) Endpoints (mg/kg/day) (mg/kg/day) Reference
Rat
(Sherman)
Rat
In feed
In feed
90
91
Body weight; 610
. histology
Body weight; 402
histology
2,920 Smyth
et al.
(1951)
770 NTP (1982)
Mouse In feed 91
Body weight; 1,075
histology
Rat
In feed 1291
Gestational
weight gain;
liver weight;
litter size
170
1,970 NTP (1982)
1,080 ICI
(1988b)
''This is a reproductive study in which males and females were fed DEHA
for 10 weeks prior to mating; pregnant dams were also dosed throughout
gestation and lactation.
VIII-9
-------
i I/day « assumed water consumption by a 10-kg child. •
100 « uncertainty factor following EPA guidelines for a
NOAEL obtained in a study using laboratory animals,
The Longer-term HA for a 70-kg adult is calculated as follows-:
(170 mq/ko7davH70 ko) =59.5 mg/L (rounded to 60,000 jig/L)
(2 L/day}{100)
where:
170 mg/kg/day
70 kg"
2 L/day
100
NOAEL, based on decreased gestational weight gain in
pregnant females, increased liver weights in both
sexes, and decreased litter size and pup weight gain.
assumed weight of an adult.
assumed water consumption by a 70-kg adult.
uncertainty factor following EPA guidelines for a
NOAEL obtained in a study using laboratory animals.
The HA values calculated exceed the water solubility of OEHA.
4. Reference Dose and Drinking Water Equivalent Level .
The teratology and reproductive studies in rats by 1C I (1988a, 1988b) are
considered adequate and suitable to serve as the basis for calculating the
Reference Dose (RfD) and Drinking Water Equivalent Level (DWEL) for DEHA. The
teratology study demonstrated that at a dose that resulted in slight maternal
toxicity (1,080 mg/kg/day), slight fetal toxicity displayed as decreased
ossification and variations in ureter development were observed. The NOAEL
was 170 mg/kg/day. The one-generation reproductive study demonstrated reduced
maternal weight during pregnancy at the high dose (same dietary concentrations
as in the teratology study), increased liver weights in parents of both sexes,
and reduced offspring weight gain during lactation at the same dose. The
LOAEL was 1,080 mg/kg/day, and the NOAEL was 170 mg DEHA/kg/day, which
supports the results of the teratology study.
VIII-10
-------
The RfD. for a 70-kg adult is calculated as follows:
RfQ » 170 mq/ko/dav = 0.56 mg/kg/day (rounded to 0.6 mg/kg/day)
3 x 100
where:
170 mg/kg/day « NOAEL, based on maternal and fetal toxicity in a
dietary teratology study in rats'
100 = uncertainty factor following EPA guidelines for a NOAEL
• obtained in a study using laboratory animals.
3 *> uncertainty factor used because the teratology study is
less-than-lifetime exposure, and there is the lack of a
multigeneration reproduction study to calculate the RfD.
Using 0.6 mg/kg/day as the RfD and assuming a 70-kg adult consumes 2 L of
drinking water per day, the DWEL is calculated as follows:
QWEL = 0.6 mo/ka/dav x 70 kg = 21 mg/L (rounded to 20 mg/L)
2 L/day
As a consequence, EPA is reproposing a Maximum Contaminant Level Goal
(MCLG) of 0.4 mg/L for OEHA based on the DWEL of 20 mg/L, an additional
uncertainty factor of 10 in accordance with Office of Water policy for Class C
Carcinogens, and an assumed drinking water contribution of 20% to total
exposure.
HCLG = 20 mg/L x 0.2 -'0.4 mq/L
10
c. QUANTIFICATION'OF CARCINOGENIC EFFECTS FOR ADIPATES
1. Categorization of Carcinogenic Potential
The National Toxicity Program (NTP, 1982) evaluated the carcinogenic
potential of DEHA in mice and rats. Two groups of 50 male and 50 female
B6C3F, mice, initially 6 weeks old, were fed diets containing 12 or 25 g
DEHA/kg food for 103 weeks. A control group of 50 male.and 50 female mice was
used. Survival rates at the end of the study (105 to 106 weeks) ranged from
•64% in treated males to 84% in control female mice. Statistically significant
increases in hepatocellular adenomas occurred in male mice at the high dose
VIII-11
-------
(p - 0.021)'and in a dose-related trend (p « 0.013). While increases in
hepatocellular carcinomas were also observed at both dose levels, the
increases were not statistically significant (p <0.05). When compared with
the incidence in historical laboratory control mice, however, the liver tumors
in male mice were not clearly compound related. Statistically significant
(p <0.001) increases in the incidences of hepatocellular carcinomas were
observed in both low- and high-dose females. In females, times' to observation
of the first hepatocellular tumor were 106, 85, and 79 weeks in the control,
low-, and high-dose groups, respectively. No other tumor types related to the
treatment were observed.
In a similar study in F344 rats, no carcinogenic effects were observed
(NTP, 1982). Fifty males and 50 females per group were fed diets containing
.0, 12, or 25 g DEHA/kg feed for 103 weeks. Survival at 105 to 107 weeks was
68% in control and low-dose males and 80% in high-dose males; and 58% in
control females, 78% in low-dose females, and 88% in high-dose females. There
was no difference in the incidence or type of tumors between treated and
control animals.
The NTP (1982) findings on liver carcinogenicity are further supported by
the structure-activity correlation reported by Kluwe (1986) and Kluwe et al.
(1985) on four compounds containing the 2-ethylhexyl moiety. Using the NTP
data, Kluwe (1986) and Kluwe et al. (1985) found that di(2-ethylhexyl)-
phthalate (DEHP), DEHA, tris(2-ethylhexyl)pha§ptiate^ (TEHP), and 2-ethylhexyl
sulfate (EHS) exhibited some evidence of hepatocarcinogenicity in rats and/or
mice ranging from very strong (DEHP) to equivocal (EHS).
No evidence for mutagenicity was observed by several investigators using
a variety of genetic toxicity assays.
EPA has categorized DEHP in Group C, Possible Human Carcinogen. This is
based on an absence of human data and increased incidence of liver tumors in
female mice. Except for a positive dominant lethal assay, there was no
evidence of genotoxicity; this compound, however, exhibits structural
relationships to other probable and possible carcinogens (IRIS, 1991).
VIII-12
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The International Agency for Research on Cancer (IARC, 1982) has not yet
evaluated the weight of evidence and classified the carcinogenic potential of
DEHA in humans.
2. Quantification ofCarcinogenic Effects
The carcinogenic potency of DEHA was estimated by a linearized multistage
model using unit risks at 1 mg/kg/day and 1 ng/L. Dose-response data used for
the extrapolation were the incidence of hepatocellular adenomas and carcinomas
combined in female B6C3F1 mice (NTP, 1982). Human equivalent concentrations
at the low and high doses were 255 and 582 mg/kg/day, and tumor incidences in
control, low-, and high-dose females were 3/50, 19/50, and 18/49,
respectively. The Oral Slope Factor (q,*) is 1.2 x 10'' (mg/kg/day)-', and the
Drinking Water.Unit Risk is 3.4 x 10" per ng/l. Risk levels of E-4, E-5, and
E-6 correspond to drinking water concentrations of 3,000, 300, and 30 ng/L,
respectively.
D. EXISTING GUIDELINES AND STANDARDS
No guidelines or standards for DEHA exposure in water or air were found.
E. SUMMARY
The recommended values for the One-day and Ten-day HAs and DWEL for both
children and/or adults and the estimated excess cancer risk are summarized in
Table VIII-3. • ^
VIII-13
-------
Table VIII-3.
Summary of Quantification of Toxicological Effects for
Di(2-ethylhexyl)adipate (DEHA)
i
Value
Drinking water
concentration
Reference
One-day HA for a child
Ten-day HA for a child
Longer-term HA for a child
Longer-term HA for an adult
DWEL (70-kg adult)
Excess cancer risk (10**)
20,000
60,000
20,000
30
ICI (1988b)
ICI (1988b)
ICI (1988a)
NTP (1982)
'All concentrations exceed the water solubility of DEHA, 0.78 ± 16 mg/L.
"The Longer-term HA value of 20,000 jig/L for a child is taken as a conservative
estimate for the One-day and Ten-day HA values.
VIII-14
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IX-3
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»
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IX-4
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January 1992
FINAL
DRINKING WATER CRITERIA DOCUMENT
FOR
DI(2-ETHYLHEXYL)ADIPATE
Health and Ecological Criteria Division
Office of Science and Technology
Office of Water
U.S. Environmental Protection Agency
Washington, DC 20460
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.1
/-Ml/b
• 3
SccM"
See A/I*
Reference Cited in the OTE Section of the Adif
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