NTP-CERHR Monograph on the 

Potential Human Reproductive and 

Developmental Effects of

Di(2-Ethylhexyl) Phthalate (DEHP)

November 2006 NIH Publication No. 06-4476


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Table of Contents

Preface..............................................................................................................................................v

Abstract.......................................................................................................................................... vii

Introduction..................................................................................................................................... ix

Developmental Toxicity and Reproductive Toxicity.........................................................................x

NTP Brief on Di(2-ethylhexyl) Phthalate (DEHP) ..........................................................................1

References.........................................................................................................................................7

Appendix I. NTP-CERHR DEHP Update Expert Panel

Preface...............................................................................................................................I-1

Expert Panel.......................................................................................................................I-2

Appendix II. Expert Panel Update on DEHP .............................................................................. II-i

Table of Contents............................................................................................................ II-iii

Abbreviations....................................................................................................................II-v

List of Tables................................................................................................................. II-viii

List of Figures...................................................................................................................II-x

Preface............................................................................................................................ II-xi

Chemistry, Usage and Human Exposure..........................................................................II-1

General Toxicology and Biologic Effects.......................................................................II-36

Developmental Toxicity Data..........................................................................................II-54

Reproductive Toxicity Data...........................................................................................II-111

Summaries and Conclusions.........................................................................................II-177

References.....................................................................................................................II-185

Health Care Without Harm............................................................................................. III-1

B. Braun Medical, Inc..................................................................................................... III-5

American Chemistry Council......................................................................................... III-6

People for the Ethical Treatment of Animals................................................................ III-72

Appendix.III..Public.Comments.on.the.DEHP.Expert.Panel.Update
iv

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v

The National Toxicology Program (NTP) established the Center for the Evaluation of Risks

to Human Reproduction (CERHR) in 1998.

CERHR is a publicly accessible resource for

information about adverse reproductive and/or

developmental health effects associated with

exposure to environmental and/or occupational

chemicals. CERHR is located at the National

Institute of Environmental Health Sciences

(NIEHS) of the National Institutes of Health

and Dr. Michael Shelby is the director.1

CERHR broadly solicits nominations of chemicals for evaluation from the public and private

sectors. Chemicals are selected for evaluation based upon several factors including the

following:

• potential for human exposure from use

and occurrence in the environment

• extent of public concern

• production volume

• extent of database on reproductive and

developmental toxicity studies

CERHR follows a formal processfor review and

evaluation of nominated chemicals that in­cludes

multiple opportunities for public comment.

Briefly, CERHR convenes a scientific expert

panel that meets in a public forum to review,

discuss, and evaluate the scientific literature on

the selected chemical. CERHR expert panels

use explicit guidelines to evaluate the scientific

literature and prepare the expert panel reports.

Public commentisinvited priortoand during the

meeting. The expert panel produces a report on

the chemical’s reproductive and developmental

toxicities and provides its opinion of the degree

to which exposure to the chemical is hazardous

to humans. The panel also identifies areas of

uncertainty and where additional data are

needed. Expert panel reports are made public

and comments are solicited.

Next, CERHR prepares the NTP brief.The goal

of the NTP brief is to provide the public, as

well as government health, regulatory, and research agencies, with the NTP’s interpretation

of the potential for the chemical to adversely

affect human reproductive health or children’s

development. CERHR then prepares the NTPCERHR monograph, which includes the NTP

brief on the chemical evaluated, the expert panel report, and public comments on the expert

panel report. The NTP-CERHR monograph is

made publicly available on the CERHR web site

and in hard copy or CD-ROM from CERHR.

Preface

1InformationabouttheCERHRis availableonitsweb

site <http://cerhr.niehs.nih.gov>or by contacting:

Dr. Michael D. Shelby

Director, CERHR

NIEHS, P.O. Box 12233, MD EC-32,

Research Triangle Park, NC 27709

919-541-3455

919-316-4511 [fax]

shelby@niehs.nih.gov

Information about the NTP is available on the web

at <http://ntp.niehs.nih.gov>or by contacting the

NTP Liaison and Scientific Review Office at the

NIEHS:

liaison@starbase.niehs.nih.gov

919-541-0530
vi

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vii

The National Toxicology Program (NTP)

Center for the Evaluation of Risks to Human

Reproduction (CERHR) conducted an updated

evaluation of the potential for DEHP to cause

adverse effects on reproduction and development

in humans. The first CERHR expert panel

evaluation of DEHP was completed in 2000 by

the Phthalates Expert Panel. CERHR selected

DEHP for an updated evaluation because of:

(1) widespread human exposure,

(2) public and government interest in adverse health effects,

(3) recently available human exposure

studies, and

(4) the large number of relevant toxicity

papers published since the earlier

evaluation.

DEHP (CAS RN: 117-81-7) is a high production

volume chemical used as a plasticizer of poly­vinyl

chloride in the manufacture of a wide variety of

consumer goods, such as building products, car

products, clothing, food packaging, children’s

products(but not in toysintended for mouthing),

and inmedical devicesmade of polyvinyl chloride.

The public can be exposed to DEHP by ingesting

food, drink or dust that has been in contact

with DEHP-containing materials, by inhaling

contaminated air or dust, or by undergoing a

medical procedure that uses polyvinyl chloride

medical tubing or storage bags. It is estimated

that the general population of the United States

is exposed to DEHP levels ranging from 1 to 30

μg/kg bw/day (micrograms per kilogram body

weight per day).

The results of this DEHP update evaluation are

published in an NTP-CERHR monograph that

includes:

(1) the NTP Brief,

(2) the Expert Panel Update on the Reproductive and Developmental Toxicity

of DEHP, and

(3) public comments on the expert panel

report.

The NTP reached the following conclusions on

the possible effects of exposure to DEHP on human development and reproduction. Note that

the possible levels of concern, from lowest to

highest, are negligible concern, minimal concern,

some concern, concern, and serious concern.

There is serious concern that certain intensive medical treatments of male infants may 

result in DEHP exposure levels that adversely 

affect devel­opment of the male reproductive 

tract. DEHP exposure from medical procedures

in infants was estimated to be as high as 6000

μg/kg bw/day.

There is concern for adverse effects on development of the reproductive tract in male offspring of pregnant and breastfeeding women 

under­going certain medical procedures that 

may result in exposure to high levels of DEHP. 

There is concern for effects of DEHP exposure 

on development of the male reproductive tract 

for infants less than one year old. Diet, mouthing

of DEHP-containing objects, and certain medical

ABSTRACT

NTP-CERHR Monograph on the Potential Human Reproductive and 

Developmental Effects of Di(2-ethylhexyl) Phthalate (DEHP)
viii

treatments may lead to DEHP exposures that

are higher than those experienced by the general

population.

There is some concern for effects of DEHP 

exposure on development of the reproductive 

tract of male children older than one year. 

As in infants, exposures of children to DEHP

may be higher than in the general population.

There is some concern for adverse effects 

of DEHP exposure on development of the 

male re­pro­ductive tract in male offspring of 

pregnant women not medically exposed to 

DEHP. Although DEHP exposures are assumed

to be the same as for the general population, the

developing male reproductive tract is sensitive

to the adverse effects of DEHP.

There is minimal concern for reproductive 

tox­icity in adults exposed to DEHP at 1–30 

μg/kg bw/day. This level of concern is not altered for adults medically exposed to DEHP. 

NTPwilltransmittheNTP-CERHRMonograph

onDEHPtofederalandstateagencies,interested

parties, and the public and it will be available

in electronic PDF format on the CERHR web

site <http://cerhr.niehs.nih.gov> and in printed

text or CD-ROM from the CERHR:

Dr. Michael D. Shelby

Director, CERHR

NIEHS, P.O. Box 12233, MD EC-32

Research Triangle Park, NC 27709

919-541-3455

919-316-4511 [fax]

shelby@niehs.nih.gov
ix

DEHP (di(2-ethylhexyl) phthalate, CAS RN

117-81-7) is a high production volume chemical used as a plasticizer of polyvinyl chloride in

the manufacturer of a wide variety of consumer

products such as building products, car products, clothing, food packaging, and children’s

products (but not in toys intended for mouthing), and in medical devices made of polyvinyl

chloride.

In 1999–2000, the CERHR Phthalates Expert

Panel evaluated DEHP and six other phthalates

for reproductive and developmental toxicities.

Between the release of the first CERHR Expert

PanelReportonDEHPin2000andtheconvening

of the present panel in 2005, approximately

70 papers relevant to human exposure and

reproductive and/or developmental toxicity of

DEHP were published. Because most people in

the United States are exposed to DEHP and it is

known to cause adverse effects on reproduction

and development in laboratory animals, there

is considerable interest in its possible health

effects on people. Forthese reasons, theCERHR

convened an expert panel to conduct an updated

evaluation of the potential reproductive and

developmental toxicities of DEHP.

This monograph includes the NTP Brief on

DEHP, a list of the expert panel members

(Ap­pen­dix I), the Expert Panel Report on

DEHP (Appendix II), and all public comments

received on the expert panel report (Appendix

III). The NTP-CERHR monograph is intended

to serve as a single, collective source of information on the potential for DEHP to adversely

affect human reproduction or development.

The NTP Brief on DEHP presents the NTP’s

opinion on the potential for exposure to DEHP

to cause adverse reproductive or developmental effects in people. The NTP brief is intended

to provide clear, balanced, scientifically sound

information. It is based on information about

DEHP provided in the expert panel report, public comments, comments from peer reviewers 2

and additional scientific information available

since the expert panel meeting.

2.Peer review of this brief was conducted by letter

review. Reviewers were:

Prof. Jürgen Angerer

Institut für Arbeits-, Sozial- und Umweltmedizin

Dr. Michael Brabec

Eastern Michigan University

Dr. Lynn Goldman

Johns Hopkins University

Dr. Mary Vore

University of Kentucky.

•

•

•

•

Introduction
x

The CERHR evaluation process addresses effects on both development and reproduction.

While there are biological and practical reasons for considering developmental toxicity

and reproductive toxicity as two separate issues, a clear separation of the two is not always possible. It is important to keep in mind

that life in mammals, including humans, is a

cycle. In brief, the cycle includes the production of sperm and eggs, fertilization, prenatal

development of the offspring, birth, postnatal development, sexual maturity, and, again,

production of sperm and eggs.

Toxic effects are often studied in a “life stage

specific” manner. Thus, developmental toxicity is typically studied by exposing pregnant

laboratory animals to the substance of interest

and looking for adverse effects on development of the resulting offspring. Developmental toxicity can be detected as death, structural

malformations, or reduced weights of the fetuses just prior to birth or abnormal structural

or functional development after birth. Reproductive toxicity is often studied by exposing

sexually mature animals to the substance of

interest and effects are detected as impaired

capacity to reproduce.

Over the years, toxicologists realized that exposure during one part of the life cycle may

lead to adverse effects that are apparent only

at a different stage of the life cycle. For example, exposure of a sexually mature animal to

a substance capable of inducing genetic damage in eggs or sperm might have no apparent

effect on the exposed individual. However, if

a genetically damaged egg or sperm from that

individual is involved in fertilization, the genetic damage might lead to death of offspring

before they are born or a genetic disorder

in the surviving offspring. In this example,

chemical-induced damage in the germ cells is

observed as a developmental disorder in the

next generation.

In contrast, development of both the male

and female reproductive systems begins well

before birth and continues until sexual maturity is attained. Thus, the exposure of sexually immature animals, either before or following birth, to agents that adversely affect

development of the reproductive system can

result in structural or functional reproductive

disorders. These effects may become apparent only when reproductive studies are conducted after the exposed individual reaches

sexual maturity.

Thus, in the case of genetic damage induced

in eggs or sperm, damage to reproductive

cells gives rise to developmental disorders.

Conversely, in the case of adverse effects on

development of the reproductive tract, developmental toxicity results in reproductive disorders. In both of these examples it is difficult

to make a clear distinction between developmental and reproductive toxicity. This issue is

important in the present evaluation because

laboratory animal studies provide evidence

that DEHP exposure before or soon after

birth can cause developmental toxicity affecting the reproductive system in later stages of

the life cycle.

Developmental Toxicity and Reproductive Toxicity
1

NTP 

Brief

What is dehp?

DEHP is an oily liquid with the chemical formula C24H38O4 and the structure is shown in

Figure 1.

Figure 1.

Chemical structure of DEHP

It is one of a group of industrially important

chemicals known as phthalates. Phthalates are

used primarily as plasticizers to add flexibility

to plastics. DEHP is used in a wide variety of

productsincluding flooring, wallpaper, auto upholstery, raincoats, toys, and food packaging. It

is not used in toys intended for mouthing such

as nipples or teething rings. DEHP is currently

the only phthalate plasticizer used in polyvinyl

chloride (PVC) medical devices such as blood

bags and tubing.

DEHP is produced by reacting 2-ethylhexanol

with phthalic anhydride. In 2002 the Agency

for Toxic Substances and Disease Registry

(ATSDR) estimated that 241 million pounds of

dioctyl phthalates (including DEHP) were produced in the United States in 1999.

Are People Exposed to DEHP?3

Yes. There are several ways that people may be

exposed to DEHP at home, at work, or through

medical procedures. Human exposure to DEHP

can occur during the manufacture of DEHP, the

manufacture of DEHP-containing products, the

use of such products, and through the presence

of DEHP in the environment.

Environmental exposures can occur through air,

water, or food. The primary source of DEHP

exposure formost people isthrough food. DEHP

migrates into foods, particularly fatty foods,

from DEHP-containing materials that are used

to process and package food. Indoor air and

dust are other common sources of exposure.

The 1999–2000 National Health and Nutrition

Examination Survey (NHANES) conducted by

the Centers for Disease Control and Prevention

reported that 78% of the 2541 urine samples

tested contained the DEHP metabolite mono (2-

ethylhexyl) phthalate (MEHP). This figure may

be an underestimate of the portion of people

exposed to DEHP because two other urinary

metabolites of DEHP not screened for in the

1999–2000 study were subsequently reported

to occur in higher concentrations in human

urine than MEHP (Silva et al., 2004).

The expert panel estimated that exposure to

DEHP in the United States general population

is approximately 1–30 μg/kg bw/day (micrograms per kilogram body weight per day). This

estimate reflects a total daily exposure of approximately 70–2100 μg DEHP for a 70-kilogram (155 pound) person.

DEHP is used in the manufacture of a number of

medical devices including blood bags and bags

and tubing used for intravenous administration

of fluids, drugs, and nutrients. DEHP can

leach from the plastic bags or tubing into the

fluids being administered. Opportunities for

high DEHP exposures occur during medical

procedures such as hemodialysis, transfusion

NTP Brief on DEHP

3 Answers to this and subsequent questions may

be: Yes, Probably, Possibly, Probably Not, No 

or Unknown

O

O

O

O
2

NTP 

Brief

of blood or blood products, extracorporeal

membrane oxygenation, heart bypass surgery,

and administration of intravenous fluids. The

highest human exposures to DEHP can occur

in newborns and infants undergoing extensive

medical procedures such as transfusions,

extracorporeal membrane oxygenation, and

total parenteral nutrition. The expert panel

estimated that newborns undergoing such

medical procedures might be exposed to DEHP

levels ranging from 130 to 6000 μg/kg bw/day.

There is sufficient evidence to conclude that

all age groups undergoing certain medical

procedures are exposed to levels of DEHP that

are far higher than those encountered by the

general population.

Can DEHP Affect Human Development 

or Reproduction?

Probably. Although there is no direct evidence

that exposure of people to DEHP adversely affects reproduction or development, studies with

laboratory rodents clearly show that exposure

to DEHP can cause adverse effects on development and reproduction (See Figures 2a & 2b).

Based on recent data on the extent to which

humans absorb, metabolize and excrete DEHP,

the NTP believes it is reasonable and prudent to

Figure 2a. The weight of evidence that DEHP causes adverse developmental 

or reproductive effects in humans

Clear evidence of adverse effects

Some evidence of adverse effects

Limited evidence of adverse effects

Insufficient evidence for a conclusion

Limited evidence of no adverse effects

Some evidence of no adverse effects

Clear evidence of no adverse effects

Developmental and Reproductive Toxicity

Figure 2b. The weight of evidence that DEHP causes adverse developmental 

or reproductive effects in laboratory animals

Clear evidence of adverse effects

Some evidence of adverse effects

Limited evidence of adverse effects

Insufficient evidence for a conclusion

Limited evidence of no adverse effects

Some evidence of no adverse effects

Clear evidence of no adverse effects

Developmental and Reproductive Toxicity
3

NTP 

Brief

conclude that the results reported in laboratory

animals indicate a potential for similar adverse

effects in human populations.

Scientific decisions concerning health risks are

generally based onwhat is known asthe “weightof-evidence.” In this case,recognizing the lack of

sufficient data on the effects of DEHP in humans

and the clear evidence of effects in laboratory

animals, the NTP judges the scientific evidence

sufficient to conclude that DEHP may adversely

affect human reproduction or development if

exposures are sufficiently high (See Figure 3).

Supporting Evidence 

The CERHR Expert Panel Update Report on

DEHP (Appendix II) provides details and citations regarding studies on the possible reproductive and developmental toxicity of DEHP.

The expert panel evaluated several human

studies but there was insufficient evidence to

conclude that DEHP causes or does not cause

developmental toxicity when exposure occurs

prenatally or during childhood. There was also

insufficient evidence to conclude that DEHP

causes or does not cause reproductive toxicity

in studies of DEHP-exposed adults. Some of

the methodological factors that limited the

usefulness of these human studies were small

sample size and, in most cases, uncertainties in

the exposure measurements.

As presented in both the earlier and present

DEHP expert panel reports, a large body of data

addressesthe adverse developmental and reproductive effects of DEHP in laboratory animals.

Results from developmental toxicity studies

in mice and rats provide a consistent pattern

of adverse effects following DEHP exposure.

Oral exposure to approximately 100–200 mg/

kg bw/day of DEHP during gestation typically

results in skeletal and cardiovascular malformations, neural tube defects, developmental delays,

Serious concern for adverse effects

Concern for adverse effects

Some concern for adverse effects

Minimal concern for adverse effects

Negligible concern for adverse effects

Insufficient hazard and/or exposure data

Male offspring exposed during pregnancy 

Male children older than one year b

Figure 3. NTP conclusions regarding the possibilities that human development or 

reproduction might be adversely affected by exposure to DEHP

Critically ill male infant a

Male infants younger than one year b,c

Male offspring of women undergoing certain 

medical treatments during pregnancy

Reproduction in adults d

aBased on estimated DEHP exposures as high as 6000 μg/kg bw/day

bBased on exposures at the high end of an estimated exposure range of 1–30 μg/kg bw/day

c Includes exposure through breast-feeding in infants younger than 1 year

dBased on estimated exposures of 1–30 μg/kg bw/day
4

NTP 

Brief

and intrauterine death of the offspring. Studies

such as these, in which pups are examined in

the prenatal or immediate postnatal period, provide only limited information on the effects of

DEHP. This is because adverse effects on the

reproductive tract may become apparent only at

laterstages of development. DEHP exposure has

been shown to adversely affect reproduction in

several species including mice, rats, guinea pigs,

and ferrets. While effects have been reported in

both males and females, the in utero and early

postnatal development of the reproductive system of males appears to be more sensitive to the

adverse effects of DEHP.

Exposure of rats to DEHP-containing feed during gestation and/or early postnatal life at 14–23

mg/kg bw/day or greater results in adverse

effects on the developing male reproductive

tract such as abnormally small or absent reproductive organs. Other studies at higher doses

show similar adverse effects on the developing

male reproductive tract. Adverse effects on the

developing female reproductive tract occur in

rats exposed to 1088 mg/kg bw/day DEHP in

the feed.

One new reproductive toxicity study in nonhuman primates was available to the DEHP

Update Panel. Marmosets were exposed orally

to DEHP at doses of 100, 500, or 2,500 mg/

kg bw/day from the juvenile stage through

young adulthood. Following 65 weeks of exposure to DEHP, no adverse changes in the male

reproductive tract were observed. Marmosets

and humans differ in intestinal lipase activity,

absorption and excretion of DEHP, and testosterone levels during development of the male

reproductive tract. These differences lead to

uncertainty as to the utility of the marmoset

as a model for studying the possible effects

of DEHP on development of the human male

reproductive tract. In addition, this marmoset

study encountered problems with the health

and growth of the study animals and did not

investigate the mostsensitive stage in the development of the male reproductive tract, i.e., the

perinatal period.

Are Current Exposures to DEHP High 

Enough to Cause Concern?

Yes. Potentially high exposures of fetuses and

infants to DEHP may lead to adverse effects on

the developing male reproductive tract. High

DEHP exposures of fetuses and infants can occur when pregnant and breast-feeding women

undergo certain medical procedures involving

DEHP-containing polyvinyl chloride medical

devices. Infants may also be exposed to high

levels of DEHP through medical procedures,

diet, and/or mouthing of DEHP-containing objects. In a recently published paper, Wormuth

et al. (2006) predicted that infants and toddlers

are exposed to higher levels of DEHP than

other subgroups in the general population. The

authors concluded that a major portion of this

exposure, perhaps as much as 35%, results from

the ingestion of DEHP-contaminated dust.

Based on the estimated high levels of exposure that can occur during intensive medical

treatments of ill infants and on the apparent

sensitivity of the developing male reproductive tract to DEHP, there is particular concern

for this subpopulation. The adverse effects on

development of the rodent male reproductive

tract are attributed to abnormally low levels of

testosterone induced by DEHP. Concerns for

such effects do not extend to female fetuses

and infants. The general adult population presently appears to be exposed to DEHP at levels

that are not expected to cause adverse effects

to the reproductive system. However, more data

are needed to better understand human DEHP

exposure levels and how these exposures vary

across the population. The NTP offers the following conclusions regarding the potential for

DEHP to adversely affect human reproduction

and development of children.
5

NTP 

Brief

The NTP concurs with the CERHR DEHP 

Update Expert Panel that there is serious concern that certain intensive medical treatments 

of male infants may result in DEHP levels that 

adversely affect development of the reproductive tract. 

This conclusion is based on the apparent sensitivity of the developing male reproductive tract

and the estimated high levels of DEHP exposure that can occur during intensive medical

treatments of ill infants. Such exposures were

estimated to be as much as 100 to 1000 times

higher than exposures in the general population.

The NTP also acknowledges, as did the expert

panel, that the health benefits of these medical

procedures may outweigh any risks. It is noteworthy that both the U.S. Food and Drug Administration (2001) and Health Canada (2002)

used the CERHR Phthalates Expert Panel Report on DEHP in conducting their own assessments of the safety of DEHP-containing medical devices. Both agencies point out that infants

and children undergoing certain medical procedures may be at increased risk for adverse effects of exposure to DEHP.

The NTP concurs with the CERHR DEHP 

Update Expert Panel that there is concern for 

adverse effects on development of the reproductive tract in male offspring of pregnant 

and breast-feeding women undergoing certain medical procedures that may result in exposure to high levels of DEHP.

DEHP exposure levels in adults undergoing

certain medical procedures can be as much as

1000 -fold greater than exposure of the general

population. Because DEHP metabolites can

crossthe placenta and enter breast milk, fetuses

and nursing infants may experience elevated

DEHP exposures if their mothers undergo such

medical procedures.

The NTP concurs with the CERHR DEHP 

Update Expert Panel that there is concern for 

effects of DEHP exposure on development of 

the reproductive tract for infants less than one 

year old. 

Thislevel of concern is based on the uncertainty

regarding DEHP exposure levels in this population, the greater activity of enzymes (lipases)

that convert DEHP to its toxic form, and the

possibility that in infants the developing male

reproductive tract will be more sensitive to the

adverse effects of DEHP than in children older

than one year. Although there is uncertainty regarding levels of DEHP exposure in this age

group, there isthe potentialfor DEHP exposures

to exceed those of the general population. Such

elevated exposures may occur through medical procedures, diet (including breast-feeding),

and/or mouthing of DEHP-containing objects.

The NTP concurs with the CERHR DEHP 

Update Expert Panel that there is some 

concern for the effects of DEHP exposure on 

development of the reproductive tract in male 

children older than one year. 

This level of concern is based on the apparent

sensitivity of the developing male reproductive

tract to the adverse effects of DEHP and the potential for DEHP exposuresin children to exceed

those of the general population. Recent studies

provide greater confidence in estimates of DEHP

exposure levels in children, exposures that may

occur through medical procedures, diet, and/or

mouthing of DEHP-containing objects.

The NTP concurs with the CERHR DEHP 

Update Expert Panel that there is some concern for adverse effects of DEHP exposure on 

development of the reproductive tract in male 

offspring of pregnant women not medically 

exposed to DEHP.

“Some concern” is a lower level of concern than

that expressed for pregnant and breast-feeding
6

NTP 

Brief

women by the previous CERHR Phthalates Expert Panel 4.Thislower level of concern is based

on a greater confidence in the estimated DEHP

exposure levels in women of childbearing age,

a greater confidence in the DEHP exposure

levels at which adverse effects are observed in

laboratory rodents, and evidence that humans

have lower levels of enzymes (lipases) that activate DEHP than rodents. Further, exposure

estimates for women of childbearing age, i.e.,

the age group that would be pregnant or breastfeeding, not medically exposed to DEHP are

the same as for the general population (1 –30

μg/kg bw/day). While studies of DEHP effects in humans and non-human primates are

not sufficient to draw conclusions, data from

recent studies in rodents provide evidence that

no adverse effects are observed in development

of the male reproductive tract following DEHP

exposure of the pregnant dams to less than 10

mg/kg bw/day.

The NTP concurs with the CERHR DEHP 

Update Expert Panel that there is minimal 

concern for reproductive toxicity in adults 

exposed at 1 –30 μg/kg bw/day. This level of 

concern is not altered for adults medically 

exposed to DEHP.

This conclusion for the general population is

based on an estimated range of DEHP exposures

of 1–30 μg/kg bw/day. Based on data from

rodent studies, the adult reproductive tract is

expected to be much less sensitive to the adverse

effects of DEHP exposure than the developing

reproductive tract. Finally, adult rodents have

higherintestinallipase activity than adult humans

and are expected to produce higher levels of

MEHP, a biologically active metabolite. Thus,

adulthumansareexpectedtobelesssensitivethan

adult rodents to the reproductive toxicity effects

of a given dose of DEHP. This expert panel did

not specifically address occupational exposures

because no significant new information on this

issue had become available since the earlier

expert panel report was released.

These conclusions are based on 

the information available at the 

time this brief was prepared. As 

new information on toxicity and 

exposure accumulate, it may form 

the basis for either lowering or 

raising the levels of concern expressed in the conclusions.

4Following the October 2005 meeting of the DEHP

update expert panel, 3 members of the panel reconsidered this conclusion and expressed the

opinion that the level of concern should not have

been lowered.
7

NTP 

Brief

References

Health Canada (2002) ExpertAdvisory Panel on

DEHP in Medical Devices. Available at: <http://

www.mindfully.org/Plastic/DEHP-HealthCanada11jan02.htm>

Silva MJ, Barr DB, Reidy JA, Malek NA,

Hodge CC, Caudill SP, Brock JW, Needham LL

and Calafat AM (2004) Urinary levels of seven

phthalatemetabolitesintheU.S.populationfrom

the National Health and Nutrition Examination

Survey (NHANES) 1999–2000. Environmental 

Health Perspectives 112(3):331–338.

U.S. Food and Drug Administration (2001)

SafetyAssessment of Di(2-ethyl­hexyl)­ phthalate

(DEHP) Released from PVC Medical Devices.

Available at: <http://www.fda.gov/cdrh/ost/dehppvc.pdf>.

Wormuth M, Scheringer M,Vollenweider M, and

Hungerbühler K. (2006) What are the sources of

exposure to eight frequently used phthalic acid

esters in Europeans? Risk Analysis 26:803–824.
I-1

Appendix I

An 11-member panel of scientists covering

disciplines such as toxicology, epidemiology,

and medicine wasrecommended by the CERHR

Core Committee and approved by the Associate

Director of the National Toxicology Program.

Prior to the expert panel meeting, the panelists

critically reviewed articles from the scientific

literature, as well as a variety of other relevant

documents.Based on thismaterial, they identified

key studies and issuesfor discussion.At a public

meeting held October 10–12, 2005, the expert

panel discussed these studies, the sufficiency

of available data, and identified data needed to

improve future assessments. The expert panel

reached conclusions on whether estimated

exposures may result in adverse effects on human

reproduction or development. Panel assessments

were based on the scientific evidence available

at the time of the final meeting.The expert panel

reports were made available for public comment

on November 21, 2005 and the deadline for

public comments was February 3, 2006 (Federal 

Register 70:220 [16 Nov. 2005] p69567; and

Federal Register 70:239 [14 Dec. 2005] p74026).

The Expert Panel Update Report on DEHP is

provided inAppendix II and the public comments

received on that report are inAppendix III. Input

from the public and interested groupsthroughout

the panel’s deliberations was invaluable in

helping to assure the completeness and accuracy

of the reports. The Expert Panel Update Report

on DEHP is available on the CERHR website

<http://cerhr.niehs.nih.gov>.

Appendix I. 

NTP-CERHR DEHP update Expert Panel
I-2

Appendix I

Robert Kavlock, Ph.D. (Chair)

U.S. Environmental Protection Agency

Research Triangle Park, NC

Dana Boyd Barr, Ph.D.

Centers for Disease Control & Prevention

Atlanta, GA

Kim Boekelheide, M.D., Ph.D.

Brown University

Providence, RI

William Breslin, Ph.D.

Eli Lilly and Company

Greenfield, IN

Patrick N. Breysse, Ph.D.

The Johns Hopkins University

Baltimore, MD

Robert Chapin, Ph.D.

Pfizer Global Research & Development

Groton, CT

Kevin Gaido, Ph.D.

CIIT Centers for Health Research

Research Triangle Park, NC

Ernest Hodgson, Ph.D.

North Carolina State University

Raleigh, NC

Michele Marcus, Ph.D.

Emory University

Atlanta, GA

Katherine M. Shea, M.D., M.P.H.

Consultant

Chapel Hill, NC

Paige L. Williams, Ph.D.

Harvard School of Public Health

Boston, MA

Appendix I. NTP-CERHR DEHP update Expert Panel 
Appendix II

NTP-CERHR EXPERT PANEL UPDATE 

ON THE REPRODUCTIVE AND 

DEVELOPMENTAL TOXICITY 

OF di(2-ethylhexyl) phthalate


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October 2005 NTP-CERHR-DEHP-05
Appendix II

II-iii

Table of Contents

Abbreviations..................................................................................................................................... v

Preface................................................................................................................................................ xi

1.0 USE AND HUMAN EXPOSURE............................................................................................. 1

1.1 General Population Exposure.............................................................................................. 3

1.1.1 Exposure Estimates Based on DEHP Levels in Environmental Samples and Foods 3

1.1.2 Exposure estimates based on biomarkers ................................................................. 8

1.2 Exposure Assessed Through Toys.....................................................................................17

1.3 Exposure Through Building Materials..............................................................................17

1.4 Exposure Through Wastewater..........................................................................................17

1.5 Medical Exposures.............................................................................................................18

1.6 Utility of Exposure Data ................................................................................................... 25

1.7 Summary of Exposure Data.............................................................................................. 26

2.0 GENERAL TOXICOLOGY AND BIOLOGIC EFFECTS.................................................. 36

2.1 Toxicokinetics................................................................................................................... 36

2.2 General Toxicity and Carcinogenicity............................................................................... 44

2.3 Summary of General Toxicology and Biologic Effects......................................................51

2.3.1 Toxicokinetics...........................................................................................................51

2.3.2 General Toxicity and Carcinogenicity......................................................................52

3.0 DEVELOPMENTAL TOXICITY DATA............................................................................... 54

3.1 Human Data ...................................................................................................................... 54

3.2 Experimental Animal Data ................................................................................................59

3.2.1 Developmental Studies Focusing on Reproductive System and Endocrine Effects .......59

3.2.2 Developmental Studies Focusing on Non-reproductive Effects.............................. 88

3.2.4 Fish .......................................................................................................................... 96

3.2.5 Abstracts.................................................................................................................. 96

3.3 Utility of Developmental Toxicity Data ............................................................................ 98

3.4 Summary of Developmental Toxicity Data....................................................................... 98

3.4.1 Human Data ............................................................................................................ 98

3.4.2 Experimental Animal Data ..................................................................................... 99

4.0 REPRODUCTIVE TOXICITY DATA..................................................................................111

4.1 Human Data ..................................................................................................................... 111

4.2 Experimental Animal Data ..............................................................................................118

4.2.1 Female ....................................................................................................................118

4.2.2 Male........................................................................................................................122

4.2.3 Male and Female ....................................................................................................145

4.2.4 Abstracts.................................................................................................................163

4.4 Summary of Reproductive Toxicity Data ........................................................................164
II-iv

Appendix II

4.4.1 Human Data ...........................................................................................................164

4.4.2 Experimental Animal Data ...................................................................................166

5.0 SUMMARY, CONCLUSIONS, AND CRITICAL DATA NEEDS.....................................177

5.1 Developmental and Reproductive Toxicity ......................................................................177

5.1.1 Developmental Toxicity (Update) ..........................................................................177

5.1.2. Reproductive Toxicity (Update) .............................................................................177

5.2 Human Exposure..............................................................................................................178

5.3 Overall Conclusions.........................................................................................................180

5.3.1. General Adult Population .......................................................................................180

5.3.2 Healthy Infants and Toddlers .................................................................................180

5.3.3 Critically Ill Infants................................................................................................181

5.3.4 Pregnancy and Lactation........................................................................................182

5.4 Critical Data Needs..........................................................................................................182

6.0 REFERENCES........................................................................................................................185
Appendix II

II-v

Abbreviations

ADP................................adenosine diphosphate

AGI.................................anogenital index

Ah...................................aryl hydrocarbon

ANCOVA .......................analysis of co-variance

ANOVA..........................analysis of variance

AUC ...............................area under the concentration–time curve

BMD10 ...........................benchmark dose, 10% effect level

BMD1 SD ........................benchmark dose, 1 control standard deviation

BMDL............................benchmark dose 95th percentile lower confidence limit

BrdU...............................bromodeoxyuridine

bw...................................body weight

CAS RN .........................Chemical Abstracts Service Registry Number

cDNA .............................complementary deoxyribonucleic acid

CERHR..........................Center for the Evaluation of Risks to Human Reproduction

CI....................................confidence interval

Cmax................................maximum concentration

CYP................................cytochrome P450

DEHA ............................diethylhexyl adipate

DEHP .............................di(2-ethylhexyl) phthalate

DMSO............................dimethylsulfoxide

DNA...............................deoxyribonucleic acid

ECMO............................extracorporeal membrane oxygenation

EPA ................................Environmental Protection Agency

F0....................................parental generation

F1....................................first filial generation

F2....................................second filial generation

F3....................................third filial generation

fasL ................................fas ligand

FDA................................Food and Drug Administration

g......................................gram(s)

FSH ................................follicle stimulating hormone

GC..................................gas chromatography

GD..................................gestation day(s)

GLP................................Good Laboratory Practice

hCG................................human chorionic gonadotropin

HPLC .............................high performance liquid chromatography

H-FABP..........................heart-fatty acid binding protein

IARC..............................International Agency for Research on Cancer

im ...................................intramuscular(ly)

ip ....................................intraperitoneal(ly)

IU ...................................international unit

iv.....................................intravenous(ly)

kDa.................................kilodalton
II-vi

Appendix II

kg....................................kilogram(s)

L.....................................liter(s)

LH ..................................luteinizing hormone

LOAEL ..........................low observed adverse effect level

LOD ...............................limit of detection

LOQ ...............................limit of quantification

m ....................................meter(s)

m3 ..............................................cubic meter(s)

M....................................molar

MEHP ............................mono(2-ethylhexyl) phthalate

mM.................................millimolar

mmol ..............................millimole(s)

mol .................................mole(s)

MRL...............................minimal risk level

mRNA ............................messenger ribonucleic acid

MS..................................mass spectrometry

MT..................................metallothionein

MTT...............................3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

NADH ............................nicotine-adenine dinucleotide, reduced

ng....................................nanogram(s)

NHANES .......................National Health and Nutrition Examination Survey

NICU..............................neonatal intensive care unit

NIEHS............................National Institute of Environmental Health Sciences

NIH ................................National Institutes of Health

nmol ...............................nanomole(s)

NOAEL ..........................no observed adverse effect level

NTP................................National Toxicology Program

OR..................................odds ratio

PARP..............................poly(ADP-ribose) polymerase

PCB................................polychlorinated biphenyl

PCNA.............................proliferating cell nuclear antigen

PMSG.............................pregnant mare serum gonadotropin

PND................................postnatal day(s)

PPAR..............................peroxisome proliferator-activated receptor

ppb..................................parts per billion

ppm ................................parts per million

PVC................................polyvinyl chloride

RAR ...............................retinoic acid receptor

RIA.................................radioimmunoassay

RNA ...............................ribonucleic acid

RT-PCR ..........................reverse transcriptase-polymerase chain reaction

RXR ...............................retinoic acid X receptor

sc ....................................subcutaneous(ly)

SD ..................................standard deviation

SDS-PAGE.....................sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Appendix II

II-vii

t1/2...................................half-life of elimination

Tmax ................................time to maximum concentration

TI....................................tolerable intake

TNF................................tumor necrosis factor

TPN................................total parenteral nutrition

TUNEL ..........................terminal deoxynucleotidal transferase-mediated dUTP nick-end labeling

UDP................................uridine diphosphate

US ..................................United States

ZnT-1..............................zinc transporter-1

μg....................................microgram(s)

μL...................................microliter(s)

μm ..................................micrometer(s)

μM..................................micromolar

μmol ...............................micromole(s)

2-EH...............................2-ethylhexanol

2-EHA............................2-ethylhexanoic acid

2-cx-MMHP...................mono(2-carboxymethyl)hexyl phthalate

5-cx-MEPP.....................mono(2-ethyl)-5-carboxypentyl phthalate

5-OH-MEHP..................mono(2-ethyl-5-hydroxyhexyl) phthalate

5-oxo-MEHP..................mono(2-ethyl-5-oxy-hexyl) phthalate

8-Br-cAMP.....................8-bromo-cyclic adenosine monophosphate
II-viii

Appendix II

List of Tables

Table 1. Markers of DEHP Exposure Measured in a Variety of Matrices to 

Assess Exposure to DEHP. ..............................................................................................2

Table 2. Environmental DEHP Concentrations Measured in the US............................................3

Table 3. Food Concentrations of DEHP ........................................................................................4

Table 4. Estimated DEHP Intake by Age Group ...........................................................................4

Table 5. DEHP Intake from Environmental and Food Sources.....................................................5

Table 6. DEHP Metabolites in Urine in the NHANES 2001–2002 Sample ................................9

Table 7. Urinary Metabolite Excretion 24 Hours after Oral Ingestion of DEHP........................12

Table 8. DEHP Metabolites in the Urine of Nursery-school Children and Adults .....................15

Table 9. Urinary MEHP in Infants in Two NICUs by DEHP Exposure Group...........................21

Table 10. FDA Estimates of DEHP Exposures Resulting from Medical Treatments ...................22

Table 11. DEHP Dose with Pheresis Procedures ..........................................................................25

Table 12. Summary of DEHP Metabolite Levels Measured in Human Urine ..............................28

Table 13. Summary of DEHP Exposure Estimates from Medical Devices ..................................32

Table 14. Summary of DEHP Metabolite Levels Measured in Medically-Exposed Infants.........34

Table 15. Toxicokinetic Parameters in Pregnant (GD 6) and Non-pregnant Female Rats 

and Mice Given Oral Radiolabeled DEHP....................................................................38

Table 16. Toxicokinetic Parameters after Oral Dosing of Marmosets at Age 3 and 

18 Months with Radiolabeled DEHP ............................................................................39

Table 17. Reproductive Organ Radioactivity Content 2 Hours after Oral Dosing 

of Marmosets with Radiolabeled DEHP .......................................................................40

Table 18. Normalized AUCs for Blood DEHP and MEHP in Rats Treated with DEHP ..............42

Table 19. AUCs for Blood DEHP and MEHP in Marmosets Treated with DEHP .......................43

Table 20. DEHP Conclusions by US, Canadian, and European Agencies....................................45

Table 21. Results Achieving Statistical Significance or Dose-response Relationships 

Following DEHP Prenatal and Lactational DEHP Exposure ........................................63

Table 22. Benchmark Dose Values for Offspring of Rats Exposed to DEHP 

During Gestation and Lactation.....................................................................................66

Table 23. Reproductive Organ Abnormalities in Combined F1+F2 Non-breeding Males

in NTP Multigeneration Study ......................................................................................70

Table 24. Outcomes after Perinatal Exposure of Rats to DEHP With or 

Without Diethylhexyl Adipate .......................................................................................73

Table 25. Testicular and Liver Weight Changes in Rats Treated with DEHP

by IV Infusion or Oral Gavage ......................................................................................78

Table 26. Relative Expression of Fetal Testis Genes after Phthalate Treatment

of Pregnant Rats.............................................................................................................85

Table 27. Summary of DEHP Doses in Mice................................................................................90

Table 28. Summary of DEHP Effects on Developmental Toxicity .............................................103

Table 29. Testosterone Metabolism after DEHP Treatment of 4 Week-old Rats ........................137

Table 30. Ovary and Uterine Weight Findings in a Marmoset 65-Week 

DEHP Feeding Study...................................................................................................147

Table 31. Results of 2-Generation Study of DEHP in Wistar Rats.............................................149
Appendix II

II-ix

Table 32. Results of Continuous Breeding Multigeneration Study of DEHP in 

Sprague-Dawley Rats ..................................................................................................155

Table 33. Reproductive Crossover Breeding Study.....................................................................162

Table 34. Summary of Male Reproductive Toxicity Data from Studies in Rats and Mice .........167

Table 35. Summary of Reproductive Toxicity Studies Involving Male and Female Rats...........170

Table 36. Estimated DEHP Dose Ranges for Selected US Population Groups ..........................179

Table 37. Dose Estimates for DEHP/MEHP Exposures from Medical Procedures

in Neonates..................................................................................................................180
II-x

Appendix II

List of Figures

Figure 1. DEHP and metabolites used to estimate DEHP exposure...............................................2

Figure 2. Age-dependent changes in primary and secondary metabolite ratios...........................10

Figure 3. Urinary MEHP Concentrations .....................................................................................30

Figure 4. Urinary 5-oxo- and 5-OH-MEHP Concentrations.........................................................31

Figure 5. DEHP Metabolism.........................................................................................................37

Figure 6. Percent of Bovine Oocytes Reaching Metaphase II after 24-hour Culture

 with MEHP.................................................................................................................121

Figure 7. Percent Apoptotic Cells in Rat Testicular Culture with MEHP...................................126

Figure 8. Testicular Caspase-3 Activity After Administration of a Single Oral Dose

of MEHP to Wistar Rats .............................................................................................131

Figure 9. Effect of DEHP Treatment in Male Rats on PND 21– 48. ..........................................139
Appendix II

II-xi

Preface

The National Toxicology Program (NTP) and the National Institute of Environmental Health Sciences 

(NIEHS) established the NTP Center for the Evaluation of Risks to Human Reproduction (CERHR) 

in June 1998. The purpose of the Center is to provide timely, unbiased, scientifically sound evaluations 

of human and experimental evidence for adverse effects on reproduction, to include development, 

caused by agents to which humans may be exposed.

Di-(2-ethylhexyl) phthalate (DEHP) was originally evaluated by the CERHR Phthalates Expert Panel 

in 1999–2000 and an expert panel report was published in 2001. DEHP was selected for re-evaluation 

by CERHR because of widespread human exposure and public and government interest in potential 

adverse health effects. Further, over 150 relevant papers on DEHP had been published since the 

first evaluation. This is the first time a CERHR expert panel was convened to update an evaluation 

conducted by a previous CERHR expert panel. 

DEHP (CAS RN: 117-81-7) is a high production volume chemical used as a plasticizer in polyvinyl 

chloride plastics. It is found in a wide variety of consumer products, such as building products, car 

products, clothing, food packaging, children’s products (but not in toys intended for mouthing), and 

in some medical devices made of polyvinyl chloride.

To obtain information about DEHP for this CERHR evaluation, the PubMed (Medline) and Toxnet 

databases were searched from January 1, 2000 through September 30, 2005, with CAS RNs for DEHP 

(117-81-7), mono-(2-ethylhexyl) phthalate (MEHP) (4376-20-9), and relevant keywords. References 

were also identified from databases such as REPROTOX®, HSDB, IRIS, and DART and from the 

bibliographies of literature being reviewed. 

This evaluation results from the effort of an eleven-member panel of government and non-government 

scientists that culminated in a public expert panel meeting held October 10–12, 2005. This report is 

a product of the Expert Panel and is intended to (1) interpret the strength of scientific evidence that 

DEHP is a reproductive or developmental toxicant based on data from in vitro, animal, or human 

studies, (2) assess the extent of human exposures to include the general public, occupational groups, 

and other sub-populations, (3) provide objective and scientifically thorough assessments of the scientific evidence that adverse reproductive/developmental health effects may be associated with such 

exposures, and (4) identify knowledge gaps to help establish research and testing priorities to reduce 

uncertainties and increase confidence in future assessments of risk. This report has been reviewed by 

CERHR staff scientists, and by members of the DEHP Expert Panel. Copies have been provided to the 

CERHR Core Committee, which is made up of representatives of NTP-participating agencies. The

findings and conclusions of this report are those of the expert panel and should not be construed

to represent the views of the National Toxicology Program.

This Expert Panel Report will be a central part of the subsequent NTP-CERHR Monograph on the 

Potential Human Reproductive and Developmental Effects of Di-(2-ethylhexyl) Phthalate. This monograph will include the NTP-CERHR Brief, the Expert Panel Report, and all public comments on the 

Expert Panel Report. The NTP-CERHR Monograph will be made publicly available and transmitted 

to appropriate health and regulatory agencies.
II-xii

Appendix II

The NTP-CERHR is headquartered at NIEHS, Research Triangle Park, NC and is staffed and administered by scientists and support personnel at NIEHS and at Sciences International, Inc., Alexandria, 

Virginia.

Reports can be obtained from the website <http://cerhr.niehs.nih.gov/> or from:

Michael D. Shelby, Ph.D.

NIEHS EC-32

PO Box 12233

Research Triangle Park, NC 27709

919-541-3455

shelby@niehs.nih.gov
Appendix II

II-xiii

A Report of the CERHR DEHP Update Expert Panel: 

Robert Kavlock, Ph.D., Chair ...........................US Environmental Protection Agency 

Research Triangle Park NC

Dana Barr, Ph.D. ...............................................Centers for Disease Control and Prevention 

Atlanta GA

Kim Boekelheide, M.D., Ph.D. .........................Brown University, Providence RI

William Breslin, Ph.D. ......................................Eli Lilly & Company, Greenfield IN

*Patrick Breysse, Ph.D......................................The Johns Hopkins University, Baltimore MD

Robert Chapin, Ph.D. ........................................Pfizer, Inc., Groton CT

Kevin Gaido, Ph.D. ...........................................CIIT Centers for Health Research

Research Triangle Park NC

Ernest Hodgson, Ph.D.......................................North Carolina State University, Raleigh NC

*Michele Marcus, Ph.D.....................................Emory University, Atlanta GA

*Katherine Shea, M.D., M.P.H..........................University of North Carolina School of Public Health, 

Chapel Hill NC

Paige Williams, Ph.D. .......................................Harvard School of Public Health, Boston MA

With the Support of CERHR Staff:

NTP/NIEHS

Michael Shelby, Ph.D. Director, CERHR

Paul M.D. Foster, Ph.D Deputy Director, CERHR

Christopher Portier, Ph.D. Associate Director, National Toxicology Program

Kristina Thayer, Ph.D. NTP Liaison and Scientific Review Office

Sciences International, Inc.

Anthony Scialli, M.D. Principal Scientist

Annette Iannucci, M.S. Toxicologist

Gloria Jahnke, D.V.M. Toxicologist

Vera Jurgenson, M.S. Research Assistant

Note to Reader:

This report is prepared according to the Guidelines for CERHR Panel Members established by NTP/NIEHS. 

The guidelines are available from the CERHR web site <http://cerhr.niehs.nih.gov/>. The format for Expert 

Panel Reports includes synopses of studies reviewed, followed by an evaluation of the Strengths/Weaknesses 

and Utility (Adequacy) of the study for a CERHR evaluation. Statements and conclusions made under 

Strengths/Weaknesses and Utility evaluations are those of the Expert Panel and are prepared according 

to the NTP/NIEHS guidelines. In addition, the Panel often makes comments or notes limitations in the 

synopses of the study. Bold, square brackets are used to enclose such statements. As discussed in the 

guidelines, [square brackets] are used to enclose key items of information not provided in a publication, 

limitations noted in the study, conclusions that differ from authors, and conversions or analyses of data 

conducted by the panel.

The findings and conclusions of this report are those of the expert panel and should not be construed

to represent the views of the National Toxicology Program.
Appendix II

II-1

1.0 USE AND HUMAN EXPOSURE

The first section of CERHR Expert Panel Reports is devoted to chemistry, use, and human exposure. 

The following conclusions regarding di(2-ethylhexyl) phthalate (DEHP) exposure were expressed by 

the Expert Panel in the CERHR Expert Panel Report released in 2000: 

While the Panel recognizes the variability and uncertainties in exposure estimates, it 

appears that for the general adult human population, ambient exposures may be on the 

order of 3−30 μg/kg bw/day. Non-dietary mouthing behaviors in infants and toddlers 

may result in exposures that are several-fold higher. The 3−30 μg/kg [bw]/d range may 

be increased by 2−3 orders of magnitude for infants undergoing intensive therapeutic 

interventions.

Since the initial CERHR Expert Panel Report on DEHP, no additional information on chemistry has 

been added.

Phthalates are used in a variety of products, including lubricants, perfumes, hairsprays and cosmetics, 

construction materials, wood finishers, adhesives, floorings, and paints. DEHP is typically added to 

building materials and medical devices made from polyvinyl chloride (PVC) to increase flexibility. 

When DEHP is used as a plasticizer in medical devices such as storage containers, bags, and tubing, 

it can leach from the device into infusate (e.g., pharmaceuticals, blood, blood products, parenteral 

nutrition solutions, air in ventilation tubing). A review by the European Commission (1) noted the use 

of DEHP in orthodontic retainers that are typically used by 7 –14-year-old children. It is not known if 

DEHP is used in orthodontic devices in the US. The Food and Drug Administration (FDA) referenced 

a study stating that DEHP has been detected as a leachate from dental composites, but that plasticizers 

other than DEHP are most often used for such applications (2).

DEHP production volume was referenced in the initial CERHR Expert Panel Report on DEHP as 

approaching 260 million pounds. No recent information on production volume was located. 

This section reviews the literature relating to human exposure studies published after the previous 

CERHR Expert Panel Report on DEHP (2000) was completed. The studies reviewed in this section 

included estimated or calculated exposures to DEHP and its metabolites from medical devices, 

residential exposures, dietary exposures, and environmental exposures. Several studies reviewed the 

effects of temperature, contact time, and solution type in medical devices such as container bags or 

tubing on exposure to DEHP and various metabolites. The specific chemicals that have been measured 

for the estimation of DEHP exposures are listed in Table 1 and shown in Figure 1.
II-2

Appendix II

Table 1. Markers of DEHP Exposure Measured in a Variety of Matrices to Assess Exposure to DEHP.

Marker Marker type Matrices Citations

DEHPa Parent diester Environmental samples, serum (3)

MEHPb Monoester metabolite Serum, urine, amniotic fluid, saliva, breast milk (4-7)

5-OH-MEHP Oxidized monoester metabolite Serum, urine, amniotic fluid, saliva, breast milk (6-8)

5-oxo-MEHP Oxidized monoester metabolite Serum, urine, amniotic fluid, saliva, breast milk (6-8)

2-cx-MMHP Oxidized monoester metabolite Serum, urine, amniotic fluid, saliva, breast milk (9, 10)

5-cx-MEPP Oxidized monoester metabolite Serum, urine, amniotic fluid, saliva, breast milk (9, 10)

DEHP

MEHP

5-OH-MEHP

5-oxo-MEHP

2-cx-MMHP

5-cx-MEPP

di(2-ethylhexyl) phthalate

mono(2-ethylhexyl) phthalate

mono(2-ethyl-5-hydroxyhexyl) phthalate

mono(2-ethyl-5-oxy-hexyl) phthalate

mono(2-carboxymethyl)hexyl phthalate 

mono(2-ethyl)-5-carboxypentyl phthalate.

a The ubiquitous presence of DEHP in both the environment and laboratory require extensive blank testing and preventative 

measures to reduce or eliminate overestimation of values from contamination. Treatment of serum samples with a preservative 

such as phosphoric acid to eliminate residual esterase/lipase activity is necessary to avoid preanalytic contamination of the 

sample leading to falsely elevated levels. In general, serum DEHP measurements are not reliable markers of exposure.

b Treatment of serum, milk, and saliva samples with a preservative such as phosphoric acid to eliminate residual esterase/lipase 

activity is necessary to avoid preanalytic contamination of the sample leading to falsely elevated concentrations.

Figure 1. DEHP and metabolites used to estimate DEHP exposure.

Abbreviations are listed in the footnote to Table 1.

O

O

O

O

DEHP

O

O

O

OH

MEHP

O

O

O COOH

OH

2-cx-MEHP

O

O

O

COOH

OH

5-cx-MEHP

O

O

O

OH

OH

5-OH-MEHP

O

O

O

OH

OH OH

COOH

HO O

Glucuronide conjugates of

all monoester and oxidative

monoester metabolites

O

O

O

OH

O

5-oxo-MEHP

Monoester

Formation

Excretion

Glucuronidation

Oxidative metabolism of monoester metabolite

Glucuronidation
Appendix II

II-3

1.1 General Population Exposure

1.1.1	 Exposure Estimates Based on DEHP Levels in Environmental Samples and

Foods

Clark et al. (11) compiled measurements of phthalate diesters in several environmental media from 

databases in Canada, the US, Europe, and Japan/Asia. [US data for DEHP are presented here.] Many of 

the measurements, including those for DEHP, were compiled by Exxon Mobil Biomedical Sciences, Inc. 

Medians and ranges are given in Table 2 for environmental samples and in Table 3 for food samples.

In a separate paper (12) the same authors presented exposure estimates using probabilistic analysis 

based on concentrations from an unpublished report prepared for industry. Log-normal distributions 

were used for most exposure sources. Estimated DEHP intakes by age group are shown in Table 4 

and Table 5. Except for intake in infants, more than 90% of estimated DEHP intake was from food. 

Formula-fed infants were estimated to derive 43.7% of DEHP intake from food, and breast-fed infants 

were estimated to derive 59.6% of DEHP intake from food. Nearly all of the remainder of DEHP 

intake in infants was estimated to arise from ingestion of dust. 

The authors indicated that exposure estimates of other authors, back-calculated based on measurements of urinary metabolites [discussed below], gave lower estimates of daily intake. They suggested 

that the current study may have overestimated food exposure to DEHP due to use of outdated food 

measurements or due to failure to account for cooking-associated loss of DEHP in food. [The Expert

Panel noted that the authorssummarized a number of recent estimates and all but 1 from Health

Canada (1996) were within the 3–30 μg/kg bw/day range assumed in the original CERHR DEHP

report.The difference in the Health Canada value is related to dust ingestion by children.]

Table 2. Environmental DEHP Concentrations Measured in the US

Medium Mean 

Concentration

Median Concentration 

(Range)

Surface water, μg/L 0.21 0.05 (<0.002–137)

Ground water, μg/L 15.7 15.7 (not detected –470)

Drinking water, μg/L 0.55 0.55 (0.16 –170)

Sediments, μg/kg 1.4 0.16 (0.00027 –218)

Soil, μg/kg 0.03 median not available (0.03 – 1280)

Outdoor air, ng/m3 5.0 2.3 (< 0.4 – 65)

Indoor air, ng/m3 109 55 (20–240)

Dust, g/kg 3.24 median not available (2.38 – 4.10)

Wastewater, μg/L 27 8.3 (0.01 –4400)

Sludge, g/kg 0.301 median not available (0.000420­ – 58.3)

Rainwater, μg/L 0.17 0.17 (0.004 – 0.68)

From Clark et al. (12).
II-4

Appendix II

Table 3. Food Concentrations of DEHP

Food Median concentration, μg/g (Range)

Beverages 0.043 (0.006 – 1.7)

Cereal 0.05 (0.02 – 1.7)

Dairy (excluding milk) 0.96 (0.059 – 16.8)

Eggs 0.12 (< 0.01 – 0.6)

Fats and oils 2.4 (0.7 –11.9)

Fish 0.001 

(0.00005 – not given [90th percentile 0.02])

Fruits 0.02 (< 0.02 – 0.11)

Grains 0.14 (< 0.1 – 1.5)

Meat, not processed 0.05 (< 0.01 – 0.8)

Milk 0.035 (< 0.005 – 1.4)

Nuts and beans 0.045 (< 0.08 – 0.8)

Poultry 0.9 (0.05– 2.6)

Processed meat 0.45 (< 0.1–4.32)

Vegetables 0.048 (0.0098– 2.2)

Infant formula, powdered 0.12 (< 0.012–0.98)

Infant formula, liquid 0.006 (< 0.005–0.15)

Breast milk 0.062 (0.01– 0.6)

Baby food 0.12 (0.01– 0.6)

Other food 0.05 (< 0.01–25)

From Clark et al. (12).

Table 4. Estimated DEHP Intake by Age Group

Age group Median DEHP intake 

(μg/kg bw/day)

Adult (20 –70 years) 8.2

Teen (12– 19 years) 10

Child (5–11 years) 18.9

Toddler (7 months –4 years) 25.8

Infant (0 – 6 months)

Formula-fed 5.0

Breast-fed 7.3

From Clark et al. (11).
Appendix II

II-5

Table 5. DEHP Intake from Environmental and Food Sources

Source Adult 

(20–70 yrs)

Teen 

(12–19 yrs)

Child 

(5–11 yrs)

Toddler 

(7 months–

4 yrs)

Infant (0–6 months)

Formula-fed Breast-fed

Outdoor air 0.0 0.0 0.0 0.0 0.1 0.0

Indoor air 1.0 0.9 1.0 0.9 1.5 1.1

Drinking water 0.1 0.1 0.1 0.1 0.7 0.0

Ingested soil 0.0 0.0 0.0 0.0 0.0 0.0

Ingested dust 4.3 4.2 5.0 6.6 54.1 39.3

Beverages a 11.2 5.2 3.3 2.2 0.0 0.0

Cereals 2.4 2.0 3.5 5.5 0.0 0.0

Dairy products b 13.2 11.7 12.2 12.9 0.0 0.0

Eggs 1.1 0.7 0.8 1.3 0.0 0.0

Fats and oils 16.9 19.1 16.5 11.1 0.0 0.0

Fish 1.6 0.8 0.7 0.4 0.0 0.0

Fruit products 0.9 0.8 1.1 1.4 0.0 0.0

Grains 13.4 16.6 18.1 11.1 0.0 0.0

Meats 5.5 5.2 3.7 3.3 0.0 0.0

Milk 3.1 6.7 8.6 12.6 0.0 0.0

Nuts and beans 1.0 1.0 0.9 0.8 0.0 0.0

Other foods 10.3 11.2 11.3 18.9 0.0 0.0

Poultry 3.9 3.5 3.5 3.6 0.0 0.0

Processed meats 3.4 3.4 3.4 2.5 0.0 0.0

Vegetable products 6.6 6.1 6.1 4.9 0.0 0.0

Formula/breast milk – – – – 43.7 59.6

Data expressed as μg/kg bw/day.

aExcluding water

bExcluding milk.

From Clark et al. (11)

Tsumura et al. (13) evaluated DEHP in prepackaged meals sold in convenience stores in Japan. In 16 

meals purchased between August, 1999, and February, 2000, DEHP levels ranges from 346 to 11,800 

ng/g food. Five of these meals contained enough DEHP that a 50-kg person would be estimated to 

receive more than the European Union tolerable daily intake value of 37 μg/kg bw/day. The authors 

evaluated 10 restaurant-prepared lunches, which are generally served in ceramic containers, and found 

DEHP levels of 12–304 ng/g food, with only 1 lunch having a DEHP level higher than 95 ng/g food. 

After an evaluation of preparation techniques, the authors concluded that higher DEHP content of the 

prepackaged meals was due to the use of PVC gloves in meal preparation. Further, spraying the gloves 

with an ethanol solution as a decontamination measure was believed to be associated with additional 

mobilization of DEHP from the gloves.
II-6

Appendix II

A Danish study (14) measured DEHP in total diet samples, baby food, and infant formulas. The total 

diet sample included foods consumed by 29 adults during a 24-hour period (excluding beverages and 

sweets). Baby food and infant formula samples were purchased in retail stores. Mean DEHP concentrations in the adult diets were 0.11–0.18 mg/kg diet. [The lower value was calculated using 0 for

samples below the limit of detection and using the limit of detection for samples that were above

the limit of detection but below the limit of quantification. The higher value used the limit of

detection for samples that were below the limit of detection and used the limit of quantification

for samples that were above the limit of detection but below the limit of quantification.] Mean 

DEHP levels in baby food were 0.36 – 0.63 mg/kg food, and mean DEHP levels in infant formula were 

0.04–0.06 mg/kg reconstituted formula.

In a review article, Latini et al. (15) estimated from European Union reports that infants consuming 

formula would be exposed to 8 – 13 μg/kg bw/day from this source. Ingestion of DEHP in human 

milk was estimated to result in intakes of 8 –21 μg/kg bw/day. This review also referred to an abstract 

(16) in which DEHP or mono(2-ethylhexyl) phthalate (MEHP) were measurable in 100% of milk or 

colostrum samples from 17 healthy mothers. Mean DEHP was 1.01 μg/mL (range 0.57 – 1.15 μg/mL). 

Mean MEHP was 0.68 μg/mL (range 0.28 –1.08 μg/mL). [Abstracts are noted for completeness but

are not used in the evaluation process.]

Main et al. (17) reported phthalate concentrations in milk collected from 65 Finnish and 65 Danish 

women as part of a study of cryporchidism and hormone levels in male children. [The relationship

between milk MEHP and infant endpoints is discussed in Section 3.1]. Women collected aliquots 

of milk at the end of a feeding starting when their infants were 1 month old. Samples were collected at 

unspecified intervals until a total sample volume of 200 mL was reached. As each sample was collected, 

it was placed in a glass bottle in the subject’s home freezer, with subsequent samples added to the same 

bottle. Mothers were instructed to collect the samples in glass or porcelain containers and to avoid 

breast pumping. [Almost half the Danish mothers used a breast pump at least once; information

on pumping was not available for Finnish mothers.The authorstested milk samplesin 1 common

Danish pump system and found no effect on phthalate monoester levels.] High-performance liquid 

chromatography (HPLC)-mass spectrometry (MS) was used to quantify milk levels of MEHP as 

well as monomethyl, monoethyl, monobutyl, monobenzyl, and mono-isononyl phthalate. MEHP was 

detected in milk from all 130 women. The median (range) concentration in Danish samples was 9.5 

(1.5–191) μg/L, and the median (range) concentration in Finnish samples was 13 (4.0–1410) μg/L. 

The difference between MEHP concentrations in Denmark and Finland was statistically significant 

(P <0.001, Mann-Whitney U test). Estimated MEHP intake was calculated using infant weight at 3 

months of age and assuming milk consumption of 0.120 L/day. For Danish children, the median (range) 

estimated MEHP intake was 1.14 (0.18–23) μg/kg bw/day and for Finnish children, the median (range) 

estimated MEHP intake was 1.56 (0.47–169) μg/kg bw/day. The authors indicated that they could 

not exclude contamination of samples with dust or other household sources of phthalates, and they 

suggested caution in interpreting the numerical values reported for milk phthalate concentrations.

Mortensen et al. (18) measured phthalates in milk collected from 36 Danish women from 1 to 3 months 

after delivery. Milk aliquots were collected in the same glass bottle at the end of a feeding and stored 

in a freezer. [The methods and collection times appear to be similar to those of Main et al. (17),

from the same group of investigators.The Main et al.study references the Mortensen et al.study
Appendix II

II-7

as involving different Danish women.] After thawing, phosphoric acid was added to half of each 

sample to inactivate milk esterases that might convert contaminant DEHP to MEHP. After clean-up, 

milk MEHP was quantified using HPLC-tandem MS. Phosphoric acid treatment was not shown to 

influence MEHP measurement. Median (range) milk MEHP was 9.5 (2.7 –13) μg/L. Seven samples 

of commercially sold cow milk were analyzed. MEHP concentrations (range) were 7.1 – 9.9 μg/L. Ten 

cow milk-based baby formulas were analyzed. MEHP concentrations (range) were 5.6–9.1 μg/L.

Yano et al. (19) measured DEHP in 27 powdered formula products obtained in 11 countries. The 

formulas had been produced in 12 countries (Japan, Taiwan, Vietnam, Turkey, the United Kingdom, 

Germany, Spain, Netherlands, New Zealand, Denmark, Ireland, and the US). Phthalate levels ranged 

from about 32 to 533 ng/g powder [estimated from a graph]. A single sample produced in Turkey 

contained the highest level of DEHP. Excluding this sample, the highest DEHP concentrations (averaged 

by country of production) were around 200 ng/g [estimated from a graph]. The authors estimated 

that a 3 kg child consuming 700 mL/d formula would receive a daily DEHP dose of 2.5– 16.1 μg/kg 

bw, below the European Commission tolerable daily intake of 37 μg/kg bw.

In Japan, the estimated dietary daily intake of several plasticizers, including DEHP, resulting from 

the preparation, packaging, and storage of food in 3 hospitals was calculated by Tsumura et al. (20). 

This study was an update of a similar study conducted in 1999 (21) that found a high level of DEHP 

contamination from disposable gloves used by the food preparers, resulting in the regulation of these 

gloves by the Japanese Ministry of Health, Labor, and Welfare. DEHP concentrations from the duplicate 

diet samples containing predetermined amounts of protein, lipids, and carbohydrates varied by hospital 

and food type, but almost all (62 of 63 samples) contained measurable amounts of DEHP. The average 

daily DEHP intake was 160 μg/day [3.2 μg/kg bw/day based on a 50 kg bw], which was lower than 

the 1999 average daily intake of 519 μg/day and lower than the tolerable daily intake range (40 – 140 

μg/kg bw/day) set by the Japanese Ministry of Health, Labor, and Welfare.

Fromme et al. (22) measured concentrations of DEHP, dibutyl phthalate, butyl benzyl phthalate, diethyl 

phthalate, dimethyl phthalate, dimethylpropyl phthalate, di-n-octyl phthalate, dipropyl phthalate, and 

dicyclohexyl phthalate in indoor air and vacuum cleaner dust in 59 apartments and in indoor air in 74 

kindergartens in Berlin. The median indoor air DEHP concentration was 156 ng/m3 (95th percentile 

390, maximum 615 ng/m3) in apartments and 458 ng/m3 (95th percentile 1510, maximum 2253 ng/m3) 

in kindergartens. Median dust DEHP content in apartments was 703.4 mg/kg (95th percentile 1542, 

maximum 1763 mg/kg). DEHP accounted for more than 80% of the phthalate content of household 

dust. The authors estimated DEHP intakes for children assuming a body weight of 13 kg, inhalation 

of 5 m3 air/day and ingestion of dust at 100 mg/day to be 24 μg/kg bw/day, of which the largest contribution was an estimated food intake of 18 μg/kg bw/day (taken from the Canadian Environmental 

Protection Act). Estimated DEHP intake for adults was 5.06 μg/kg bw/day, assuming a 70 kg body 

weight, 23 m3/day inhaled air, and ingestion of 10 mg/day dust. The food contribution to this estimate 

was 4.9 μg/kg bw/day. [No source was given for the estimates of dust ingestion.]

Another evaluation of DEHP and 5 other phthalates in household dust was conducted in Sweden as part 

of a case-control study of children with allergic disease and asthma (23). Dust samples were obtained 

from children’s rooms in 346 homes. In the 343 samples with DEHP levels above the limit of detection, 

the geometric mean DEHP dust concentration was 0.789 mg/g. In samples from the homes of 173 
II-8

Appendix II

case children, the geometric mean DEHP dust concentration (95% CI) was 0.836 (0.724– 0.964), and 

in samples from the homes of 176 control children, the geometric mean dust concentration (95% CI) 

was 0.741 (0.643– 0.855). There was no significant difference between the DEHP dust concentration 

in the homes of cases and controls (P=0.232, t-test on log-transformed data).

Koo and Lee (24) measured DEHP in 42 perfumes, 8 deodorants, 21 nail polishes, and 31 hair care 

products marketed in Korea. DEHP was detected in 2 (4.8%) of the perfumes, 2 (9.5%) of the nail 

polishes, and none of the deodorants or hair products. The maximum DEHP detected in perfume was 

18.315 mg/L, and the maximum detected in nail polish was 25.077 mg/L. Based on questionnaires 

probing cosmetic use in the community, models were constructed for the estimation of DEHP exposure 

from these products. The 3 different models gave median exposure values of 0.6 – 26 ng/kg bw/day 

and 90th percentile values of 1.3–69 ng/kg bw/day.

1.1.2	 Exposure estimates based on biomarkers

Estimates of DEHP exposure are often based on urinary concentrations of DEHP metabolites, particularly MEHP or its oxidation products. Urinary measures of metabolites provide an integrative measure 

across routes of exposure. By contrast, blood serum DEHP and MEHP have been found below or 

at limit of detection (5.7 ng/mL) in healthy adults when environmental contamination is minimized 

through the use of HPLC-tandem MS (25). MEHP has also been measured in saliva (7) at up to 4.9 

ng/mL, comparable to serum. The median saliva value was lower than the limit of detection.

A study of the reproducibility of urinary MEHP concentrations was conducted by Hoppin et al. (26). 

The study sample consisted of 46 African American women between the ages of 35 and 49 years. The 

women collected first-morning urine samples on each of 2 consecutive days, timed to the onset of 

menses. Urine samples were frozen until analyzed. MEHP was determined using HPLC- tandem MS 

with both urine samples from each woman evaluated in the same laboratory run. The median (range) 

urinary MEHP concentration was 7.3 (1.0–143.9) ng/mL. Adjusted for urinary creatinine, the median 

(range) MEHP concentration was 6.4 (0.4 –77.3) μg/g creatinine. The intraclass correlation coefficient 

(95% CI) for urinary MEHP was 0.52 (0.32 –0.68). The intraclass correlation coefficient (95% CI) 

for creatinine-adjusted urinary MEHP was 0. 67 (0.49–0.79). Interperson variability was greater than 

intraperson variability. The authors indicated that the spot urine samples were a reliable biomarker of 

individual exposure, but because the urine collections were first-morning voids from consecutive days, 

the reproducibility represented in this study was a best-case example. Most women’s patterns of exposure 

may be sufficiently stable to assign an exposure level based on a single first-morning urine biomarker 

measurement. However, the authors also noted that no data exist to correlate these monoester urinary 

markers to total exposure over time since the biological half-life of MEHP is around 12 hours.

The National Health and Nutrition Examination Survey (NHANES) 1999 –2000 measured monoester 

metabolites of 7 phthalate esters in 2540 urine samples from adults and children older than 6 years (4). 

NHANES was updated in 2005 with data for the period 2001 – 2002 (n=2782), and phthalate levels in 

the 2 periods were similar (27). In 1999 – 2000, MEHP was found in more than 75% of the samples: 

87% from 6 – 11 year olds (n=328), 84% from 12 – 19 year olds (n=753), and 76% from adults ≥ 20 

years old (n=1461). [The Expert Panel noted that no children under age 6 were tested in either

time period, and it is most likely that MEHP would be detected in younger children.] Data from 

the 2001–2002 samples are summarized in Table 6. 
Appendix II

II9

Table 6. DEHP Metabolites in Urine in the NHANES 2001 –2002 Sample

Group

n Geometric mean (95% CI) 

Total Creatinine Corrected

MEHP 5-OH MEHP 5-oxo-MEHP

μg/L μg/g creatinine μg/L μg/g creatinine μg/L μg/g creatinine

Total 

Sample

— 2782 2772 4.27 (3.80–4.79) 3.99 (3.57–4.46) 20.0 (17.8–22.5) 18.8 (17.0–20.8) 13.5 (12.0–15.0) 12.6 (11.5–13.9)

Age 

Group 

(years)

6–11 393 392 4.41 (3.90–5.00) 5.02 (4.47–55.64) 33.6 (29.7–37.9) 38.3 (34.3–42.6) 23.3 (20.9–26.1) 26.6 (24.0–29.4)

12–19 742 742 4.57 (3.96–5.27) 3.53 (3.09–4.03) 24.9 (21.3–29.1) 19.2 (17.0–21.8) 17.5 (15.1–20.3) 13.5 (12.0–15.2)

20+ 1647 1638 4.20 (3.63–4.86) 3.96 (3.48–4.50) 18.1 (15.7–20.9) 17.2 (15.2–19.4) 12.0 10.5–13.9) 11.4 (10.2–12.8)

Sex

Male 1371 1367 4.31 (3.84–4.83) 3.49 (3.06–3.98) 22.0 (19.5–24.7) 17.9 (16.2–19.7) 14.5 (13.0–16.2) 11.8 (10.7–13.0)

Female 1411 1405 4.23 (3.67–4.86) 4.53 (4.01–5.11) 18.3 (15.7–21.4) 19.7 (17.3–22.5) 12.5 (10.8–14.6) 13.5 (11.9–15.3)

Race/

Ethnicity

Mexican American 677 674 4.32 (3.75–4.98) 4.05 (3.57–4.61) 18.5 (16.2–21.1) 17.5 (15.9–19.2) 13.1 (11.6–14.9) 12.4 (11.4–13.5)

Non-Hispanic Black 703 702 6.60 (5.57–7.82) 4.63 (3.95–5.42) 29.8 (26.1–34.1) 21.0 (18.8–23.3) 19.6 (17.1–22.5) 13.8 (12.3–15.4)

Non-Hispanic White 1216 1211 3.85 (3.37–4.40) 3.80 (3.33–4.33) 19.1 (16.7–21.9) 19.0 (17.1–21.1) 12.8 (11.2–14.6) 12.7 (11.4–14.1)

From Centers for Disease Control and Prevention (27).
II-10

Appendix II

NHANES noted that urinary MEHP levels were roughly comparable to those in previous reports [discussed below] for US residents (28), pregnant women in New York (29), and in men from an infertility 

clinic (30). The 2001 –2002 report indicated that levels of MEHP, 5-oxo-MEHP, and 5­‐OH-MEHP, the 

last 2 of which were evaluated for the first time in this report, were similar to or up to 2-fold higher 

than samples obtained in German adults and children (31-33). [The Expert Panel noted that in the

NHANES reports, levels below the detection limit were imputed by dividing the limit of detection

by the square root of 2. The procedure was unlikely to skew conclusions for children, because

most levels were above the limit of detection, but the Panel noted a possibility for error. The

Expert Panel considersthe NHANES data representative and thus generalizable to the entire US

population. From the publicly available NHANES 2001–2002 data (www.cdc.gov/nchs/nhanes),

the Expert Panel calculated the ratio of the oxidative monoester metabolites 5-OH-MEHP and

5oxo-MEHP to the monoester metabolite MEHP using standard procedures for analyzing

NHANES data (e.g., SAS and SUDAAN PROC DESCRIPT procedure). The ratio of oxidative

metabolites to monoester metabolites changed almost linearly with age group but not with sex

and race/ethnicity (Figure 2). Children aged 6–11 years produced a larger fraction of oxidative

metabolitesthan adolescents or adults.A further analysis of children and adolescents aged 6–15

yearsstratified by year of age showed a similar though less pronounced relation with the metabolite

ratios.Although the differences were most notable at the high end of the ratio distribution (95th

percentile) the trend was clearly still evident even at the median of the distribution.]

Figure 2. Age-dependent changes in primary and secondary metabolite ratios

0

4

6

8

R

a

tio

6 -11 12 -19 20+ 12

Age (yrs)

B

J

B

B

B

B

B

B B B

B

B B

B

B

B

B B B

B

B

J

J J

J

J

J

J

J J

J J J J

J

J J J

J J

B

B

B

J

J

J

BJ B

B

J

J

2

10

6 7 8 9 10 11 13 14 15

50th Percentile

5-OH-MEPH / MEPH

5-oxo-MEPH / MEPH

95th Percentile

5-OH-MEPH / MEPH

5-oxo-MEPH / MEPH

Age (yrs)

Obtained from the publicly available NHANES 2001 –2002 data <www.cdc.gov/nchs/nhanes>.

Itoh et al. (34) measured MEHP in urine samples collected from 36 Japanese adults. HPLC-tandem 

MS was used after enzymatic deconjugation. Estimates of DEHP exposure were based on the method 

of David (discussed below). The median (range) MEHP urine concentration was 5.1 (0.76– 25) μg/L. 

The creatinine-adjusted median (range) urine level was 4.5 (0.79 –27) μg/g creatinine. The estimated 
Appendix II

II-11

median DEHP intake±geometric SD (range) was 1.8±2.17 (0.37 –7.3) μg/kg bw/day. 

Brock et al. (35) measured urinary phthalate monoesters in 19 children aged 12– 18 months at a 

clinic visit and about 4 weeks later at a home visit. Phthalate-free adhesive collection bags were used 

to obtain the samples. Determinations were made using HPLC-tandem MS. Eight samples from 6 

children had detectable levels of MEHP ranging from 6.1 to 47.3 ng/mL [12 –202 μg/g creatinine,

calculated from data presented in the study].

Koch et al. (31, 36) estimated exposures to DEHP based on first-morning urine samples from 85 urban 

Germans aged 7 – 34 years (median age 33 years). Concentrations of MEHP and of the secondary 

metabolites 5-OH- and 5-oxo-MEHP were used with metabolite excretion factors to estimate exposure. 

Levels of the DEHP metabolites measured in urine are summarized in Section 1.7. MEHP concentrations predicted a median DEHP daily intake level of 10.3 μg/kg bw/day. The range of estimated DEHP 

daily intake was from the limit of quantification to 165 μg/kg bw/day, with a 95th percentile estimate 

of 38.3 μg/kg bw/day. The authors believed that the primary metabolite, MEHP, was susceptible to 

contamination, and that the low urinary MEHP concentrations made it difficult to estimate accurately 

DEHP exposures. Concentrations of the secondary metabolites were 3– 5 times higher than MEHP 

concentrations and gave a median DEHP intake estimate of 13.8 μg/kg bw/day with a 95th percentile 

estimate of 52.1 μg/kg bw/day. The secondary metabolites were considered by the authors to give a 

more accurate estimate of DEHP exposure, and any fluctuation in 1 metabolite was also seen in the 

other. Men had higher daily intake estimates than women (95th percentile 65.0 μg/kg bw/day for men 

and 27.4 μg/kg bw/day for women). No significant relationships were found between estimated DEHP 

daily intake and lifestyle habits obtained from a questionnaire.

David (37) argued in a letter-to-the-editor that Koch’s daily intake estimate was too high. David’s 

estimation of DEHP, based on a different MEHP molar excretion fraction, was approximately 5 times 

lower (median daily intake 1.76 μg/kg bw/day compared to the Koch et al. estimate of 10.3 μg/kg bw/

day). Koch responded stating that conservative fractions were used because there were limited studies 

regarding molar extraction fractions (38). In addition, Koch noted that if the higher molar extraction 

values were chosen and the secondary metabolites also considered, the metabolite dose would exceed 

100% of the DEHP dose. Koch also pointed to his conclusion that the secondary metabolites were 

better predictors of DEHP exposure than was MEHP.

Koo and Lee (39) measured DEHP, MEHP, and other phthalates (diethyl, dibutyl, and benzyl butyl) in 

the urine of 150 Korean women 20 –73 years old and 150 Korean children 11 – 12 years old [method

ofsubjectselection notspecified except as “hospital visitors”]. Geometric mean urinary DEHP was 

12.5±17 μg/L in women and 9.5±8 μg/L in children [error assumed to be geometric SD]. Geometric 

mean urinary MEHP was 41.3±50 μg/L in women and 13.3±24 μg/L in children. Geometric mean 

DEHP adjusted for creatinine (μg/g creatinine) was 16.0 in women and 7.8 in children. Geometric 

mean MEHP adjusted for creatinine (μg/g creatinine) was 39.6 in women and 9.6 in children. The 

authors estimated median daily DEHP intake to be 21.4 μg/kg bw in women and 6.0 μg/kg bw in 

children with a 95th percentile estimated daily DEHP intake of 158.4 μg/kg bw in women and 37.2 

μg/kg bw in children. They noted that more than 40% of women had an estimated daily intake above 

the tolerable intake level of 37 μg/kg bw/day set in 1998 by the EU Scientific Committee for Toxicity, 

Ecotoxicity, and the Environment.
II-12

Appendix II

Koch et al. (40) evaluated urine and serum levels of DEHP metabolites after a single oral dose of 

deuterium-labeled DEHP. A 61-year-old male volunteer weighing 75 kg (the senior author) consumed 

48.10 mg [641 μg/kg bw] labeled DEHP. The DEHP was incorporated into butter and eaten on bread. 

Urine samples were collected prior to dosing and for 44 hours thereafter. Blood samples were collected 

prior to dosing and every 2 hours thereafter, for a total of 5 blood samples, the final of which was 8 

hours post-dosing. Blood was immediately centrifuged. Urine and serum samples were frozen until 

analyzed. MEHP, 5-oxo-MEHP, and 5-OH-MEHP were determined by reverse phase HPLC-tandem 

MS. The peak urine concentration of labeled MEHP was 3.63 mg/L, 2 hours after the dose. The peak 

urine concentration of labeled 5-OH-MEHP was 10.04 mg/L, and the peak urine concentration of 

5-oxo-MEHP was 6.34 mg/L. The peak urinary concentrations of these MEHP oxidation products 

occurred 4 hours after the dose. Over the course of the 2-day study period, 47% of the DEHP dose was 

represented in urine (on a molar basis) by 1 of the 3 measured metabolites. On a molar basis, 7.34% of 

the administered DEHP dose appeared in the urine as MEHP, 24.7% of the administered DEHP dose 

appeared in the urine as 5-OH-MEHP, and 14.9% of the administered DEHP dose appeared in the urine 

as 5-oxo-MEHP. Serum concentrations of MEHP were higher than those of its oxidation products at 

all time points, consistent with the more rapid urinary elimination of the polar metabolites. Estimated 

serum elimination half-lives for the 3 measured DEHP metabolites were all less than 2 hours.

Koch et al. (9) published a further characterization of DEHP urinary metabolites that may be useful 

in estimating DEHP exposure. The focus of the study was 2 ω-oxidation products, mono(2-ethyl-5-

carboxypentyl) phthalate and mono[2-(carboxymethyl)hexyl]­ phthalate. This paper presented urine 

and blood measurements of 5 DEHP metabolites obtained from a single 61-year-old German male 

(the senior author) after oral ingestion of 3 different doses of deuterium ring-labeled DEHP in butter 

(doses were separated by 1 week). The proportional metabolite excretion relative to the DEHP dose 

did not vary by dose (Table 7). 

Table 7. Urinary Metabolite Excretion 24 Hours after Oral Ingestion of DEHP

Metabolite

Estimated 

Elimination 

t1/2 (h)

DEHP dose (μg/kg bw)

4.7 28.7 650

MEHP 5 6.2 4.3 7.3

5-OH-MEHP 10 23.1 22.7 24.1

5-oxo-MEHP 10 17.3 13.0 14.6

mono(2-ethyl-5-carboxypentyl)phthalate 12 – 15 15.5 19.4 20.7

mono[2-(carboxymethyl)hexyl]phthalate 24 3.7 5.2 3.8

Total percent of DEHP dose 65.8 64.6 70.5

t1/2=half-life. 

Data expressed as percent of administered deuterium-labeled DEHP on a molar basis. 

From Koch et al. (9).

Over the first 2 days, 74.3% of the administered DEHP dose was excreted as metabolites, the most 

abundant of which, on a molar basis, was 5-OH-MEHP (24.7% of the DEHP dose), followed in 

descending order by mono(2-ethyl-5-carboxypentyl) phthalate (21.9%), 5-oxo-MEHP (14.9%), MEHP 

(7.34%), and mono[2-(carboxymethyl)hexyl] phthalate (5.4%). The authors suggested that the use of 
Appendix II

II-13

secondary DEHP metabolites in urine would give a more accurate estimate of DEHP exposure and dose 

than MEHP in blood or urine. The study authors noted that serum MEHP is not a useful biomarker of 

DEHP exposure due to its short half-life. However, they stated that serum levels were present at the 

same orders of magnitude as in animal studies, despite the fact that the human dose was 50–1000 times 

lower than in animal studies. The authors noted that if it is assumed that MEHP in blood is a surrogate 

for toxic potential, DEHP would be 15–100 times more toxic in humans than in marmosets or rats. 

Barr et al. (8) conducted a urinary metabolite study to evaluate whether the metabolites 5-OH-MEHP 

and 5-oxo-MEHP were better biomarkers than MEHP of DEHP exposure. In the 50 (of 62) urine samples 

of adults and children that had detectable levels of all 3 metabolites, the average concentration of 5-OHMEHP was 4.3 times higher than the average concentration of MEHP; 5-oxo-MEHP concentration was 

approximately 3 times higher than the MEHP concentration. The median concentration of 5-OH-MEHP 

was 36 ng/mL, the median concentration of 5-oxo-MEHP was 28 ng/mL, and the median concentration 

of MEHP was 4.5 ng/mL. Concentrations of 5-OH- and 5-oxo-MEHP were highly correlated to one 

another (r2=0.984), and both were correlated with MEHP (r2=0.944 for 5-oxo-MEHP and 0.892 for 

5-OH-MEHP). 5-OH- and 5-oxo-MEHP appeared to be formed consistently within each individual 

subject (5-OH/5-oxo ratio 1.4, relative standard deviation [SD] 22%), but there appeared to be variations 

between individuals in the oxidization of MEHP (5-OH-MEHP/MEHP ratio 8.2, relative SD 80%; 

5-oxo-MEHP/MEHP ratio 5.9, relative SD 74%). The authors concluded that 5-OH- and 5-oxo-MEHP 

are “more sensitive indicators” than MEHP due to higher urinary concentrations and frequency of 

detection, although MEHP was considered a valid biomarker for health endpoints. The authors also 

noted that because NHANES used only MEHP as a biomarker for DEHP, exposure levels may have 

been higher than previously calculated.

Kato et al. (6) analyzed 127 paired human serum and urine samples for MEHP and the secondary 

metabolites 5-OH- and 5-oxo-MEHP. The volunteers in this experiment were aged 6 years and older 

and had no known previous DEHP exposure (Silva, M personal communication June 29, 2005). The 

concentrations of the secondary metabolites were 10 times the concentrations of MEHP in urine; 

metabolite levels are summarized in Section 1.7. 5-OH- and 5-oxo-MEHP were excreted primarily as 

their glucuronide conjugates, and their concentrations were highly correlated with one another (r=0.928, 

P<0.0001). Fewer than half of the serum samples had detectable levels of 5-OH- and 5-oxo-MEHP, 

and unlike the urinary samples, sera contained higher concentrations of MEHP than of 5-OH- and 

5-oxo-MEHP. The authors noted that because lipases that convert DEHP to MEHP are present in the 

serum samples, MEHP concentrations may have been artifactually increased by any DEHP introduced 

during blood collection and storage. The authors’ conclusions were similar to those of Barr et al. (8) 

that 5-OH- and 5-oxo-MEHP appear to be more sensitive urinary biomarkers than MEHP of DEHP 

exposure, but that MEHP remains important in studying the health effects of DEHP exposure.

Becker et al. (33) measured MEHP, 5-OH-MEHP, and 5-oxo-MEHP in first-morning urine samples 

collected from 254 German children aged 3–14 years. House dust samples were collected from ordinary 

vacuum cleaner bags and extracted with toluene for analysis of DEHP. Questionnaires were used to collect 

information on age, gender, nutrition, time spent on the floor, floor coverings, furniture, urban versus 

rural residence, diet, and the presence of orthodontic braces. The non-creatinine-adjusted geometric 

mean urinary MEHP concentration was 7.91 μg/L (range 0.74–226 μg/L), the geometric mean urinary 

concentration of 5-OH-MEHP was 52.1 μg/L (range 1.86 –2590 μg/L), and the geometric mean urinary 
II-14

Appendix II

concentration of 5-oxo-MEHP was 39.9 μg/L (range < 0.5 –1420 μg/L). As in the previous studies 

by Koch et al. (31, 36), urinary concentrations of 5-OH- and 5-oxo-MEHP were higher than those of 

MEHP and correlated with one another (r=0.98). MEHP concentrations correlated significantly but 

less closely with 5-OH-MEHP (r=0.72). Geometric mean concentrations of all 3 metabolites were 

19 –34% higher in boys than girls. When 2-year age blocks were considered, children at 13–14 years 

of age had the lowest geometric mean urinary concentration of 5-OH- and 5-oxo-MEHP. The ratios 

of secondary metabolites to MEHP also decreased with increasing age, suggesting age-dependent 

metabolism. None of the factors identified by questionnaire were significant determinants of urinary 

DEHP metabolites. House dust contained a geometric mean DEHP concentration of 508 mg/kg [ppm]

(range 22 – 5530 mg/kg). There was no correlation between house dust concentration of DEHP and 

urinary concentrations of MEHP (r=0.06) or 5-OH-MEHP (r=0.00). The authors concluded that failure 

to show a correlation between house dust DEHP and urinary DEHP metabolites may have been due to 

consideration of the entire sample of children (ages ranging from 3 to 14 years). They proposed that 

evaluation of very young children, who are more likely to spend time on or near the floor, might show 

such a correlation. They indicated that their study had too few children in this age group to evaluate 

this possibility. [The Expert Panel noted that an alternative explanation for the lack of correlation

between dust and urinary measures is that dust is not the only exposure medium.]

Koch et al. (32) measured MEHP, 5-oxo-MEHP, and 5-OH-MEHP in first-morning urine samples collected from 36 German nursery-school children aged 2.6–6.5 years. Four teachers and 15 parents also 

provided urine samples. Determinations were made using multidimensional liquid chromatography and 

tandem MS. Urinary concentrations in adults and children were compared using the Mann-Whitney 

U-test. The results are shown in Table 8. The authors interpreted these results as demonstrating that 

DEHP exposure was greater [double] among children than adults living in the same environment. The 

difference between children and adults was particularly evident when creatinine adjustment was used. 

The authors indicated that there was no relationship between urinary DEHP metabolite concentration 

in children and parental reports (by questionnaire) of mouthing activities. The authors speculated that 

the difference between children and adults might be attributable to dust inhalation or to differences in 

food phthalate exposures. The study authors concluded that exposure of children was twice as high 

as adults when body weight was considered, and that measures to reduce exposure of children need 

to be considered. The authors also suggested that using 5-oxo- and 5-OH-MEHP as biomarkers of 

exposure in children may be preferable to using MEHP because the oxidation products are present 

at higher concentrations and less likely to be affected by environmental contamination. [The Expert

Panel noted that thisimportantstudy suggested double the exposure in pre-school aged children

compared to adults living in the same environment. However, it was noted that highly significant

differences between creatinine-adjusted child and adult concentrations were likely due to natural

differencesin creatinine excretion between children and adults.Because creatinine excretion is a

function of lean muscle mass,smaller individuals excrete lessthan larger individuals, and children

excrete half the levels of adults.Thus, exposures based upon creatinine-corrected results would

lead to overestimation of exposure in children compared to adults.However, creatinine-corrected

results may be a reasonable surrogate for body weight-adjusted dose. The study demonstrated

no correlation between biomarker-measured exposure and mouthing behavior (by report, no

data shown). Oxidation to secondary metabolites was observed to be much higher in children

compared to adults. Study authors noted the lack of toxicity data on secondary metabolites at

the time this report was written.]
Appendix II

II-15

Table 8. DEHP Metabolites in the Urine of Nursery-school Children and Adults

Metabolite

Urine Concentration, Median 

(Range)

P (children vs 

adults)

Unadjusted 

(μg/L)

Adjusted (μg/g 

creatinine)

Unadjusted/

Adjusted

MEHP

Children 6.6 

(1.5 –18.3)

8.7 

(1.7 –48.4) 0.045/0.908

Adults 9.0 

(2.6 –43.1)

8.6

 (3.8 –26.6)

5-OH-MEHP

Children 49.6 

(2.7 – 129)

55.8 

(15.4 –258) 0.038/< 0.0001

Adults 32.1 

(10.7 – 103)

28.1 

(10.9 –63.6)

5-oxo-MEHP

Children 33.8 

(2.2 – 90.6)

38.3

(10.2 –158) 0.015/< 0.0001

Adults 19.6 

(4.9 – 55.1)

17.2 

(4.5 –40.9)

Sum of the 3 

metabolites

Children 90.0 

(6.3 – 221)

98.8

(28.7 –464) 0.074/< 0.0001

Adults 59.1 

(21.1 – 201)

50.9 

(20.5 –124)

From Koch et al. (32).

Ten men participated in a study by Hauser et al. (41) in which 8 phthalate monoesters were measured 

monthly in urine for 3 consecutive days over the course of 3 months. The measured monoesters were the 

monoethyl, -butyl, -benzyl, -methyl, -octyl, -isononyl, and -cyclohexyl phthalates and MEHP. Five of 

the phthalates were identified in more than 90% of the urine samples with substantial variation between 

day-to-day and month-to-month levels. More variation was observed for urine samples collected 1 – 3 

months apart compared to 1 –2 days apart. The authors concluded that the predictive value of a single 

urine measurement in characterizing exposure as high, medium, or low over the course of 3 months 

was highest for monoethyl phthalate and lowest for MEHP. Of men in the highest tertile for MEHP 

exposure based on the 3 months of urine monitoring, 56% would have been identified by a single 

urine sample. A single MEHP measurement would have identified 83% of men in the lower 2 tertiles. 

The study authors noted that the poor predictive value of MEHP levels in single urine samples could 

lead to misclassification of exposure over a 3-month period and bias towards the null hypothesis when 

assessing exposure-response relationships.

Adibi et al. (29) measured phthalate diesters in 48-hour air monitoring samples collected by 30 pregnant 

women in New York city and 30 pregnant women in Krakow, Poland. The New York women were 

Dominican or African-American, were of low socioeconomic status, and were in the third trimester 

of pregnancy. The Polish women had been residents of the city for at least a year, were middle-class, 

and were in the second trimester. Spot urine samples were collected at the end of the personal air 

sampling period from 25 of the New York women and analyzed for monoester metabolites of the 

phthalate diesters. Spearman rank correlation was used to evaluate the relationship between the 

diester concentration in air and the corresponding monoester concentration in urine. All 60 personal 
II-16

Appendix II

air samples contained measurable concentrations of diethyl, di-n-butyl, di-isobutyl, and butyl benzyl 

phthalate and DEHP. The median DEHP air concentrations (ranges) were: New York 0.22 (0.05–0.41) 

μg/m3, Krakow 0.37 (0.08 – 1.1) μg/m3. Median (range) urine MEHP in the subset of 25 New York 

women was 4.60 (1.80 – 449) μg/g creatinine. The study authors noted that urinary MEHP levels 

reported for the New York group were similar to values reported for the NHANES sample. There was 

no significant relationship between urinary MEHP and air sample DEHP concentrations (Spearman 

correlation coefficient 0.37). [The Expert Panel noted that the lack of association between air

levels and MEHP urine levels may be due to the fact that air is not the only exposure medium.]

Significant correlations were reported for personal air samples and urinary concentrations of butyl 

benzyl, dibutyl, and diethyl phthalate. 

Latini et al. (42) reported placental transfer of DEHP and MEHP. They found either DEHP or MEHP in 

87.5% of 24 maternal plasma samples and 76% of 25 umbilical cord samples (1 set of twins). Samples 

were collected at delivery at 35 –42 weeks gestation. DEHP was measurable in 17 (71%) of the 24 

maternal samples and 11 of 25 cord samples (P=0.024, chi-squared). MEHP was measurable in 18/24 

(75%) of maternal samples and 18/25 (72%) cord samples (P NS). Mean±SD DEHP concentrations 

were higher in cord plasma than in maternal plasma (2.05±1.47 μg/mL [n=11] compared to 1.15±0.81 

μg/mL [n=11], P=0.042, t test). MEHP concentrations in maternal and umbilical cord plasma were 

similar with mean±SD values of 0.68±0.85 for maternal plasma and 0.68±1.03 μg/mL for cord 

plasma. The authors concluded that variation in plasma concentrations could have been due to different 

environmental exposures, and that fetal bioaccumulation may have been due to lack of maturation of 

excretory organs. Although no statistical correlations were found between DEHP and MEHP levels in 

either the mother or infant, the authors noted that exposure to phthalates begins prenatally and suggested 

that fetal exposure is “closely related to maternal exposure.” [The Expert Panel was concerned that

there had been pre-analytic contamination in this study because the measured levels of MEHP

were 3 orders of magnitude above levels obtained in other studies.]

An abstract (43) reported maternal and cord blood phthalate concentrations in samples collected from 

50 maternal-child pairs at cesarean section. MEHP and its oxidative metabolites were said to be present 

at higher concentrations in fetal than maternal blood. [Data were not given.Abstracts are noted but

are not used in the evaluation.]

Silva et al. (44) measured phthalate monoesters, including MEHP and its oxidative metabolites, in 

amniotic fluid samples from 54 women. The samples were described as having been obtained during 

“routine amniocentesis.” [The Expert Panel notes that amniocentesis is performed for clinical

indications and is never routine.] No demographic or clinical information, including gestational age, 

was available for any of the samples. Quantification was performed using HPLC-tandem MS. MEHP 

was detected in 24% of the samples, but its oxidative metabolites were not present above the limit of 

detection in any sample. The median MEHP amniotic fluid level was below the limit of detection, with 

a maximum detected vale of 2.8 ng/mL. The authors noted that MEHP levels in amniotic fluid, which 

is fetal urine, were lower than NHANES reported for urine in adults and children. They further noted 

that the fetus may not be able to biotransform MEHP to its oxidative metabolites due to immaturity 

of the liver.
Appendix II

II-17

1.2 Exposure Assessed Through Toys

Bouma et al. (45) measured DEHP released from 47 PVC-containing toys bought in the Netherlands 

after mixing with a saliva stimulant [composition not specified]. DEHP was found in 20 (43%) of 

the 47 toys at 3–44% by weight. Migration of DEHP into saliva simulant increased with increasing 

DEHP content. Six toys exceeded the Dutch guidance release value (2.3 μg/min/10 cm2) for children 

younger than 1 year. Six toys also exceeded the Scientific Committee on Toxicity, Ecotoxicity and the 

Environment migration guidance of 1.7 μg/min/10 cm2; 5 of these toys were intended for children 

older than 3 years of age.

Niino et al. (46) identified migration into simulated saliva of DEHP from a sample of a PVC ball 

that contained DEHP 190 mg/g. The simulated saliva contained sodium chloride 4.5 g/L, potassium 

chloride 0.3 g/L, sodium sulfate 0.3 g/L, ammonium chloride 0.4 g/L, urea 0.2 g/L, and lactic acid 

3.0 g/L dissolved in distilled water and adjusted to pH 6.5– 7.0 with 5 M sodium hydroxide. The 

ball portion was shaken with the simulated saliva at 35oC for 15 minutes, yielding DEHP 315±25.0 

μg/hour/10 cm2 (mean±SD, n=5). When 4 volunteers [age not specified] chewed a ball segment for 

four 15-minute sessions separated by rest periods, the amount of mobilized DEHP measured in saliva 

was 44.4±12.3 μg/hour/10 cm2. Hydrolysis to MEHP occurred to a limited extent. Over the 60-minute 

chewing period, salivary DEHP decreased from about 42 to 32 nmol while MEHP increased from 0 

to about 2 nmol [estimated from a graph].

1.3 Exposure Through Building Materials

Otake et al. (47) measured indoor air concentrations of common phthalates and phosphate esters in 

Japanese homes. Twenty-seven homes in the Tokyo metropolitan area had indoor air concentrations 

of DEHP ranging from < 0.001 to 3.13 μg/m3. The mean±SD DEHP concentration was 0.32±0.6 

μg/m3, the second highest concentration next to dibutyl phthalate, which was 0.75±1.17 μg/m3. DEHP 

levels were 100 – 1000 times higher than ambient outdoor air concentrations reported in articles cited 

by the author: 2.0 ng/m3 in Sweden and 16 ng/m3 in Japan. 

Danish authors (48, 49) studied the DEHP emission and sorption characteristics of PVC flooring material 

in an emission cell and in an emission test chamber. Airborne DEHP concentrations increased up to 

150 days, at which point emissions leveled off at approximately 1 μg/m3. Dust on soiled PVC material 

increased the emission rate of DEHP; dust sorbed 3700 μg of DEHP compared to 900 μg emitted from 

unsoiled PVC material over 68 days. The authors concluded that resuspended dust may be an important 

route of DEHP exposure. A similar evaluation of PVC-coated wall coverings showed variable chamber 

air DEHP concentrations with a maximum air concentration of just under 1 μg/m3 (50).

1.4 Exposure Through Wastewater

Marttinen et al. (51, 52) found DEHP to be the most frequently encountered phthalate in sewage in 

Finland, with DEHP concentrations of 98 – 122 μg/L compared to <5 μg/L for other phthalates. The 

highest concentration, 160 – 166 μg/g, was found in treated sewer sludge; this concentration exceeded 

the maximum value set by the European Commission for the use of sludge in agriculture. In 4 sewage 

treatment plants, 80 – 96% of DEHP was removed, but the remaining DEHP accumulated in treated 

sludge. In leachate from 11 landfills, DEHP was the most commonly identified of 14 measured waste 

chemicals. When landfill leachate was handled in the same treatment plants as sewage, the contribution 

of leachate DEHP to total sewage leachate was low, on the order of 1%.
II-18

Appendix II

Sewage sludge in Spain contained DEHP at levels of 180 –267 ppm dry matter [μg/g] (53). Composts 

made with sludge had DEHP contents of 38–99 ppm dry matter, and soil mixtures with sludge or 

compose contained DEHP 3­ – 21 ppm at mixing, declining to 0.4 – 2.5 ppm 9 months later. A study 

from Scotland did not find a difference in tissue levels of DEHP in sheep grazing on pastures fertilized 

with sewage sludge compared to pastures treated with inorganic fertilizer (54). 

1.5 Medical Exposures

Loff et al. (55) quantified DEHP leaching from PVC infusion set tubing during infusion of parenteral 

nutrition, blood products, and selected pharmaceuticals at room temperature (27oC) using neonatal 

intensive care (NICU) protocols employed in treating sick neonates. The highest DEHP concentration 

was found in lipid-containing solutions used for parenteral nutrition (424.4 μg/mL over 24 hours) 

resulting in an exposure of 5 mg/kg bw for a 2 kg infant (25 mL solution). Small amounts of DEHP 

were found in an amino acid/glucose solution (0.83 μg/mL, 24 hours). Blood products stored in 20 mL 

PVC bags contained 7 –339 μg/mL DEHP. When the blood product in the PVC bags was administered 

through PVC tubing, a single 20 mL dose of a blood product for a 2 kg baby was estimated to contain 

608 μg DEHP for packed red blood cells, 928 μg DEHP for platelet-rich plasma, and 552 – 8108 μg 

DEHP for fresh frozen plasma. [MEHP levels were not measured.] Administration of 1% propofol 

(10 mL) resulted in a daily DEHP dose of 6561 μg. Administration of 28.8 mL fentanyl resulted in 

a DEHP dose of 132.5 μg and administration of 24 mL midazolam resulted in a DEHP dose of 26.4 

μg. The study authors concluded that the dose of DEHP for a typical preterm neonate requiring total 

parenteral nutrition (TPN) and additional therapy can range from 10 to 20 mg/day.

Data from the Loff et al. (55) study were used by theFDA (2) to estimate infant exposures to DEHP 

through administration of sedatives (discussed below). Because propofol is not approved for sedation 

in pediatric patients, the intake value from fentanyl (0.03 mg/kg bw/day) was used as the upper-bound 

estimate of DEHP exposure of 4 kg neonates receiving conscious sedation. [The Expert Panel notes

that the FDA report was dated 2001 on the FDA web site, although the cited publication in

Government Reports,Announcements, & Index was dated 2004.]

Loff et al. (56) updated their previous study (55) by evaluating the effects of temperature on DEHP 

release. Temperature and contact time greatly affected the release of DEHP from PVC-infusion lines 

into a lipid-containing infusion solution. An increase in temperature from 27oC (the temperature used in 

the earlier study) to 33oC increased the amount of released DEHP by approximately 30% (422 μg/mL 

at 27oC and 540 μg/mL at 33oC). The administration of 24 mL of this infusion to a 2-kg newborn 

resulted in a DEHP dose of 13 mg (6.5 mg/kg bw) at 33oC compared to 10 mg (5 mg/kg bw) at 27oC. 

The rate of extraction of DEHP from PVC tubing was directly related to the length of contact time 

between the solution and the tubing. The concentration of DEHP in the infusion solution increased 

from 25 μg/mL at 4 hours to 478 μg/mL between 20 and 24 hours. The authors noted that these findings 

were important because neonatal ICUs are typically maintained at 30o, and incubator temperatures can 

reach 37oC. Loff et al. (56) also noted that these exposure estimates were only from 1 type of medical 

device, and that newborns in these units can be exposed through other devices as well. 

[The Expert Panel noted limitations in the Loff et al. studies (55, 56). First, the authors did not

address prevention of DEHP contamination or mention if contamination was a problem. Second,

because blood products contain enzymes that can metabolize DEHP to MEHP, measurement
Appendix II

II-19

of DEHP alone may underestimate total blood product exposure to DEHP-related chemicals.

Several important observations were made by the Expert Panel.The first isthat even though glass

containers were used to store lipophilic substances that were slowly infused through PVC tubing,

significant DEHP exposure was estimated. Second, data demonstrated that at NICU temperatures

(33oC), perfusion ofTPN through PVC tubing increased DEHP extraction by 20–30%, compared

to extraction observed at room temperature (27oC).A study from another group also showed the

influence of temperature on DEHP release from PVC (57).Third, extraction rates increased for

the first 12 hours and then slowed. Lastly, rinsing lines did not affect leaching.]

Loff et al. (58) reported the extraction by lipid emulsions of DEHP from different brands of PVC 

infusion tubing and different lengths of tubing. Emulsions were run through the lines at 1 mL/minute 

for 24 hours and were collected in glass flasks. Glassware was rinsed with solvents and heated for 

removal of possible DEHP contamination. After infusion through PVC tubing, DEHP was present in 

emulsions at concentrations of 69–117 mg/L. When PVC tubing with a polyurethane lining was used, 

the post-infusion emulsion concentration was 67– 78 mg/L, and when PVC with a polyethylene liner 

was used, the emulsions concentration was 32–52 mg/L. The amount of DEHP in the emulsion after 

infusion increased with tubing length.

Another study (59) measured the extent of DEHP leaching into a lipid-containing TPN solution from 

ethyl vinyl acetate bags with PVC connectors and tubing. The bags and tubing were stored at 4oC for 

24 hours or 1 week prior to simulated use. The 1-week storage period simulated conditions that can 

occur in home parenteral nutrition programs. For both storage periods, DEHP concentrations were 

highest in solutions with the highest lipid content (3.85%) and decreased for simulated infusions as 

flow rate decreased. After storage for 24 hours, DEHP content per infusion set ranged from 0.2 to 0.7 

mg in the ethyl vinyl acetate bags and from 0.8 to 2 mg in the outlet tubing. The authors concluded 

that the DEHP dose from a TPN infusion could range from 0.8 to 2 mg/day for an infant or child 

depending on the lipid content and flow rate. [The Expert Panel noted that the study provides new

information on DEHP leaching during home TPN use.]

Kambia et al. (60) used an HPLC method to measure the amount of DEHP leaching into lipid-containing TPN solutions stored in ethyl vinyl acetate bags with PVC outlets and infused through PVC 

tubing. The amount of DEHP leaching into TPN solutions was estimated at 0.2±0.008 to 0.7±0.02 

mg from bags and 0.8±0.09 to 2±0.07 mg from tubing. [Variances assumed to be SD as reported in

other parts of the paper.] DEHP was measured at 0.3–6.9 μg/mL in blood samples from 4 children 

receiving TPN. [Levels of DEHP metabolites in blood were not measured.]

PVC tubing designed to reduce DEHP leaching by using an “inert” polyethylene inner lining did 

not show significant differences in the amount of DEHP released into solution compared to standard 

PVC tubing (61). Three types of multi-layer tubing (PVC, PVC/polyethylene, and PVC/ethyl vinyl 

acetate/polyethylene) were tested using an etoposide solution containing a polysorbate excipient. DEHP 

concentration increased nonlinearly with polysorbate concentration and linearly with temperature and 

contact time. DEHP leaching was particularly evident during the first 2 hours of contact. The authors 

concluded that polysorbate was responsible for the release of DEHP into etoposide solutions, and that 

the polyethylene linings did not prevent the release of DEHP into solutions. They noted that DEHP was 

found on the inert lining even before coming in contact with either solution and suggested that DEHP 
II-20

Appendix II

might gain access to the tubing lumen through pores in lining materials. The authors suggested the use 

of polyethylene-only tubing for infants in incubators (37oC) who receive solutions with polysorbate. 

[The Expert Panel noted that the study demonstrates that multilayer tubing does not prevent

leaching as marketed and is not a solution to DEHP exposure problems. However, failure to

address possible contamination could have been the reason why no differences were observed

between lined and unlined tubing.]

Haighton et al. (62) published an abstract in which DEHP exposure from a closed inhalation spray 

container was estimated at 0.0037 μg/kg bw/day. Details of the assumptions made in this estimation 

were not available in the abstract. [The Expert Panel notes this abstract for completeness, but the

abstract will not be considered in the evaluation.]

Calafat et al. (5) conducted a study to measure DEHP exposures in infants receiving multiple treatments 

in the NICU. Six premature newborns undergoing intensive care interventions for more than 2 weeks 

were tested for the 3 DEHP urinary biomarker metabolites MEHP, 5-oxo-MEHP, and 5-OH-MEHP. 

All 3 metabolites were found in 33 of the 41 urine samples collected from these infants. 5-OH- and 

5-oxo-MEHP were found in all 41 samples, and measurements of these 2 metabolites were an order 

of magnitude higher than those for MEHP. Urinary concentrations varied widely among the infants. 

[The Expert Panel noted that the high variability in the ratio of MEHP to oxidative metabolites

suggests metabolic variation. Less than 25% of the metabolites were present in “free form,”

which is the putative biologically active species.] Geometric mean 5-oxo-MEHP was 1617 ng/mL, 

5-OH-MEHP was 2003 ng/mL, and MEHP was 100 ng/mL. Urinary concentrations of 5-OH-MEHP 

and 5-oxo-MEHP were highly correlated. The author notes that the geometric means found in this 

study were several-fold higher than the MEHP geometric mean in the general US population 6 years 

and older (3.43 ng/mL). [The Expert Panel notes the high importance of this study because it is

the first to quantify real-world exposures resulting from the use of multiple DEHP-containing

devices in a contemporary NICU. The study documents DEHP exposures that are orders of

magnitude higher than the general population, including children ≥ 6 years. Three metabolites

were examined, and metabolism in premature infants was partially elucidated. Resultssuggested

that MEHP may not be the best marker of exposure. The study also enumerated persistent

data gaps regarding possibly increased susceptibility of children, including the effects of high

gastric lipase activity, lower capacity for glucuronidation, increased permeability of the bloodtestis barrier, and possibly increased absorption in the gastrointestinal tract. Limitations of the

study included small sample size, no measurement of exposure from individual sources, and no

discussion of primary MEHP exposure (i.e., MEHP infused directly into the patient).]

Green et al. (63) measured urinary DEHP metabolites in 54 infants in a NICU. The infants were 

hospitalized in 1 of 2 hospitals. One of the investigators observed the care of each infant for a total of 

3–12 hours (1–4 hours/day on 1–3 days) and noted the products used in the care of the infants. DEHP 

exposure was rated low, medium, or high based on the kind of medical devices used and the length of 

time used. Medical records were not consulted in evaluating infant exposures. Urine was collected from 

diaper liners or from cotton gauze placed in the diaper. The urine was collected during the observation period. Some infants had 2 or 3 urine specimens collected; in these instances, the urinary MEHP 

concentrations were highly correlated within infants. Urine was assayed for MEHP, 5-oxo-MEHP, and 

5-OH-MEHP using HPLC-tandem MS. [Only MEHP results were given in the paper.] Specimens 
Appendix II

II-21

with MEHP levels below the limit of detection were assigned a value of half the limit of detection. 

Statistical analysis of urinary MEHP by sex, institution, and DEHP exposure group was performed 

using the Mann-Whitney-Kruskal-Wallis test, multiple linear regression, and quartile regression.

Urinary MEHP levels are shown in Table 9. DEHP exposure group was described as a substantial 

predictor of urinary MEHP levels (P=0.09 after adjusting for infant sex and institution). Infants in 

the medium-exposure group had urinary MEHP concentrations twice as high (calculated from the 

regression model) as infants in the low-exposure group (P=0.3), and infants in the high-exposure group 

had urinary MEHP concentrations 5.1 times as high as infants in the low-exposure group (P=0.03). 

[The Expert Panel noted that urinary MEHP levels were quite different between infants at the

two hospitals and suggested that it may be due to different products used at the two hospitals.]

Table 9. Urinary MEHP in Infants in Two NICUs by DEHP Exposure Group

Exposure Group (n)

Urinary MEHP, μg/L, by percentile

25th Median 75th

Low (13) < 0.87 4 18

Medium (24) 3 28 61

High (17) 21 86 171

Exposure status assigned by observing use of medical device during 3 –12 

hours of the child’s care.

P=0.001 for exposure class (i.e., low, medium, high)

From Green et al. (63).

The Expert Panel is aware of recent reviews in which exposure to DEHP through medical devices was 

estimated. The most thorough estimates were conducted by the FDA (2) and are summarized in Table 

10. [The Expert Panel notes that the estimates were conducted using data that were available to

the CERHR Expert Panel during their first phthalates review and does not provide new data.]

The FDA noted a lack of data to estimate exposure through breast milk for infants of mothers who 

had undergone or were undergoing medical procedures like hemodialysis. The FDA believed that few 

infants were exposed to breast milk from women undergoing these kinds of medical procedures. 

A 1 m segment of PVC tubing was used to measure DEHP release into polysorbate 80 solutions 

(64). Physiological saline and distilled water solutions of polysorbate 80 resulted in greater DEHP 

release from tubing than did glucose solutions. Use of a flow rate of 90 mL/hour resulted in greater 

DEHP release than did 60 mL/hour. After 5 hours of infusion of 2 mg/mL polysorbate 80 at a rate 

of 90 mL/hour, the cumulative amount of DEHP recovered was 850 μg [estimated from a graph]. 

Recovery of DEHP was greater at 90 mL/hour than at 60 mL/hour, even when the concentration of 

polysorbate 80 was increased 1.5-fold at the lower flow rate, suggesting to the authors that the amount 

of polysorbate passing through the tube segment was less important than the speed of the polysorbate 

micelles interacting with the walls of the tubing. [The Expert Panel noted that the study is pertinent

considering the very slow flow rate of TPN administered to neonates in NICU settings.]
II-22

Appendix II

Table 10. FDA Estimates of DEHP Exposures Resulting from Medical Treatments

Medical procedure

Estimated DEHP 

dose (mg/kg bw/day)

70 kg 

adult

4 kg 

neonate

Crystalloid intravenous (iv) solution infusion 0.005 0.03

Infusion of pharmaceuticals with solubilization vehicles

Administered according to manufacturer instructions 0.04 0.03

Mixed and stored at room temperature for 24 hours 0.15

TPN administration

Without added lipid 0.03 0.03

With added lipid 0.13 2.5

Administered via ethyl vinyl acetate bag and PVC tubing 0.06

Blood transfusion

Trauma patient 8.5

Transfusion/extracorporeal membrane oxygenation 

(ECMO) in adult patients 3.0

Exchange transfusion in neonates 22.6

Replacement transfusions in neonates in NICU 0.3

Replacement transfusions to treat anemia in chemotheraphy and sickle cells disease patients 0.09

Replacement transfusions in patients undergoing coronary 

artery bypass grafting 0.28

Treatment of cryodisorders with cryoprecipitate 0.03

Cardiopulmonary bypass

Coronary artery bypass grafting 1

Orthotopic heart transplant 0.3

Artificial heart transplant 2.4

ECMO 14

Apheresis 0.03

Hemodialysis 0.36

Peritoneal dialysis < 0.01

Enteral nutrition 0.14 0.14

Aggregate exposures of NICU infants undergoing iv administration of sedatives, iv administration of TPN, and replacment 

transfusion

2.83

From FDA (2).

Polyethoxylated hydrogenated castor oil, an emulsifier used in pharmaceuticals to increase solubility, 

was found to increase the release of DEHP from PVC tubing when given in distilled water, glucose, or 

physiological saline (65, 66). Release appeared to increase linearly over time, reaching an approximate 

cumulative value of 776 μg DEHP after 4 hours. Sugar solutions (ribose, fructose, or glucose) containing 
Appendix II

II-23

polyethoxylated hydrogenated castor oil resulted in less DEHP release from tubing. DEHP levels 

increased with increasing polyethoxylated hydrogenated castor oil concentrations in all solutions. A 

decrease in release of DEHP from tubing was shown when paclitaxel in polyethoxylated hydrogenated 

castor oil was replaced by paclitaxel in polymeric micelles (67). Cyclosporine preparations, which use 

polyethoxylated hydrogenated castor oil in ethanol, have been shown to contain DEHP at concentrations 

of 3 –4 mg/L after storage in PVC bags for 12 hours (68).

Demore et al. (69) studied the release of DEHP from containers when the antineoplastic drug etoposide 

was stored. Etoposide was evaluated because it is prepared with the surfactant polysorbate 80, which is 

believed to release DEHP from PVC containers. After 24 hours at room temperature in PVC containers, 

etoposide in saline contained 18– 25 μg/mL DEHP, and etoposide in 5% dextrose contained 17– 25 

μg/mL DEHP. Etoposide in glass or polyolefin containers did not contain detectable levels of DEHP 

after similar time periods. Another study using etoposide infused through PVC tubing found that flow 

rate, tubing length, and etoposide concentration influenced DEHP leaching, with DEHP concentrations 

in the solutions of 54 –155 mg/L after 6 hours of infusion (70). As noted above, triple-layer tubing, 

with a PVC outer layer, a polyvinyl acetate middle layer, and a polyethylene inner layer, offered no 

advantage in preventing access of DEHP to the solution. Haishima et al. (71) evaluated the relationship of DEHP released from medical-grade PVC and physical chemical properties of 53 medications 

that are administered by injection. The most important predictor of DEHP release was lipid solubility 

of the medication preparation, which could be easily assessed by solubility of the lipophilic pigment 

methyl yellow. 

DEHP and MEHP in stored whole blood or red blood cells have been simultaneously measured and 

showed ranges of 6.8– 83.2 mg/L for DEHP and 0.3 –9.7 mg/L for MEHP (72). Platelets and fresh 

frozen plasma contained lower concentrations of both phthalates. The concentration in blood products 

increased with storage time.

In Japan, DEHP was measured in a blood circuit system used to simulate hemodialysis and pumpoxygenation therapy using heparin-coated and uncoated PVC tubing (73). In the hemodialysis system, the 

bovine blood used as the simulant had a baseline DEHP concentration of 249 ppb [μg/L]. After 4 hours 

of circulation, the DEHP concentration was 1718 ppb [μg/L], a 7-fold increase. In the pump-oxygen 

system, PVC tubing with covalently bonded heparin coating resulted in DEHP levels approximately 

50% lower than tubes with ionic-associated heparin coating or no coating at all. The DEHP daily dose 

for an 11 kg child exposed to 6 hours of pump-oxygenation therapy without heparin-coated tubes was 

estimated (using bovine blood) at 0.7 mg/kg bw/day and using heparin-coated tubing between 0.3 and 

0.6 mg/kg bw/day. An adult exposure was estimated at approximately 0.3 mg/kg bw/day for uncoated 

tubing and between 0.16 and 0.3 mg/kg bw/day for heparin-coated tubings. These values were noted 

by the authors to be above the upper limit of the tolerable daily intake established by the Japanese 

Ministry of Health, Labor and Welfare. In the pump-oxygenation system, the authors estimated that 

3–4% of DEHP was converted to MEHP. MEHP was also found to decrease with the use of covalently 

bonded heparin-coated PVC tubing, but concentrations increased over time. In the hemodialysis system, 

approximately 80 ppb [μg/L] MEHP was measured after 4 hours. The authors concluded that the use of 

PVC tubing for high-risk patients and for long-term therapy should be questioned. [The Expert Panel

noted that this study does not provide much new information, with the exception that covalent

and not ionic bonding of heparin to tubing is necessary to prevent significant leaching.]
II-24

Appendix II

Mettang et al. (74) compared serum, urine, and dialysate levels of phthalate acid esters in 5 adult 

peritoneal dialysis patients before and 42 days after the use of plasticizer-free bags and tubing. Following 

the switch to plasticizer-free materials, significant changes included reductions in phthalic acid levels 

in serum and MEHP and phthalic acid levels in effluent dialysate. Serum concentrations of DEHP 

decreased non-significantly. There was no effect on levels of MEHP or 2-ethylhexanol in serum or 

phthalic acid in urine. The study authors concluded that peritoneal dialysis patients are likely exposed 

to sources of phthalates in addition to dialysis equipment.

In an effort to simulate exposure during respiratory therapy, Hill et al. (75) measured the concentrations 

of phthalates including DEHP in air after passage through PVC medical tubing. DEHP was detectable 

in an unspecified proportion of samples but was not above limit of quantification or was not present at 

concentrations demonstrably higher than background. The presence of 2-ethylhexanol was interpreted 

as due to DEHP breakdown. The authors concluded that for most adults, exposure from respiratory 

therapy is small compared to other exposures, but that sensitive populations, particularly those with 

allergies to plasticizers or with asthma, may be at “significant risk” from respiratory therapy exposures.

[The Expert Panel noted that findings are consistent with previous studies.]

Platelet pheresis donors (n=36) were evaluated for DEHP exposure by measuring serum DEHP concentrations before and after pheresis sessions of 38–89 minutes (76). In 4 donors, additional serum 

samples were evaluated for up to 48 hours after the pheresis session. Median (range) serum DEHP 

increased from a baseline of 92.2 ng/mL (5.9– 219.6 ng/mL) to 213.8 ng/mL (7.3– 716.1 ng/mL). 

The authors estimated a median (range) DEHP dose of 6.46 (1.8 – 20.3) μg/kg bw. In the subjects 

with further serum monitoring, DEHP serum concentrations returned to baseline within 3 hours of 

the procedure. Serum triglyceride concentration was correlated with the relative increase in serum 

DEHP (r2=0.24, P=0.03).

Koch et al. (77) measured urinary concentrations of the DEHP metabolites MEHP, 5-OH-MEHP, 

5-oxo-MEHP, 5-cx-MEPP, and 2-cx-MMHP in 1 platelet pheresis donor before and for 24 hours 

after the pheresis procedure. Over the 24-hour period, the total molar excretion of DEHP metabolites 

was 4.508 μmol. Urinary excretion ratios from different authors yielded estimates of DEHP dose of 

2.6 –4.0 mg or 31.6 –48.1 μg/kg. The same authors evaluated 18 pheresis donors and 5 non-donor 

controls using 24-hour urine samples for measurement of MEHP, 5-OH-MEHP, and 5-oxo-MEHP (78). 

The first urine samples in the donors were collected just prior to the pheresis procedure. There were 

6 donors who underwent plasma pheresis and 12 donors who underwent platelet pheresis (6 with a 

dual-needle continuous-flow technique and 6 with a single-needle discontinuous flow technique). Mean 

metabolite concentrations in urine shortly after pheresis were about twice as high for continuous flow 

techniques as for discontinuous flow techniques. Most metabolite excretion occurred during the first 

5 hours after the pheresis procedure. Using metabolite excretion factors, DEHP doses were calculated 

as summarized in Table 11. The authors compared the weight-adjusted dose with the European Union 

tolerable daily intake values of 20–48 μg/kg bw. They suggested that the DEHP dose associated with 

plasma pheresis may not be elevated above background because the lipid-rich plasma removed by the 

procedure may contain most of the DEHP associated with exposure to the pheresis tubing.
Appendix II

II-25

Table 11. DEHP Dose with Pheresis Procedures

Procedure Median Dose 

(mg)

Mean Dose (Range)

μg/kg bw

Platelet pheresis Continuous technique 2.10 32.1 (28.2 – 38.1)

Discontinuous technique 1.18 18.1 (14.3 – 23.8)

Plasma pheresis 0.37 5.7 (3.1 –9.6)

Controls (no procedure) 0.41 6.2 (3.0 –11.6)

From Koch et al. (78).

A study of recipients of platelet concentrate, derived from pheresis procedures, identified an increase 

in serum DEHP from a median (range) of 192 (10 –532) ng/mL to 478 (142 –1236) ng/mL 5 minutes 

after transfusion (79). Storage time of the platelet concentrates was related to DEHP concentration 

in the product, increasing from a median (range) of 1.88 (0.41 – 3.2) mg/L shortly after collection to 

6.59 (2.09–10.67) mg/L 5 days later. Washing of platelets 5 days after collection with resuspension 

in saline resulted in a 31– 80% reduction in DEHP concentration in the preparation.

The amount of DEHP retained by dialysis patients during a 4-hour dialysis treatment was estimated by 

measurement of DEHP blood levels in blood coming to the patient from the dialysis machine and in 

blood coming from the patient to the dialysis machine (80). In all patients, a higher concentration of 

DEHP was present in blood entering the patient than in blood leaving the patient. The mean amount of 

DEHP retained by the patient after 4 hours of dialysis was 16.4 mg (range 3.6–59.6 mg). The authors 

used their data to construct a toxicokinetic model of DEHP transfer during dialysis. [There was no

discussion of MEHP infused during dialysis or created in vivo during dialysis.]

Ito et al. (81) noted that release of DEHP from medical-grade PVC could be reduced by ultraviolet 

irradiation without altering the material’s strength or flexibility. The authors attributed the reduction 

in DEHP release to alterations in the surface structure of the material. [The Expert Panel notes that

ultraviolet-irradiated PVC is not currently used in medical devices.]

1.6 Utility of Exposure Data

Estimates of DEHP exposures from medical devices have been made using simulated medical procedures 

with a variety of media and by measuring urinary metabolites in patients undergoing medical procedures. 

General population exposures have been estimated from urinary concentrations of DEHP metabolites. 

Other exposure estimates have been derived from measurements of DEHP or MEHP in food, blood, 

air, or environmental media.

Exposures to DEHP can be estimated using environmental contamination/exposure data coupled with 

estimates of inhalation and ingestion rates. This probabilistic method will provide accurate estimates 

of exposure in situations where the routes of exposure and environmental concentrations are well 

characterized. In the case of DEHP, for which it has been estimated that more than 90% of the intake is 

from food, probabilistic models are more straightforward, requiring accurate data on food contamination 

and intake rates. There is considerable variability in the degree of DEHP contamination of foods based 

upon packaging and processing practices and lipid content of foods. There are situations in which 

non-food exposure pathways may contribute significantly to exposure, including medical exposures, 
II-26

Appendix II

occupational exposures, some indoor air exposures, and, potentially, exposure from mouthing of DEHPcontaining objects. Probabilistic models are attractive because they provide a distribution of probable 

intakes; however, the uncertainties described above can lead to large variability in dose estimation.

An alternate approach is to use direct measures of DEHP metabolites in urine samples and back 

calculate to the DEHP dose (or dose reconstruction). There are uncertainties to this approach as well. 

The metabolite back calculation approach, when it relies on a single urine measure, assumes a steady 

state exposure and cannot differentiate between peak levels and background, which is particularly 

important in small studies. This method also assumes that the metabolite excretion fraction is known 

and is constant across and within populations with diverse demographic characteristics such as age, 

sex, and ethnicity. A study by Hauser et al. (41) indicated that there is large intra-person variability 

in excretion of MEHP. Kohn et al. (82), cited in the initial CERHR Expert Panel Report on DEHP, 

concluded that fractional metabolite excretion is highly variable and that exposure estimates based on 

metabolite excretion calculations provide order of magnitude estimates of exposure. Another limitation 

of urinary measurements is that spot samples vary in the degree of dilution based upon hydration state of 

individuals. Several methods have been evaluated to “correct” for the variability in urine dilution across 

spot samples, the most popular being creatinine (83). Creatinine excretion varies due to many factors, 

including the size of the participant, so inter-individual variation, especially among diverse populations, 

is large. Thus, creatinine-adjusted DEHP metabolite concentrations should never be compared among 

individuals of vastly different age groups (i.e., children versus adults); however, creatinine-adjusted 

measurements may serve as a surrogate for a weight-related dose. Changes in creatinine excretion 

during pregnancy should be thoroughly evaluated before comparing to other women in similar age 

groups. Similarly, creatinine adjustment has not been standardized for neonates or small children. The 

validity of creatinine adjustment may also be metabolite-dependent based upon the renal excretion of 

the metabolite. Thus, caution should be exercised when using creatinine-adjusted concentrations for 

comparisons among exposure populations or dose reconstruction using urinary metabolite data.

Both methods for estimating dose (probabilistic and dose reconstruction) suffer from uncertainty. Dose 

calculations using urinary measures tend to be lower than probabilistic estimates. However both methods 

tend to agree within an order of magnitude, suggesting that the probabilistic methods account for the 

major routes of exposure in the general population or in known exposure scenarios. The Expert Panel 

finds current dose estimates robust because both methods provide estimates within a close range. 

1.7 Summary of Exposure Data

General population intake estimates for DEHP have been developed using probabilistic analysis (11). 

More than 90% of estimated daily DEHP intake in people over the age of 6 months is from food. 

Median estimates are given in Table 4 and range from 8.2 μg/kg bw/day in adults to 25.8 μg/kg bw/day 

in toddlers. These estimates are similar to those assumed for the general population, not occupationally 

exposed, by the first DEHP Expert Panel.

Since the first Expert Panel Report on DEHP, 2 population-based surveys of DEHP exposure have 

been conducted on representative samples of the US population over age 6 years have been completed. 

NHANES 1999 – 2000 measured MEHP, and NHANES 2000 – 2001 measured MEHP as well as 

5-OH-MEHP and 5-oxo-MEHP. Mean MEHP, 5-OH-MEHP, and 5-oxo-MEHP vary by age with 

younger ages groups having both higher MEHP concentrations (both corrected for creatinine and whole 
Appendix II

II-27

volume) and higher proportions of secondary metabolites (5-OH-MEHP and 5-oxo-MEHP) than older 

children and adults. In addition, a number of investigators have evaluated urinary metabolites in small 

populations for a variety of purposes, and these are summarized in Table 12 and shown graphically 

in Figure 3 and Figure 4. Which metabolite(s) are optimum for estimation of exposure is an issue 

that is currently being discussed, but 5-oxo- and 5-OH-MEHP may be more sensitive predictors of 

DEHP exposure due to their relatively high concentration in urine and their lack of susceptibility to 

contaminants in the sample collection process (8, 31, 36). Calculations of population exposure based on 

urinary metabolites are generally within the original range assumed of 3– 30 ug/kg/day, but estimates 

made from the upper 95th percentile of measured ranges exceed this range by up to a factor of 2 in 

some studies. For example, estimates of general population exposures (95th percentile) using these 

urine metabolites are 65.0 μg/kg bw/day for men and 27.4 μg/kg bw/day for women (31, 36). Not all 

investigators agree with the methods used to derive these estimates, and alternative estimates have been 

as much as 5-fold lower (37). Exposure to DEHP from medical devices is summarized in Table 13.
II-28

Appendix II

Table 12. Summary of DEHP Metabolite Levels Measured in Human Urine

Population Data Presentation

Urinary levels of metabolites 

(μg/L unless otherwise specified)

MEHP 5-OH-MEHP 5-oxo-MEHP Reference

46 women 

35–49 years old from the US Median (Range) 7.3 (1.0 –143.9 6.4 (0.4 – 77.3) a Hoppin et al. (26)

2540 urine samplesAdults and children older than 6 years from the US

Children 

6–11 years old

Geometric Means

5.12 

(5.19 a) NHANES 

1999 – 2000

Presented by 

Silva et al. (4)

Adolescents 

12 – 19 years old

3.75 

(2.53 a)

Adults ≥ 20 years

3.21 

(3.03) a

2782 urine samplesAdults and children older than 6 years from the US

Children 

6– 11 years old

Geometric Means

4.41

(5.02) a

33.6

(38.3) a

23.3

(26.6) a

NHANES 

2001 – 2002 

(27)

Adolescents 

12 – 19 years old

4.57

(3.53) a

24.9

(19.2) a

6 – 11, 17.5

(13.5) a

Adults ≥ 20 years

4.20

(3.96) a

18.1

(17.2) a

12.0

(11.4) a

150 Korean women 150 children Women 20– 73 years Geometric Means 41.3±50 Koo and Lee (39) Children

11– 12 years old 13.3±24

19 children 12–18 months old in the US 6.1 to 47.3 Brock et al. (35)85 volunteers Age 7 –34 years in Germany Median (Range)

10.3 

(<LOQ– 177)b

9.2 (<LOQ–123) a

46.8 (0.5 – 818)

 40.2 (6.9 –449) a

36.5 (0.5 – 544)

30.4 (6.4 – 262) a

Koch et al. 

(31, 36)

62 urine samplesAdults and children in the US Median 4.5 36 28 Barr et al. (8)127 adults or children ≥ 6 years in the US

Median (5th–95th 

percentile)

<LODc

(<LOD– 20.4) 17.4 (<LOD–220) 15.6 (<LOD– 243) Kato et al. (6)
Appendix II

II-29

Population Data Presentation

Urinary levels of metabolites 

(μg/L unless otherwise specified)

MEHP 5-OH-MEHP 5-oxo-MEHP Reference

254 children age 3–14 years in Germany Geometric Means

7.91 (range 

0.74–226) 52.1 (1.86 – 2590) 39.9 (< 0.5 – 1420) Becker et al. (33)

36 children (age 2.6 –6.5 years) in 

Germany Median (Range)

6.6 (1.5 –18.3)

8.7 (1.7 – 48.4) a 49.6 (2.7 –129) 55.8 (15.4– 258) a 33.8 (2.2 – 90.6) 38.3 (10.2 – 158) a Koch et al. (32)

19 adults in Germany Median (Range) 9.0 (2.6 –43.1) 8.6 (3.8 – 26.6) a 32.1 (10.7 – 103) 28.1 (10.9– 63.6) a 19.6 (4.9 – 55.1) 17.2 (4.5 – 40.9) a Koch et al. (32)25 pregnant women in the US Median (Range) 4.60 (1.80–449) a Adibi et al. (29)Urine samples randomly selected from 289 adults Median (Range)

Total urinary 2 

ethylhexyl metabolites: 

2.7 (<1.2 to 66.6)

Blount et al. 

(28)

234 young Swedish males Median (Range) <15 (<15–150) Jönsson et al. (85)369 men presenting for a fertility examination 

Median (5th –95th 

percentile)

5.2 (0.1 –110);

Specific gravityadjusted 

6.5 (0.8 – 120) 

Hauser et al. 

(41)

aμg/g creatininebLOQ=limit of quantification cLOD=limit of detection.
II-30

Appendix II

Figure 3. Urinary MEHP Concentrations

LOD1101001000

UrinaryMEHP

Concentration(μg/L)

Adults, Adolescents, Mixed Ages Children

Hoppinetal.(26)

Adults,n=46

Median(range)

NHANES1999-2000(4)

Adolescents,n=752

Geometricmean(95thpercentile)

NHANES1999-2000(4)

Adults,n=1461

Geometricmean(95thpercentile)

NHANES2001-2002(27)

Adolescents,n=742

Geometricmean(95thpercentile)

NHANES2001-2002(27)

Adults,n=1647

Geometricmean(95thpercentile)

KooandLee(39)

Adults,n=150

Mean(95thpercentile)

Kochetal.(31,36)

Adults,n=85

Mean(range)

Katoetal.(6)

Childrenandadults,n=127

Median(5th-95thpercentiles)

Kochetal.(32)

Adults,n=19

Median(range)

Jönssonetal.(85)

Adults,n=234

Median(range)

Hausereta.(41)

Adults,n=369

Median(5th-95thpercentile)

NHANES1999-2000(4)

Children≥6years,n=328

Geometricmean(95thpercentile)

NHANES2001-2002(27)

Children≥6years,n=393

Geometricmean(95thpercentile)

KooandLee(39)

Children11-12years,n=150

Mean(95thpercentile)

Brocketal.(35)

Childrenage12-18months,n=19

Range

Beckeretal.(33)

Childrenage3-14years,n=254

Geometricmean(range)

Kochetal(32)

Childrenage2.6-6.5years,n=36

Median(range)
Appendix II

II-31

Figure 4. Urinary 5-oxo- and 5-OH-MEHP Concentrations

LOD1101001000

Urinary5-OH-MEHP

Concentration(μg/L)

Adults, Adolescents,

Mixed Ages Children

NHANES2001-2002(27)

Adolescents,n=742

Geometricmean(95thpercentile)

NHANES2001-2002(27)

Adults,n=1647

Geometricmean(95thpercentile)

Kochetal.(31,36)

Adults,n=85

Mean(range)

Katoetal.(6)

Childrenandadults,n=127

Median(5th-95thpercentiles)

Kochetal.(32)

Adults,n=19

Median(range)

NHANES2001-2002(27)

Children≥6years,n=393

Geometricmean(95thpercentile)

Kochetal.(32)

Childrenage2.6-6.5years,n=36

Median(range)

LOD1101001000

Urinary5-oxo-MEHP

Concentration(μg/L)

NHANES2001-2002(27)

Adolescents,n=742

Geometricmean(95thpercentile)

NHANES2001-2002(27)

Adults,n=1647

Geometricmean(95thpercentile)

Kochetal.(31,36)

Adults,n=85

Mean(range)

Katoetal.(6)

Childrenandadults,n=127

Median(5th-95thpercentiles)

Kochetal.(32)

Adults,n=19

Median(range)

NHANES2001-2002(27)

Children≥6years,n=393

Geometricmean(95thpercentile)

Kochetal.(32)

Childrenage2.6-6.5years,n=36

Median(range)

Adults, Adolescents,

Mixed Ages Children
II-32

Appendix II

Table 13. Summary of DEHP Exposure Estimates from Medical Devices

Population Medical device Medium Estimated DEHP exposure Reference

Infants

IV tubing

Lipid-containing solutions used for parenteral nutrition 

424.4 μg/mL over 24 hours;5 mg/kg bw for a 2 kg infant receiving 25 mL solution

Loff et al. (55)

Amino acid/glucose solution 0.83 μg/mL, 24 hours1% propofol, continuous 6561 μg for a 2 kg infantFentanyl solution 28.8 mL 132.5 μg for a 2 kg infant Midazolam 24 mL 26.4 μg for a 2 kg infant

Blood bag + tubing

Packed red blood cells, 20 mL 608 μg for a 2 kg infantPlatelet-rich plasma 928 μg for a 2 kg infantFresh frozen plasma 552 – 8108 μg for a 2 kg infant

IV tubing

Lipid-containing infusion solution, 27°C

422 μg/mL

10 mg for a 2 kg infant receiving 24 mL Loff et al. (56) Lipid-containing infusion 

solution, 33°C

540 μg/mL

13 mg for a 2 kg infant receiving 24 mL

Not specified IV tubing

Hydrogenated castor oil in saline or water 775 μg after 4 hours

Hanawa et al. (65) Hydrogenated caster oil 

in sugar solutions 150 μg over 4 hours

Not specified PVC bags Etoposide in polysorbate 80-containing solution 17 – 25 μg/mL after 24 hours Demore et al. (69)Children

Ethyl vinyl acetate bags with PVC connectors and tubing

Lipid-containing parenteral nutrition solution stored at 4oC for 24 hours or 1 week

0.8­ – 2 mg/day Kambia et al. (59)

Not specified PVC multi-layer iv tubing Polysorbate solution 26 – 30 mg/mL after 2 hours; 62 – 70 mg/mL after 24 hours Bourdeaux et al. (61)
Appendix II

II-33

Population Medical device Medium Estimated DEHP exposure Reference

Children and adults

Hemodialysis simulation

Bovine blood

1718 μg/L after 4 hours, compared to 249 μg/L at baseline. MEHP 80 μg/L after 4 hours. 

Estimated adult dose 0.067 mg/kg bw/day. Haishima et al. (73)

Pump-oxygenation simulation Estimated child’s dose of 0.3­ –0.7 mg/kg bw/day Estimated adult dose of 0.16– 0.3­ mg/kg bw/day. MEHP 200–400 μg/L after 4 hours.

Adults Hemodialysis Blood from 11 patients on therapy Patients retained DEHP 16.4 mg (range 3.6 – 59.6 mg) after a 4-hour dialysis session Dine et al. (80)Adults Platelet pheresis Blood from 36 healthy donors Median dose retained after pheresis session was 6.46 μg/kg bw (range 1.8– 20.3 μg/kg bw) Buchta et al. (76)Adults Platelet pheresis Blood from 12 healthy donors Median dose retained after pheresis session was 18.1 – 32.3 μg/kg bw (range 14.3– 38.1 μg/kg bw) Koch et al. (84)Infants All ICU 

exposures Aggregate exposures 2.83 mg/kg bw/day FDA (2)

Adults Medical treatment 0.005– 8.5 mg/kg bw/dayAdults ECMO, multiple transfusions Blood ≥  4 mg/kg bw/dayNot specified Respirator PVC 

tubing

Air Below limit of quantification Hill et al. (75)
II-34

Appendix II

At the time of publication of the first DEHP Expert Panel report, there was concern that infants undergoing multiple medical procedures might have exposures 3 orders of magnitude higher than the population 

exposure level. The previous report based this concern upon studies that were either several decades old 

or in which exposures were calculated based upon single source DEHP exposure. Since then, 3 studies, 2 

from the US (5, 63) and 1 from Germany (84) have confirmed that doses assessed using urinary metabolites 

do indeed reach levels up to about 6 mg/kg bw/day. These studies are summarized in Table 14.

Table 14. Summary of DEHP Metabolite Levels Measured in Medically-Exposed Infants

Population 

Urinary levels of metabolites (μg/L)

MEHP 5-OH-MEHP 5-oxo-MEHP Reference

54 neonates admitted to NICU 

for at least 2 days

Presented as median 

(25th – 75th percentile)

Total Group:

Female: 20 (3 –64)

Male: 39 (19 – 75)

DEHP Exposure Group:

Low: 4 (<Lod – 18)

Medium: 28 (3 –61) 

High: 86 (21– 171)

Green et al. 

(63)

41 urine samples from 6 pre- mature newborns who were 

potentially given IV infusions 

for >2 weeks

Presented as median 

(5th – 95th percentile) 

129

(6.22-704)

2221

(290 – 13,161)

1697

(243 – 10,413)

Calafat et 

al. (5)

45 neonates treated with 

various medical procedures:

• Blood transfusions

• Intubation

• Continuous positive 

airway pressure

• Intralipids feeding

• Orogastric tubing

• IV nutrition

Presented as 95th percentile

557 406 Koch et al. 

(84)

Another data gap that has been filled since the last report is represented by the work by Loff (55, 56)

showing that DEHP leaches from PVC iv tubing used to deliver TPN at levels in the 5 –10 mg/kg 

bw/day dose range. DEHP extraction from iv tubing increases with increasing temperature, of note 

since most NICUs are kept warm and babies are often under warmers or in heated isolettes, and varies 

with the nature of the solution being administered. Infant ICU exposures were estimated by FDA (2) at 

2.83 mg/kg bw/day from only 3 potential sources using only data available at the time of the original 

Expert Panel Report on DEHP. Blood transfusion is an important source of DEHP exposure, and FDA 

(2) estimated adult exposures at ≥ 4 mg/kg bw/day resulting from ECMO or multiple transfusions.

General population exposures of concern can include fetal and neonatal exposure via general exposures 

to pregnant and lactating women. The initial Expert Panel Report on DEHP assumed both placental 
Appendix II

II-35

and mammary transfer of DEHP based upon experimental animal studies. Human data are now available and document both placental transfer in humans (44) as well as breast milk transfer (5, 17). [The

Expert Panel notes that another potential source of infant exposure is breast milk expressed

using DEHP-containing breast pumps.] These data are still scant, but may be of particular concern 

if the toxic metabolites of DEHP are present in breast milk or amniotic fluid in free (unconjugated) 

form. DEHP is also present in some infant formulas (12, 14, 18, 19).

Dietary intake has been identified as an important route of exposure (12-14, 20-22). Reported daily 

intakes are variable and generally cover the range of exposures expected for the general population 

(i.e., 1–30 μg/kg bw/day).

Since the initial Expert Panel Report on DEHP, 2 studies have estimated DEHP release from toys due 

to mouthing behavior. Bouma et al. (45) measured DEHP released from 47 toys containing PVC after 

mixing with a saliva simulant. DEHP was found in 20 (43%) of the 47 toys at 30 – 45% by weight. 

Six toys exceeded the Dutch guidance release value (2.3 μg/min/10 cm2) for children younger than 

1 year. Niino et al. (46) identified migration into simulated saliva of DEHP from a sample of a PVC 

ball that contained DEHP 190 mg/g. DEHP leaching rate was found to be 315±25.0 μg/hour/10 cm2

(mean±SD, n=5). 

Three publications reported on inhalation as a route of exposure. Otake et al. (47) measured concentrations of common phthalates in 27 homes in the Tokyo metropolitan area. Indoor air concentrations 

of DEHP ranged from < 0.001 to 3.13 μg/m3. The mean±SD concentration was 0.32±0.6 μg/m3. 

DEHP levels were 100 – 1000 times higher than ambient outdoor concentrations Adibi et al. (29)

measured phthalate diesters in 48-hour personal air samples collected by 30 pregnant women in New 

York city and 30 pregnant women in Krakow, Poland. The median DEHP air concentrations (ranges) 

were: New York 0.22 (0.05 – 0.41) μg/m3 and Krakow 0.37 (0.08 – 1.1) μg/m3. The median indoor 

air concentration reported by Fromme et al. (22) in German apartments was 0.16 μg/m3 and 0.458 

μg/m3 in kindergartens.

DEHP has also been shown to be a constituent of dust in households. Fromme et al. (22) found DEHP 

775.5 mg/kg in dust collected from 30 apartments in Germany. Bornehag et al. (23) measured DEHP 

concentration in dust samples from children’s bedrooms in 346 homes. The geometric mean was 0.789 

mg/g of dust.
II-36

Appendix II

2.0 GENERAL TOXICOLOGY AND BIOLOGIC EFFECTS

Section 2 of this report contains summaries of toxicokinetics, general toxicity, or carcinogenicity 

studies that may be especially relevant to the interpretation of developmental and reproductive effects 

associated with DEHP exposure. Since the initial CERHR Expert Panel Report on DEHP, there have 

been additional studies on toxicokinetics in rats and marmosets. There have also been studies using 

systems designed to assess the anti-androgenicity and estrogenicity of DEHP.

2.1 Toxicokinetics

The metabolism of DEHP in humans is discussed in Section 1 with respect to the use of urinary 

metabolite concentrations in the estimation of DEHP exposures and is illustrated in more detail in 

Figure 5. Metabolism has also been summarized by Koch et al. (84). By 24 hours after an oral DEHP 

dose, about 70% appears in the urine as 5 major metabolites (see Table 7). According to this review, 

metabolism is similar after iv exposure to DEHP.

It is important to distinguish between in vivo and in vitro metabolism. The former is based by

analogy on excreted metabolites and provides no definitive information on reaction mechanisms,

on the enzymes involved, or their polymorphic forms. In vitro studies, on the other hand, provide

this information and, in addition, information on the proper sequence of secondary and tertiary

metabolites, as well as revealing reactive, butshort-lived, metabolites.A significant data gap isthe

lack in vitro metabolic studies in general but particularly in the case of in vitro studies of human

metabolism.Although such studies have been carried out extensively for other xenobiotics, they

have not yet been carried out for DEHP. Only human studies can reveal the extent of metabolic

variation within the human population and shed light on the metabolic parameters involved in

the identification of populations or individuals at greater or lesser risk.These studies may also

help in the selection of the most appropriate surrogate animal for in vivo studies and help in

estimating uncertainty factors in risk analysis.]

Calafat et al. (86) measured MEHP in maternal urine and amniotic fluid after gavage administration 

of DEHP [purity not specified] in corn oil to pregnant Sprague-Dawley rats on gestation day (GD) 

8, 10, 15, 16, and 17. [The abstract indicates administration also on GD “5/7”.] Doses were 0, 

11, 33, 100, and 300 mg/kg bw (n=2/dose group). Urine was collected approximately 6 hours after 

dosing, and amniotic fluid was collected at necropsy on GD 18. MEHP was analyzed by HPLC-tandem 

MS after solid-phase extraction and enzymatic hydrolysis. There was no temporal trend in urinary 

MEHP levels over the collection period, and the 5 urine MEHP levels were combined for each animal. 

Creatinine-corrected and uncorrected urinary MEHP and uncorrected amniotic fluid MEHP were 

highly correlated with maternal DEHP dose (r values 0.964 –0.998). [Data were presented only in

graphic form.At the 300 mg/kg maternal DEHP dose level, urinary MEHP was estimated from

a graph at 16.4 mg/L and amniotic fluid MEHP was estimated at 2.8 mg/L.] Maternal urinary 

MEHP was only 13.3% unconjugated, while amniotic fluid MEHP was 88.2% unconjugated. The 

authors observed that the finding that MEHP was largely conjugated in urine did not agree with reports 

of other studies on urinary MEHP in rats. The authors also indicated that the lack of measurement 

of more oxidized MEHP metabolites may lead to an underestimation of exposure to DEHP and its 

biotransformation products.
Appendix II

II-37

Figure 5. DEHP Metabolism

CH2 – CH2 – OH

R– CH2 – CH– (CH2)3 – CH3

|

2-(2-Hydroxyethyl)-

hexylphthalate

OH

O–

O

O

R =

R– CH2 – CH– (CH2)3 – CH2 – OH

CH2 – CH3 |

2-Ethyl-6-hydroxyhexylphthalate

R– CH2 – CH– (CH2)2 – CH– CH3

CH2 – CH3 |

OH

|

2-Ethyl-5-hydroxy-hexylphthalate

(5-OH-MEHP)

R– CH2 – CH– (CH2)3 – CH3

CH2 – CH3 |

OH

|

2-(1-Hydroxyethyl)-

hexylphthalate

R– CH2 – CH– CH2 – CH– CH2 – CH3

CH2 – CH3 |

OH

|

2-Ethyl-4-hydroxy-hexylphthalate

R– CH2 – CH– CH2 – COOH

CH2 – CH3 |

2-Ethyl-3-carboxypropylphthalate

OCH2 – CH– (CH2 )3 – CH3

CH2 – CH3

|

OCH2 – CH– (CH2 )3 – CH3

CH2 – CH3 |

O

O

Di(2-ethylhexyl)phthalate

(DEHP)

OH

OCH2 – CH– (CH2 )3 – CH3

CH2 – CH3 |

O

O

Mono(2-ethylhexyl)phthalate

(MEHP)

OH – CH2 – CH– (CH2 )3 – CH3

CH2 – CH3 |

2-Ethylhexanol

OH

OH

O

O

Phthalic Acid

OH – CH2 – CH– (CH2 )3 – CH3

CH2 – CH3 |

2-Ethylhexanol

CH2 – COOH

R– CH2 – CH– (CH2)3 – CH3

|

[2-(Carboxymethyl)-

hexyl]phthalate

(2-cx-MMHP)

R– CH2 – CH– (CH2)3 – COOH

CH2 – CH3 |

2-Ethyl-5-carboxypentylphthalate

(5-cx-MEPP)

R– CH2 – CH– (CH2)2 – C– CH3

CH2 – CH3 |

O

||

2-Ethyl-5-oxy-hexylphthalate

(5-oxo-MEHP)

R– CH2 – CH– (CH2)3 – CH3

CH2 – CH3 |

O

||

2-(1-Oxyethyl)-

hexyl]phthalate

R– CH2 – CH– CH2 – C– CH2 – CH3

CH2 – CH3 |

O

||

2-Ethyl-4-oxy-hexylphthalate

CH2 – COOH

R– CH2 – CH– (CH2)3 – CH3

|

2-Carboxyhexyl-phthalate

R– CH2 – CH– (CH2)2 – COOH

CH2 – CH3 |

2-Ethyl-4-carboxybutylphthalate

The metabolites discussed in this report are circled. From Koch et al. (84), 

used with kind permission of Springer Science and Business Media and of Prof. Dr. J. Angerer. 
II-38

Appendix II

Toxicokinetic studies using radiolabeled DEHP by gavage in pregnant and non-pregnant female 

Wistar rats and CD-1 mice appeared in unpublished reports sponsored by the European Council for 

Plasticizers and Intermediates (87-91). Determinations were made after single doses of 200 or 1000 

mg/kg bw and after 5 daily doses at these levels. Results are summarized in Table 15. 

Table 15. Toxicokinetic Parameters in Pregnant (GD 6) and Non-pregnant Female 

Rats and Mice Given Oral Radiolabeled DEHP

Model Cmax

(nmol-eq/mL)

AUC0–48

(nmol-eq-h/mL) t 1⁄2 (hours)

Single 200 mg/kg bw Dose

Non-pregnant rat 64 1426 7.1

Pregnant rat 58 (32.1/36.4) a 983 (217/511) 7.8 (82.1/5.9)

Non-pregnant mouse 154 2069 7.1

Pregnant mouse 91 (28/84) 1078 (171/816) 7.3 (–/4.0)

Single 1000 mg/kg bw Dose

Non-pregnant rat 353 5825 10.2

Pregnant rat 249 (90.8/146.3) 6254 (1180/2445) 5.5 (–/14.0)

Non-pregnant mouse 1339 6838 10.3

Pregnant mouse 227 (103/215) 6745 (1107/3526) 9.7 (–/14.2)

Repeated 200 mg/kg bw Dose

Non-pregnant rat 77 1007 8.7

Pregnant rat 98 1606 10.3

Non-pregnant mouse 197 2252 7.1

Pregnant mouse 90 1083 7.3

Repeated 1000 mg/kg bw Dose

Non-pregnant rat 405 6398 13.5

Pregnant rat 518 7410 6.6

Non-pregnant mouse 396 5672 7.9

Pregnant mouse 551 4890 11.4

aFigures are given for total radioactivity with (DEHP/MEHP) in parentheses, as determined 

by gas chromatography (GC). 

Cmax=maximum concentration

 tmax=time to maximum concentration

 t1/2=half-life

AUC=area under the concentration –time curve

From Laignelet and Lhuguenot (87-91).

An unpublished report from Mitsubishi Chemical Safety Institute, Ltd. (92) described a 65-week oraldose toxicity study of DEHP in marmosets (discussed in Section 4.2.3) and included a toxicokinetic 

study. [Some data from thisstudy were published in abstract (93).] The study was sponsored by the 

Japan Plasticizer Industry Association. Ring-labeled 14C-DEHP (99.6% purity) in corn oil was given to 

3 groups of marmosets. The first group was treated at 3 months of age. The second group was treated 
Appendix II

II-39

at 18 months of age. The third group was treated for 65 weeks from 3 months of age with unlabeled 

DEHP and studied at 18 months of age. There were 3 animals of each sex in each treatment group. 

Treatments were by gavage at dose levels of 100 or 2500 mg/kg bw. Blood samples were collected 1, 

2, 4, 8, 12, 24, 48, 72, 120, and 168 hours after dosing. Spontaneous urine and feces were collected 

for radioactivity determination. At least 2 weeks after the kinetic studies, animals were dosed again 

and tissues collected 2 hours later for determination of radioactivity. Radioactivity determination was 

by liquid scintillation counting. Toxicokinetic parameters are shown in Table 16. 

Table 16. Toxicokinetic Parameters after Oral Dosing of Marmosets at 

Age 3 and 18 Months with Radiolabeled DEHP

Dose group

Cmax

(μg eq/mL)

tmax

(hour)

t1/2

(hour)

AUC (μg 

eq-hour/mL)

AUC/dose 

Age (hour-kg/L)

(months)

Dose 

(mg/kg bw) Sex

3

100

Male 6.86±4.86 4.0±3.5 8.0±4.0 37.4±19.3 0.374±0.193

Female 17.08±10.69 1.3±0.6 6.0±3.5 78.7±67.2 0.787±0.672

2500

Male 36.00±37.47 10.0±12.2 21.3±23.1 270.2±194.5 0.108±0.078

Female 66.00±22.34 4.0±0.0 8.0±0.0 347.7±66.5 0.139±0.027

18

100

Male 13.53±6.07 2.3±1.5 5.3±2.3 99.0±57.4 0.990±0.574

Female 19.49±16.71 1.0±0.0 4.7±3.1 150.8±137.8 1.508±1.378

2500

Male 50.00±39.23 1.0±0.0 2.7±1.2 444.7±197.8 0.178±0.079

Female 62.67±38.73 1.3±0.6 4.7±3.1 952.8±1093.3 0.381±0.437

18 

(after 65 weeks 

pretreatment)

100

Male 14.77±17.04 2.3±1.5 5.3±2.3 83.0±104.9 0.830±1.049

Female 4.81±3.61 2.3±1.5 6.7±4.6 48.2±46.0 0.382±0.460

2500

Male 32.33±6.43 1.0±0.0 2.7±1.2 153.7±18.9 0.061±0.008

Female 4.33±2.31 1.0±0.0 2.7±1.2 11.5±10.3 0.004±0.004

Data are mean±SD, n=3 animals/sex/group. 

From Mitsubishi Chemical Safety Institute, Ltd. (92).

Reproductive organ radioactivity contents 2 hours after dosing are shown in Table 17. The authors 

found the highest level of radiation in the kidneys after a single oral dose and considered that high 

radioactivity levels in the prostate and seminal vesicles of some animals may have been due to urine 

contamination. Repeated dosing for 65 weeks did not appear to alter the distribution of DEHP in 

18-month-old animals. The authors called particular attention to the small amount of label distributed 

to the testis and postulated that differences in access of DEHP metabolites to the testis may explain 

a lack of testicular toxicity in marmosets compared to rodents, in which large amounts of MEHP are 

distributed to the testis after DEHP treatment.
II-40

Appendix II

Table 17. Reproductive Organ Radioactivity Content 2 Hours after Oral Dosing of Marmosets 

with Radiolabeled DEHP

Dose group Concentration 

(μg eq/mL or μg eq /mg) Organ/

Plasma 

Ratio

Distribution 

Age (% of dose)

(months)

Dose 

(mg/kg bw) Tissue Male Female Organ

3

100

Plasma 26.54±35.56 33.55±29.22

Testis 5.47±7.35 0.21 0.002±0.003

Epididymis 10.25±13.28 0.39 0.002±0.002

Prostate 13.04±8.79 0.49 0.001±0.002

Seminal 

vesicle 8.03±4.57 0.30 0.001±0.001

Ovary 4.84±4.33 0.14 0.000±0.000

Uterus 11.28±13.16 0.24 0.003±0.004

2500

Plasma 45.51±31.47 45.50±45.92

Testis 10.52±4.63 0.23 0.000±0.000

Epididymis 14.09±2.54 0.31 0.000±0.000

Prostate 34.22±30.49 0.75 0.000±0.000

Seminal 

vesicle 23.14±15.18 0.51 0.000±0.000

Ovary ND – –

Uterus 8.91±9.49 0.20 0.000±0.000

18

100

Plasma 9.01±9.97 16.88±13.48

Testis 0.83±0.81 0.09 0.003±0.004

Epididymis 1.97±1.29 0.22 0.001±0.001

Prostate 4.59±4.52 0.51 0.002±0.002

Seminal 

vesicle 16.26±21.74 1.80 0.005±0.006

Ovary 5.93±4.25 0.35 0.004±0.004

Uterus 3.79±2.64 0.22 0.002±0.002

2500

Plasma 65.48±95.58 123.74±33.78

Testis 8.47±11.95 0.13 0.001±0.001

Epididymis 15.98±20.15 0.24 0.000±0.001

Prostate 10.64±14.53 0.16 0.000±0.000

Seminal 

vesicle 13.84±15.79 0.21 0.000±0.001

Ovary 36.11±11.39 0.29 0.001±.000

Uterus 33.52±12.62 0.27 0.001±0.001
Appendix II

II-41

Dose group Concentration 

(μg eq/mL or μg eq /mg) Organ/

Plasma 

Ratio

Distribution 

Age (% of dose)

(months)

Dose 

(mg/kg bw) Tissue Male Female Organ

18 

(65 weeks 

pretreatment)

100

Plasma 29.92±6.61 47.28a

Testis 3.03±0.97 0.10 0.011±0.004

Epididymis 9.79±6.91 0.33 0.008±0.008

Prostate 7.34±3.34 0.25 0.002±0.002

Seminal 

vesicle 12.60±11.95 0.42 0.004±0.004

Ovary 14.12 a 0.30 0.006a

Uterus 9.24 a 0.20 0.004a

2500

Plasma 102.78±81.44 41.70±29.53

Testis 12.40±9.07 0.12 0.002±0.002

Epididymis 27.98±20.66 0.27 0.001±0.001

Prostate 20.38±14.74 0.20 0.000±0.000

Seminal 

vesicle 23.73±18.80 0.23 0.000±0.000

Ovary 13.18±8.51 0.32 0.000±0.000

Uterus 9.97±6.12 0.24 0.000±0.000

Data are mean±SD, n=3 animals/sex/group, ND=Not determined. aThere were only 2 females in this group.

From Mitsubishi Chemical Safety Institute, Ltd. (92).

Kessler et al. (94), sponsored in part by the American Chemistry Council, compared blood levels of 

DEHP and MEHP in pregnant and non-pregnant Sprague-Dawley rats and marmosets [strain not

indicated] in a Good Laboratory Practice (GLP) study. DEHP or deuterium-labeled DEHP were 

dissolved in an aqueous Tween 80/Methocel/saccharose solution that was fed to marmosets through a 

syringe following their first meal and administered to Sprague-Dawley rats by gavage. In most cases, the 

deuterated-DEHP was administered in at least 1 dose group on the days that time-course experiments 

were conducted in order to differentiate between background DEHP and MEHP. Non-pregnant female 

rats (n=3–4 group) were dosed with 30, 500, or 1000 mg/kg bw. Rats in the 500 mg/kg bw group were 

dosed for 7 days, and time-course experiments were conducted on study days 1, 4, and 7. Pregnant 

rats were dosed with 30 or 500 mg/kg bw/DEHP on GD 14–20, and concentration time courses were 

determined on GD 14 and 19 [of a 21–22 day gestation]. In rats, blood samples were collected over 

a 24– 48 hour period following dosing. Non-pregnant marmosets (n=8/dose) were treated with 30 or 

500 mg/kg bw/day DEHP for 29 days; concentration time courses were determined on treatment days 

1 and 29. Pregnant marmosets (n=4/dose) were dosed with 30 or 500 mg DEHP/kg bw/day on GD 

96–125; concentration time courses were determined GD 96, 103, 117, and 124 [of a 140–148 day

gestation]. On days when concentration-time courses were determined, blood samples were drawn over 

15 hours following exposure of non-pregnant marmosets and 8 hours following exposure of pregnant 

marmosets. Because blood could be drawn only once per week from the arm vein of the marmosets, 

each time point of the blood sampling curve was represented by 1 animal. Blood levels of DEHP and 

MEHP were determined by GC/MS.
II-42

Appendix II

Area under the concentration – time curve (AUC) values determined in rat studies are listed in Table 

18. Authors concluded that concentration time courses were similar in pregnant and non-pregnant 

rats and that repeated dosing had no marked effects on kinetics in either group of rats. In both groups 

of rats, MEHP blood AUCs were about 2 orders of magnitude higher than DEHP blood AUCs. For 

the non-pregnant rats, maximum concentrations for DEHP were obtained at about 1 hour following 

dosing; maximum concentrations of MEHP following dosing were reached at 30 minutes in the 30 

mg/kg bw group, 2 hours in the 500 mg/kg bw group, and 4 hours in the 1000 mg/kg bw group. Based 

on normalized AUCs that were not dose dependent, the authors concluded that kinetics were linear 

for DEHP. The authors concluded that kinetics for MEHP were saturated based on AUC values and 

increased time to reach maximum concentration at higher doses. 

Table 18. Normalized AUCs for Blood DEHP and MEHP in Rats Treated with DEHP

Rats DEHP dose 

(mg/kg bw/day)

Treatment 

Day

MEHP Cmax

(μM)

DEHP AUC

(nmol-h/mL per 

mmol DEHP/kg) a

MEHP AUC

(nmol-h/mL per 

mmol DEHP/kg) a

Non-Pregnant

30 1 10 NDb 695±113

500

1 210 5.9±3.1 1058±60

4 7.1±3.1 1104±423

7 4.7±0.7 1237±636

1000 c 1 500 8.4±4.4 1756±838

Pregnant

30d 1 (GD 14) 8.5±3.6 606±77

6 (GD 19) 21.0±7.9 646±42

500 d 1 (GD 14) 10.0±5.4 1537±158

6 (GD 19) 12.7±6.3 1106±230

aTotal normalized AUC presented as mean±SD.

bND=Not determined. cDeuterated DEHP administered to 2 of 4 animals.

dDeuterated DEHP administered to all animals.

From Kessler et al. (94).

AUC values for marmosets are listed in Table 19. Concentration time courses were similar in pregnant 

and non-pregnant marmosets with the exception that MEHP values in the 500 mg/kg bw DEHP group 

were lower compared to the non-pregnant animals at GD 103 and beyond. In the non-pregnant marmosets, 

DEHP concentrations peaked at 2 hours following dosing; MEHP concentrations returned to starting 

levels within 15 hours following dosing. MEHP AUCs in pregnant and non-pregnant marmosets were 

more than an order of magnitude higher than DEHP AUCs and were independent of dose. 

In a comparison of species differences, maximum concentrations of MEHP in rats were an average 

of 3.2 times higher (range 1.3–7.5) than those of marmosets. MEHP AUCs were an average of 7.3 

times higher (range 2.6–15.6) in rats compared to marmosets. Based on maternal blood levels, the 

study authors concluded that the MEHP burden in marmoset fetuses is lower than in rat fetuses. [The

Expert Panel notes that the burden to rats compared to marmosets was not determined. The

Expert Panel also notes that species differences in Cmax and AUC between marmosets and rats

are less at the lower dose levels.]
Appendix II

II-43

Table 19. AUCs for Blood DEHP and MEHP in Marmosets Treated with DEHP

DEHP dose 

(mg/kg bw/day) Treatment Day MEHP

Cmax (μM)

DEHP AUC 

(nmol-h/mL 

per mmol 

DEHP/kg) a

MEHP AUC 

(nmol-h/mL 

per mmol 

DEHP/kg) b

MEHP AUC 

(nmol-h/mL 

per mmol 

DEHP/kg) a

Non-pregnant Marmosets

30c 1 8 8.9 172 181

29 6.5 112 118

500

1 66 1.2d 100 d ND

29 2.5 123 130

Pregnant Marmosets

30c

1 

(GD 96) 5.6 178 ND

8

(GD 103) 5.2 258 ND

22

(GD 117) 3.5 154 ND

29

(GD 124) 6.4 245 ND

500

1

(GD 96) 12.3 170 ND

8

(GD 103) 4.1 31 ND

22

(GD 117) 2.8 63 ND

29

(GD 124) 3.4 71 ND

aTotal normalized AUC (unless otherwise indicated).

bNormalized AUC (up to 8 hours unless otherwise indicated).

cDeuterated DEHP administered to all animals.

dNormalized AUC up to 6 hours.

ND=Not determined.

From Kessler et al. (94).

Ito et al. (95) evaluated enzyme activities in tissues from rats, mice, and marmosets to assess possible 

specifies differences in the biotransformation of DEHP. CD-1 mice and Sprague-Dawley rats were 11 

weeks old and Common marmosets were 18 months old when liver, kidney, lung, and small intestine 

were harvested. Tissues were stored at −85oC until used. Tissue homogenates or microsomal fractions 

were assayed for lipase activity based on hydrolysis of DEHP to MEHP and uridine diphosphate 

(UDP)-glucuronyl transferase by measuring glucuronidation of MEHP, naphthol, and bisphenol A. 

Alcohol dehydrogenase was measured using 2-phenoxyethanol and 2-ethylhexanol as substrates, and 

aldehyde dehydrogenase was measured using 2-phenylpropionaldehyde and 2-ethylhexanal as substrates. 

Lipase activity was highest in liver, small intestine, and kidney in mice. The lowest lipase activity was 

found in marmosets. Marmoset hepatic lipase activity was 4–5% that of mouse activity, and small 

intestine lipase activity in marmosets was <1% of mouse small intestine activity. Rat lipase activities 
II-44

Appendix II

in these organs were intermediate between mouse and marmoset. Lipase activities were comparably 

low in rat and mouse lung and were undetectable in marmoset lung. UDP-glucuronyl transferase was 

detectable only in liver in the 3 species. Although activity was greater in mouse than marmoset, the 

difference between species was not as great as for lipase. Alcohol and aldehyde dehydrogenases were 

higher in marmoset than in rodents; however, the authors concluded that the possible increased ability 

of marmosets over rodents to convert MEHP to its ω-oxidation products was unlikely to be important 

given the small amount of MEHP that would be expected to be generated in marmosets from oral or 

IV exposures.

An earlier study (96) evaluated the hydrolysis of phthalates, including DEHP, in rat, ferret, baboon, 

and human liver and intestine. While the rates for intestinal hydrolysis in rat, ferret, and human were 

similar, with ferret>rat>human, the rate for baboon intestine was some 3-fold higher than that of 

the ferret.

Ono et al. (97) evaluated the testicular distribution of DEHP in 8-week-old Sprague-Dawley rats. The 

rats were given a single gavage dose of DEHP 1000 mg/kg bw, radiolabeled either in the ring or the 

aliphatic side chains. The animals were perfusion-fixed with paraformaldehyde and glutaraldehyde under 

anesthesia 6 or 24 hours after DEHP administration (n=4 animals/time point). Testis, liver, and kidney 

were collected and processed for light and electron microscopic autoradiography. After ring-labeled 

DEHP was given, light microscopy showed preferential distribution of grains to the basal portions of 

stage IX–I tubules at 6 hours. Grain counts were high in the kidney at 6 hours at the epithelial brush 

border and the abluminal cytoplasm of the proximal tubule. At 24 hours, grain counts in testis and 

kidney were much reduced, and hepatic grain counts were increased in a centrilobular distribution 

in the liver. Electron microscopic autoradiography of Stage IX–I seminiferous tubules 6 hours after 

ring-labeled DEHP showed grains in Sertoli cell smooth endoplasmic reticulum and mitochondria. 

There were also grains at cell-junctions involving neighboring Sertoli cells and Sertoli-germ cells. 

Fewer grains were seen in the Sertoli cell Golgi apparatus and lysomes and in spermatocyte cytoplasm. 

By contrast, administration of side arm-labeled DEHP resulted in few grains in the seminiferous 

epithelium and 6 hours and no grains in any tissue examined at 24 hours. The authors concluded that 

phthalic acid is transported into tissue after DEHP administration and is responsible for the testicular 

toxicity of both DEHP and MEHP.

2.2 General Toxicity and Carcinogenicity

Conclusions in recent reviews by the FDA, Health Canada, and the European Commission are 

summarized in Table 20. [The Expert Panel notes the conclusions in this table are based on the

presumed lack of peroxisome proliferator-activated receptor (PPAR)α-mediated toxicity.It may

be premature to decide that effects mediated through PPARα are not relevant in humans (FDA,

2004 #200).Although peroxisome proliferation, mediated by PPARα, occurs in rodents but not

in humans, nevertheless, humans do have a functional PPARα nuclear receptor.]
Appendix II

II-45

Table 20. DEHP Conclusions by US, Canadian, and European Agencies

Topics

Agency

FDA (2) ATSDR (101) Health Canada (102) European Commission (1)

Most sensitive target organ

Testis Testis Testis and conceptus Testis

Other possible targets of toxicity

One study suggested that DEHP could contribute to hyaline membrane disease in mechanically ventilated children.Factors such as poor bowel perfusion more likely contribute to necrotizing enterocolitis in newborns than DEHP.

Although confounded, there is some evidence suggesting that DEHP released from PVC tubing during respiratory ventilation can cause lung disorders in children.

Suspicions about development of polycystic kidney disease in patients undergoing hemodialysis have not been confirmed by clinical evidence.Causation cannot be determined for the role of DEHP in pathological lung effects in ventilated preterm infants. Evidence suggests that DEHP is not a causative agent of he­patoblastoma.

Genetic toxicity The weight of evidence indicates 

that DEHP is not genotoxic. 

Cancer Mechanisms of liver cancer in 

rats and mice are not relevant to humans.

Concurs with IARC conclusion that mechanisms of liver tumors in rodents are not relevant to humans.

There are no concerns about carcinogenicity in humans, based on animal studies.

Sensitive populations

Children receiving some medical treatments may receive a higher dose on a mg/kg bw basis than adults. Compared to adults, children may absorb greater amounts of DEHP due to greater intestinal permeability, may more effectively convert DEHP to MEHP (the toxic metabolite) due to higher levels of intestinal lipases, and may less effectively excrete MEHP due to reduced glucuronidation.

Infants have higher levels of gastric lipases and may be more able to convert DEHP to MEHP.Permeability of blood-testis barrier is higher in children.There appear to be few indications of biological polymorphisms that increase sensitivity.Younger animals appear to be more sensitive to DEHP-induced toxicity than older animals.

Populations at highest risk ofDEHP toxicity include newborns, infants, toddlers, and children with critical illnesses.Populations with unknown risk of toxicity include breast-fed children, the fetus, and pre-pubescent males.

There is evidence of greater DEHP sensitivity in immature compared to mature animals.
II-46

Appendix II

Topics

Agency

FDA (2) ATSDR (101) Health Canada (102) European Commission (1)

Sensitive populations (continued)

Children may be more pharmacodynamically sensitive to DEHP than adults (e.g., increased permeability of the blood-testis barrier).DEHP may exacerbate zinc and vitamin E deficiencies, which are not uncommon in preterminfants.There are polymorphisms in genes coding for pancreatic lipase.There are polymorphisms in several UDP-glycuronyltransferase genes.

Species differences

Liver effects mediated through PPARα do not appear relevant to humans. [The Expert Paneldoes not necessarily concur withthis conclusion inasmuch as afunctionalPPARαreceptor doesoccur in humans.]

Liver effects mediated through PPARα do not appear relevant to humans.DEHP hydrolysis rates are highest in mouse>rat >guinea pig>hamster>humans and primates.Primates are more efficient at glucuronidating metabolites but less effective at oxidizing metabolites than rodents.

It is believed that DEHP toxicity in rodents is mediated through the PPARα receptor, which is less relevant in humans; however, there is also evidence that PPARα-independent toxicity also occurs.Mechanisms of adverse effects in rodents do not appear to be of great significance in non-human primates; evidence that those mechanisms apply to humans is lacking.

Acceptable limits

TI (oral)=0.04 mg/kg bw/day.

TI (parenteral)=0.6 mg/kg bw/day.

MRL=0.1 mg/kg bw/day for oral exposures of intermediate duration (15–364 days).MRL=0.06 mg/kg bw/day for oral exposures of chronic duration (≥ 365 days).

No Tolerable Intake Value can be recommended regarding use of DEHP in medical devices.
Appendix II

II-47

Topics

Agency

FDA (2) ATSDR (101) Health Canada (102) European Commission (1)

Situations where DEHP exposures may be of toxicological concern

“. . . children undergoing certain medical procedures may represent a population at increased risk for the effects of DEHP.”Medical procedures of possible concern include: TPN in infants and pregnant women, enteral nutrition, exchange transfusions in infants, ECMO in infants and adults, aggregate exposures of neonates in NICU; cardiopulmonary by-pass surgery may lead to high exposure but exposures vary widely depending on use of heparin-coated tubing.

Subpopulations at greatest risk: ECMO patients, cardiopulmonary by-pass patients, infants, and children receiving exchange transfusions, patients receiving some iv therapies such as TPN and lipophilic drug formulations.Subpopulations with possible but undetermined risk: trauma patients receiving multiple blood transfusions, hemodialysis patients, patients receiving oxygen therapy.

Premature infants are a particular risk group because they can be exposed to high DEHP concentrations through blood transfusions, ECMO, and respiratory therapy.

Situations not likely to result in toxicological concern

Infusion (iv) of crystalloid fluids and drugs.TPN in adults.Blood transfusions. Hemodialysis and peritoneal dialysis.

Ambient levels in environment. DEHP levels do not pose a danger to the environment on which 

human life is dependent.

ECMO extra corporeal membrane oxygenationMRL minimal risk levelTI tolerable intake.
II-48

Appendix II

Stroheker et al. (98) evaluated the anti-androgenic activity of DEHP in a modified Hershberger assay 

using Wistar rats. Male offspring were weaned and randomized by weight at 20 days of age [day of

birth not defined]. On the following day, the animals were castrated and allowed to recover for 1 

week. DEHP (>99% purity) in corn oil was given by gavage for 10 days at 0, 200, 400, 600, 800, or 

1000 mg/kg bw/day in the first experiment and 0, 4, 20, or 100 mg/kg bw/day in the second experiment 

(n=8/treatment group). In both experiments, testosterone propionate 0.4 mg/kg bw/day was given 

subcutaneously (sc) on the same days as the DEHP treatments. The animals were weighed and killed 

24 hours after the last treatment and relative weights were determined for the seminal vesicles, prostate, 

and bulbocavernosus/levator ani muscles. As expected, testosterone propionate treatment produced 

a significant increase in the relative weight of all accessory sex organs compared to vehicle-treated 

control. A significant impairment of the testosterone propionate-induced organ weight increase occurred 

with DEHP treatment beginning at 100 mg/kg bw/day for the bulbocavernosus/levator ani muscles, 

200 mg/kg bw/day for the prostate, and 400 mg/kg bw/day for the seminal vesicles. The authors 

concluded that DEHP treatment has anti-androgenic effects but does not inhibit 5α-reductase because 

bulbocavernosus/levator ani muscles, the most sensitive organs, are only testosterone-responsive, 

whereas prostate is only dihydrotestosterone-responsive, and seminal vesicles are responsive to both 

androgens. [The Expert Panel noted that it is not clear if testosterone propionate data were

combined or compared separately for the two blocks.The lack of dose-response was noted.]

In the same report, Stroheker et al. (98) evaluated DEHP, MEHP, 5-oxo-MEHP, and 5-OH-MEHP in 

an androgen receptor-positive breast cancer cell line stably transfected with a luciferase reporter gene. 

The cell line showed an 81% decrease in dihydrotestosterone-induced luciferase activity after exposure 

to the positive control nilutamide, an androgen receptor antagonist, at 10–6 M. DEHP and MEHP 

were added to cultures at log unit concentrations ranging from 10 –10 to 10–5 M. The secondary 

DEHP metabolites 5-oxo- and 5-OH-MEHP were added at 10–10 to 10–8 M, limited by solubility or 

cytotoxicity. [The method for evaluating cytotoxicity was not described.] There was no inhibition of 

dihydrotestosterone stimulation of luciferase activity at any tested concentration of DEHP or MEHP. Both 

5-oxo- and 5-OH-MEHP inhibited luciferase activity at all tested concentrations to 40–70% of control 

levels [estimated from a graph]. The authors concluded that although in vivo anti-androgenic activity 

of DEHP could be indirect, due to increased catabolism of testosterone, it might alternatively be due to 

the anti-androgenic activity of the 5-oxo- and 5-OH-MEHP metabolites. [The lack of a dose response

with the oxidative metabolites, combined with a lack of clear understanding of the mechanism by

which these compounds reduced luciferase activity, reduces the usefulness of these data.]

Roy et al. (99) evaluated DEHP in a recombinant cell-based in vitro assay for anti-androgenicity. Chinese 

Hamster ovary cells were stably transfected with human androgen receptor and an androgen-dependent 

luciferase reporter. The androgen receptor agonist R1881 was used at a half maximally stimulating 

concentration of 0.1 nM. Cyproterone acetate and hydroxyflutamide were used to check that the assay 

responded to anti-androgens. A panel of 60 compounds was tested, including DEHP, which was negative 

in the assay. [The report did not give the tested concentration(s) of DEHP.The Expert Panel notes

that testing of MEHP would have been preferable to the testing of DEHP in this assay.]

Hwang et al. (100) evaluated DEHP in a novel double-transgenic mouse assay for anti-androgenicity. 

The transgenic animal co-expressed the tetracycline-controlled transactivator and human CYP1B1. 

Expression of human CYP1B1 in this model was high during the neonatal period and decreased in 
Appendix II

II-49

adult males. Castration resulted in an increase in CYP1B1, which could be suppressed with testosterone 

treatment. Flutamide, an anti-androgen, was shown to increase CYP1B1 in intact adult transgenics. 

DEHP [purity not given] in corn oil was administered as a single sc dose to 10-week-old transgenic 

mice at 0, 100, 500, or 1000 mg/kg bw (5 mice/group). Total ribonucleic acid (RNA) was extracted 

from livers 3 days later and amplified by reverse-transcriptase polymerase chain reaction (RT-PCR). 

Microsomal protein was harvested, and human CYP1B1 was detected by Western blotting. CYP1B1 

activity was determined by measurement of the dealkylation of benzyloxyresorufin. Statistical analysis 

used 1-way analysis of variance (ANOVA) [post hoc test not specified]. In a separate experiment 

[described in the Resultssection], transgenic mice were treated with DEHP 0 or 1000 mg/kg bw/day 

on days 1, 3, and 9 or for 1, 3, or 9 consecutive days. [The text of the Results section describes the

first dosing schedule, and a figure legend describes the second dosing schedule. Evaluation of

RNA, protein, and activity were performed at unspecified times after dosing.]

There was a dose-related increase in CYP1B1 transcript, CYP1B1 protein, and CYP1B1 activity, with 

a significant increase in transcript at 500 and 1000 mg/kg bw and an increase in protein and activity 

at all doses compared to the control values for each of the assays. Transcript, protein, and activity 

showed a duration-related increase with treatments labeled 1, 3, and 9 day. [The Expert Panel notes

that di(n-butyl) and diethyl phthalate were tested in the same model and showed responses in

the graphic representation of the 1, 3, and 9-day results that were similar to the DEHP response.

Linuron also showed a duration-related increase in response.]

The authors concluded that the double-transgenic model they described was a useful test for antiandrogenic activity. [The Expert Panel notes lack of a readily discernable androgen-dependent

link between the double-construct and its response to anti-androgens; the Panel was unable to

discern why this construct should react to anti-androgens.The lack of flutamide in the group of

test compounds, as well asthe absence of any metabolism-requiring androgen-receptor negatives,

only raises the level of concern that this construct is really reporting a metabolic need and has

nothing to do with androgenicity. Diethyl phthalate should have served as a negative control in

the Hwang paper (100), and the fact that it did not indicates that the assay is not specific for

anti-androgenic activity.]

Kim et al. (103) evaluated DEHP and butyl benzyl and dibutyl phthalate for the ability to inhibit tamoxifen-induced apoptosis in MCF-7 cells in culture. Tamoxifen caused a concentration-related decrease in 

MCF-7 cell viability. The phthalates increased MCF-7 cell proliferation with DEHP 10 μM [3.9 mg/L]

for 24 hours, giving rise to 133% of the control number of cells [estimated from a graph]. 17b-Estradiol, 

the positive control, gave rise to 158% of the control number of cells [estimated from a graph] at a 

concentration of 1 nM. By contrast, none of the treatments affected the number of estrogen receptornegative MDA-MB-231 cells. Coadministration of DEHP 10μM and tamoxifen for 24 hours resulted 

in 72% survival compared to the control culture, compared to 93% survival after coadministration of 

17b-estradiol 1 nM and tamoxifen. Tamoxifen alone resulted in 59% survival. Tamoxifen was shown 

to decrease the anti-apoptotic Bcl-2 protein and increase the pro-apoptotic Bax protein in the MCF-7 

cells. The Bcl-2:Bax ratio was increased by 17b-estradiol and by the phthalates, including DEHP. 

Hong et al. (104) evaluated the activity of DEHP and diethyl, benzylbutyl, dibutyl, and dicyclohexyl 

phthalate on MCF-7 cells in culture and on uterine calbindin-D9k in preweaning Sprague-Dawley rats. 
II-50

Appendix II

Both MCF-7 proliferation and an increase in uterine calbindin-D9k were considered to be estrogenic 

endpoints. In the MCF-7 assay, ethinyl estradiol and 17b-estradiol were used as positive controls 

and induced a 9-fold increase in cell proliferation (relative to vehicle control) at concentrations of 

10- 5 M. DEHP produced a 6-fold increase in proliferation at a concentration of 10- 4 M [39 mg/L]

and no significant increase in proliferation at 10 - 5 M. In the calbindin-D9k assay, DEHP in corn oil 

was given at 0 or 600 mg/kg bw/day on postnatal day (PND) 14– 16 and uteri were harvested on 

PND 17. Calbindin-D9k messenger RNA (mRNA) and protein were assayed. Ethinyl estradiol and 

diethyl­stilbestrol, the positive controls, increased calbindin-D9k mRNA and protein, but DEHP and the 

other phthalates had no effect. The authors suggested that the phthalates may have been metabolized 

in the tissues of the intact rats with consequent loss of their estrogenic activity. [The Expert Panel

notes that these authors did not evaluate whether the MCF-7 cell response to phthalates was

estrogen receptor-mediated.]

Voss et al. (105) administered DEHP (>99% purity) in the diet to male Sprague-Dawley rats beginning 

at an age of 90– 110 days and continuing for the entire lifetime of the animals (up to 159 weeks). 

DEHP was administered in feed at 0, 600, 1897, and 6000 mg/kg diet, given in 5 g feed/100 g bw/day 

6 days/week. On the 7th day of the week, animals received DEHP-free feed after their DEHP-treated 

feed had been completely consumed. DEHP dose levels were 0 (n=390), 30 (n=180), 95 (n=100), 

and 300 (n=60) mg/kg bw/day [6 days/week unless residual treated feed was consumed on the 7th

day. Daily feed consumption was not reported.] The number of animals in each group was chosen 

based on anticipated tumor incidence, with larger numbers of animals in groups expected to have a 

lower incidence of tumors. Animals were killed when moribund if they did not die spontaneously, 

and all animals were necropsied after death. Brain, liver, adrenals, testes, thyroid, lungs, spleen, and 

macroscopic lesions were fixed in 7% formalin, sectioned in paraffin, and examined by light microscopy 

after staining with hematoxylin and eosin. Livers were weighed, and liver slices were fixed in Carnoy 

fluid. In addition to hematoxylin and eosin-stained sections, liver evaluation included treatment with 

periodic acid-Schiff with orange G and iron hematoxylin counterstaining. Statistical evaluation was 

performed with Kruskal-Wallis and chi-squared tests. 

The animals fed DEHP in all dose groups experienced a transient absolute weight reduction compared 

to control animals about 300 days after the beginning of the experiment, but weights were comparable 

thereafter. The authors described a dose-dependent increase in liver weight, reaching 108% of control 

values in the highest dose group but indicated that liver weights were not statistically different from 

controls. There was no effect of DEHP treatment on survival time of the animals. The proportion of 

animals with malignant and benign tumors, overall, was not affected by treatment; however, detailed 

evaluation of livers of the sacrificed animals showed a 29.0% incidence of all neoplasms in the highest 

DEHP dose group compared to 9.0% of control animals (P=0.005). Although the lower 2 DEHP dose 

groups did not have a statistically increased incidence of hepatic neoplasms on pair-wise comparison 

with the control, trend testing showed a significant trend over the dose ranges (P=0.001). 

Leydig cell tumors occurred in 28.3% of animals in the highest dose group compared to 16.4% of 

control animals (P=0.038), and a dose-related trend was identified in Leydig cell tumors over the dose 

range (P=0.019). The association of DEHP treatment with Leydig cell tumors extended to analysis of 

unilateral, bilateral, and multifocal unilateral tumors. When the lifetimes of the animals were divided into 

3 periods (0–750, 750–950, and 950–1250 days), the associations between total and unilateral Leydig 
Appendix II

II-51

cell tumors and DEHP dose level were most evident during the middle period. Bilateral and multifocal 

unilateral tumors showed dose-related DEHP increases during the third period. The authors postulated 

that the DEHP-associated increase in Leydig cell tumors might be due to an increase in gonadotropin 

production secondary to decreased testosterone synthesis or increased testosterone aromatization.

2.3 Summary of General Toxicology and Biologic Effects

2.3.1 	 Toxicokinetics

As discussed in Section 1, exposure studies in humans measuring primary and secondary urinary 

metabolites (MEHP, 5-OH-MEHP, 5-oxo-ME HP) suggest aged-related differences in production 

and/or clearance. Younger children produce higher proportions of 5-OH-MEHP and 5-oxo-MEHP 

compared to MEHP, and this difference increases with decreasing age. Furthermore, as noted in the 

first Expert Panel Report on DEHP, premature and term infants have reduced renal clearance based on 

decreased glomerular filtration rates and immature glucuronidation, which may increase the internal 

doses of toxic metabolites. Data from Calafat (106) and Silva (44) (reviewed in Section 1) show that 

oxidative metabolites are present in free (unconjugated) form in breast milk and amniotic fluid, which 

may pose additional risk from these metabolites. Finally, the Expert Panel notes, as mentioned in the 

initial Expert Panel Report on DEHP, that neonates have lingual, gastric, and intestinal lipases that 

would need to be quantified in comparison to adults levels in order to assess DEHP conversion rates. 

Breast milk also contains lipases. The relative activities of these combined systems would determine 

gut absorption in the newborn and young infant and need to be elucidated.

An unpublished report from Mitsubishi Chemical Safety Institute, Ltd. (92) included a toxicokinetic 

study in marmosets at 3 and 18 months of age. The 18-month-old animals included a group that had 

been pretreated with radiolabeled DEHP for 65 weeks and a treatment-naïve group. Blood was drawn 

at intervals during the week following a single dose of 100 or 2500 mg/kg bw. Two weeks later, an 

additional oral dose was given and tissues were sampled 2 hours later. The report presented radioactivity contents, expressed as μg equivalents, without characterization of unchanged DEHP or DEHP 

metabolites. There was no apparent effect of chronic DEHP treatment on toxicokinetic parameters or 

organ distribution at 18 months of age. Concentrations of radiolabel in testis were 9 – 23% of plasma 

concentrations, and the authors remarked that the small amount of DEHP and metabolites distributed 

to the marmoset testis might explain the lack of testicular toxicity noted by them in a 65-week feeding 

study (discussed in Section 4.2.3). [The Expert Panel noted that given the large variability in tmax

(1 –10 hours,Table 16), the organ to plasma ratio of radiolabeled DEHP (Table 17) collected at a

fixed time (2 hours) may not accurately reflect age- and dose-related differences.Furthermore,the

smallsample size combined with the large inter-individual variability complicatesinterpretation

of the data and may not accurately reflect dose- and age-related differences.]

A GLP study compared blood levels of DEHP and MEHP in pregnant and non-pregnant rats and 

marmosets dosed with 30 or 500 mg/kg bw/day DEHP (94). The study authors concluded that 

concentration time courses were similar in pregnant rats receiving both dose levels by gavage on GD 

14–20 and non-pregnant rats given 500 mg/kg bw/day for 7 days; repeated dosing had no marked 

effects on kinetics in either group of rats. In both groups of rats, MEHP AUCs were about 2 orders 

of magnitude higher than DEHP AUCs. The authors concluded that kinetics for DEHP were linear, 

while kinetics for MEHP were saturated. In marmosets fed 30 or 500 mg/kg bw/day DEHP through 
II-52

Appendix II

a syringe, concentration time courses (based on 1 marmoset per time point) were similar in pregnant 

animals dosed on GD 96 –125 and non-pregnant animals dosed for 29 days; the exception was that 

MEHP values in the 500 mg/kg bw DEHP group were lower compared to the non-pregnant animals 

on GD 103 and beyond. MEHP AUCs in pregnant and non-pregnant marmosets were more than an 

order of magnitude higher than DEHP AUCs and were independent of dose. In a comparison of species 

differences, maximum blood concentrations of MEHP in rats were an average of 3.2 times higher 

(range 1.3–7.5) than those of marmosets. MEHP AUCs were an average of 7.3 times higher (range 

2.6 – 15.6) in rats compared to marmosets. [However, the small marmoset sample size prevents

a lack of a good understanding of inter-individual variability and reduces confidence in the

comparisons of Cmax andAUC between marmosets and rats, which is reflected by the wide range

in rat to marmosetAUC ratios.The Expert Panel also notes that species differences in Cmax and

AUC between marmosets and rats are less at the lower dose levels.]

A study using tissues from rats, mice, and marmosets evaluated the activities of enzymes involved in 

the metabolism of DEHP (95). Marmoset small intestine, liver, and kidney appear unable to convert 

DEHP to MEHP to any great extent, based on enzyme activities in vitro. [The Expert Panel believes

that it would have been better, from an experimental point of view, if lipase secreted into the

lumen of the gut had been measured.]

2.3.2 	 General Toxicity and Carcinogenicity

The anti-androgenic activities of DEHP and some of its metabolites were evaluated by Stroheker et 

al. (98). Treatment of castrated 21-day-old Wistar rats with DEHP prevented an increase in accessory 

sex-organ weight after testosterone propionate. The bulbocavernosus/levator ani muscles were the 

most sensitive to this DEHP effect, and the prostate was the least sensitive, leading the authors to 

conclude that DEHP did not inhibit 5α-reductase. Evaluation of DEHP, MEHP, and the secondary 

DEHP metabolites, 5-oxo- and 5-OH-MEHP, in an androgen receptor-positive cell line showed no 

antagonism by DEHP or MEHP of the activity of the androgen receptor agonist dihydrotestosterone. 

Both 5-oxo- and 5-OH-MEHP showed significant dihydrotestosterone antagonism. The authors 

concluded that the in vivo anti-androgenic effects of DEHP could be mediated through the secondary 

metabolites. [The lack of a dose response with the oxidative metabolites combined with a lack of

clear understanding of the mechanism by which these compounds reduced luciferase activity

reduces the usefulness of these data.]

Voss et al. (105) administered DEHP at 0, 600, 1897, and 6000 mg/kg feed in the diet to male 

Sprague-Dawley rats beginning at an age of 90–110 days and continuing for the entire lifetime of 

the animals. DEHP dose levels were 0, 30, 95, and 300 mg/kg bw/day [6 days/week unless residual

treated feed was consumed on the 7th day]. The animals fed DEHP experienced a transient weight 

reduction compared to control animals about 300 days after the beginning of the experiment, but 

weights were comparable thereafter. The authors described a dose-dependent increase in liver weight, 

reaching 108% of control values in the highest dose group, but indicated that liver weights were not 

statistically different from controls. There was no effect of DEHP treatment on survival time of the 

animals. The proportion of animals with malignant and benign tumors, overall, was not affected by 

treatment; however, detailed evaluation of livers of the sacrificed animals showed a 29% incidence 

of all neoplasms in the highest DEHP dose group compared to 9% of control animals (P=0.005). 

Although the lower 2 DEHP dose groups did not have a statistically increased incidence of hepatic 
Appendix II

II-53

neoplasms on pair-wise comparison with the control, trend testing showed a significant trend over the 

dose ranges (P=0.001). Leydig cell tumors occurred in 28.3% of animals in the highest dose group 

compared to 16.4% of control animals (P=0.038), and a dose-related trend was identified in Leydig 

cell tumors over the dose range (P=0.019). The authors postulated that the DEHP-associated increase 

in Leydig cell tumors might be due to an increase in gonadotropin production secondary to decreased 

testosterone synthesis or increased testosterone aromatization.
II-54

Appendix II

3.0 DEVELOPMENTAL TOXICITY DATA

3.1 Human Data

Since the initial CERHR Expert Panel Report on DEHP, there have been several studies in humans in 

which development of the male reproductive system has been evaluated with respect to estimates of 

DEHP exposure during pregnancy or early childhood. There has also been a study addressing premature 

breast development and DEHP exposure.

Latini et al.(107), funding not indicated, conducted a study to examine the effects of prenatal exposure 

to DEHP and MEHP. Cord blood samples were collected from 84 consecutive newborns (including a set 

of twins) delivered at an Italian hospital. Ages of mothers ranged from 18 to 42 years. DEHP and MEHP 

levels were measured in cord blood serum by HPLC. Glass equipment was used in sample preparation and 

analyses to avoid phthalate contamination. Analyses were conducted to determine possible relationships 

between phthalate exposure and adverse neonatal outcomes. Relationships between phthalates in cord 

blood and outcomes in infants were assessed by Fisher exact test and unpaired t-tests. Significance 

levels for multiple t-tests were Bonferroni corrected. Multivariable logistic regression models were 

used to evaluate significant differences in univariate analyses and effects from potential confounders. 

DEHP and/or MEHP were detected in 74 of 84 cord blood samples. Mean (range) cord blood serum 

concentrations were 1.19 (0 –4.71) μg/mL for DEHP and 0.52 (0 –2.94) μg/mL for MEHP. Mean 

gestational age was significantly lower in MEHP-positive neonates (38.16±2.34 [SD] weeks) versus 

MEHP-negative neonates (39.35±1.35 weeks; P=0.033). There were no significant associations between 

DEHP or MEHP concentrations and infant sex, delivery mode, maternal smoking, premature membrane 

rupture, cord loops, neonatal jaundice, small infant size, birth weight, 1- or 5-minute Apgar scores, or 

maternal age. [With the exception of birth weight, data were not shown for these endpoints.]

The study authors concluded that their study demonstrated the presence of DEHP/MEHP in most 

newborns, and that phthalate exposure is associated with shorter pregnancy duration.

Strengths/Weaknesses: Use of cord blood, which reflects infant exposure in utero, is a strength as is 

the use of consecutive births at same hospital. Outcome assessment determination blind to exposure 

status is a strength. However, the levels measured in blood were unusually high and led the Expert 

Panel to wonder whether pre-analytic contamination occurred or the wrong units were reported. In 

addition, the blood samples were not pretreated and in a previous publication on a subgroup of the 

current study (42), these authors saw no significant correlations between maternal DEHP/MEHP and 

cord blood DEHP/MEHP.

Utility (Adequacy) for CERHR Evaluation Process: Because the Expert Panel cannot resolve whether 

the unusually high reported blood levels represent an error in units (μg instead of ng) or were the result 

of pre-analytic contamination, this study cannot be used in the evaluation process.

Swan et al. (108), supported by the Environmental Protection Agency (EPA), National Institutes of 

Health (NIH), and the state of Iowa, evaluated anogenital distance in children and maternal urinary 

phthalate monoester concentrations. Pregnant women in 1 of 3 US cities were recruited as part of a larger 

study. Urine samples were collected from women at a mean gestational age of 28.3 weeks. Data from 

the 134 sons of 172 women who were eligible for this part of the study were used to assess the associa-
Appendix II

II-55

tion between anogenital index (AGI; anogenital distance adjusted for body weight) and other genital 

parameters such as testicular descent. Exclusion criteria included mother-son dyads with incomplete 

information, lack of consent for genital examination, and child age greater than 18 months, considered to 

make anogenital distance measurement unreliable due to movement. Of these 134 sons, 85 had phthalate 

measurements used in the analyses of association between phthalates and anogenital index. The urine 

analytes included metabolites of DEHP (MEHP, 5-oxo-MEHP, and 5-OH-MEHP) and monobutyl, 

monobenzyl, mono-3-carboxypropyl, monoethyl, mono-isobutyl, and monomethyl phthalate. Analysis 

was performed by HPLC-tandem MS after enzymatic deconjugation. Infant evaluation included height, 

weight, head circumference, skin-fold thickness, anogenital distance (measured from the midpoint of 

the anus to the anterior base of the penis), anoscrotal distance (measured from the midpoint of the anus 

to the posterior insertion of the scrotum), and detailed examination of the breasts and genitalia. 

The relationship between maternal pregnancy urinary phthalate concentration (logarithmically transformed) 

and anogenital distance was evaluated using general linear models. Analyte concentrations below the 

limit of detection were considered to be the limit of detection value divided by the square root of 2. 

Data were also analyzed using a categorical approach based on 25th and 75th percentiles for age- and 

weight-adjusted anogenital distance and 25th and 75th percentiles for analyte concentrations. [Categorical

analysis was reported only for the 4 analytes associated with decreased anogenital index.] Potential 

confounders considered in regression analysis included mother’s ethnicity, smoking status, time and 

season of urine collection, gestational age at time of urine collection, and infant weight at examination. 

[Confounding by clinic site, calendar time, and maternal education was not assessed.] In addition to 

using individual urinary phthalate monoester concentrations in the analysis, the authors constructed a total 

phthalate score based on quartiles of individual phthalate concentrations [using only the concentrations

of monobutyl, monobenzyl, monoethyl, and mono-isobutyl phthalate, which had been shown to

be associated with anogenital index, discussed below]. Individual phthalate concentrations in the 

lowest quartile made no contribution to the total phthalate score. One point was given for each quartile 

above the lowest quartile. The calculation of total phthalate score led to the trichotomizing of samples 

into categories of low (score 0–1), intermediate (2–10), and high (11–12).

The final regression model, including only age and age-squared as covariates, showed an inverse 

relationship between logarithmically transformed analyte concentration and weight-adjusted anogenital 

distance for monobutyl (P=0.031), monobenzyl (P=0.097), monoethyl (P=0.017), and mono-isobutyl 

phthalate (P=0.007). The regression coefficient for MEHP was – 0.051 (P=0.833). The regression 

coefficient for 5-oxo-MEHP was –0.412 (P=0.114), and the regression coefficient for 5-OH-MEHP 

was – 0.398 (P=0.145). The authors noted that the regression coefficients for the oxidative MEHP 

metabolites were of similar magnitude to the coefficients for monobutyl and monobenzyl phthalate 

(–0.592 and–0.390). The authors indicated that DEHP shortens anogenital distance in rodents and that 

it was not possible to tell if the urinary concentrations of MEHP and its oxidative metabolites failed 

to be associated significantly with anogenital index in children because of sample size limitations or 

because humans and rodents responded differently to DEHP.

The relationship between testicular descent (normal or normal-retractable versus one or both testicles 

incompletely descended) and anogenital index was assessed in 134 boys. The proportions of boys with 

one or both testicles incompletely descended were 20.0, 9.5, and 5.9% for boys with short AGI (below 

the 25th percentile), intermediate (25th–75th percentile), and long (75th percentile or higher) (P value 
II-56

Appendix II

for short AGI vs. all others <0.001.) [The Expert Panel was not able to confirm this P value from

the data presented in the paper.]

Strengths/Weaknesses: The prospective nature of the study and the collection of urine for exposure 

assessment during pregnancy, reflecting in utero exposure, are strengths as is the measurement of 

urinary metabolites rather than parent compounds, avoiding contamination issues. The masking of 

clinicians measuring anogenital distance to the laboratory assessment of phthalates and vice versa are 

additional strengths. The choice of anogenital distance as an endpoint is consistent with a sensitive 

endpoint in rodents. However there were no data presented on the reliability of the measurement of 

anogenital distance or other variables that may be associated with anogenital distance. Methods used 

to determine independent or combined effects of various phthalates (creation of summary score) were 

not appropriate for that purpose. A weakness of the study is that potential confounding by clinic, 

education, and calendar time was not assessed.

Utility (Adequacy) for CERHR Evaluation Process: This study is useful for the evaluation process.

Main et al. (17), supported by the European Commission, the Danish Medical Research Council, the 

Svend Andersen and Velux Foundation, the Turku University Central Hospital, and the Academy of 

Finland, studied the association of breast milk levels of MEHP and other phthalates and blood levels of 

reproductive hormones in 3-month-old boys. [Milk concentrations were discussed in Section 1.1.1.]

Pooled milk samples were obtained from each of 130 women (half from Denmark and half from Norway) 

when their children were 1–3 months old. Milk was analyzed using HPLC-MS for MEHP as well 

as monomethyl, monoethyl, monobutyl, monobenzyl, and mono-isononyl phthalate. Cryptorchidism 

was identified in 62 of the 130 children of these women; however, there was no significant association 

between milk phthalate concentrations and cryptorchidism. The children had venous blood sampled at 3 

months of age for determination of sex hormone-binding globulin, total and free testosterone, luteinizing 

hormone (LH), follicle stimulating hormone (FSH), and inhibin B. Individual hormone levels were 

used to calculate LH/testosterone, LH/free testosterone, and FSH/inhibit B ratios. Multiple regression 

analysis was used to explore relationships between log-transformed milk phthalate concentrations and 

hormone levels using gestational age at birth, weight for gestational age, parity, smoking, diabetes, 

and country of origin as potential confounders. Only country of origin was retained as a confounder. 

Associations between milk phthalate levels and hormone levels were then tested with country-adjusted 

partial Spearman correlations with exact P-values obtained using Monte Carlo permutation.

MEHP was found in all milk samples. Milk concentration of MEHP was observed to have a marginally 

significant correlation with free testosterone (Spearman r=−0.169, P=0.107) and inhibin B (r=0.185, 

P=0.075). In addition, associations were observed with three ratios: LH/testosterone (r=0.180, 

P=0.087), LH/free testosterone (r=0.175, P=0.095), and FSH/inhibin B (r=−0.204, P=0.050). [The

Expert Panel places more weight on individual hormone measures rather than the hormone

ratios (LH/testosterone, LH/free testosterone), because these ratios are not biologically relevant

in a non-clinical setting. The Expert Panel notes that 9 hormones or ratios of hormones were

evaluated for each of 6 phthalate monoesters, yielding multiple comparisons without adjustment.

The Expert Panel also has concerns about the adequacy of control for country differences both

as a potential confounder and as an effect modifier. The Expert Panel is also concerned about

possible contamination by use of breast pumps after the feeding of the infant.]
Appendix II

II-57

The authors concluded that there were “subtle, but significant, dose-dependent associations between 

neonatal exposure to phthalate monoesters in breast milk and levels of reproductive hormones in boys 

at three months of age.”

Strengths/Weaknesses: Strengths included collection of breast milk to assess exposure during the first 

3 months of life, drawing of blood samples at the 3-month visit to assess hormone levels, the analyses 

of phthalates conducted blind to case status and hormone levels, and the assessment of hormone levels 

conducted blind to case status and phthalate levels. Weaknesses include possible contamination of breast 

milk samples. Women who used a breast pump in Denmark had significantly higher levels of monoethyl 

and monobutyl phthalate. Breast pump-associated levels of other phthalates were not significantly different, 

but data were not shown and breast pump use was not reported for the Finnish population. Confounding 

was not assessed for comparison of phthalate levels between cryptorchid cases and controls, although the 

authors stated that there was no significant difference stratified by country. The small sample size may 

have yielded limited power for stratified analyses, and confounding was not assessed by other variables. 

(It appears that cases and controls may have differed on prevalence of maternal diabetes and gestational 

age even though differences did not reach statistical significance.) Statistical analyses of associations 

between phthalate levels and hormone levels were not presented clearly. Confounding was assessed with 

multiple regression on log transformed data, but it appears that associations were assessed with a rankbased model adjusting for country. It was also not stated what criteria were used to assess confounding. 

The authors stated that parity, maternal smoking, gestational age, and weight for gestational age were 

not “significant” confounders. Confounding is different than statistical significance; therefore, it is not 

clear that confounding was adequately assessed. The sample size was small.

Utility (Adequacy) for CERHR Evaluation Process: This study is of some utility for suggesting an 

association between testosterone and MEHP, but concerns remain about assessment of confounding 

and contamination by breast pump use. 

Rais-Bahrami et al.(109), sponsored by NIH, examined onset of puberty and sexual maturity parameters in 14– 16-year-old adolescents who had been subjected to ECMO as neonates; the procedure 

potentially led to high DEHP exposure. The adolescents included 13 males and 6 females. Measurements 

taken during physical examinations included height, weight, head circumference, testicular volume, 

and phallic length. Pubertal staging was conducted according to the Tanner method. Laboratory tests 

were conducted to assess thyroid, liver, and renal function. LH and FSH levels were measured in both 

sexes, estrogen levels were measured in females, and testosterone levels were measured in males. 

Except for 1 female with Marfan syndrome, growth percentiles were normal for age and sex. Pubertal 

development was stated to be normal. [The authors did not state whether testicular volume and

phallic length were normal.] Laboratory results indicated normal thyroid, liver, and renal function. 

LH, FSH, testosterone, and 17b-estradiol levels were normal for stage of pubertal development. [A

control group of children who did not receive ECMO treatment as neonates was not included

for comparison. In addition, the authors did not state the criteria they used for determining if

parameters were within normal ranges.] The study authors concluded that their study “. . . did not 

show long-term adverse outcome related to physical growth and pubertal development in adolescents 

previously exposed to DEHP in the neonatal period.”

Strengths/Weaknesses: The extensive assessment of endocrine function to supplement Tanner stages 
II-58

Appendix II

is a strength of this study; however, there were no measurements of phthalate exposure, and there was 

no comparison group to compare to children presumed to be exposed. Another weakness is the very 

small sample size (13 males and 6 females) and the inability to detect changes in hormone levels that 

were still within the normal range.

Utility (Adequacy) for CERHR Evaluation Process: Given the small sample size and lack of a comparison group, this study is of minimal utility for the evaluation process.

Colón et al (110), supported in part by the EPA Minority Academic Institutions Traineeship programs, 

compared blood phthalate levels in premature thelarche patients and controls. Cases consisted of girls 

between the ages of 6 months and 8 years with premature breast development. Controls consisted of 

females aged 6 months to 10 years who displayed no evidence of premature sexual development or 

other endocrine disease. Blood samples from premature thelarche patients were taken between January 

1994 and April 1998. [It is not specified if blood samples were collected from controls during

the same time period.] Forty-one samples were obtained from premature thelarche patients and 35 

samples from control patients. [It was not stated if each sample was obtained from a different

subject.] Levels of phthalates, including DEHP, were measured in serum by GC/MS; numerous blank 

samples were analyzed to rule out contamination through solvents, water, or medical or laboratory 

equipment. Phthalates were detected in 28 of 41 samples from premature thelarche patients. DEHP 

was detected in 25 of the samples at concentrations ranging from 187 to 2098 μg/L (ppb); average 

concentration was reported at 450 μg/L. MEHP was detected in 5 of the samples at concentrations of 

6.3–38 μg/L. In the control group, DEHP was detected in 5 of 35 blood samples at concentrations of 

276–719 μg/L; average concentration was reported to be 70 μg/L. Di-n-octyl phthalate was the only 

other phthalate detected in 1 control sample. The difference in average blood DEHP level in cases 

versus controls was found to be statistically significant using the 95% confidence interval. [Methods

of statistical analyses were not discussed.] Study authors concluded “This study suggests a possible 

association between plasticizers with known estrogenic and antiandrogenic activity and the cause of 

premature breast development in a human female population.” 

[The Expert Panel notes a letter by McKee (3) in response to the Colón et al. study. This letter

identified the blood DEHP and MEHP concentrations as being difficult to reconcile with published

studies on phthalate blood levels. The very high blood levels of DEHP and low blood levels of

MEHP were described as “anomalous” and consistent only with sampling immediately after

introduction ofsubstantial amounts of DEHP into the bloodstream, as might occur after a medical

procedure.Thisscenario would not be consistent with a picture of chronic DEHP exposure levels

such as might be hypothesized to affect thelarche.The Expert Panel agrees with McKee that the

DEHP concentrations reported in this study are unreliable.]

Strengths/Weaknesses: This study used a clinically relevant outcome, but phthalates detected in 

serum specimens may have been unreliable due to laboratory contamination or to medical procedures 

conducted because of the diagnosis. It was not stated whether phthalate laboratory analyses were 

conducted blind to case status.

Utility (Adequacy) for CERHR Evaluation Process: This report is not useful because of the lack of 

confidence in the reported DEHP measurements.
Appendix II

II-59

3.2 Experimental Animal Data

Since the initial CERHR Expert Panel Report on DEHP, several experimental animal studies have 

addressed the mechanisms by which fetal and neonatal DEHP exposure interferes with development 

of the male reproductive system in rodents. There has also been a multigeneration continuous-breeding 

study in rats using 8 dietary dose levels to evaluate dose-response relationships for developmental 

and reproductive endpoints.

3.2.1 Developmental Studies Focusing on Reproductive System and Endocrine Effects

This section examines reproductive or endocrine effects occurring in animals dosed during gestation 

or during the pre-weaning stage. Studies examining reproductive effects in animals dosed subsequent 

to the lactational stage (≥21 days of age) are summarized in Section 4. 

3.2.1.1 In Vivo Exposures

Akingbemi et al. (111), supported by NIEHS, evaluated the effect of DEHP on Leydig cell function 

in male Long-Evans rats exposed in utero, during nursing, or during post-weaning stages (the postweaning results are presented in Section 4.2.2.2). Pregnant rats were exposed to DEHP (>99% purity) 

on GD 12–21 [plug day not indicated], and lactating rats were exposed on PND 1– 21 (day after 

birth=PND 1). DEHP was administered to dams by gavage in corn oil at 0 or 100 mg/kg bw/day. 

Males were obtained for evaluation on PND 21, 35, or 90 (n=7 dams/group/stage) [no information

was provided on culling or rearing]. Male offspring were decapitated within 24 hours of the final 

dose, and trunk blood was collected for measurement of LH and testosterone by radioimmunoassay 

(RIA). Testes and seminal vesicles were weighed, and testicular interstitial fluid was collected for 

measurement of fluid testosterone by RIA. Testicular histology was evaluated. Cultures of Leydig 

cells or, in 21-day-old animals, progenitor Leydig cells, were prepared from testes by Percoll density 

gradient preceded in 90-day-old rats by centrifugal elutriation. The resulting preparations were 90% 

pure for progenitor Leydig cells and 95–97% pure for PND 35 or 90 Leydig cells, as evaluated by 

staining for 17-hydroxysteroid dehydrogenase. Cultures were evaluated after 3-hour incubation with 

and without a maximally stimulating concentration of ovine LH. Testosterone was measured in the 

medium. [According to the methods section, the activity of different enzymes in the testosterone

biosynthesis pathway were evaluated by incubating Leydig cells for 3 hours with saturating

concentrations ofsubstrate for the enzyme of interest; however, results of these experiments were

given only for males treated during the post-weaning period (summarized in Section 4.2.2.2).]

Statistical analysis was by ANOVA and Duncan multiple range test. 

There were no effects of treatment during gestation or lactation on dam weight or weight gain or on 

offspring weight. Offspring testis and seminal vesicle weights were also not affected by treatment 

during either developmental period. Serum testosterone was reduced 31– 33% and serum LH was 

reduced 50– 64% [estimated from a graph] in 21- and 35-day-old males exposed to DEHP during 

gestation. There were no prenatal DEHP effects on serum testosterone or LH in 90-day-old males. 

Prenatal exposure to DEHP resulted in decreased testosterone production by cultured progenitor 

Leydig cells obtained from 21-day-old males. Basal testosterone production was reduced 47%, and 

LH-stimulated testosterone production was reduced 56%. There were no treatment effects on cultured 

Leydig cells derived from 35- and 90-day-old offspring. Lactational exposure to DEHP was associated 

with a 13% decrease in serum testosterone on PND 21. There were no significant changes in serum 

LH on PND 21 or in testosterone or LH on PND 35 or 90. [No results were presented for cultured
II-60

Appendix II

Leydig cells derived from males exposed during lactation.] Testicular histology was described as 

normal in all treatment groups. 

The authors concluded that exposure to DEHP during gestation or lactation resulted in suppression of 

pituitary LH in the presence of reduced serum testosterone, and that growing rats were more susceptible 

to the effects of developmental exposure than adult rats.

Strengths/Weaknesses: This report contains good descriptions of experimental design and methods with 

some exceptions. The studies used an appropriate route and time of exposure, and chemical source and 

purity were described. Multiple dose levels in the second study (discussed in Section 4.2.2.2) allowed 

for dose-response analyses. The comparison of responses to postnatal exposure at 3 different ages is a 

strength. Weaknesses include inadequate detail on the numbers of animals and numbers of litters per 

group used for histopathologic examination. The litter was not utilized as the unit of analysis following 

maternal/gestational exposure. For prepubertal and young adult rats, animals were randomly selected 

and assigned to treatment groups, but correction for potential litter effects was not conducted. Although 

the lack of control for litter effects is a weakness in this study, it is less compromising for prepubertal 

rats directly dosed with DEHP, because litter effects diminish somewhat with age post-weaning, and 

the animals were given a standardized dose based on individual body weight. The single, high dose 

level used in the first experiment is a weakness.

Utility (Adequacy) for CERHR Evaluation Process: The histopathology results are of limited value 

because no data were presented, and it was not clear how many animals from each treatment group 

were evaluated. The gestational and lactational results from this study are not useful for the evaluation 

because the study design did not control for litter effect following maternal exposure. The lactational 

data were not presented. The ex vivo testosterone production data are not useful for the evaluation 

process due to the artificial in vitro environment in which these data were generated, which has an 

uncertain application to human risk. The enzyme activity and testosterone production information are 

useful for providing insight into potential mechanisms of action (Section 4.2.2).

Shirota et al. (112), support not indicated, evaluated testicular pathology after intrauterine exposure 

of Sprague-Dawley rats to DEHP. In experiment 1, pregnant rats were treated by gavage with DEHP 

[purity not given] in corn oil at 0, 500, or 1000 mg/kg bw/day on GD 7–18 [plug=GD 0]. Ethinyl 

estradiol 0.25 or 0.5 mg/kg bw/day was used as a positive control. There were 28 – 30 dams/treatment group. Six dams/treatment/time point were killed on GD 12, 14, 16, 18, or 20 and live fetuses 

processed for light or electron microscopic examination. An additional 5 dams/treatment group given 

DEHP 500 or 1000 mg/kg bw/day were permitted to deliver and raise their young. Male offspring 

from these litters were killed at 7 weeks of age for histologic evaluation of testes and epididymides. 

In experiment 2, designed to identify a no-effect level, 11 or 12 pregnant rats/treatment group were 

given DEHP in corn oil at 0, 125, 250, or 500 mg/kg bw/day on GD 7 – 18. Fetuses were delivered by 

cesarean section on GD 20 in 3 dams/treatment group. The remaining dams were permitted to deliver 

and rear their offspring. Four male offspring per treatment group per time point were killed at 5 or 10 

weeks of age for light microscopic examination of testes and epididymides, 2 male offspring/treatment 

group/time point were killed at 5 or 10 weeks for electron microscopic examination of the testes, 

and 4 male offspring/treatment group were killed at 10 weeks of age for evaluation of testicular and 

epididymal sperm. [Litter of origin of the offspring at 5 and 10 weeks was not mentioned and the
Appendix II

II-61

data tables suggest that each offspring was considered an independent treatment unit.] Light 

microscopy was performed after fixation of testes in Bouin fluid and then formalin. Tissues were 

embedded in paraffin and stained with hematoxylin and eosin. In experiment 2, a testicular section was 

also stained with periodic acid Schiff to confirm acrosomal status of sperm. Immunohistochemistry 

was performed with antibody to androgen receptor. Epididymal sperm were assessed in experiment 

2 using computer-assisted sperm motion analysis. Epididymal sperm counts were also assisted using 

an automated method. Statistical analysis was performed using ANOVA or Kruskal-Wallis rank-sum 

test with post hoc Dunnett test.

Dam weight was decreased about 10% by DEHP 1000 mg/kg bw/day and by 17b-estradiol. There 

were no effects of lower DEHP dose levels on dam weight. Fetal weight and mortality were increased 

by DEHP treatment of the dam at 1000 mg/kg bw/day. The developmental lowest-observed adverse 

effect level (LOAEL) was 1000 mg/kg bw/day based on increased intrauterine mortality and decreased 

live fetuses/litter). [Statistical differences were not marked in the data table in the paper, but

were apparent by ANOVA with post hoc Dunnett test performed by CERHR.The BMD10

1 was

734–755 mg/kg bw/day for the developmental endpoints.The BMDL10 was 334 mg/kg bw/day for

the decrease in live fetuses.BMD1 SD was 846–874, and the BMDL1 SD was 490 mg/kg bw/day for

decrease in live fetuses. Due to the large SD for the litter percent intrauterine mortality, BMDLs

computed for this endpoint were not meaningful.]

In experiment 2, pup birth weight was increased in the groups exposed to DEHP at 250 and 500 mg/kg 

bw/day. On PND 4, there were no group differences in pup weight. Histologic examination of GD12 

fetuses did not show identifiable testicular tissue. On GD 14, germinal ridges with germ cells were 

distinguishable. There were no treatment-related effects at this time point. On GD 16, testicular cords 

were evident and germ cell degeneration was apparent in 1 of the 12 fetuses of the DEHP 1000 mg/kg 

bw/day group. Germ cell degeneration was shown by electron microscopy [whether in this fetus or

in others was not stated]. On GD 18 and 20, fetal testes in the DEHP-treated groups were small and 

showed hyperplasia of interstitial cells and multinucleated germ cells. Testes from 17b-estradiol exposed 

fetuses were also small and contained multinucleated germ cells. At 7 weeks of age, 1 offspring in the 

DEHP 500 mg/kg bw/day group showed multinucleated giant cells in the seminiferous tubules, but 

otherwise, testicular histology was normal. Testes from the DEHP 1000 mg/kg bw/day group showed 

branched and dilated tubules, atrophic tubules, multinucleated giant cells, and dilatation of the rete 

testis at 7 weeks. There was also epididymal atrophy, dilatation, and inflammation. 

In experiment 2, there were multinucleated germ cells in fetal testes from all groups exposed to DEHP. 

Interstitial hyperplasia was also seen in the groups exposed to DEHP 250 and 500 mg/kg bw/day, with 

some degenerated germ cells in the 500 mg/kg bw/day group. Androgen receptor immunohistochemistry in the fetal testes was consistent with Leydig cell hyperplasia in the 500 mg/kg bw/day group. 

At 5 and 10 weeks of age, there were no abnormalities in the testes in any of the treatment groups by 

1Benchmark doses are used commonly in a regulatory setting; however, they are used in this report when the 

underlying data permit their calculation, and are only supplied to provide 1 kind of description of the doseresponse relationship in the underlying study. Calculation of a benchmark dose in this report does not mean 

that regulation based on the underlying data is recommended, or even that the underlying data are suitable for 

regulatory decision-making.
II-62

Appendix II

light or electron microscopy. Epididymal sperm counts and sperm motility parameters did not show 

treatment effects. 

The authors concluded that DEHP was toxic to the fetal testis with histologic findings of germ cell 

degeneration and interstitial cell hyperplasia. These effects were seen at maternal dose levels of 

250 mg/kg bw/day but not 125 mg/kg bw/day, which the authors identified as a no-observed effect 

level. [The Expert Panel notes that multinucleated germ cells were identified in 0/15 fetuses in

the control group, 6/16 fetuses in the 125 mg/kg bw/day group, 15/19 fetuses in the 250 mg/kg

bw/day group, and 25/28 fetuses in the 500 mg/kg bw/day group. Benchmark dose analysis for

this endpoint gives a BMD10 of 73 mg/kg bw/day and a BMDL10 of 54 mg/kg bw/day.]

Strengths/Weaknesses: The multiple exposure levels allow for a dose-response evaluation. The presentations of most methods and data are fairly good. The study shows the developmental progression for 

testicular injury following in utero exposure and recovery postnatally, through sexual maturity. This study 

also evaluates lower doses in order to establish a no adverse effect level. The statistical evaluation and 

study design are weak. Although the investigators started with sufficient animals within the treatment 

groups of experiment 1 (28–30 dams/group), breaking the groups into multiple sampling time points 

resulted in relatively small group sizes per time point (5 or 6 dams for experiment 1 and 3–9 dams 

for experiment 2). In experiment 2, the GD 20 groups were limited to 3 dams/group. Since the dams 

were directly dosed, the dam or litter should have been the unit of analysis. The investigators failed 

address the unit of analysis in the statistical analyses section of the Methods and appeared to use the 

fetus or offspring as the unit of analysis for most parameters. The endpoints that appear to be analyzed 

correctly, with dam as the unit of analysis, included number of implantations, intrauterine mortality, 

survival indices, number of live fetuses, and sex ratio; all other endpoints were either analyzed on a 

fetal/offspring basis or it could not be determined how the endpoints were analyzed. For pathologic 

observations noted at high incidences (high percentage of fetuses), the unit of analysis deficiency has 

little impact in drawing conclusions regarding clear effect levels, due to the lack of similar findings in 

the control animals. However, for extrapolating no-observed adverse effect levels (NOAELs), benchmark 

dose, or LOAELs, these data are not useful. The investigators failed to identify how offspring within 

a group, or tubules within a tissue section, were selected for evaluation (random, 1st 4/5?). For the 

“recovery” evaluation in experiment 2, low confidence is placed in these conclusions due to the small 

sample size (4 offspring/group). In addition, it could not be determined if each offspring was from 

a different dam or all were from the same dam, further confounding the interpretation. The number 

of dams in each group did not add to the number stated to be assigned to study. Under experimental 

design, the authors state “In experiment 1, 28 –30 dams per group were given... Each 6 of these dams 

were killed...on G12, 14, 16, 18 and 20... In addition, each 5 dams... were allowed to deliver...” Given 

this assignment (5 gestation day kills of 6 dams/day=30, plus one delivery group of 5 dams) a total 

of 35 dams/group appear to have been used, not 28­ –30. This confusion may be due to the wording of 

the text, as a result of translation from Japanese.

Utility (Adequacy) for CERHR Evaluation Process: The data on number of implantations, intrauterine 

mortality, survival indices, number of live fetuses and sex ratio are useful for the evaluation process. 

However, the number of dams/group is somewhat small, reducing the confidence in the NOAELs. 

Although the pathology data were not presented on a litter basis, the high incidence findings can be 

used to establish effects levels, but should not be used for benchmark dose calculations.
Appendix II

II-63

Moore et al. (113), supported by NIH and University of Wisconsin, examined rat sexual development 

in offspring of dams dosed with DEHP during gestation and lactation. In an experiment conducted in 

2 blocks, at least 8 pregnant Sprague-Dawley rats/group were orally dosed [presumed gavage] with 

DEHP (99% purity) at 0 (corn oil vehicle), 375, 750, or 1500 mg/kg bw/day from GD 3 (GD 1=day 

after sperm detected) to PND 21. One group of rats was dosed with 3000 mg/kg bw/day in the first 

block of the study, but that dose was not used in the second block due to excess toxicity consisting 

of nearly complete prenatal or postnatal mortality. Dams were allowed to litter, and the litters were 

adjusted to 10 pups 1–2 days following birth. Litters were maintained at 10 pups by replacing any 

pups that died with pups from litters exposed to the same or lower concentrations of DEHP; data 

from replacement pups were not reported. Parameters examined in all pups (time period examined) 

included pup weight (PND 1, PND 7, and then weekly), anogenital distance (PND 1), presence of 

areolas (from PND 11), vaginal opening (from PND 24), time to first estrus (starting from vaginal 

opening), preputial separation (from PND 38), and male sex organ weight (PND 21, 63, and 105). In 

PND 63 rats, 1 epididymis and testis were fixed in neutral-buffered formalin, and the other testis and 

epididymis were used to determine daily sperm production. Sexual behavior with a sexually receptive 

female rat was assessed in males that were later necropsied on PND 105. The litter was considered 

the experimental unit in statistical analyses that included Levene test for homogeneity of variance, 

ANOVA, least significant difference test, chi-squared test, and/or Fisher exact test.

Results achieving statistical significance or displaying dose-response relationships are summarized 

in Table 21. 

Table 21. Results Achieving Statistical Significance or Dose-response Relationships 

Following DEHP Prenatal and Lactational DEHP Exposure

Endpoint Maternal DEHP dose (mg/kg bw/day)

0 375 750 1500

Maternal prenatal weight gain, g 

(% of control value) 128±4 123±7 (96)

99±10* 

(77)

87±13* 

(68)

Parturition incidence (%) 100 100 89 75

Pups born/dam 

(% of control value) 12.5±1.0 11.4±0.8 (91)

9.6±1.3 

(77)

7.7±1.4* 

(62)

Pups surviving/dam 

(% of control value) 10.9±1.0 9.8±0.8 (90)

7.5±1.3* 

(69)

5.0±1.3* 

(46)

Mean male anogenital distance, mma 3.5 3.3 3* 2.5*

Mean no. areolas per male a 0 2 7* 9.5*

Mean % litters containing males with:

Areolas or nipples on PND 14a 0 62* 100* 100*

Areolas or nipples as adults a 0 50* 85* 100*

Incomplete preputial separationa 0 10 25 80*

Undescended testis on PND 21a 0 42 75* 100*

With undescended testis as adults 0 25 58 40

Daily sperm production, 106/testes 

(% of control value) 34.2±1.5 36.5±1.2 (107)

25.6±4.5 

(75)

24.4±5.4 

(71)
II-64

Appendix II

Endpoint Maternal DEHP dose (mg/kg bw/day)

0 375 750 1500

Epididymal sperm number in 106/cauda 

(% of control value) 55.5±3.7 46.5±5.1 (84)

29.8±8.7* 

(54)

19.3±7.5* 

(35)

Litters (pup) with abnormality/no. examined

Ventral prostate agenesis 0/8 (0/42) 1/8 (1/32) 2/8 (5/29) 2/5 (3/12)

Dorsolateral prostate agenesis 0/8 (0/42) 1/8 (1/32) 0/8 (0/29) 1/5 (2/12)

Anterior prostate agenesis 0/8 (0/42) 1/8 (1/32) 5/8* (9/29) 4/5* (6/12)

Seminal vesicle agenesis 0/8 (0/42) 0/8 (0/32) 0/8 (0/29) 2/5 (2/12)

Litters with reproductive defects, %a, b 0 65* 88* 100*

Index of abnormalities, %a, c 0 18* 55* 75*

Absolute testes weight, % of control value

PND 21 90 78* 62*

PND 63 103 73* 59*

PND 105 99 71 39

Absolute epididymides weight, % of control value

PND 63 98 66* 69*

PND 105 91 61* 55*

Absolute glans penis weight, % of control value

PND 21 90 82* 71*

PND 63 97 89* 83*

PND 105 97 86* 79*

Age at vaginal opening, days 31.1±0.9 29.7±0.9 29.7±1.2 27.2±1.1

Body weight at vaginal opening, g 

(% of control value) 94±6 87±6(92)

86±9 

(91)

64±9*

 (68)

Age at first estrus in days 33.5±0.3 33.1±0.4 35.8±2.0 34.4±0.7

Body weight at first estrus, g 

(% of control value) 108±4 105±2 (97)

116±11 

(107)

98±2 

(91)

From: Moore et al. (113). Mean±SEM unless otherwise stated.

*P<0.05. aEstimated from graph by CERHR; 

bReproductive defects included missing, malformed, or small sex organs; incomplete preputial separation; 

or undescended testis

c Index of abnormalities is score based on missing, pathological, or small reproductive organs; presence of 

nipples or undescended testis; incomplete preputial separation; and failed ejaculation.

DEHP treatment reduced prenatal maternal weight gain at the middle and high dose. There was no 

significant effect on implantation sites, though the number appeared to be slightly reduced by DEHP 

treatment. All the rats with implantation sites gave birth to litters except for 1 mid-dose and 2 high-dose 

rats. Number of pups born was reduced at the high dose, and postnatal survival was decreased at the 

middle and high dose. Adult male offspring exposed to DEHP experienced a 6% reduction in body 

weight at the middle dose and 12% reduction at the high dose [data not shown]. An 8% reduction in 

body weight of adult female offspring of the high-dose group was reported as not significant. [Data

were not shown. A significant reduction in female body weight was reported for day of vaginal

opening, as discussed below.]
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II-65

DEHP treatment caused numerous effects on the reproductive systems of male rats, outlined in detail 

in Table 21. Areolas or nipples were not observed in any control male rats but were increased according 

to dose in all treated rats. Effects first noted in male rats of the mid-dose group were reduced anogenital 

distance, increased numbers of undescended testes, reduced sperm counts, and agenesis of anterior 

prostate. Incidence of incomplete preputial separation was significant at the high dose, but the authors 

considered the effect to be biologically significant at all doses due to the rarity of the effect in rats. 

Agenesis of prostate, seminal vesicles, and epididymis was noted in some treated rats. Agenesis of 

anterior prostate was significant at the middle and high dose; the study authors suggested that the 

effect was biologically significant at the low dose due to rarity of prostate agenesis. DEHP treatment 

reduced absolute weights of testes, epididymides, and glans penis at PND 21, 63, and/or 105, and in 

most cases, statistical significance was obtained at the middle and high dose. Absolute weight effects 

are outlined in Table 21. In most cases, effects on relative weights were similar in terms of direction 

and statistical significance of effect. Weights of accessory male organs, which are not illustrated in 

Table 21, were also reduced by DEHP treatment. The organs affected (day and dose that statistical 

significance was achieved for absolute organ weight) were ventral prostate (PND 21: middle and high 

dose; PND 105: mid dose), dorsolateral prostate (PND 21 and 105: middle and high dose; PND 63: 

high dose), anterior prostate (PND 21: all doses; PND 63 and 105: middle and high dose), and seminal 

vesicles (PND 21: middle and high dose). Effects on relative organ weights were similar in most cases. 

In sexual behavior tests, there were fewer rats from all dose groups that did not mount, intromit, and/or 

ejaculate. The authors stated that statistical significance was not obtained due to the small numbers of 

animals tested (n=7–8 at control and 2 lower dose levels and n=2 at high-dose level). 

Statistically significant and dose-related effects for female offspring are also listed in Table 21. In female 

pups, DEHP treatment had no effect on anogenital distance. At the high dose, vaginal opening was 

described as having occurred slightly earlier than in control rats, but age at first estrous was described as 

slightly higher; neither effect was statistically significant. Body weight of high-dose females was 68% 

that of control body weight on the day of vaginal opening, and the effect was statistically significant. 

The study authors attributed the effect to DEHP-induced toxicity and not to an estrogenic effect.

[CERHR estimated benchmark doses for endpoints when there was evidence of a dose-response

relationship and for which the authors reported sufficient data for benchmark dose modeling.

Benchmark dose values are presented in Table 22.] The study authors identified a LOAEL of 375 

mg/kg bw/day for this study based on a significant decrease in anterior prostate weight and an increase 

in permanent nipple retention. Other biologically significant effects observed by study authors at 375 

mg/kg bw/day were non-descent of testes, incomplete preputial separation, and agenesis of accessory sex 

organs. The study authors noted that DEHP exposure adversely affected reproductive system development 

and sexual behavior in male rats, but there was no evidence of estrogenic activity in female rats. 

Strengths/Weaknesses: This paper includes a good, detailed description of methodology and statistical 

analyses using the litter as the unit of analysis. The study was well-designed, using an appropriate route 

and timing of exposure and multiple dose levels. Even thought the sample size was small, there was sufficient magnitude of effects to obtain statistical significance. The study demonstrated a pattern of effects 

consistent with other study findings and the establishment of NOAELs/LOAELs. The dose-response 

data are appropriate for benchmark dose evaluation. It is a strength that animals were followed into early 

adulthood. The observation of effects on reproductive behaviors in the absence of gross external changes 
II-66

Appendix II

suggested additional effects on the central nervous system. This study reinforced the increased sensitivity of 

the fetal male compared to the pubertal or adult male, although only by reference to existing literature. 

Table 22. Benchmark Dose Values for Offspring of Rats Exposed to DEHP 

During Gestation and Lactation

Endpoint Benchmark dose (mg/kg bw/day) a

BMD10 BMDL10 BMD1 SD BMDL1 SD

Maternal prenatal weight gain 433 317 754 509

Pups born/dam 378 269 872 565

Pups surviving/dam 269 205 696 479

Absolute testis weight 

PND 21 384 298 562 400

PND 63 421 283 452 258

PND 105 374 204 342 203

Epididymis weight

PND 63 394 291 395 233

PND 105 262 196 220 135

Glans penis weight 

PND 21 503 393 526 379

PND 63 806 623 445 321

PND 105 642 500 278 171

Daily sperm production/testes 490 289 686 389

Epididymal sperm number 213 167 612 428

Body weight at vaginal opening 780 343 1157 611

From: Moore et al. (113). aThe BMD10 is the benchmark dose associated with a 10% effect, estimated from a curve fit to the 

experimental data. The BMDL10 represents the dose associated with the lower 95% confidence 

interval around this estimate. A 10% alteration in a continuously distributed parameter is an 

arbitrary benchmark that may not be comparable to a similar alteration in any other endpoint. The 

BMD1 SD, which represents an alteration equivalent to 1 SD of the control distribution, may permit 

more appropriate comparisons of the responses of continuously-distributed parameters. Benchmark 

doses are used commonly in a regulatory setting; however, they are used in this report when the 

underlying data permit their calculation, and are only supplied to provide 1 kind of description of 

the dose-response relationship in the underlying study. Calculation of a benchmark dose in this 

report does not mean that regulation based on the underlying data is recommended, or even that the 

underlying data are suitable for regulatory decision-making. Values were calculated using the power 

model by CERHR using EPA Benchmark Dose Software version 1.3.2. The program offers models 

based on homogeneity of variance, and CERHR was guided by the program in this regard. 

Strengths/Weaknesses: Weaknesses include the small sample size, and negative effects cannot be accepted 

with high confidence. For example, there appeared to be a significant biological effect of reduced 

implantation sites at 1500 mg/kg but no statistical significance. Professional experience and judgment 

would lead to the conclusion of an effect on the number of implantation sites. The use of relatively 

high dose levels is another weakness. The authors used a post hoc statistical design for grouping of 

all reproductive abnormal­ities/defects and for behavioral effects. Post hoc (after generation and visual 

assessment of the data) statistical analyses are generally considered inappropriate, however, because 

the signal for reproductive abnormalities/defects is overwhelming (60–80% of litters affected in the 
Appendix II

II-67

DEHP group compared to zero in the control), this oversight has no significance for these endpoints. 

It is a common practice when conducting embryo/fetal toxicity studies to group malformations and 

variations by system or type (external, visceral, or skeletal; organ system). For the behavioral assessment, the post hoc grouping of findings across DEHP treatment groups was inappropriate and not valid 

for risk assessment but was valuable for hypothesis generation. In addition, no data were presented for 

the behavioral evaluation.

Utility (Adequacy) for CERHR Evaluation Process: This study was well conducted and used a comprehensive battery of relevant developmental endpoints, multiple dose levels, and a relevant route of 

exposure during critical periods of sexual development. Even though sample size and power were 

limited, clear treatment-related (and in many cases dose-responsive) effects on maternal toxicity, 

reproductive parameters, measures of sexual differentiation/development, and sexual function were 

identified. Given the variability in response, small sample size, post hoc grouping of results across 

treatments, and lack of presentation of data, the behavioral data are not appropriate for the evaluation 

process. The negative findings are not viewed with high confidence given the small sample size.

The National Toxicology Program (NTP) (114) sponsored a multigeneration continuous breeding 

study in rats. [Because developmental effects were reported, particularly on the male reproductive

system, the study is included in this section. This summary with additional details concerning

the reproductive effects is also presented in Section 4.2.2.2.] Sprague-Dawley rats (17/sex/group) 

were randomly assigned to diets containing 1.5 (control group exposed to background DEHP levels 

in feed), 10, 30, 100, 300, 1000, or 7500 ppm DEHP (99.8% pure) from the first day of the study until 

the day of necropsy. Due to a lack of reproductive effects in the first litter produced, the study was 

repeated with 2 additional doses, 1.5 (control) and 10,000 ppm. Ranges of DEHP intake in the F0, F1, 

and F2 animals were estimated at 0.09 –0.12, 0.47–0.78, 1.4–2.4, 4.8–7.9, 14–23, 46–77, 392–592, 

and 543–775 mg/kg bw/day. At about 5 weeks of age, F0 rats were fed the DEHP-containing diets for 

6 weeks prior to mating and were then cohabitated for 9 weeks. Concentrations of dosing solutions 

were verified. The first 2 litters delivered during the cohabitation period (F1a and F1b) were counted, 

weighed, assessed for anogenital distance, and discarded. The third litter (F1c) was raised by the dam 

until weaning on PND 21 [designation for day of birth not specified]. Following weaning of pups, 

vaginal cytology was monitored in F0 females for 14 days. After completion of crossover studies 

described below, at least 10 F0 rats sex/group were necropsied. Sperm analyses were conducted, and 

organs were collected for histopathological evaluation. Ovaries were preserved in Bouin fluid. Testes 

and epididymides were preserved in 2% paraformaldehyde/3% glutaraldehyde. F1 pups were counted, 

weighed, and examined for anogenital distance and nipple retention during the lactation period. On 

PND 16, 1 female per litter was evaluated for vaginal opening, and a second was selected for F1 mating. 

One male per litter was selected for mating, and 4 or 5 males per litter were evaluated for testicular 

descent and preputial separation; both groups of rats were necropsied. At weaning (PND 21), pups 

were given diets containing the same DEHP concentrations as their parents. On PND 81, the F1 rats 

chosen for mating (17/sex/group) were randomly assigned to breeding pairs (preferably non-sibling) and 

cohabited for 9 weeks. The study conducted in F0 parents and F1 offspring was repeated in F1 parents 

and F2 offspring, except that the third F3 litter born (F3c) did not undergo the continuous-breeding 

protocol. Selected F3c males were necropsied on PND 63–64 and selected females on PND 60–74. 

Statistical analyses included Jonckheere test to determine if data should be analyzed by Shirley or 

Dunn test. Shirley test was used to evaluate data that consistently increased or decreased according 
II-68

Appendix II

to dose. Dunn test was used to evaluate data with severe departures from monotonicity. Additional 

statistical analyses included Wilcoxon, Cochran-Armitage, and chi-squared tests. 

Some systemic effects were consistent across all generations. During numerous time periods of the 

study and especially at necropsy, body weight gains were decreased in rats from the 7500 and 10,000 

ppm groups. Dam body weights during delivery and lactation were decreased by 8– 20% in the F0

10,000 ppm group. Increases and decreases in feed intake were observed at most dose levels. In the 

F0 7500 and 10,000 ppm groups, feed intake was decreased during lactation. The liver was identified 

as a target of toxicity, with increases in liver weight and hepatocellular hypertrophy observed at dose 

levels ≥1000 ppm. Changes in organ weights and lesions were also observed in kidney at ≥7500 ppm 

and adrenal gland at 10,000 ppm.

The lowest dose level producing dose-related effects in F1 offspring was 7500 ppm, and those effects 

included decreases in number of live pups/litter, reduced male anogenital distance, and delays in vaginal 

opening, preputial separation, and age of testicular descent. Additional effects noted in the F1 offspring 

from the 10,000 ppm group included decreased live pup weight at birth and during the lactation period 

and increased ratio of female anogenital distance to body weight. In the non-mating F1 adult males of 

the 300 ppm group, there was a small increase in the number of animals (3 of 45 with small testes and/or 

epididymides. The effects were not observed at the next higher dose (1000 ppm), but small testes were 

observed in 10 of 30 males of the 7500 ppm non-mating group. Small testes and epididymides were 

observed in 21 of 21 animals of the 10,000 ppm non-mating group. A small percentage (3 –7%) of 

non-mating rats treated with ≥1000 ppm had small ventral prostates. In rats that were mated, the only 

decreases in reproductive organ size occurred in testes at 7500 and 10,000 ppm (8 of 10 and 10 of 10 

affected at each dose) and epididymides at 7500 ppm (2 of 10). Histopathological findings observed 

in all animals of the 7500 and 10,000 ppm groups were consistent with those observed in the F0

generation and included minimal-to-marked seminiferous tubule atrophy and occasional sperm release 

failure. Minimal seminiferous tubule atrophy was observed in 1 of 10 males in the 100 and 300 ppm 

groups. Reductions in numerous reproductive organ weights were observed in mating and non-mating 

F1 males treated with ≥7500 ppm. Additional reproductive effects observed in F1 rats were reduced 

sperm counts at 7500 ppm and higher and increased uterus and ovary weights at 10,000 ppm. Estrous 

cycle length was slightly increased at 10,000 ppm. In the F2 pups, delays in preputial separation and 

testicular descent occurred at every dose level above the control. [In no other generation did delaysin

preputialseparation and testicular descent occur atsuch low doses, but the study authors did not

offer any explanationsfor this observation.The Expert Panel believesthese findings are consistent

with a problem with the control group in that generation.] All other effects occurred in F2 pups of 

the 7500 ppm group and included delayed vaginal opening and reductions in live pup weight at birth 

and during the lactation period, male anogenital distance, and survival during the lactation period. 

In non-mating F2 male rats, small testes and epididymides were observed at ≥300 ppm (1/21), 1000 

ppm (1/25), and 7500 ppm (7 – 11/20). However, in males that were mated, small epididymides and 

testes were only observed at the 7500 ppm dose level (8/10). Seminiferous tubule atrophy was observed 

in 10/10 males of the 7500 ppm group. In F3 pups, a decrease in postnatal survival of females was 

observed only on PND 7 in the 300 ppm group but was not observed on any other day or dose level. 

All other effects in F3 pups occurred at 7500 ppm and included decreases in male anogenital distance, 

delayed vaginal opening, preputial separation, and testicular descent, and an increase in male pups with 
Appendix II

II-69

nipples. F3 pups were the only generation of rats to experience an increase in males with nipples. At 

necropsy of adult F3 rats, effects were only observed at the 7500 ppm dose level and included reduced 

sperm counts and weights of dorsolateral prostate, testis, and epididymis.

The study authors discussed the relevancy of small male reproductive organ sizes observed in both 

F1 and F2 rats of the 300 ppm groups. They noted that although incidences were low, the effects were 

consistent with phthalate-induced developmental toxicity. The incidence of small testes and epididymides exceeded historical control data from the laboratory. Therefore, the study authors considered 

the effects as potentially treatment-related. However, the study authors concluded that the overall 

significance of the effects could not be determined due to lack of histopathological data and lack of 

adverse reproductive effects at 300 and 1000 ppm. 

A crossover breeding study was conducted to investigate the decrease in F3 pup body weight in the 

7500 ppm group. High-dose rats of each sex (n=17/sex/group) were mated with naïve animals for 7 

days or until a vaginal plug was detected. Pups were counted, weighed, assessed for anogenital distance, 

and discarded. Implantation sites were examined in naïve females. The crossover study demonstrated 

a decrease in pup weight and male anogenital distance in offspring born to females treated with 7500 

and 10,000 ppm DEHP and mated to naïve males. 

The study authors concluded, “The findings obtained in this study indicate that DEHP is clearly a 

reproductive and developmental toxicant at 7500 and 10,000 ppm based upon changes in fertility and 

pregnancy indices, litter data, sperm parameters, sexual development, and/or histopathological changes 

in testes.” Intake at 7500 ppm was estimated at 392 –592 mg/kg bw/day, and intake at 10,000 ppm was 

estimated at 543 – 775 mg/kg bw/day. [The lowest BMD10 is 787 ppm based on F3 sperm/cauda.The

BMDL10 for this endpoint is 728 ppm.The BMD1 SD is 1188 ppm and the BMDL1 SD is 970 ppm.

Extrapolating from the authors’ estimates of intakes at 1000 mg/kg bw/day,the BMD10 intake level

is 36–61 mg/kg bw/day, the BMDL10 intake level is 33–56 mg/kg bw/day, the BMD1 SD intake level

is 54–92 mg/kg bw/day, and the BMDL1 SD is 45–75 mg/kg bw/day. The Expert Panel carefully

considered the finding ofsmall reproductive organ sizes by gross observationsin both F1 and F2 rats.

The combined F1 and F2 data were reviewed to determine the occurrence of these alterations on a

per animal and per litter basis across the dose range, as shown inTable 23. Based on the incidence

of small reproductive organ size at necropsy, the Expert Panel considered 300 ppm (about 14–23

mg/kg bw/day) to be an effect level, giving a NOAEL of 100 ppm, about 3–5 mg/kg bw/day.]

Strengths/Weaknesses: Clearly, a major strength of this study is the number of doses evaluated. The 

relatively small group sizes were compensated by the unusually large numbers of groups and the 

very low doses used. An additional strength is the fact that more offspring were evaluated early for 

alterations in the development of the reproductive system; a weakness might be that not all animals 

were so evaluated. The quality of the histology is another strength. 

Utility (Adequacy) for CERHR Evaluation Process: These data are adequate for the evaluation process 

and show that 10,000 and 7500 ppm are clearly toxic to the developing reproductive system in rats. 

The Expert Panel considers 300 ppm and 1000 ppm to represent the tail of the dose-response curve in 

this study based on the incidence of testicular abnormalities, which would put the NOAEL for these 

developmental effects at 100 ppm, in the 3 –5 mg/kg bw/day range.
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Appendix II

Table 23. Reproductive Organ Abnormalities in Combined F1+F2 Non-breeding Males

 in NTP Multigeneration Study

Organ

DEHP dose level, ppm in feed (n)

1.5

(39)

10

(36)

30

(39)

100

(41)

300

(45)

1000

(43)

7500

(30)

Testis 0 0 0 0 4 3 21

Epididymis 0 0 0 0 3 3 7

Seminal vesicles 0 1 0 0 2 0 0

Prostate 0 0 0 0 0 4 1

Any reproductive organ 0 1 (1) 0 0 5 (4) 7 (5) 22 (14)

Data expressed as number of animals (litters) affected. From NTP (114)

Borch et al. (115), support not indicated, conducted a series of studies in Wistar rats to examine 

anti-androgenic effects of DEHP (99% purity) alone or in combination with diisononyl phthalate 

or diethylhexyl adipate. Diisononyl phthalate was examined alone in some cases, but this summary 

focuses on DEHP. Dams were gavage dosed with vehicle (peanut oil) or DEHP in peanut oil during 

gestation and/or lactation. Endpoints examined in male offspring included hormone levels in testes or 

blood and ex vivo testicular testosterone production. To measure ex vivo testosterone production, the 

left testis was incubated in media for 3 hours and the supernatant was saved for testosterone analysis. 

Following extraction from incubation media, testis, or blood, quantification of hormone levels was 

performed using fluorometric or immunofluorometic methods. Testicular testosterone production was 

measured in testes from 2 males per litter, and testicular testosterone content was measured in testes 

from 1 male per litter. Plasma samples were pooled from 1 or 2 litters in the case of fetal or immature 

offspring or were obtained from 9–16 males per group in the case of mature offspring. Data were 

evaluated by ANOVA, analysis of covariance (ANCOVA), Dunnett test, and /or Pearson correlation. 

Litter was included as an independent random factor in ANOVA analyses. 

The first study appears to have been previously reported in an abstract (116). Approximately 8 dams/

group were gavage dosed during gestation with vehicle, 300 mg/kg bw/day DEHP, or 300 mg/kg 

bw/day DEHP+750 mg/kg bw/day diisononyl phthalate on GD 7 – 21. [Criteria for determining

day of gestation were not stated, but it is assumed that GD 1 was the day following mating, as in

study 2 described below.] Endpoints examined in GD 21 male fetuses included testicular testosterone 

content and production and plasma testosterone and LH levels. Compared to control values, testicular 

testosterone content and production were significantly reduced in the DEHP and DEHP+diisononyl 

phthalate groups. Plasma testosterone was significantly reduced, and plasma LH was significantly 

increased in the DEHP+diisononyl phthalate group; similar effects on plasma testosterone and LH 

levels were described in groups receiving DEHP or diisononyl phthalate alone, but statistical significance was not achieved. Factorial statistical analyses revealed no significant interactions between 

DEHP and diisononyl phthalate. 

In the second study, 16 pregnant Wistar rats per group were gavage dosed with vehicle, 750 mg/kg 

bw/day DEHP, or 750 mg/kg bw/day DEHP+400 mg/kg bw/day diethylhexyl adipate from GD 7 to 

PND 17. GD 1 was the day following mating, and PND 1 was the day following birth. Eight dams per 
Appendix II

II-71

group were randomly selected and killed on GD 21. The fetuses were also killed for collection of blood 

and testes. The remaining 8 dams per group were allowed to litter; anogenital distance was measured in 

their male offspring on PND 3, and nipple retention was assessed on PND 13. In GD-21 male fetuses 

exposed to DEHP or DEHP+diethylhexyl adipate, testicular testosterone content and production and 

plasma testosterone levels were significantly reduced compared to the control group. Plasma LH level 

was significantly increased in GD 21 fetuses exposed to DEHP. Plasma LH levels were not measured in 

the DEHP+diethylhexyl adipate group. Anogenital distance was significantly reduced in PND 3 male 

offspring from the DEHP and DEHP+diethylhexyl adipate groups, and the effect was not related to birth 

weight. Numbers of nipples per animal were significantly increased in PND offspring from the DEHP 

group, but the increase in the DEHP+diethylhexyl adipate group was not statistically significant. 

In the third study, 80 Wistar rats were randomly assigned to 1 of 4 groups that were treated with DEHP 

0, 300, or 750/kg bw/day or DEHP 750 mg/kg bw/day+diethylhexyl adipate 400 mg/kg bw/day from 

GD 7 to PND 17. Blood and testes were collected from male offspring killed on PND 22 and PND 190. In 

PND 22 offspring treated with 750 mg/kg bw/day DEHP, there was a dose-related, significant reduction in 

serum inhibin B level. A non-significant “tendency” for increased serum FSH levels in the DEHP-treated 

groups was described by the study authors. There were no significant findings for serum LH levels, but 

about half the litters in the 750 mg/kg bw/day DEHP-treated group had LH levels several-fold higher than 

those of control rats. Correlations were noted between serum LH and serum FSH (r=0.61, P<0.001) 

and serum FSH and serum inhibin B (r=−0.51, P<0.001). There were no significant differences in 

plasma testosterone levels on PND 22. No statistically significant differences in serum testosterone levels, 

testicular testosterone content, or serum inhibin B levels were observed in PND 190 rats. 

The study authors concluded that in neonatal rats, endocrine-modulated effects following gestational 

or lactational exposure to DEHP were similar to those previously reported in the literature, including 

a reduction in anogenital distance and an increase in nipples. However, hormonal effects were less 

evident in prepubertal and adult animals. Administration of diisononyl phthalate or diethylhexyl adipate 

in combination with DEHP caused no significant modulation of endocrine effects. 

Strengths/Weaknesses: The use of the Wistar rat was a strength in extending the evaluation of DEHP to 

a strain different from that used in most other studies. The paper included a good, detailed description 

of methods including test material source and purity, appropriate statistical evaluation of the data using 

the litter as the unit of analysis, and nesting of individual animals/litter. Study 2 demonstrated clear treatment-related effects on plasma testosterone and LH in fetuses following maternal gestational exposure 

to DEHP 750 mg/kg bw. Although the sample size was small (n=2–6 samples/group), the magnitude 

of the DEHP effect was sufficient to show statistical significance. Gestational DEHP exposure to 750 

mg/kg bw also resulted in decreased anogenital distance and increased number of nipples per male. Study 

3 employed multiple dose groups (300 and 750 mg/kg DEHP), allowing for a dose-response evaluation. 

Maternal animals were dosed from GD 7 through PND 17, the period of sensitivity for male sexual 

development. The only treatment-related effect noted following maternal exposure from GD 7 through 

PND 17 was decreased inhibin B on PND 22, demonstrating a Sertoli cell effect. The small sample 

size (<8 litters/group/time point) was a weakness in these studies. Ex vivo testosterone production is of 

questionable relevance to human risk, especially when no in vivo plasma testosterone or LH changes 

were noted. Study 1 demonstrated no treatment-related effects on plasma testosterone and plasma LH in 

21-day-old rat fetuses following maternal gestational DEHP exposure at 300 mg/kg bw. However, given 
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Appendix II

the small sample size (n=3–7), there is a low level of confidence in the lack of findings. In studies 1 and 

2, a single dose level of 300 or 750 mg DEHP/kg bw was used, precluding a dose-response evaluation.

Utility (Adequacy) for CERHR Evaluation Process: Studies 1 and 2 utilized single doses of DEHP 

and have low value for a quantitative evaluation. The results from these studies support findings 

reported by other authors. The ex vivo testosterone production data are of questionable relevance to 

human risk assessment, having no direct human correlate. Study 3 utilized multiple doses of DEHP 

(300 and 750 mg/kg bw/day) during a critical period of male sexual development and is useful in the 

evaluation process. This assay is best used for studying potential mechanisms/modes of action and 

for screening for potential activity.

Jarfelt et al. (117), supported by the Denmark Directorate for Food, Fisheries and Agro Business, 

evaluated the effects of perinatal exposure of Wistar rats to DEHP with or without diethylhexyl adipate. 

Timed-mated pregnant animals were treated by gavage from GD 7 to PND 17 [plug day unspecified;

date of birth=PND 0]. Dose groups were vehicle control, DEHP 300 mg/kg bw/day, DEHP 750 mg/kg 

bw/day, and DEHP 750 mg/kg bw/day+diethylhexyl adipate 400 mg/kg bw/day (n=20/group). Chemical 

purity was 99%. Unadjusted litters were raised by their dams until weaning on PND 21, after which 1 

male and 1 female per litter were retained. Anogenital distance was assessed on PND 3, and retention 

of nipples/areolae was assessed on PND 13. Retained offspring were observed for vaginal opening 

and balano-preputial separation, and males underwent evaluation of epididymal sperm parameters 

and testicular histopathology on PND 190. [Later in the Methods section, histopathologic evaluation was described for 14 – 16 adult males representing 14–16 litters.] Non-retained pups and 

dams were killed on PND 22 and evaluated for macroscopic lesions, and 3 –5 males/litter underwent 

histopathologic evaluation of the testes. [Later in the Methods section, histopathologic evaluation

of testes was described for about 5% of PND 22 males, representing 10 litters.The resultssection

presents histolopathology data for 18–21 PND 22 males/dose group.] Testes were fixed in Bouin 

fluid (half of PND 22 testes and all adult testes) or in formalin (half of PND 22 testes). Embedding 

material was not specified for testes. Staining was hematoxylin and eosin. Immunohistochemistry for 

3b-hydroxysteroid dehydrogenase and smooth muscle actin was performed using 1 section/testis. Brain, 

liver, kidney, adrenal, testis, epididymis, seminal vesicle, ventral prostate, bulbourethral gland, and 

levator ani/bulbocavernosus muscle weights were recorded in all males. Histologic sections of accessory sex organs were prepared for 10 males representing 10 litters. Statistical analysis was performed 

using ANOVA with post hoc Dunnett test or, for data not satisfying conditions for ANOVA, KruskallWallis or Fisher test. Litter was included in the analysis. [Data were shown in the results for 11–15

litters/dose group, with 12 litters in the control group, although 20 timed-mated animals/dose

group had been treated.In the group receiving DEHP+diethylhexyl adipate, there were 3 dams

with total litter loss.The other missing litters were not explained.]

Results are summarized in Table 24. Although there were no significant alterations in sperm count 

and motility parameters, the study authors reported that “a few animals” were severely affected with 

regard to these parameters. The study authors indicated that males exposed to DEHP with or without 

diethylhexyl adipate had histologically normal testicular tissue with small foci of malformed tubules 

associated with interstitial cell hyperplasia. Within these malformed tubules, the seminiferous epithelium 

was disorganized with decreased spermatogenesis and tubular anastomoses. Immunohistochemistry for 

3b-hydroxysteroid dehydrogenase and smooth muscle actin showed Leydig cells inside the malformed 
Appendix II

II-73

tubules. The authors called attention to the higher incidence of malformed tubules among young animals, 

and they concluded that “dysgenic” testicular tissue was surrounded by normal tissue during pubertal 

testis growth. There were no abnormal histopathologic findings in accessory sex organs in PND 22 males 

and only 4 males with mild prostate changes among the 3 DEHP-exposed dose groups on PND 190.

The authors concluded that their study confirmed the anti-androgenic effects of DEHP identified in 

previous studies, and they called attention to the variability in response among their animals.

Table 24. Outcomes after Perinatal Exposure of Rats to DEHP 

With or Without Diethylhexyl Adipate

Endpoint

Treatment Group

(mg/kg bw/day to the dam)

DEHP Benchmark Dose a

(mg/kg bw/day to the dam)

DEHP 

300

DEHP 

750

DEHP 750+

DEHA 400 BMD10 BMDL10 BMD1SD BMDL1SD

Maternal pregnancy weight 

gain ↔ ↔ ↓ 21%

Birth 

weight

Males ↔ ↓ 8% ↓ 9% 770 701 735 405

Females ↔ ↓ 8% ↓ 11% 758 638 750 533

Litter size ↔ ↔ ↓ 28%

Postnatal death “↑” 4-fold “↑” 5-fold ↑ 17-fold 622 127 754 504

Postimplantation loss ↔ ↑ 2.3-fold ↑ 3-fold 72 13 1127 574

Male anogenital distance ↓ 14% ↓ 17% ↓ 17% 432 315 338 179

Male retained nipples ↑ 39-fold ↑ 52-fold ↑ 39-fold BMD computation failed

Sperm/g cauda ↔ ↔ ↔

Sperm motility parameters ↔ ↔ ↔

Macroscopic malformations ↔ ↔ ↔

PND 22

Body weight ↔ ↔ ↔

Paired testis weight ↔ ↓ 8% ↔ 748 538 761 652

Other organ weights ↔ ↔ ↔

Abnormal testis histology ↑ 2.6-fold ↑ 2.6-fold ↔

PND 190

Body weight ↔ ↔ ↔

Ventral prostate weight ↓ 20% ↓ 21% ↓ 17% 388 250 753 454

Sexual muscle weight ↓ 15% ↓ 18% ↓ 16% 428 290 635 406

Other organ weights ↔ ↔ ↔

Abnormal testis histology b 44% 27% 31%

DEHA=diethylhexyl adipate.

↑ , ↓ , ↔ Statistical increase, decrease, no change compared to control. 

“↑” Increase identified by study authors, although not statistically different from control. aBenchmark dose calculations performed using the groups treated with control and DEHP only. See footnote to Table 

22 for information on the benchmark dose calculations

b control rate 0%.

From Jarfelt et al. (117).
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Appendix II

Strengths/Weaknesses: Multiple doses of DEHP were used (300 and 750 mg/kg), allowing for doseresponse analyses. This study provided a moderately comprehensive evaluation of reproductive and 

developmental effects following maternal gestational and lactational exposure during the critical period 

of male sexual development. Assessment of offspring was carried out through sexual maturity. The paper 

presented good detail on general methodology and test material source and purity, with the exception 

of suboptimal tracking of litters and lack of consistent use of the litter as the unit of analysis. When 

multiple pups/litter were used, the litter was included as an independent random and nested factor; 

however, it was not clear in the paper when, and for what specific parameters, litter was used as an 

analysis factor. Clear treatment-related findings in male offspring (anogenital distance, nipple retention, reproductive organ weights) were apparent and adequately analyzed. These results also supported 

findings from other investigators. The use of the Wistar rat was a strength in extending the evaluation 

of DEHP to a strain different from that used in most other studies. Weaknesses include the relatively 

high DEHP dose levels that were used. Because the pathology data apparently were not analyzed on 

a litter basis, these data are not optimal for benchmark dose evaluation. There is significant confusion 

as to the sample size used for specific parameters and the lack of litter as the unit of analysis for the 

pathologic data. The authors stated that 1 male and 1 female/litter were kept after weaning (PND 21) 

to investigate sexual maturation, sperm quality, and histopathology of the testes at adulthood, but this 

statement contradicts a subsequent statement that “sixteen animals per group were used, one to two 

males per litter,” in reference to sperm count evaluations and the statement that 1 –4 males per litter 

from 10 –16 litters were used to analyze sperm quality at PND 190 and terminal body weight. Table 

1 of the paper lists only 11 –15 litters/group, yet the methods describe males representing 14 – 16 litters. With the exception of addressing 3 litters with total loss of pups, there is no mention of the loss 

of litters in the remaining dams. A total of 27 litters are unaccounted for, assuming the 3 litters with 

total pup loss are included in Table 1 of the paper. 

Utility (Adequacy) for CERHR Evaluation Process: The dose-response data for reproductive results 

(postnatal death, postimplantation loss), offspring growth, nipple development, anogenital distance, 

sperm quality, and organ weights are suitable for use in the evaluation process. Although NOAELs 

were not observed for some of these endpoints, the benchmark dose methodology can be applied. The 

pathology data cannot be used for the benchmark dose evaluations because the litter effect was not 

controlled and findings in the DEHP groups did not demonstrate a dose-response.

Borch et al. (118), supported by the Denmark Directorate for Food, Fisheries and Agro Business, 

evaluated early testicular effects of perinatal exposure to DEHP with or without diethylhexyl adipate 

in Wistar rats. In the first experiment, pregnant females were treated by gavage with vehicle, DEHP 

750 mg/kg bw/day or DEHP 750 mg/kg bw/day+diethylhexyl adipate 400 mg/kg bw/day beginning on 

GD 7 (plug=GD 0; n=18/dose group). Chemicals were of 99% purity. On GD 21, 8 dams/group were 

killed and fetal testes were harvested. The remaining 8 dams/group continued to receive treatment until 

PND 17. These animals were permitted to litter. Male offspring were killed on PND 26 (birth=PND 0), 

and testes were harvested.

A second experiment used 20 pregnant animals in each of 4 dose groups: vehicle control, DEHP 300 

mg/kg bw/day, DEHP 750 mg/kg bw/day, and DEHP 750 mg/kg bw/day+diethylhexyl adipate 400 

mg/kg bw/day. Treatment was from GD 7 through PND 17. On PND 22, 3 males/litter were killed and 

testes were harvested. On PND 190, 1 or 2 males/litter were killed and testes harvested.
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Of the testes collected on GD 21, 14– 19/dose group (2–4/litter) were fixed in formalin, embedded in 

paraffin, and sections were stained with hematoxylin and eosin for light microscopy. Ten testes/dose 

group (1 or 2/litter) from PND 22 and PND 26 animals were processed in the same manner. Another 

10 testes from these age groups as well as 16 testes/dose group (1 or 2/litter) were fixed in Bouin fluid, 

and stained with hematoxylin and eosin for light microscopy [embedding material not specified]. 

Tubule diameters were measured, and a 10% increase over the control maximum was defined as 

enlarged. Terminal deoxynucleotidal transferase-mediated dUTP nick-end labeling (TUNEL) staining 

was performed using a commercial kit, and immunostaining was performed for caspase-3, proliferating 

cell nuclear antigen (PCNA), histone H3, anti-Müllerian hormone, 3b-hydroxysteroid dehydrogenase, 

vimentin, and smooth muscle actin. Caspase-3 activity was measured in 10 testes/dose group (from 

5–10 litters/group) at GD 21, PND 22, and PND 26. [The method was described only by reference

to another paper.] DNA laddering was assessed based on relative fluorescence of DNA ladders on 

gels. Statistical analysis was by ANOVA with post hoc Dunnett test or by Kruskall-Wallis test. Litter 

was included as a factor in the ANOVA.

In testes evaluated on GD 21, vacuolization of Sertoli cells, shedding of gonocytes, reduced interstitial 

cell cytoplasm, and enlarged tubules were identified in offspring of all dams exposed to DEHP 750 

mg/kg bw/day, regardless of diethylhexyl adipate co-exposure, compared to 0 –14% of dams exposed 

to vehicle. Leydig cell hyperplasia was identified in offspring of more dams with DEHP treatment 

than control dams [statistical analysis not shown]. The number of histone H3-positive cells per testis 

section was not altered by treatment. [Other immunohistochemistry results were not quantified

but were not reported as affected by treatment.] Staining for anti-Müllerian hormone to identify 

Sertoli cells showed positive cells within Leydig cell clusters, outside the tubules. DNA laddering was 

increased by DEHP treatment, although TUNEL-positive cells and caspase-3-positive cells were not 

increased by maternal DEHP 750 mg/kg bw/day. 

On PND 26, tubules without spermatocytes were found in all litters exposed to DEHP compared to 29% of 

control litters [statistical analysis notshown]. Malformed tubules were identified in 17–29% of DEHPexposed litters compared to none of the control litters. There were no effects of DEHP treatment on any 

of the measures of apoptosis on PND 22, 26, or 190, although the authors indicated that “a few animals 

in the treated groups had very high numbers of TUNEL positive cells, presumably spermatocytes.”

The authors concluded that the development of dysgenic tubules in response to DEHP exposure 

was related to interstitial changes occurring during gestation, including the presence of Sertoli cells 

in the interstitium. They believed that Sertoli cell dysfunction in the fetal period might underlie the 

focal testicular dysgenesis seen in older animals. The authors proposed that the lack of alteration in 

Sertoli cell structure in prepubertal rats in this study might reflect recovery from DEHP, which was 

last administered on PND 17.

Strengths/Weaknesses: This study included a detailed description of methods, source and purity 

of test material, and appropriate statistical analyses, when utilized, employing nesting of offspring 

within litter. Animals were dosed during a relevant period of male sexual development. Both litter and 

individual offspring incidence for histopathology were presented in Study 1. This single-dose study 

at 750 mg/kg bw demonstrated clear effects on the incidence of Sertoli cell vacuolization, shedding 

of gonocytes, multinucleated gonocytes, and reduced cytoplasm in interstitial cells in 21-day-old 
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Appendix II

rat fetuses, and malformed tubules and tubules lacking sprematocytes in 26-day-old (postnatal) rat 

pups. This effect was clear even though statistical evaluations were not conducted on these endpoints. 

Study 2 explored a dose-response relationship (DEHP at 300 and 750 mg/kg bw) with 20 time-mated 

females/group. The number of litters/group, however, was not reported. The use of the Wistar rat 

was a strength in extending the evaluation of DEHP to a strain different from that used in most other 

studies. Weaknesses include the small sample size (5 –8 litter/group) in the first study, although effects 

in treated animals were observed at a high incidence and were clearly distinguishable from control. 

The lack of dose-response data in the first study was a weakness. The second study, which permitted 

dose-response evaluation, focused primarily on mechanism/mode of action and either did not present 

the data in a format useful for the evaluation process or included few endpoints useful for assessing 

risk. In addition, the dose levels used in the second study were relatively high.

Utility (Adequacy) for CERHR Evaluation Process: The single-dose study is useful in supporting 

previous study results but is of limited use in a quantitative evaluation. The multi-dose study is more 

useful for the evaluation process, although it is limited in the evaluated endpoints.

Li et al.(119), supported by NIH, examined the effects of DEHP and 2 of its metabolites on neonatal rat 

gonocytes and Sertoli cells. Male Sprague-Dawley rat pups from 4–7 litters were pooled and randomly 

placed into groups of 4–5 pups. On PND 3 (day of birth=PND 1), the rats were gavage dosed with DEHP 

(> 95% purity) at 0 (corn oil vehicle), 20, 100, 200, or 500 mg/kg bw. Pups were killed 24 hours after 

dosing. One testis was collected and preserved in 2% glutaraldehyde for a morphological examination. 

The other testis was collected to examine Sertoli cell proliferation through bromodeoxyuridine (BrdU) 

uptake. Serum FSH was measured by RIA. In a second experiment, Sertoli cell proliferation was 

measured and a morphological examination of testis was conducted at 6, 9, 12, 24, or 48 hours after 

rats were dosed with 0 or 200 mg/kg bw DEHP. Statistical analyses included one-way ANOVA and 

Student-Newman-Keuls t-test. In rats treated with 100 –500 mg/kg bw DEHP, there was a dose-related 

increase in abnormally large gonocytes containing 2– 4 nuclei. Multi­nucleated gonocytes were first 

detected at 12 hours following exposure to 200 mg/kg bw DEHP, and their numbers increased with 

time. Sertoli cell proliferation was reduced in rats treated with ≥100 mg/kg bw DEHP, as noted by 

significant decreases in BrdU labeling. BrdU labeling indices were reported at 27.03% in control rats, 

20.83% in the 100 mg/kg bw group, 9.95% in the 200 mg/kg bw group, and 4.13% in the 500 mg/kg 

bw group. There was a rebound in Sertoli cell proliferation at 48 hours following treatment with 200 

mg/kg bw DEHP; at that time point, the labeling index was 30.5% in the DEHP group and 24.7% in 

controls. Dosing with up to 500 mg/kg bw DEHP did not affect serum FSH levels. 

To determine the role of DEHP metabolites, measurement of Sertoli cell proliferation and a morphological 

examination of testis were conducted in 4 rats/group that were gavage dosed with vehicle, 393 mg/kg bw 

MEHP (> 95% purity) in corn oil, or 167 mg/kg bw 2-EH (> 95% purity) in phosphate-buffered saline. 

The doses of MEHP and 2-ethylhexanol were equivalent to 500 mg/kg bw DEHP on a molar basis (1.28 

mmol/kg bw). Like DEHP, MEHP caused an increase in large multinucleated gonocytes and a decrease 

in BrdU labeling. Those effects were not observed following treatment with 2-ethylhexanol. 

To determine if inhibited Sertoli cell proliferation is due to altered expression of cell cycle regulators, 

expression of D1, D2, D3, and p27kipl proteins and cyclin D2 mRNA was measured in 4 –5 rats/group 

gavage dosed with 0, 200, or 500 mg/kg DEHP; rats were killed at 6, 8, 12, or 24 hours following 
Appendix II

II-77

dosing. Statistically significant effects included a small but reproducible decrease in cyclin D2 protein 

level at 8 and 12 hours following treatment with 200 mg/kg bw DEHP. [It was not clear if protein

expression was also examined in rats treated with 500 mg/kg bw DEHP.] The decrease in D2 

protein expression was confirmed by a dose-related reduction in D2 mRNA expression in the 200 

and 500 mg/kg bw groups. 

The conclusions of the study authors were that DEHP-induced transient reductions in Sertoli cell 

proliferation and changes in gonocyte morphology are mediated through MEHP, alterations in Sertoli 

cell proliferation do not occur as a result of changes in FSH levels, and developing testes are especially 

vulnerable to phthalate-induced toxicity. 

Strengths/Weaknesses: An appropriate route of exposure (oral) was used, and the multiple DEHP dose 

groups were a strength. There was a strong dose-response with statistical significance for decreased 

Sertoli cell proliferation as measured by the BrdU labeling index. This study has some serious design 

and reporting deficiencies. Sample sizes were very small for most endpoints, 4 or 5 pups per group, 

with a limited dosing duration (single dose) during the postnatal period that may have been too late 

for many of the developmental endpoints. It was not clear how many pups/group were evaluated for 

serum FSH or if serum samples were pooled. A quantitative evaluation of FSH did not demonstrate an 

effect of treatment; however, the small sample size, single-dose exposure, and uncertainty regarding 

pooling of samples resulted in low confidence for this conclusion. There was no quantification of the 

pathologic findings in the testes, such as numbers of animals with specific lesions. Without incidence 

data or quantitative evaluations, the effect and no-observed-effect levels for most pathologic observations 

cannot be substantiated. 

Utility (Adequacy) for CERHR Evaluation Process: Although this study utilized multiple dose levels 

for dose-response evaluation, the study suffered significant design and reporting deficiencies and is 

not useful for the evaluation process, with the possible exception of the Sertoli cell proliferation data. 

Low sample size, lack of control for litter effect, and lack of quantification of pathology findings make 

these data unsuitable for a quantitative assessment. The primary value of these data is to characterize 

the known pathologic effects on the testes and provide information on mechanistism/mode of action 

(changes in Sertoli cell proliferation and cell cycle regulators) for these effects.

Cammack et al.(120), in a GLP study commissioned by the Advanced Medical Technology Association, 

examined reproductive development of Sprague-Dawley rats treated IV or orally with DEHP. Beginning 

at 3–5 days of age, rats were treated for 21 days with DEHP (99.8% purity) at 0 (vehicle), 60, 300, or 

600 mg/kg bw/day by IV infusion or 0 (vehicle) or 300 mg/kg bw/day by oral gavage. Another set of 

rats was dosed with 600 mg/kg bw/day DEHP by oral gavage for 19 days; this group replaced a previous 

group that suffered high mortality rates following gavage dosing with 1000 mg/kg bw/day. Concentration 

and stability of dosing solutions were verified. The dosing vehicle was Intralipid®, a 10% fat emulsion solution for IV use. Each dose group in this study consisted of 16 animals. Seven rats/group were 

scheduled to be killed following the dosing period, and 9/group were to be held for a recovery period until 

90 days of age, at which time they were also killed and necropsied. During each necropsy period, brain, 

liver, spleen, heart, kidneys, and testes were weighed. A testis from each rat was fixed in Bouin fluid. 

Histopathological examinations of testes were conducted during both necropsy periods; histopathological 

analyses of prostate, seminal vesicle, and epididymis were conducted only in 90-day-old animals. Sperm 
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Appendix II

count, motility, and morphology were examined in the 90-day-old rats. Sperm data were evaluated by 

Kruskal-Wallis nonparametric ANOVA, and if there was a significant finding, a Mann-Whitney U test 

was used for pair-wise comparisons. Body weight and organ weight data were assessed by determining 

group differences followed by pair-wise comparisons in the case of significant findings. 

The only significant body weight effect reported was reduced body weight gain in rats given 600 

mg/kg bw/day by IV infusion and oral gavage. Percent changes in testes and liver weight compared 

to control are outlined in Table 25. 

Table 25. Testicular and Liver Weight Changes in Rats Treated with DEHP

 by IV Infusion or Oral Gavage

Weight 

Change

Percent of Control Value in Each Dose Group 

(mg/kg bw/day)

60 IV 300 IV 600 IV 300 oral 600b oral

Period immediately after dosinga

Testis 91a 67*** 52*** 59*** 36***

Liver 99 126* 133*** 125 108

Following recovery period a

Testis 101 88** 74*** 69*** 58***

Liver 98 96 89 94 89

From: Cammack et al. (120).

*P<0.01; **P<0.001; ***P<0.001. aRats were examined immediately after the 21-day dosing period or following a 

recovery period at 90 days of age.

bRats in this group were dosed for 19 days, while the other dose groups were 

dosed for 21 days.

[Several organ weight effects were noted, but data were presented only for absolute liver and testis

weight. Relative weights are discussed only when differences were noted from absolute weights.]

In animals killed immediately after the dosing period, absolute testis weights were significantly reduced 

in the 300 and 600 mg/kg bw/day oral and IV groups. [For testis weights, CERHR calculated a

BMD10

2 of 122 mg/kg bw/day, a BMDL10 of 106 mg/kg bw/day, a BMD1 SD of 179 mg/kg bw/day,

and a BMDL1 SD of 125 mg/kg bw/day in animals treated by IV infusion. After oral exposure,

the BMD10 was 90.7 mg/kg bw/day, the BMDL10 was 77.4 mg/kg bw/day, the BMD1 SD was 875

mg/kg bw/day, and the BMDL1 SD was 628 mg/kg bw/day.] Absolute liver weights were increased 

in rats given 300 or 600 mg/kg bw/day DEHP by IV infusion. [For liver effects in IV-dosed rats,

CERHR calculated a BMD10 of 163 mg/kg bw/day, BMDL10 of 122 mg/kg bw/day, a BMD1 SD

of 101 mg/kg bw/day, and a BMDL1 SD of 66 mg/kg bw/day.In orally dosed rats, the BMD10 was

712 mg/kg bw/day, the BMDL10 was 196 mg/kg bw/day, the BMD1SD was 585 mg/kg bw/day,

and the BMDL1 SD was 193 mg/kg bw/day.] Liver weight relative to body weight was reportedly 

increased in the 300 and 600 mg/kg bw/day oral DEHP groups. Other absolute and relative organ 

weight effects reported were increased spleen weight in the IV 600 mg/kg bw/day group and decreased 

kidney weight in the oral 600 mg/kg bw/day group. Depletion of germinal epithelium and/or decreased 

2 See footnote to Table 22 for definitions and a discussion of the use of benchmark doses in this report.
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II-79

seminiferous tubule diameter was noted in all animals from the 300 and 600 mg/kg bw/day oral and IV 

dosing groups. [It was not clear if all 7 rats from each dose group were examined, or how many

rats were affected with each type of lesion.] Germinal epithelium depletion was rated as moderate 

(51 –75% reduction in thickness) in the 600 mg/kg bw/day oral group and mild (25 –50% change) in 

all other dose groups given ≥300 mg/kg bw/day DEHP. Reduced tubule diameter was rated as mild 

(25 –50% reduction in diameter) in the 600 mg/kg bw/day oral group and minimal (<25% change) 

in the other dose groups treated with ≥300 mg/kg bw/day DEHP. 

In animals killed at 90 days of age, testicular weights in the 300 and 600 mg/kg bw/day IV and oral 

groups remained lower than those of controls. [CERHR estimated a BMD10 of 222 mg/kg bw/day,

a BMDL10 of 190 mg/kg bw/day, a BMD1 SD of 169 mg/kg bw/day, and a BMDL1 SD of 105 mg/kg

bw/day for testicular weights in the IV group. In the orally dosed group, the BMD10 was 138

mg/kg bw/day, the BMDL10 was 125 mg/kg bw/day, the BMD1 SD was 85 mg/kg bw/day, and the

BMDL1 SD was 65 mg/kg bw/day.] Earlier increases in absolute liver weights did not persist in 90-

day-old animals. The only persisting testicular lesion, apart from reduced testis weight, was a minimal 

(<25%) decrease in seminiferous tubular diameter in 2 of 5 rats in the 300 mg/kg bw/day oral group 

and 3 of 7 rats in the 600 mg/kg bw/day oral group. [The number of animals examined in other dose

groups was not indicated.] No lesions were observed in prostate, epididymis, or seminal vesicles. 

There were no adverse effects on sperm count, motility, or morphology. 

The study authors concluded that sperm and testicular histologic parameters resolved after DEHP 

treatment was discontinued. They stated that “lack of residual effects on sperm parameters found 

in this study will be important in the understanding of potential health risks from DEHP in patients 

undergoing critical procedures, such as ECMO in infants, and the management of these risks.”

[The Expert Panel notes that an acknowledgement in this paper indicates that advice on design

was received from an Expert Panel member “representing the NTP/CERHR Phthalate Expert

Panel”; however, this Expert Panel member was rendering advice as an individual scientist and

not as a representative of the Expert Panel.]

Strengths/Weaknesses: This GLP study was conducted using relevant routes of exposure, both IV 

and oral, during a relevant period of postnatal development. Multiple dose levels allowed for doseresponse and benchmark dose assessment. Test material source and purity were provided. The dosing 

emulsification was characterized and evaluated for stability. Although the characterization and stability 

data for the dosing emulsification were not presented, the wording in this section suggested that DEHP 

concentration and stability were confirmed. This study demonstrated dose-responsive effects in a 

variety of parameters following both IV and oral exposure. Weaknesses included inadequate detail on 

statistical analyses; only sperm endpoints were addressed with specific details on analyses methods. 

The description of analyses of body and organ weights included reference to “standard operating 

procedures.” The use of an alpha level of 0.01 is a weakness. The authors did not address assignment 

of animals to treatment or whether litter effect was controlled. The pathology incidence data were 

not presented, making it difficult to confirm effect levels and compare to background lesions in the 

control. However, even though no control data were presented, the incidence of findings was high in 

the treated animals and the types of lesions observed were fairly rare in untreated control animals. 

The variability in onset of dosing (PND 3–5) is an additional weakness.
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Appendix II

Utility (Adequacy) for CERHR Evaluation Process: Portions of this study can be used for the evaluation. 

This study provides important dose-response information following both IV and oral exposure in young 

animals. The testes and liver weight and sperm assessment parameters provide quantitative multi-route doseresponse data. Due to incomplete presentation of data, there is less confidence in the conclusion regarding 

the pathologic findings; however, pathological changes consistent with disruption of spermatogenesis 

are supported by the findings of other investigators. The relatively small sample size also reduces the 

confidence level of these conclusions, especially with regard to lack of treatment-related effects. 

Gray et al. (121), from the EPA, examined the effect of perinatal phthalate exposure in rats. It appears 

that data from this study were also reported in an abstract by Ostby et al. (122). Sprague-Dawley rats 

were gavage dosed with 0 (corn oil vehicle) or 750 mg/kg bw/day DEHP (99% purity) from GD 14 

(GD 1=day sperm detected) to PND 3 (PND 1=postcoital day 23). The experiment was repeated 

with a second block of animals. In each block of the experiment, there were 7–9 treated dams and 

9–10 control dams. Parameters examined in pups (period examined) included body weight (PND 2), 

anogenital distance (PND 2), testicular histology (PND 2, 9–10, and 13, 3–5 months, and 4–7 months), 

areolas/nipples (PND 13), preputial separation (beginning on PND 28), mating behavior (adulthood), 

abnormalities of reproductive organs (3 –5 months and 4 –7 months), and sperm counts. Statistical 

analyses were based on litters, and blocks were pooled in cases of identical results. Analyses included 

1-way ANOVA followed by post hoc t-tests when statistical significance was obtained. Anogenital 

distance and organ weight data were covaried with body weight. Categorical data were analyzed by 

Fisher exact test or chi-squared test.

DEHP treatment resulted in a small reduction in maternal body weight gain. Litter weight at birth was 

significantly reduced by 15% in the DEHP group, but there was no effect on number of live pups at 

birth. In DEHP-treated males on PND 2, anogenital distance was significantly decreased by ~30%, with 

or without adjustment for body weight, and paired testis weights were significantly decreased by 35%. 

There was no effect on anogenital distance in female pups. Histological examination of testes from 

DEHP-treated rats on PND 2–3 revealed focal interstitial hemorrhage and multinucleated gonocytes 

containing 3 –5 nuclei or undergoing degenerative changes. Hemorrhagic testes were observed in 7 

DEHP-treated males from 3 litters at PND 8 – 9. Histological examination of testes on PND 9 – 10 

revealed evidence of focal hemorrhage in some testes and extensive coagulative necrosis in other 

testes of DEHP-treated rats; loss of seminiferous epithelium was observed in areas with hemorrhage 

or necrosis. Areolas were observed in 87% of DEHP-treated male pups versus none in control pups. 

DEHP treatment did not delay the age of preputial separation, but preputial separation was incomplete 

due to malformations in 19 of 56 treated pups. 

DEHP did not appear to affect sexual behavior in adult rats, except that males with malformed penises 

were unable to achieve intromission. At necropsy, 45 DEHP-treated adult rats from 15 litters were 

assessed for malformations of reproductive organs, which were observed in 82% of DEHP-treated 

males. The types of malformations included permanent nipples, clefting of phallus and hypospadias, 

vaginal pouches, agenesis of prostate, seminal vesicles, or coagulating glands. Sperm production and 

numbers were said to be unaffected by DEHP treatment [data not shown]. Testicular defects included 

hemorrhage, granuloma, fibrosis, reduced size or atrophy, and non-descent associated with abnormal 

gubernacula or ligaments. Significant reductions in weight were observed for all male reproductive 

organs including testis, levator ani plus bulbocavernosus muscle, seminal vesicle, prostate, penis, and 
Appendix II

II-81

epididymis. Liver, pituitary, kidney, and adrenal weights were not affected by DEHP treatment. Serum 

testosterone levels were unaffected in DEHP-treated rats. The study authors concluded that 750 mg/kg 

bw/day DEHP severely alters sexual differentiation in an anti-androgenic manner.

Strengths/Weaknesses: This paper reports a high quality comprehensive evaluation of the potential 

anti-androgenic effects of DEHP in rats when administered at a single dose during a period of critical 

sexual differentiation, from GD 14 to PND 3. The experiment included a relevant route of exposure, and 

the authors provided the source and purity of test material. Strengths include good detailed description 

of methodology, robust numbers of litters evaluated (as high as 16 litters) for the “active” phthalates, 

appropriate statistical analyses using the litter as the unit of analysis for most endpoints, randomization 

of animals into blocks, and near complete presentation of data with standard errors where appropriate. 

Most treatment-related changes were robust and clearly distinguishable from controls, resulting in high 

confidence for the findings. The authors described a characteristic phthalate phenotype, although they 

were not the first to do so. The suggestion of a similar mechanism of action for fetal and pubertal male 

effects based on the structure of active and inactive phthalates was an important contribution. Weaknesses include the single, high dose level, the small sample size for most of the “inactive” phthalate 

exposures, and the presentation of only fetal incidence for malformation data.

Utility (Adequacy) for CERHR Evaluation Process: The majority of these data are adequate for 

consideration. The study robustly demonstrates multiple adverse effects on sexual development in 

males, fetal growth, and maternal toxicity. Because only one dose level was used, a NOAEL was not 

obtained and a dose-response and benchmark dose evaluation cannot be conducted. The presentation 

of the pathology findings is troublesome in that no data or comparison to the controls were presented, 

diminishing the value of this information for the evaluation. However, given the severity of the findings 

and the support of other endpoints affected, this oversight may be of less importance. The behavioral 

evaluation is insufficient and cannot be used for risk assessment.

Parks et al. (123), from the EPA, conducted a series of in vivo, ex vivo, and in vitro studies to examine 

mechanisms of DEHP-induced malformations in rat reproductive organs. A competitive androgenbinding study was conducted in monkey cells transfected with the human androgen receptor vector 

pCMVhAR. Radioactivity was measured following incubation of the cells for 2 hours with 5 nM 

3H-R1881 (a synthetic androgen ligand) and DEHP or MEHP at concentrations of 0 or 0.05 –10 μM. 

Neither DEHP nor MEHP competed with R1881 for androgen receptor binding. 

In the in vivo study, Sprague-Dawley rats were randomly assigned to groups that were gavage dosed 

with 0 (corn oil vehicle) or 750 mg/kg bw/day DEHP from GD 14 (GD 1=day after mating) until 

necropsy. Rats were killed and necropsied on GD 17, 18, or 20 or PND 2 (PND 1=day after birth). The 

study was conducted in 2 blocks, and a total of 4 –5 litters per group were examined at each necrospy 

period. At GD 17, 18, and 20 and PND 2, 1 testis from 2 or 3 males/litter was incubated in media for 

3 hours to determine ex vivo testosterone production, and the other testis was used to measure testosterone content. In GD 17, 18, and 20 males, testosterone levels were also measured in the carcasses 

from which testes were removed (n=18 – 20 per group). Testosterone levels were measured by RIA. 

One testis from each of 4 DEHP-treated and 6 control PND 2 males was fixed in 5% glutaraldehyde 

for histopathological examination. One testis from each of 4 control and 5 DEHP-exposed PND 20 

males and an unspecified number of DEHP-exposed PND 3 males from a parallel study was stained 
II-82

Appendix II

for 3b-hydroxysteroid dehydrogenase, which is specific for Leydig cells. Anogenital distance was 

measured in all male and female offspring on PND 2. Litter means were used in statistical analyses. 

Data were analyzed by ANOVA followed by 2-tailed t-tests if ANOVA resulted in significant findings. 

Testicular histopathological findings were analyzed by Fisher exact test. 

Maternal weight gain during gestation was significantly reduced in the DEHP-treated group. Number 

of live pups at birth was not significantly affected by DEHP treatment. Ex vivo testicular testosterone 

production in GD 17, 18, and 20 and PND 2 offspring from DEHP-exposed groups was significantly 

lower compared to control groups. Testicular testosterone content in DEHP-exposed offspring and 

pups was reduced by 60 –85% compared to controls examined at each necropsy period; the effect was 

statistically significant at all time points except GD 20. [It appears a footnote regarding GD 20 is

missing in Table 1 of the study.] Whole body testosterone levels were significantly lower in DEHPexposed fetuses on GD 17 (71% lower than controls) and 18 (47% lower than controls), but the reduction 

on GD 20 was not significant. Significant reductions in testis weight were noted in the DEHP group 

on GD 20 (18% lower than controls) and PND 2 (49% lower than controls). Body weights of DEHPexposed pups were described as 23% lower than controls on PND 2, but statistical significance was 

not achieved. Testis weights adjusted for body weights were significantly decreased in PND 2 pups 

exposed to DEHP. Anogenital distance was significantly reduced by 36% in PND 2 males compared 

to controls but was not affected in female pups exposed to DEHP. Histopathological examination of 

PND 2 testes of DEHP-treated rats revealed an increased number of enlarged and multinucleated 

gonocytes and aggregates of hyperplastic Leydig cells. 3b-Hydroxysteroid dehydrogenase staining 

confirmed the presence of Leydig cell aggregates in DEHP-exposed males on GD 20 and PND 2. In 

contrast, 3b-hydroxysteroid dehydrogenase staining revealed an even dispersion of Leydig cells and 

less intense staining in testes of control fetuses and pups. 

The study authors concluded that treatment with 750 mg/kg bw/day DEHP inhibited testosterone 

production in male pups during the period of sexual differentiation, and this inhibition was a likely 

cause of malformations observed in other studies. Malformations likely result from a mechanism that 

does not directly involve the androgen receptor. 

Strengths/Weaknesses: This report includes a good, detailed description of the methods, appropriate route 

of exposure, and statistical methods using the litter as the unit of analysis. The authors provided excellent 

use of controls in the evaluation of pathology. An appropriate exposure period was used for male effects. 

This study demonstrated clear effects on fetal whole body and fetal and postnatal testicular testosterone 

levels in male offspring, testes weight, anogenital distance, and histopathology of the testes following 

maternal gestational or gestational and postnatal exposure, and provided clear evidence that DEHP and 

its principal metabolite do not bind to the androgen receptor. The description of the time course for fetal 

testosterone alterations is an additional strength. Weaknesses include the single, high dose level and the 

small sample size. The samples size was sufficient to demonstrate robust effects of treatment but not to 

provide high confidence in lack of effect. There was no purity information on the test material. 

Utility (Adequacy) for CERHR Evaluation Process: This study is adequate in providing mechanistic 

information for use in the evaluation process. Because only one dose level was used, the study cannot 

establish a NOAEL and is not suitable for dose-response or benchmark dose evaluation. Given the 

small sample size, there is low confidence in negative findings. 
Appendix II

II-83

Wilson et al. (124), from the EPA, conducted 2 in vivo studies to determine if gubernacular lesions 

induced by DEHP and other chemicals result from inhibition of insulin-like hormone 3 (insl3), a hormone produced by fetal Leydig cells and considered to be a marker of cell maturation. In the first study, 

10 pregnant Sprague-Dawley rats were gavage dosed with 0 (corn oil vehicle) or 750 mg/kg bw/day 

DEHP (99% purity) on GD 14–18 (GD 1=day sperm detected). In the second study, 3 dams/group 

were dosed with 0 or 1000 mg/kg bw/day DEHP on GD 14–18. The DEHP concentrations were found 

to induce gubernacular lesions in previous studies. Dams were killed on GD 18, and fetal testes were 

removed. The testes were pooled by litter for examination of insl3 expression by a polymerized chain 

reaction method. The second study also examined ex vivo testosterone and progesterone production by 

incubating the testes in media for 3 hours, measuring hormone levels in media by RIA, and pooling 

the data by litter. Statistical analyses were conducted on a litter basis. Analyses included ANOVA, 

followed by paired t-test if statistical significance was obtained by ANOVA. DEHP treatment significantly reduced insl3 expression by about 80% in the first study and [~60%] in the second study. Ex 

vivo testosterone production was reduced [~50%] in testes from the 1000 mg/kg bw/day DEHP group 

compared to the control group. There was no effect on ex vivo progesterone production. 

The study authors proposed that DEHP intake in rodents results in delayed fetal Leydig cell maturation, 

which leads to reduced testosterone production and insl3 production. It was stated that reduced insl3 

expression results in gubernacular malformations and undescended testis, while reduced testosterone 

leads to malformations in testosterone-dependent tissues. 

Strengths/Weaknesses: Use of the oral route of exposure and the appropriate exposure window are 

strengths of this study. Statistical methods were appropriate, using the litter as the unit of analysis. The 

data are consistent with previous findings with evaluation of a possible mode of action for impaired 

testis descent. The use of a single dose level is a weakness, and the ex vivo endpoints are not directly 

relevant to human risk assessment.

Utility (Adequacy) for CERHR Evaluation Process: This paper has limited utility in the evaluation 

process.

Liu et al. (125), supported by NIH, evaluated gene expression profiles in GD 19 fetal testes after 

GD 12 – 19 gavage treatment of dams with 1 of 7 phthalates (n=5/group) or with corn oil vehicle 

(n=10; vaginal sperm=GD 0). The phthalates were DEHP or diethyl, dimethyl, dioctyl tere-, dibutyl, 

dipentyl, or benzyl butyl phthalate. [Purity was said to have been verified but was not specified.]

The dose of each phthalate was 500 mg/kg bw/day. After removal by cesarean section, pups were 

evaluated for anogenital distance and testes were harvested from male offspring. Total RNA was 

extracted from the testes of 3 pups/treatment group, each from a different litter, and hybridized to a 

microarray gene chip. ANOVA and post hoc Dunnett test were used to evaluate differences in gene 

expression in phthalate-exposed and control samples. A Bonferroni adjustment was used for multiple 

comparisons. Selected genes were investigated further using RT-PCR on total testis RNA from 6 

control fetuses and 3 phthalate-treated fetuses/group. Relative expression ratios were calculated with 

respect to glyceraldehyde-3-phosphate dehydrogenase and compared to control using ANOVA and 

post hoc Dunnett test. Comparisons between different phthalates were also evaluated using ANOVA. 

Immunohistochemistry was performed in formalin-fixed testicular sections from control and dibutyl 

phthalate-exposed fetuses [not discussed here].
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Appendix II

Anogenital distance was significantly reduced by pregnancy treatment with DEHP, dibutyl phthalate, 

benzyl butyl phthalate, and dipentyl phthalate. Dimethyl, diethyl, and dioctyl terephthalate did not 

affect anogenital distance. Of 391 significant gene probe sets, there were 167 characterized sequences. 

Genes related to lipid, sterol, and cholesterol homeostasis accounted for 31 of these 167 genes. There 

were also 10 genes involved in lipid, sterol, and cholesterol transport, 12 genes involved in steroidogenesis, 9 transcription factor genes, 22 signal transduction genes, 11 genes involved in oxidative 

stress, and 13 genes related to the cytoskeleton. Eighteen of these genes were evaluated using RT-PCR, 

and 16 of the 18 were affected by those phthalates that altered anogenital distance in comparison to 

the phthalates that did not alter anogenital distance (Table 26). There were few differences in relative 

expression between the individual phthalates in the group that altered anogenital distance. The authors 

concluded that the developmentally toxic phthalates were “indistinguishable in their effects on gene 

expression in the developing fetal testis.” These phthalates were described as targeting pathways 

directly or indirectly related to Leydig cell production of testosterone and pathways important for 

Sertoli cell-gonocyte interaction.

[The Expert Panel notesthat there is a study by Lehmann et al.(126) in which di(n-butyl) phthalate

was administered to pregnant Sprague-Dawley rats on GD 12 – 19 at gavage doses of 0.1, 1.0,

10, 50, 100, or 500 mg/kg bw/day. Fetal testes were evaluated for testosterone and mRNA and

protein concentration for key steroidogenic enzymes. Decreases in mRNA for key steroidogenic

enzymes occurred at maternal exposure levels below those associated with a decrease in testicular

testosterone. To the extent that di(n-butyl phthalate) and DEHP share molecular mechanisms

of action, this study may offer insights relevant to DEHP testicular toxicity.]

Strengths/Weaknesses: This study was a well-conducted and reported and investigated the potential 

mechanisms of “testicular dysgenesis” and male reproductive tract abnormalities in rats following 

gestational exposure to DEHP and other phthalates. The dams were treated during a critical period 

of male reproductive tract development (GD 12 –19) by an appropriate route (oral) of exposure. The 

statistical design was appropriate, using the litter as the experimental unit of analysis and incorporating 

body weight as a cofactor in the anogenital distance analysis. The use of both “active” and “inactive” 

phthalates was a strength, permitting a comparison of gene expression profiles. Weaknesses include 

the use of only a single dose level of 500 mg/kg bw, and small sample size (5/treatment); however, the 

sample was sufficient to detect strong effects. The study used only 1 endpoint (anogenital distance) 

that is classically used for risk assessment. The remaining endpoints were mechanistic in nature (gene 

expression). Evaluation at a single time point is a weakness inasmuch as the noted effects could be 

the consequences rather than the causes of the altered differentiation.

Utility (Adequacy) for CERHR Evaluation Process: The anogenital distance data from this study 

are useful for hazard identification at the relatively high dose level of 500 mg/kg bw and the study 

supports/confirms other reports of adverse effects on this endpoint following gestational exposure 

to DEHP. Because only one dose level was used, a dose-response evaluation and NOAELs/LOAELs 

cannot be established. The gene expression assays are valuable in helping to understand potential 

mechanisms of action. Based on the affected genes, a complex mode of action is suggested involving 

Leydig cell, Sertoli cell, and Sertoli-gonocyte interactions.
Appendix II

II-85

Table 26. Relative Expression of Fetal Testis Genes after Phthalate Treatment of Pregnant Rats

Gene Direction of Alteration

Lipid, sterol, and cholesterol transport

Epididymal secretory protein 1 ↓

Low density lipoprotein receptor ↓

Steroidogenesis

17b-Hydroxysteroid dehydrogenase 7 ↓

17b-Hydroxysteroid dehydrogenase 3 ↔

LH/chorionic gonadotropin receptor ↓

Transcription factors

CCAAT/enhancer binding protein, beta ↓

Early growth response 1 ↑

Nuclear receptor subfamily 4, group A, member 1 ↑

Nuclear factor, interleukin 3, regulated ↑

Nuclear receptor subfamily 0, group B, member 1 ↓

Transcription factor 1

Signal transduction

Insulin-induced gene 1 ↓

Cytoskeleton

Fasciculation and elongation protein ↑*

Unclassified

Decay-accelerating factor ↔

DOPA decarboxylase ↓

Seminal vesicle secretion 5 ↓

Testis-derived transcript (testin) ↑

↑,↓,↔ Statistically increased, decreased, or unchanged on comparison of the 

phthalates that decreased anogenital distance (DEHP, dibutyl phthalate, benzyl 

butyl phthalate) with the phthalates that did not (dimethyl, diethyl, and dioctyl 

terephthalate). 

*DEHP did not show a response, although the other anogenital distance-altering 

phthalates did. From Liu et al. (125).

Kobayashi et al. (127) presented an abstract describing treatment of pregnant Sprague Dawley rats 

by gavage with DEHP 0, 25, 100, or 400 mg/kg bw/day on GD 6–PND 20. There were no treatment 

effects on offspring body weight, body length, tail length, organ weights, or plasma levels of thyroxine 

or tri-iodothyronine at 1, 3, or 9 weeks of age. [Abstracts are noted but are not used in reaching

conclusions.]

Wang et al.(128) presented an abstract describing treatment of pregnant Sprague Dawley rats by gavage 

with DEHP 0, 25, 100, or 400 mg/kg bw/day on GD 6–PND 20. Blood testosterone and progesterone in 
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Appendix II

male offspring were described as showing a dose-related increase at 9 weeks of age, although differences 

were not statistically significant. There were no treatment effects on relative testis or prostate weight 

at 9 or 36 weeks of age. Human chorionic gonadotropin was used to stimulate testosterone production 

[age unspecified]; blood testosterone increases were numerically lower in DEHP-exposed offspring 

than in control offspring. [Abstracts are noted but are not used in reaching conclusions.]

3.2.1.2 In Vitro Exposures

Iona et al. (129), supported by “EU BIO-CT96-0183,” “MURST,” and the Italian Public Health Ministry, conducted in vitro studies to examine the effect of MEHP and 2 other chemicals on primordial 

mouse germ cells. In the first and second studies, primordial germ cells were obtained from sexually 

undifferentiated gonadal ridges of CD-1 mouse embryos at 11.5 days post coitum and were seeded 

onto STO, an embryonic mouse fibroblast cell line, to allow the germ cells to proliferate. Cultures were 

treated with MEHP [purity not indicated] at 0 (dimethylsulfoxide [DMSO] vehicle), 100, 300, or 600 

μM [0, 27.8, 83.4, or 167 mg/L] for 2 hours or 1 day. [It was not specified if MEHP was rinsed from

cells following the 2-hour and 1-day treatments.] Numbers of primordial germ cells were counted 

after 1 and 3 days of culture. MEHP treatment for 2 hours did not affect numbers of primordial germ 

cells at any dose. Following treatment with MEHP for 1 day, there was a “slight but not significant” 

decrease in primordial germ cell number at 100 and 300 μM MEHP and cytotoxicity at 600 μM MEHP. 

It was determined that 600 μM MEHP reduced the viability of supporting STO cells by about 50%. 

In the third study, primordial germ cells were incubated in suspension with 100–300 μM MEHP for 

2 hours. MEHP was washed from the cells, and the germ cells were then seeded onto STO cells. The 

numbers of primordial germ cells were measured after 1 and 3 days of culture. Numbers of germ cells 

were reduced by about 38% [estimated from a graph] at ≥300 μM MEHP [statistical significance

not indicated, but based on the graphed standard errors, the results appear to be significant]. In 

a fourth study, apoptosis was not significantly increased following incubation of the primordial germ 

cells in 500 μM MEHP for 6 hours. In a fifth study, a short-term adhesion assay was used to determine 

that exposure to 300–600 μM MEHP for 2 hours reduced by about 40% the percentages of primordial 

germ cells capable of binding to STO cells. The study authors concluded that MEHP affected adhesion 

of primordial germ cells to STO cells without inhibiting growth or survival of the germ cells. 

A sixth study was conducted to determine if in vitro effects of MEHP could be replicated in vivo. 

Pregnant CD-1 mice were gavage dosed with 1000 or 2000 mg/kg bw MEHP on day 8.5, 10.5, or 

11.5 post coitum. [One control group was used, but it was not specified if or when the control was

gavaged.] Three mice per dose were killed 12.5 days post coitum, and gonadal ridges were taken from 

each embryo. Sections from at least 3 randomly selected embryos/sex were examined to determine 

primordial germ cell numbers. Apparent reductions in primordial germ cell numbers were noted in 3 

of 5 female embryos and 2 of 5 male embryos treated with 2000 mg/kg bw DEHP 8.5 and 11.5 days 

post coitum, but mean differences were not statistically significant in any dose group. 

Strengths/Weaknesses: A range of dose levels was used for in vitro (100–600 μM; 28–167 mg/L) and in 

vivo (1000–2000 mg/kg) studies, but it is a weakness that the in vitro levels were not related to in vivo

exposure levels. In addition, the endpoints are not typical examples of phthalate toxicity, which limits 

the importance of the experiments. Although the studies were insightful, potentially useful experiments 

to help understand the mechanisms of action of reproductive toxicants, the in vitro studies were primarily an exercise in methods development/methods utility. The in vitro systems were not standardized or 
Appendix II

II-87

validated for their predictive value for in vivo effects (i.e., the artificial environments used may not be 

relevant to in vivo exposure). This opinion is supported by the authors’ conclusion that their in vitro results 

were not always exactly predictive of the effects in vivo. The in vivo study used an insufficient number 

of litters, employed a single dose, and did not directly address any relevant functional outcome. 

Utility (Adequacy) for CERHR Evaluation Process: This paper has no utility in a quantitative assessment, although it may provide supplemental mechanism information.

Li and Kim (130), supported by NIEHS, examined the effects of MEHP on cultured fetal and neonatal 

rat testes during 3 periods: GD 13 (plug=GD 0), GD 18, and PND 3. GD 13 is the period when testicular 

cord formation and Sertoli cell differentiation begin. GD 18 represents the period when Sertoli cells are 

proliferating and gonocytes are mitotically quiescent. PND 3 represents the period when Sertoli cells 

continue proliferating and gonocytes migrate to the basal side of the seminiferous tubule and become 

mitotically active. Gonads were obtained from fetuses or pups of Sprague-Dawley rat dams (at least 3 

dams/group and 4 offspring/dam) during each of the 3 periods of development. A testis from each animal 

was cultured for 3 days in medium containing MEHP (≥99% purity) 50, 100, or 200 μM [13.9, 27.8, or

55.6 mg/L] and the other in medium containing the DMSO vehicle. Assays were conducted to determine 

cell counts, cell differentiation and proliferation, and expression of testicular cell marker proteins. Data 

were analyzed by one-way ANOVA followed by pair-wise comparison. In GD 13 testes, MEHP had no 

effect on testicular cord formation or number of gonocytes. According to the study authors, the lack of 

effect on testicular cord formation meant that MEHP did not affect differentiation of indifferent gonad 

to testis. MEHP treatment also had no effect on expression patterns of Müllerian-inhibiting substance 

or GATA-4, Sertoli cell markers, or on mouse vasa homolog protein, a germ cell marker. In GD 18 

testes, Sertoli cells were targets of MEHP as indicated by decreased Müllerian-inhibiting substance and 

GATA-4 expression and impaired Sertoli cell proliferation at ≥100 μM MEHP. Numbers of gonocytes 

and mouse vasa homolog protein expression were unaffected by MEHP treatment. Both Sertoli cells and 

gonocytes were targets of MEHP in PND 3 testes. There were significant dose-dependent reductions of 

Müllerian-inhibiting substance and GATA expression; Sertoli cell proliferation was inhibited at ≥100 

μM MEHP. Reduced gonocyte numbers and Sertoli and germ cell disorganization were observed at 

≥100 μM MEHP. Gonocyte proliferation was not inhibited, but apoptosis was increased by treatment 

with ≥100 μM MEHP. Seminiferous cord structure remained intact following MEHP exposure. 

The study authors concluded that these results suggest that MEHP targets testicular cells during periods 

when they are mitotically active. 

Strengths/Weaknesses: The use of multiple concentrations of MEHP allowed a dose-response evaluation. 

In general, the methods and results were well presented. The culture system was unique, without a 

history of use in other laboratories, and the absence of positive and negative controls was a weakness. 

The MEHP concentrations that were used were not related to in vivo exposure levels, and the assumption 

that exposure levels would be constant over a 3-day culture period was not verified. There were limited 

numbers of dams (3) and uncertain control for litter effect. 

Utility (Adequacy) for CERHR Evaluation Process: The study provides evidence for a potential mode of 

action for adverse effects on the testes noted after in vivo embryonic or prepubertal exposure to DEHP. 

Because these studies were conducted in vitro, they are not directly useful in the evaluation process.
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Appendix II

3.2.2 	 Developmental Studies Focusing on Non-reproductive Effects

Magliozzi et al. (131), supported by Italian “CNR” and “MURST” grants, examined the effects of 

prenatal DEHP exposure on rat neonatal lung. Wistar rats were fed diets containing 0 or 1% (w/w) 

DEHP from the week prior to delivery through 2 days following delivery. Five dams that ate at least 

1000 mg DEHP/kg bw/day and 5 control dams were used for the study. Two days following delivery, 

pups were weighed and killed. One pup per litter was used to measure DEHP in blood by GC/MS. Lungs 

from 5 pups per litter were fixed in Bouin fluid for examination by light microscopy, and lungs from 

1 pup per litter were fixed in 4% formaldehyde for examination by electron microscopy. Lungs from 

2-day-old rat pups were stated to have similar histological features as lungs from premature infants at 

gestation weeks 26 –36. A catalase-immunoreactivity method was used to measure number and sizes 

of pneumocytes, cells that are a major source of surfactant and contain high numbers of peroxisomes. 

Pup livers were removed and weighed. [Statistical methods were not discussed.] DEHP in blood 

was measured at 4.7±0.46 μg/mL [error not specified] in treated pups and 1.9±0.57 in control 

pups. Relative liver weight was significantly increased in the DEHP-treated pups, thus confirming 

DEHP exposure. Examination of lungs by light microscopy revealed reduced respiratory surface in 

DEHP-treated animals as a result of fewer airspace units that were dilated and units that were less 

branched than spaces of untreated animals. In treated compared to control pups, relative number of 

type II pneumocytes increased by 187% and mean diameter increased by 120% (P<0.01). Pneumocyte 

peroxisomes were unaffected in DEHP-treated pups. The study authors concluded that the relevancy 

of these results to intubated preterm infants inhaling DEHP is not known due to differences in routes 

of exposure and interspecies metabolism. However, they stated that their study indicates a need to 

examine possible injury related to forced ventilation of infants. 

Strengths/Weaknesses: For most procedures, this paper included excellent presentations of methodology and results. The quantitative representation of pathology using morphometrics provided value; 

however, no description of the statistical analyses was noted. The investigation of a new target tissue 

(lung) was a strength. Weaknesses included the use of a single very high dose level without an indication that the effect is relevant to lower exposure levels. Only animals consuming at least 1 g/kg bw/day 

were selected, resulting in low sample size. Plasma extraction validation was conducted with human 

blood rather than rat blood. Significant contamination of control animals with DEHP was apparent 

with DEHP blood levels in controls about one-third those of treated animals. In addition, only DEHP 

was measured, leading to the possibility that evaluation of other analytes would have suggested even 

greater control animal contamination. The relatively high levels of DEHP in control pups raises serious 

questions regarding dosing errors and quality of study conduct and/or plasma level evaluation. Because 

exposure of the dams continued until the pups were killed, the pups from the DEHP-treated dams 

should have had markedly higher levels of DEHP than the control pups. Methods for the statistical 

analyses were not presented. 

Utility (Adequacy) for CERHR Evaluation Process: Although the evaluation of a new target tissue 

is potentially valuable, due to the deficiencies in presentation of statistical analyses and the potential 

contamination of the control pups, this study is of low utility.

Masuo et al.(132), from the Japanese National Institute of Advanced Industrial Science and Technology, 

studied the effects of DEHP and other chemicals on motor activity in rats. At 5 days following birth, 

male Wistar rats (~10 g) received DEHP [purity not specified] at 0 (olive oil vehicle) or 87 nmol [3.4
Appendix II

II-89

mg/kg bw] by intracisternal administration. Groups of 7 control pups and 5 DEHP-treated pups were 

nursed by lactating dams until weaning at 3 weeks of age. Spontaneous motor activity was measured in 

all 4-week-old pups using a Supermex activity-monitoring system. The rats were killed at 8 weeks of 

age. Multiple gene expression in striatum and midbrain of 3 rats/group was determined using an array, 

and an immunohistochemical technique was used to measure tyrosine hydroxylase levels in sectioned 

brains from 8-week-old rats. Statistical analyses included ANOVA, followed by post hoc Scheffé test. 

DEHP caused a significant increase in spontaneous motor activity during the dark phase, light phase, 

and entire 24-hour period. DEHP treatment did not appear to affect stereotyped behavior. There was 

no effect on tyrosine hydroxylase immunoreactivity (data not shown). In midbrain, DEHP treatment 

up-regulated expression of genes for glutamate/aspartate transporter, superoxide dismutase 1, heat 

shock 90-kilodalton (kDa) protein beta, neuropeptide Y, fibroblast growth factor 10, and natriuretic 

peptide precursor C. DEHP treatment down-regulated midbrain expression of genes for c-fos protooncogene, cytochrome P450 17, heat shock 70-kDa protein 1A, dopamine receptor 1A, galanin 

receptor 2, arginine vasopressin receptor 2, and glutamate ionotropic receptor. In striatum, DEHP 

treatment down-regulated expression of genes for c-fos proto-oncogene, heat shock 70-kDa protein 

1A, galanin receptor 2, glutamate ionotropic receptor, and PDGF B polypeptide. Based on analyses of 

gene expression changes, the study authors postulated that an inhibition of glutamatergic transmission 

in midbrain and striatum may be a mechanism of DEHP-induced changes in motor activity.

Strengths/Weaknesses: The hypothesis that DEHP would alter brain dopaminergic system, as has been 

reported for estrogenic chemicals, was reasonable. The comparison of a positive control (6-hydroxydopamine) and 4 chemicals considered capable of endocrine alterations (bisphenol A, p-nonylphenol, 

p-octylphenol, and DEHP), all of which caused a similar increase in diurnal spontaneous motor activity, 

is a strength. Weaknesses include the route of administration (intracisternal), the high dose level, the 

lack of information on offspring body weights, the apparent lack of control for litter effects, the small 

sample sizes (5 –7 litters), and the use of a single dose level. It is not clear to what extent DEHP would 

be metabolized after intracisternal administration.

Utility (Adequacy) for CERHR Evaluation Process: This study is not useful in the evaluation process.

Tanaka (133), support not indicated, examined neurobehavioral toxicity in mice exposed to DEHP 

(> 97% purity) during prenatal development. At 5 weeks of age, 10 CD-1 mice/sex/group were fed 

diets containing 0, 0.01, 0.03, or 0.09% DEHP for 4 weeks prior to mating and during a 5-day mating 

period that began at 9 weeks of age. Females continued to receive the control or DEHP-containing 

diets throughout the gestation and lactation periods. The authors converted DEHP doses to a mg/kg 

bw/day basis, and those values are summarized in Table 27. Each female was mated to 1 male, and the 

females were allowed to litter and rear their offspring. At birth (PND 0), litter size, litter weight, and 

sex ratio were determined. Offspring were individually weighed, and postnatal survival was monitored 

during the lactation period. Neurobehavioral parameters examined in all offspring during the lactation 

period included surface righting (PND 4 and 7), negative geotaxis (PND 4 and 7), cliff avoidance 

(PND 7), swimming behavior (PND 4 and 14), and olfactory orientation (PND 14). Weaning occurred 

at 4 weeks of age, and 1 male and 1 female per litter were selected to continue receiving treatment 

until 9 weeks of age. [Though not specified, it is assumed that the offspring from each treatment

group received the same doses as their parents.] Doses on a mg/kg bw/day basis for offspring are 
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Appendix II

also included in Table 27. At 7 weeks of age, the F1 mice were tested using a Biel type water T-maze. 

Exploratory behavior was assessed using an animal movement analyzing system in 3-week-old mice 

from the F1 generation and 8-week-old mice from the F0 and F1 generations. Statistical analyses 

included Bonferroni multiple comparison, ANOVA, Kruskall-Wallis test, chi-squared test, Fisher 

exact test, Wilcoxon sign test, and/or Jonckheere test. [It does not appear that statistical analyses

were conducted on a per litter basis.]

Table 27. Summary of DEHP Doses in Mice

Generation and 

Study Period

Mean DEHP Doses, 

mg/kg bw/day a

Diet group

0.01% 0.03% 0.09% 

F0 males premating 16 47 142

F0 females

Premating 20 56 168

Mating 15 40 126

Gestation 17 47 140

Lactation 60 172 493

F1 males 16 48 145

F1 females 19 56 171

From: Tanaka (133).

aValues were presented as mean±SD by study authors. The values 

presented here are means rounded to whole numbers because that 

information is sufficient for the CERHR evaluation process.

In F0 mice, DEHP treatment had no effect on body weight gain, movement, or exploratory activity. 

As a result of non dose-related failures to become pregnant or abortions in 1 –2 dams of the low- and 

mid-dose groups, 8– 10 litters were available for evaluation in each treatment group. There were no 

significant effects on sex ratio or litter size or weight at birth. A 7% decrease in body weight in male 

offspring of the low-dose group compared to control males on PND 0 was the only significant body 

weight effect observed in offspring. Significant reductions in survival were noted in the high-dose group 

for female offspring from PND 4 to 14 and for total offspring from PND 4 to 21. Percentages of total 

surviving offspring at PND 21 were 98.4% in the control group and 92.8% in the high-dose group. Time 

for surface righting was significantly delayed in females of the low- and mid-dose groups on PND 4, 

in males of the high-dose group on PND 7, and in females of the low-dose group on PND 7. There 

were no other significant findings in neurobehavioral parameters examined during the lactation period 

[data not shown]. Compared to controls, there were no adverse effects in water T-maze performance 

in treated animals at 7 weeks of age, and movement and exploratory behavior were not affected by 

treatment at 3 or 8 weeks of age. The study authors concluded that “few adverse effects on several 

behavioral parameters were produced at the high-dose level of DEHP in the present study.”

Strengths/Weaknesses: Source and purity of test material were provided and an appropriate route of 

exposure (diet) was used. The use of relatively low dietary exposure levels was a strength. Additional 
Appendix II

II-91

strengths included the use of multiple dose levels and multigenerational exposure in mice from 5 weeks 

of age for the starting F0 generation through 9 weeks of age for the F1 generation, encompassing the 

premating, gestation, lactation, and sexual maturation periods. For the F0 generation animals, the postweaning evaluations were controlled for litter effect by selecting one male or female per litter. Litter 

means were also used in the evaluation of pup weight and litter size. It is unfortunate that animals were 

not evaluated for the classic phenotype of prenatal phthalate exposure, which would have extended the 

observations to another species. Sexually dimorphic behaviors should have been evaluated, given the 

presumed mode of action involving a reduction in fetal steroid hormone levels. There are weaknesses 

in the presentation and analysis of data. The preweaning data for surface righting, negative geotaxis, 

cliff avoidance, swimming behavior, and olfactory orientation were analyzed on an individual animal 

basis and were not controlled for litter effect. Of these endpoints, the authors only presented data for 

surface righting. Exploratory behavior data at 8 weeks of age were also not presented. Preweaning 

survival indices were also not analyzed on a litter basis. There was a limited set of parameters evaluated; 

endpoints of sexual development, fertility, and pathology were not evaluated. Sample size was limited 

for this type of experiment.

Utility (Adequacy) for CERHR Evaluation Process: This study has limited utility for the specific 

endpoints for which data were presented and litter effect was controlled (litter size, average pup 

weight, and T-maze performance). None of these endpoints demonstrated a significant dose-responsive 

treatment effect; however, there is low confidence in these results due to use of relatively small group 

sizes (8– 10 litters/group). Other endpoints from the F1 offspring are not useful due to deficiencies in 

statistical evaluation and/or lack of data presentation. 

James (134) wrote a letter to the editor to question Tanaka’s (133) conclusion that sex ratios were not 

affected in offspring of mice fed diets containing 0.01–0.09% DEHP during pregnancy. James noted 

that there were a total of 190 male offspring and 152 female offspring in all DEHP treatment groups and 

that the difference in sex ratio was significantly higher than equality (P<0.05). It was stated that DEHP 

can have opposite effects on endocrine parameters (e.g., testosterone and estradiol levels) in male versus 

female rats, and that it could potentially affect offspring sex ratio differently in treated males versus 

females. Because Tanaka mated treated female mice with treated male mice, James concluded that lack of 

effect of DEHP on sex ratio could not be ruled out. A later study by Tanaka (135) did examine sex ratios 

in DEHP-treated male and female mice mated with untreated control animals, and that study is addressed 

below. [The James letter is noted for completeness but is not used in the evaluation process.]

Tanaka (136) responded to the James letter (134) regarding sex ratios in the Tanaka (133) study. Tanaka 

re-analyzed the data on a litter basis using Steel multiple comparison test and demonstrated there were 

no significant differences in sex ratios of offspring from DEHP-treated versus control mice. It was 

noted that variations in sex ratio were much higher among litters than among treatment groups. Tanaka 

noted that other studies in rodents also failed to demonstrate an effect on sex ratio following DEHP 

treatment. Lastly, Tanaka stated that sex ratios in all treated groups were within ranges observed in 

control mice in his laboratory during the past 10 years. Tanaka concluded that there was no experimental 

evidence that DEHP adversely affects sex ratio in offspring of DEHP-treated mice.

Strengths/Weaknesses: This re-evaluation of data from the previous study used a more appropriate 

litter-based analysis. The re-analysis and discussion of additional studies referenced in the literature 
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Appendix II

and historical control data from the author’s laboratory fully support the lack of an effect on sex ratio 

following exposure (as studied) in mice. 

Utility (Adequacy) for CERHR Evaluation Process: This information supports previous conclusions 

(133).

Tanaka (135), support not indicated, examined the effects of prenatal DEHP exposure on sex ratio 

in mice. It appears that the study was conducted to address concerns about DEHP effects on sex 

ratio that were raised in letter by James (134). Starting at 5 weeks of age, 20 male and female CD-1 

mice/sex/group were fed diets containing 0 or 0.03% DEHP (purity >97.0%). At 9 weeks of age, 

each female was mated for 5 days with a male from the same or opposite treatment group (i.e., crossmating). There were 4 treatment groups consisting of 10 mice/sex: control females × control males, 

control females × treated males, treated females × control males, and treated females × treated males. 

Females continued to receive the DEHP-containing or control diets during the mating period and 

throughout gestation. The study authors estimated that intake of DEHP was ~47– 49 mg/kg bw/day 

in males and ~55–58 mg/kg bw/day in females during the preconception period. Intakes by females 

were estimated at ~45 mg/kg bw/day during the mating period and ~50 mg/kg bw/day during the 

gestation period. Females were allowed to litter, and endpoints examined on day of birth were litter 

size, litter weight, individual offspring weight, and sex ratio. Statistical analyses included ANOVA or 

Kruskal-Wallis test followed by Bonferroni multiple comparison to assess food intake, litter size, and 

litter and body weights. Chi-squared test was used to evaluate sex ratio based on offspring, and the 

Steel test was used to assess sex ratio based on litter. As a result of pregnancy failures or abortions, 

there were 8 –10 litters delivered in each treatment group. Compared to the group consisting of control 

males and females, mean body weights of male offspring were increased in all groups containing a 

treated female and/or male parent. No significant effects were noted for litter size, litter weight, total 

or average sex ratio, or female offspring weights. The study authors concluded that the concentrations 

of DEHP used in this study did not produce adverse effects on sex ratios.

Strengths/Weaknesses: Source and purity of test material was provided, and an appropriate route of 

exposure (diet) was used. Exposures were conducted during the critical period of sexual differentiation. 

Mice were exposed from 5 weeks of age from the start of the F0 generation through delivery of the 

F1 offspring, encompassing the premating and gestation periods. Strengths are that a factorial design 

was used based on exposure of one sex bred with an unexposed or exposed mate and that litter was 

the unit of analysis for most reproductive and developmental endpoints evaluated. Weaknesses are 

that only a single dose level was used (0.03% in the diet) and that classical measures of phthalate 

toxicity in male offspring were not evaluated. Sample size was limited (8– 10 litters/group) for this 

type of experiment, resulting in low confidence in negative outcomes or marginal effects. For example, 

statistically significant increases in male offspring weight were observed in all groups with DEHP 

parents. However, in two other studies by this author utilizing a similar study design exposing mice 

to equal or higher doses of DEHP, no effect on pup weights or an increase in female pup weight was 

found. (The author concluded that the male pup weights in the concurrent control were unusually low 

and the effect of increased male pup weight was not treatment-related. The Expert Panel agrees).

Utility (Adequacy) for CERHR Evaluation Process: This study is limited in scope to litter size, litter weight, 

pup weight and sex ratio, and to a single dose level of 0.03% in the diet (40–56 mg/kg bw/day).
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II-93

Tanaka et al. (137), support not indicated, gave DEHP (>97% purity) to CD-1 mice in the diet from 

5 weeks of age in the F0 generation to 9 weeks of age in the F1 generation. A single dietary dose level 

of 0.03% was used, with control animals receiving untreated basal feed (n=20/sex/treatment group). 

At 9 weeks of age, 10 DEHP-treated females were paired with DEHP-treated males, 10 DEHP-treated 

females were paired with control males, 10 control females were paired with DEHP-treated males, 

and 10 control females were paired with control males. The females’ diet was available to males 

during the 5-day cohabitation phase. Females reared their own unadjusted litters, which were weaned 

at 4 weeks of age. One female and male from each litter were retained and fed their dam’s diet until 

9 weeks of age. 

All F1 offspring underwent neurobehavioral testing during the lactation period, including surface 

righting and negative geotaxis on PND 4 and 7, cliff avoidance on PND 7, swimming behavior on 

PND 4 and 14, and olfactory orientation on PND 14. Exploratory behavior was assessed in 1 male 

and 1 female from each litter at 3 weeks of age. Post-weaning tests included multiple-T water maze 

at 7 weeks of age and exploratory behavior at 8 weeks of age. Statistical analyses were performed 

using ANOVA or Kruskal-Wallis test followed by Bonferroni multiple comparison test. Proportions 

were evaluated using chi-squared or Fisher test. [It is not stated whether litter was considered in

the analysis of the preweaning neurobehavioral tests.]

Based on measured feed consumption, mean DEHP intake by treated males [rounded by CERHR]

was 46 mg/kg bw/day. Treated females received 53–57 mg/kg bw/day during the preconception period, 

~43 mg/kg bw/day during mating, 46 –49 mg/kg bw/day during gestation, and 154 –171 mg/kg bw/day 

during lactation. DEHP had no effect on feed consumption or dam body weight. As repeated in Section 

4.2.3, there were no significant treatment effects on the number of pregnant females, number of litters, 

number of offspring, average litter size or weight, or offspring sex ratio. Offspring body weight during 

the lactation period was similar between groups except for an 8% decrease in body weight on PND 14 

in female offspring when the parents both had received DEHP. The author did not consider this isolated 

alteration to be treatment related. Swimming ability was accelerated in PND 4 female offspring when 

the dam received DEHP. The number of movements in the test of exploratory behavior was decreased 

in male offspring the parents of which both received DEHP. There were isolated differences in T-maze 

performance by sex, trial, and treatment group that were not considered to represent treatment-related 

alterations in maze-learning. None of the other behavioral tests revealed effects of DEHP treatment.

The author concluded that “few adverse effects on several behavioral parameters were produced at 

the dose level of DEHP in the present study.”

Strengths/Weaknesses: Source and purity of test material were provided, and an appropriate route of 

exposure (diet) was used. The use of multigenerational exposure in mice from 5 weeks of age for the 

F0 generation through 9 weeks of age for the F1 generation encompassed the premating, gestation, 

lactation, and sexual maturation periods. The use of factorial design based on exposure of one sex bred 

with an unexposed or exposed mate was a strength. For the F0 generation animals, the post-weaning 

evaluations were controlled for litter effect by selecting one male or female per litter. Litter means 

were also used in the evaluation of pup weight and litter size. It is a weakness that only a single dose 

level was used, although it is a strength that this dose level was relatively low. Sample size was limited 

(8 –10 litters/group), resulting in low confidence in negative outcomes. For example, the author states 
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Appendix II

that no effect was observed on pup weight, with the exception of decreased female pup weights on 

PND 14 in group 4. However, the data suggest that decreased weight in both sexes at all preweaning 

time points would have been evident had a sufficient sample size been used. Sexually dimorphic 

behaviors were not evaluated. Other weaknesses in the presentation of data and conduct of behavioral 

testing are as discussed above for Tanaka (133).

Utility (Adequacy) for CERHR Evaluation Process: This study has limited use only for specific endpoints 

in which data were presented and litter effect controlled (litter size, mean pup weight, post-weaning 

exploratory behavior, and T-maze performance). None of these endpoints demonstrated a significant 

dose-responsive treatment effect; however, there is low confidence in these results due to use of a 

relatively small group sizes (8– 10 litters/group). All other endpoints on the F1 offspring are not useful 

for the evaluation due to deficiencies in statistical evaluation and/or lack of data presentation.

Lee et al. (138), supported by the South Korean Ministry of Environment, examined the expression 

of zinc-metabolizing enzymes in mouse dams and embryos exposed to DEHP. One hypothesis is that 

altered zinc homeostasis is a cause of teratogenicity following DEHP exposure. On GD 9 (9 days 

post-coitus), CD-1 mice were given corn oil (vehicle) or DEHP 800 mg/kg bw by gavage. Dams were 

killed at 3, 4.5, or 6 hours following exposure, and maternal liver, visceral yolk sac, and embryonic 

forebrain were collected. Polymerase chain reaction and Western blotting techniques were used to study 

expression of zinc-metabolizing enzymes in the collected tissues. Results were analyzed by Student 

t-test. Maternal liver expression of metallothionein (MT)-I and MT-II, enzymes that sequester zinc in 

liver and thus lower blood levels, were increased at 3.0–4.5 hours following DEHP exposure and then 

began returning to baseline levels at 6 hours following exposure. Maternal liver expression of zinc 

transporter-1 (ZnT-1), a transmembrane protein involved in zinc efflux, was not affected by DEHP 

exposure. Exposure to DEHP resulted in a down-regulation of MT-I, MT-II, and ZnT-1 expression in 

embryonic brain from 3 to 6 hours following exposure. There was no effect on visceral yolk sac. 

A dose-response study was conducted in which pregnant mice were gavaged on GD 9 with 0, 50, 200, 

or 800 mg DEHP/kg bw. Dams were killed, and maternal liver and embryonic brains were collected 

at 3 hours following exposure. According to the text of the study, up-regulation of MT-I and MT-II 

in maternal liver reached statistical significance at 200 mg/kg bw/day DEHP. [Table 1 of the study

indicates that the increase in MT-II expression reached statistical significance at 800 mg/kg bw.]

The study authors calculated a BMD5 of 6.7 mg/kg bw for MT-1 and 5.6 mg/kg bw for MT-II. BMDL5s 

(lower 95% confidence limit) were calculated at 3.7 mg/kg bw for MT-I and 3.2 mg/kg bw for MT-II. 

In embryonic brain, reductions in MT-I and ZnT-1 were significant at 200 mg/kg bw, and reductions 

in MT-II were significant at 50 mg/kg bw. Study authors calculated BMD5 responses of 11.6 mg/kg 

bw for MT-I, 8.9 mg/kg bw for MT-II, and 6.6 mg/kg bw for ZnT-1. BMDL5s (lower 95% confidence 

limit) were calculated at 7.1 mg/kg bw for MT-I, 5.2 mg/kg bw for MT-II, and 3.9 mg/kg bw for ZnT1. The study authors concluded that exposure of dams to DEHP during periods of organogenesis can 

alter the expression of key fetal enzymes involved in zinc homeostasis. 

Strengths/Weaknesses: This paper is based on a strong hypothesis and includes good presentation of 

methods and results. Multiple dose levels were evaluated, and a dose-response was demonstrated. The 

paper lacked direct measurement of zinc levels in sensitive tissue to correlate with the gene expression/protein data. MT-I/MT-II and ZnT-1 are not validated biomarkers for the endpoints of concern 
Appendix II

II-95

(affected by DEHP). Changes in gene expression alone are not adverse, may be adaptive, and must be 

linked directly to an adverse outcome, which was not done. The authors conclude “How this mechanism 

contributes to the overall developmental toxicity of phthalates in general and DEHP in particular 

remains to be further examined.” This statement clearly indicates the authors believe their information 

is useful for hypotheses generation, preliminary in nature, and in need of further development before 

it can be used directly in risk assessment. An additional limitation is the time of dosing, which is not 

the most critical period for phthalate adverse effects on male reproductive organ development.

Utility (Adequacy) for CERHR Evaluation Process: This paper is not directly useful in the evaluation 

process, although it provides good hypothesis-generating information for explaining potential 

mechanisms.

Rhee et al. (139), supported by the Korean Food and Drug Administration, evaluated the developmental effects of DEHP using in vitro tests. Whole embryo culture was performed by explanting 

GD 9.5 Wistar rat embryos into serum-based media treated with DEHP [purity notspecified] in 0.5% 

DMSO. DEHP concentrations were 1, 10, or 100 μg/mL [mg/L], n=15­ –28 embryos/concentration. 

Comparisons were made to untreated (n=35 embryos) and DMSO vehicle-treated (n=30 embryos) 

controls. Cultures were maintained for 48 hours, following which embryos were evaluated for yolk 

sac diameter, crown-rump length, head length, somite number, and Maele-Fabry morphology score. 

Data were analyzed using ANOVA with post hoc Bonferroni or Dunnett test. Yolk sac diameter, 

somite number, and Maele-Fabry score were significantly decreased at all concentrations of DEHP. 

Crown-rump and head length were decreased by the 2 highest concentrations of DEHP. The authors 

also performed micromass cultures using dissociated limb bud and midbrain cells from GD 12.5 

embryos. Cell suspensions were allowed to attach for 2 hours, following which they were exposed to 

DEHP at concentrations ranging from 7.81 to 1000 μg/mL for 96 hours. Cytotoxicity was determined 

using neutral red. Differentiation was determined in limb bud cells using Alcian blue uptake and in 

midbrain cells using hematoxylin staining followed by quantification of differentiated foci with an 

image analyzer. The planned endpoint was a comparison of the concentrations at which differentiation 

and cell survival were 50% inhibited compared to a control value. The authors reported that the planned 

inhibitory concentration comparison could not be carried out. [The authors do not give a reason for

not being able to report a result, but inspection of a graph in the paper suggeststhat a 50% effect

was not achieved for both cytotoxicity and differentiation in either the limb or midbrain culture

system.] The authors concluded that the absence of evaluable effect in the micromass assay in the face 

of prominent effects in the whole embryo culture system was consistent with lack of metabolism of 

DEHP to toxic intermediate(s) in the cell culture system.

Strengths/Weaknesses: The use of multiple in vitro screening methods and multiple exposure levels are 

strengths. Comparison to other phthalates was a strength, but failure to include “inactive” phthalates 

was a weakness. The assays are used for potential screening and mechanistic studies, however, rather 

than for risk assessment. No mention was made of how the embryos were assigned to groups and 

whether litter effect was controlled. There was no analytical support for metabolite formation in culture 

media to support the hypothesis on toxic metabolite formation in the whole embryo culture. 

Utility (Adequacy) for CERHR Evaluation Process: This paper is not useful in the evaluation process.
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Appendix II

3.2.4 	 Fish

Chikae et al. (140), support not indicated, evaluated the hatching, survival, and sex ratio of Japanese 

medaka (Oryzias latipes) exposed to DEHP [purity not given] in ethanol at nominal DEHP concentrations of 0, 0.01, 0.1, 1.0, and 10.0 μg/L. Final ethanol concentrations were <100 μg/L. Fish were 

exposed during the “embryo stage” [exact time of exposure not indicated]. Observations were made 

of hatching time, hatching success, mortality, body weight [age not specified, but possibly at 5 – 6

months,when the experiment wasterminated], sex ratio, and gonadosomic index [not defined; other

authors have used this term for relative gonad weight]. There were significant delays in hatching 

among the eggs exposed to DEHP 0.1 and 1.0 μg/L but not among the eggs exposed to DEHP 10.0 

μg/L. The percent of embryos showing eye development and the percent successfully hatching were not 

altered by DEHP exposure. Adult mortality was increased in the groups exposed to DEHP 0.01, 0.1, 

and 1 μg/L but not in the group exposed to DEHP 10.0 μg/L. Sex ratio was reduced (fewer males) in 

the 0.01 μg/L group only. Body weight was reduced in a DEHP concentration-dependent manner with 

significant difference from control at and above 0.1 μg/L. The authors concluded that DEHP exposure 

of Japanese medaka embryos “negatively affected some biological parameter[s] in both embryo and 

adulthood.” They acknowledged that the effects were not necessarily dose-dependent.

Strengths/Weaknesses: Multiple exposure levels allowed for a dose-response evaluation. The quantitative presentation of data was fairly good. There were clear effects on some endpoints, but changes 

in many parameters appear random and unrelated to dose. Findings were not interpreted in light of 

postulated mode of action in the development of mammalian males. Evaluation of embryo levels of 

the active fish androgen (11-ketotestosterone) would have been helpful.

Utility (Adequacy) for CERHR Evaluation Process: This paper is not useful in the evaluation process.

3.2.5	 Abstracts

CERHR retrieved several abstracts reporting developmental toxicity associated with DEHP exposure. 

Although information from abstracts is not considered by the Expert Panel in reaching final conclusions, 

the abstracts are briefly summarized for the sake of completeness. 

Borch et al.(141) gavage dosed pregnant Wistar rats on GD 7 – 21 with DEHP 0 or 750 mg/kg bw/day 

or with a combination of DEHP 750 mg/kg bw/day plus di(2-ethylhexyl)adipate 400 mg/kg bw/day. 

Testosterone production by offspring testes over a 3-hour ex vivo incubation period was reduced from 

2.9 ng in controls to 0.4 ng in the DEHP-exposed group and to 0.9 ng in the group exposed to DEHP 

plus di(2-ethylhexyl)adipate. Testicular testosterone was reduced from 1.8 ng/testis in controls to 0.4 

ng/testis in the group exposed to DEHP and 0.3 ng/testis in the group exposed to DEHP plus di(2-

ethylhexyl)adipate. Plasma testosterone was reduced in male offspring from 188 pg/mL in controls 

to 25 pg/mL in the group exposed to DEHP and 18 pg/mL in the group exposed to DEHP plus di(2-

ethylhexyl)adipate. There were no significant differences between the effects of DEHP alone and DEHP 

plus di(2-ethylhexyl)adipate. Similar treatment of dams during gestation and lactation resulted in no 

significant alterations in serum testosterone in male offspring evaluated on PND 27.

Phokha et al. (142) evaluated the toxicokinetics of DEHP and MEHP in pregnant and nonpregnant 

Sprague-Dawley rats. Nearly all orally administered DEHP was hydrolyzed in the intestine to MEHP. 

The AUC of DEHP was 0.7% that of MEHP. The AUC did not change in non-pregnant rats with 
Appendix II

II-97

repeated administration and was 950, 970, and 1070 nmol-h/L per mmol/kg dose on the first, fourth, 

and seventh consecutive days of treatment. In pregnant rats, administration on GD 14–19 resulted in 

AUC values (nmol-h/L per mmol/kg dose) of 1250 on GD 14 and 970 on GD 19.

Foster and Barlow (143) gavage dosed 10 pregnant Sprague Dawley rats per group on GD 12– 21 

with corn oil vehicle, 100 mg/kg bw/day DEHP, 100 mg/kg bw/day dibutyl phthalate, or 100 mg/kg 

bw/day DEHP in combination with 100 mg/kg bw/day dibutyl phthalate. Treatment with DEHP+dibutyl 

phthalate reduced anogenital distance on PND 1 and increased retained areolae on PND 13 in male 

offspring; there were no significant effects in rats treated with either phthalate alone. Only a small number 

of reproductive organ lesions were observed in the phthalate groups. There were no consistent changes 

in organ weights. The study authors concluded that the effects of the 2 phthalates were additive. 

Lambright et al. (144) treated pregnant Sprague-Dawley rats with DEHP 1000 mg/kg bw/day on 

GD 14 –18. Fetal testes were harvested on GD 18 and placed in culture for 3 hours. Testosterone and 

progesterone production in culture were decreased by gestational DEHP treatment.

Gray et al. (145) gavage dosed Sprague-Dawley rats with DEHP at 0, 11, 33, 100, or 300 mg/kg 

bw/day from GD 8 to PND 17. Dosing continued in half of the male offspring from PND 18 to 63–65, 

at which time they were necropsied. In those offspring necropsied on PND 63 – 65, liver and adrenal 

weights were affected at ≥11 mg/kg bw/day, puberty was affected at ≥100 mg/kg bw/day, and reproductive organ weights were affected at 300 mg/kg bw/day DEHP [no further details were provided

regarding effects]. The other half of male offspring were not dosed further after PND 17 and were 

necropsied upon reaching full maturity. Permanent effects noted in those offspring treated with 300 

mg/kg bw/day DEHP were reductions in anogenital distance, reduced reproductive organ weights, and 

increased incidence of nipples. Testicular and/or epididymal abnormalities were observed in 25% of 

offspring from the 300 mg/kg bw/day group; a low incidence of malformations was observed at the 

lower dose levels. 

Gray et al. (146) treated pregnant and lactating Sprague-Dawley rats with DEHP from GD 8 to day 

17 of lactation at dose levels of 0, 11, 33, 100, or 300 mg/kg bw/day, given by gavage. Dosing was 

continued in 2 or 3 males/litter from PND 18 until PND 63– 65. Puberty was delayed and males were 

heavier at the time of preputial separation in the 100 and 300 mg/kg bw/day groups. The authors 

concluded that DEHP is similar to dibutyl and benzyl butyl phthalate in exerting anti-androgenic 

effects on the developing androgen-signaling pathway.

Wilson et al. (147) treated pregnant Sprague-Dawley rats with oral DEHP 0, 100, 300, 600, or 900 

mg/kg bw/day on GD 8–18. GD-18 fetal testes were harvested and incubated for 3 hours in 500 μL 

medium, following which media were evaluated for hormone concentrations. Harvested testes were 

investigated using RT-PCR for assessment of gene expression. DEHP treatment resulted in a decrease 

in testicular testosterone production in the 300, 600, and 900 mg/kg bw/day groups and a decrease 

in testicular progesterone production in the 900 mg/kg bw/day group. Insl3 gene expression was 

decreased in a dose-dependent manner.

Hass et al. (148) treated pregnant and lactating Sprague-Dawley rats (n=8/group) with gavage doses 

of DEHP at 0, 10, 30, 100, 300, 600, and 900 mg/kg bw/day from from GD 7 to PND 17. Anogenital 
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Appendix II

distance was decreased and nipple retention increased at all DEHP doses compared to the control, 

although the anogenital distance difference from control was not statistically significant in the groups 

exposed to less than 300 mg/kg bw/day. The authors suggested that nipple retention may be a more 

sensitive indicator of anti-androgenic effects on development.

Numtip et al. (149) evaluated the toxicokinetics of DEHP in pregnant (n=4) and nonpregnant (n=8) 

marmosets. Animals were treated orally with 30 or 500 mg/kg bw/day for 29 days (GD 96 – 124). The 

AUCs for DEHP were about 20 times less than the AUCs for MEHP. The concentration-time curve 

for MEHP showed an oscillating pattern that the authors concluded represented enterohepatic cycling 

of MEHP glucuronide, which is not encountered in rats. Pregnancy did not alter the AUC of MEHP 

after the low dose of DEHP, but some reduction in MEHP AUC occurred in late pregnancy at the high 

DEHP dose. Maximum MEHP concentrations and AUCs were lower in pregnant marmosets than has 

been reported for pregnant rats.

Regnier (150) incubated GD 9 rat embryos for 48 hours in serum obtained from CD rats treated with 

1000 mg/kg bw DEHP on GD 6 – 11. The DEHP-exposed embryos had increased malformations and 

reduced crown-rump and head lengths, somite numbers, and morphological scores. In a second in vitro

experiment comparing embryotoxicity of DEHP and its metabolites, the order of potency (highest to 

lowest) was MEHP oxidized metabolites>DEHP=MEHP=2-ethylhexanol>2-ethylhexanoic acid. 

3.3 Utility of Developmental Toxicity Data

Since the initial CERHR evaluation of DEHP, 4 human studies were published that examined associations 

between in vivo DEHP or metabolite levels and adverse development outcome or premature thelarche. 

Another human study examined puberty in children who had received ECMO as neonates. Eight 

multiple dose-level animal toxicity studies have been published since the original DEHP evaluation. 

The studies focused on the effects of gestational or neonatal DEHP exposure on reproductive organ 

toxicity, pulmonary toxicity, and neurobehavioral endpoints. One study compared testicular toxicity 

following neonatal exposure to DEHP through the oral or IV route. Numerous studies focused on 

mechanistic aspects of DEHP-induced toxicity, such as effects on testosterone production, identification 

of target cells, interaction with the androgen receptor, and effects on zinc metabolism.

3.4 Summary of Developmental Toxicity Data

3.4.1 	 Human Data

Only 3 useful studies have been conducted among humans assessing developmental toxicity. Each 

study measured a different endpoint, and each had limitations. The Main et al. paper (17) suggested 

possible subtle effects in male infants associated with MEHP, and the Swan et al. paper (108) suggested 

subtle effects associated with the presence of MEHP metabolites. Replication of these studies with 

more extensive consideration of confounding and with larger sample sizes should be undertaken. 

Swan et al. (108) measured MEHP and its oxidative metabolites in the urine of pregnant women. 

Anogenital distance was evaluated at 2 –18 months of age in the male children born to these women. 

There was no significant association between maternal urinary MEHP concentration and infant 

anogenital index (anogenital distance adjusted for weight). The regression coefficients for 5-oxo- and 

5-OH-MEHP, while not significant, were of similar magnitude to regression coefficients for other 
Appendix II

II-99

phthalate monoesters (monobutyl, monoethyl, and mono-isobutyl phthalate) that were significantly 

associated with reduced anogenital index.

Main et al.(17) studied the association of breast milk levels of MEHP and other phthalates and cryptorchidism and blood levels of reproductive hormones in 3-month-old boys in Denmark and Finland. No 

association between phthalate exposure and cryptorchidism was found. Milk concentration of MEHP 

was observed to have a marginally significant correlation with free testosterone (Spearman r=−0.169, 

P=0.107) and inhibin B (r=0.185, P=0.075). This conclusion is tempered by concern about possible 

contamination by use of breast pumps and limited evaluation of confounders and effect modifiers.

Rais-Bahrami et al.(109) examined onset of puberty and sexual maturity parameters in 14 –16-yearold adolescents (13 males and 6 females) who had been potentially exposed to high DEHP levels as a 

result of receiving ECMO as neonates. Except for 1 female with Marfan syndrome, growth percentiles 

were normal for age and sex. Pubertal development was stated to be normal. Laboratory results 

indicated normal thyroid, liver, and renal function. LH, FSH, testosterone, and 17b-estradiol levels 

were normal for stage of pubertal development. No control children were evaluated, and exposure to 

phthalates was assumed rather than measured. While this study was supportive of no dramatic effect 

in ECMO-treated children, its small size and the wide range of normal values for adolescents limit 

the power to detect effects.

3.4.2 	 Experimental Animal Data

Experimental animal studies using multiple dose levels and thus providing dose-response information 

are summarized in Table 28.

In a number of studies, developing rats were exposed to DEHP and subsequently examined for effects on 

reproductive and endocrine systems. Five studies were notable, 4 that examined dose-related effects of 

DEHP exposure, and a fifth study that compared testicular toxicity in rats dosed by the oral versus the IV 

route. The remaining studies largely focused on mechanisms of developmental reproductive toxicity. 

Moore et al. (113) orally dosed at least 8 pregnant Sprague-Dawley rats/group with DEHP at 0, 375, 

750, or 1500 mg/kg bw/day from GD 3 (GD 1=day after sperm detected) to PND 21. Parameters 

associated with sexual development were observed through puberty or adulthood in male and female 

offspring, and male offspring were tested for sexual behavior. DEHP treatment reduced prenatal 

maternal weight gain at the middle and high dose. There was no significant effect on implantation 

sites. Number of pups born was reduced at the high dose, and postnatal survival was decreased at the 

middle and high dose. The most sensitive DEHP effect on males was an increase in areolae or nipples, 

which occurred at all dose levels and persisted through adulthood. Incomplete preputial separation, 

non-descent of testes, and agenesis of anterior prostate and other accessory reproductive organs 

did not attain statistical significance at the lowest dose level, but the authors considered them to be 

biologically significant at all doses due to the rarity of the effects. Reproductive effects observed in 

males exposed to higher doses included reduced anogenital distance, agenesis of seminal vesicle and 

epididymis, decreased sperm count, and reduced testis, epididymis, glans penis, prostate, and seminal 

vesicle weights that often persisted through adulthood. In female pups, DEHP treatment had no effect 

on anogenital distance. Body weight of high-dose females was 68% that of control body weight on 

the day of vaginal opening, and the effect was statistically significant. The study authors attributed 
II-100

Appendix II

the effect to DEHP-induced toxicity and not to an estrogenic effect. The study authors identified a 

LOAEL of 375 mg/kg bw/day for this study based on a significant decrease in anterior prostate weight 

and increase in permanent nipple retention.

Li et al. (119) examined dose-related effects of DEHP on neonatal rat gonocytes and Sertoli cells. 

Male Sprague-Dawley rat pups from 4 – 7 litters were pooled and randomly placed into groups of 4 

or 5 pups. On PND 3 (day of birth=PND 1), the rats were gavaged with DEHP at 0, 20, 100, 200, or 

500 mg/kg bw. Pups were killed 24 hours after dosing, and testes were collected for morphological 

examination and measurement of Sertoli cell proliferation through BrdU uptake. The time course 

of effects was examined in a second experiment in which rats were dosed with 0 or 200 mg/kg bw 

DEHP and examined between 6 and 48 hours following exposure. In rats treated with 100–500 mg/kg 

bw DEHP, there was a dose-related increase in abnormally large gonocytes containing 2– 4 nuclei. 

Multinucleated gonocytes were first detected at 12 hours following exposure to DEHP 200 mg/kg 

bw, and multinucleated gonocyte numbers increased with time. Sertoli cell proliferation was reduced 

in rats treated with ≥100 mg/kg bw DEHP. Sertoli cell proliferation rebounded at 48 hours following 

treatment. DEHP did not affect serum FSH levels. [Due to reporting deficiencies, only the Sertoli

cell proliferation data were considered to be of utility.]

Cammack et al. (120) examined reproductive development of Sprague-Dawley rats (n=16/group) 

treated IV or orally with DEHP. Beginning at 3– 5 days of age, rats were treated for 19– 21 days 

with DEHP at 0, 60, 300, or 600 mg/kg bw/day by IV infusion or 0, 300, 600 mg/kg bw/day by oral 

gavage. Seven rats/group were scheduled to be killed following the dosing period, and 9/group were 

scheduled to be held for a recovery period until 90 days of age. Histopathological analyses of prostate, 

seminal, vesicle, and epididymis and an evaluation of sperm count, motility, and morphology were 

conducted in the 90-day-old rats. Body weight gain was decreased in rats given 600 mg/kg bw/day 

by IV infusion and oral gavage. In animals killed immediately after the dosing period, absolute testis 

weight was significantly reduced at IV and oral doses ≥300 mg/kg bw/day. Absolute liver weight was 

increased in rats given ≥300 mg/kg bw/day DEHP by IV infusion. Depletion of germinal epithelium 

and/or decreased seminiferous tubule diameter was noted in all animals from the 300 and 600 mg/kg 

bw/day oral and IV dosing groups. Germinal epithelium depletion was rated as moderate (51 –75% 

reduction in thickness) in the 600 mg/kg bw/day oral group and mild (25–50% change) in all other 

groups given ≥300 mg/kg bw/day DEHP. Reduced tubule diameter was rated as mild (25 –50% reduction in diameter) in the 600 mg/kg bw/day oral group and minimal (<25% change) in the other groups 

treated with ≥300 mg/kg bw/day DEHP. In animals killed at 90 days of age, reduced testicular weights 

persisted in IV and oral groups given ≥300 mg/kg bw/day. The only persisting testicular lesion was 

a minimal (<25%) decrease in seminiferous tubular diameter in 2 of 5 rats in the 300 mg/kg bw/day 

oral group and 3 of 7 rats in the 600 mg/kg bw/day oral group. No prostate, epididymis, or seminal 

vesicles lesions and no adverse effects on sperm count, motility, or morphology were observed. 

A multigeneration DEHP toxicity study conducted in rats also provided some information on developmental toxicity (151). The study is described in detail in Section 4. Briefly, offspring of rats that 

were fed DEHP in diet at 3000 and 9000 ppm (340 and 1088 mg/kg bw/day) during gestation and 

lactation experienced an increase in stillbirth, an increase in PND 0 –4 pup mortality, retardation of 

F2 pup body weight, altered male anogenital distance, and retained nipples/areolae. A delay in sexual 

maturation was also noted in F1 offspring at the 9000 ppm exposure level.
Appendix II

II-101

The NTP (114) multigeneration continuous breeding study in rats evaluated effects of DEHP in feed 

at dose levels of 1.5 (control group exposed to background DEHP levels in feed), 10, 30, 100, 300, 

1000, 7500, and 10,000 ppm. Ranges of DEHP intake in the F0, F1, and F2 animals were estimated 

at 0.09 – 0.12, 0.47–0.78, 1.4– 2.4, 4.8–7.9, 14 –23, 46 –77, 392–592, and 543 –775 mg/kg bw/day. 

[Because developmental effects were reported, particularly on the male reproductive system,

the study is included in this section.This summary with additional details concerning the reproductive effects is also presented in Section 4.2.2.2.] The lowest dose level producing dose-related 

effects in breeding F1 offspring was 7500 ppm, and those effects included decreases in number of 

live pups/litter, reduced male anogenital distance, and delays in vaginal opening, preputial separation, and age of testicular descent. Additional effects noted in the F1 offspring from the 10,000 ppm 

group included decreased live pup weight at birth and during the lactation period and increased ratio 

of female anogenital distance to body weight. [The Expert Panel carefully considered the finding

of small reproductive organ sizes by gross observations in both F1 and F2 rats. The combined

F1 and F2 data were reviewed to determine the occurrence of these alterations on a per animal

and per litter basis across the dose range, as shown in Table 23. Based on the incidence of small

reproductive organ size at necropsy, the Expert Panel considered 300 ppm (about 14– 23 mg/kg

bw/day) to be an effect level, giving a NOAEL of 100 ppm, about 3 –5 mg/kg bw/day.]

A multiple dose study in rats by Shirota et al.(112) was designed to evaluate testicular pathology after 

intrauterine exposure to DEHP. Pregnant Sprague-Dawley rats were given gavage doses of DEHP in 

corn oil on GD 7–18 at 0, 500, or 1000 mg/kg bw/day in 1 experiment and 0, 125, 250, or 500 mg/kg 

bw/day in a second experiment. Decreased fetal weight and increased intrauterine mortality were 

noted at 1000 mg/kg bw/day. Postnatal findings included changes in pup weight at 250 and 500 mg/kg 

bw/day and increased incidences of multinucleated germ cells at ≥125 mg/kg bw/day and interstitial 

hyperplasia at 250 and 500 mg/kg bw/day.

Jarfelt et al. (117) evaluated the effects of perinatal exposure of groups of 20 Wistar rats to DEHP 

with or without diethylhexyl adipate. Timed-mated pregnant animals were treated by gavage from 

GD 7 to PND 1 with vehicle control, DEHP 300 mg/kg bw/day, DEHP 750 mg/kg bw/day, or DEHP 

750 mg/kg bw/day+diethylhexyl adipate 400 mg/kg bw/day (n=20/group). Litters were raised by their 

dams until weaning on PND 21, after which 1 male and 1 female per litter were retained. Recorded 

endpoints included anogenital distance on PND3, retention of nipples/areolae on PND13, onset of 

vaginal opening and balano-preputial separation, and epididymal sperm parameters and testicular 

histopathology on PND 190. Non-retained pups and dams were killed on PND 22 and evaluated for 

macroscopic lesions, and 3–5 males/litter underwent histopathologic/immunocytochemical examination 

of the testes. Increased postimplantation loss, reduced anogenital distance, and increased incidence of 

retained nipples were significantly different at all levels of DEHP exposure. There was also evidence 

of increased incidence of abnormal testes histology at both levels of DEHP exposure.

Effects noted in numerous single dose-level studies were consistent to those observed in the multiple 

dose-level studies summarized above. Although testicular weight and histology were not affected in 

offspring of rats treated with 100 mg/kg bw/day DEHP during pregnancy or lactation (111), treatment 

of rats with DEHP 750 mg/kg bw/day in late pregnancy and/or early lactation resulted in decreased 

testicular weights and testicular lesions in offspring (121, 123). Nipples and reduced anogenital 

distance were repeatedly observed in male offspring exposed to 750 mg/kg bw/day DEHP during 
II-102

Appendix II

gestation or lactation (115, 121). Additional observations in male offspring of rats dosed with 750 

mg/kg bw/day during late pregnancy and early lactation were lack of testicular descent, agenesis of 

accessory reproductive organs, and incomplete preputial separation (121). 

A number of studies examined mechanisms of DEHP toxicity. Single dose-level studies with exposures 

during gestation and/or lactation and examination of fetal or immature rats consistently demonstrated 

reductions in blood testosterone levels at ≥100 mg/kg bw/day DEHP (111, 115), Leydig cell testosterone production at ≥100 mg/kg bw/day (111), testicular testosterone content at ≥300 mg/kg bw/day 

(115, 123), and ex vivo testicular testosterone production at ≥750 mg/kg bw/day (115, 123, 124). One 

of the studies indicated that reductions in testosterone production observed shortly after exposure in 

neonatal or weanling rats were no longer present in adulthood (111). 

Evidence that DEHP targets Leydig cells, gonocytes, and Sertoli cells was noted following gestational 

and lactational exposure of rats to ≥100 mg/kg bw/day DEHP (111, 119, 123). MEHP, but not 2-ethylhexanol, was found to cause increases in large multinucleated gonocytes and to inhibit Sertoli cell 

proliferation (119). An in vitro study demonstrated that gonocytes and Sertoli cells are susceptible to 

MEHP-induced toxicity during periods of proliferation (130). In 1 study, DEHP doses ≥750 mg/kg 

bw/day during gestation in rats reduced testicular expression of insulin-like hormone 3, a hormone 

produced by Leydig cells and possibly involved in development of the gubernaculum (124). 

Liu et al.(125) evaluated gene expression profiles in the GD 19 fetal testis after GD 12–19 gavage treatment of dams with 1 of 7 phthalates (n=5/group) or with corn oil vehicle (n=10; vaginal sperm=GD 0). 

The phthalates were DEHP or diethyl, dimethyl, dioctyl tere-, dibutyl, dipentyl, or benzyl butyl phthalate 

at a dose level of 500 mg/kg bw/day. On GD19, pups were evaluated for anogenital distance and testes 

were processed for gene expression profiles for 3 pups/treatment group, each from a different litter. 

Anogenital distance was significantly reduced by pregnancy treatment with DEHP, dibutyl phthalate, 

benzyl butyl phthalate, and dipentyl phthalate. Dimethyl, diethyl, and dioctyl terephthalate did not 

affect anogenital distance. Of 391 significantly altered gene probe sets, there were 167 characterized 

sequences. Genes related to lipid, sterol, and cholesterol homeostasis accounted for 31 of these 167 

genes. There were also 10 genes involved in lipid, sterol, and cholesterol transport, 12 genes involved 

in steroidogenesis, 9 transcription factor genes, 22 signal transduction genes, 11 genes involved in 

oxidative stress, and 13 genes related to the cytoskeleton. In general, there was a similar pattern of 

gene expression profile with those phthalates that altered anogenital distance as compared those that 

did not, suggesting that these phthalates operate by a common mode of action on the developing testes. 

Targeted pathways were directly or indirectly related to Leydig cell production of testosterone and 

pathways important for Sertoli cell-gonocyte interaction.
Appendix II

II-103

Table 28. Summary of DEHP Effects on Developmental Toxicity

Species and 

Strain Treatment

Effect Levels (mg/kg bw/day)

NOAEL Maternal LOAEL Developmental Reference LOAEL BMDLa

Sprague-DawleyRat

Oral0, 375, 750, or 1500 mg/kg bw/day GD 3 to PND 21.

Maternal: 375Developmental: NAb

750(↓prenatal weight gain)

375 (↑in areolae or nipples, 

incomplete preputial separation, non-descent of testes, and agenesis of anterior prostate)

10% Level: 167 (↓epididymal sperm)

1 SD Level: 135 

(PND 105 epididymis weight)

Moore et al. 

(113)

Sprague-DawleyRat

Gavage0, 20, 100, 200, or 500 mg/kg bw PND 3 (Day of birth=PND1)

Developmental: 20 NA 100 

(↓Sertoli cell pro­lifera­tion)

Li et al. (119)

WistarRat

Feed0, 1000, 3000, and 9000 ppm(0, 113, 340, and 1088 mg/kg bw/day) During gestation and lactation

Maternal: 340Developmental: 113

1088(↓feed intake and body weight gain)

340 (↑pre- and postnatal mortality, ↓body weight, altered male anogenital distance, and retained nipples/areolae)

10% Level: 231 (F2 pup survival on 

PND 0–4)

Schilling et 

al. (151)

Discussed in 

Section 4.

Sprague-DawleyRat

Gavage0, 300, or 600 mg/kg bw/day19–21 days beginning at 3–5 days of age. 

Examination immediately after dosing: Cammack et 

al. (120) NA 300 

(depletion of germinal epithelium and/or ↓seminiferous tubule diameter and ↓absolute testes weight)

10% Level: 77.4

1 SD Level: 628 

(testis weight) 

Examination at 90 days of age:Developmental: <300 300 

(↓in seminiferous tubular diameter, ↓absolute testis weight)

10% Level: 125

1 SD Level: 65
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Appendix II

Species and 

Strain Treatment

Effect Levels (mg/kg bw/day)

NOAEL Maternal LOAEL Developmental Reference LOAEL BMDLa

Sprague-DawleyRat

IV0, 60, 300, or 600 mg/kg bw/day 21 days beginning at 3–5 days of age.

Examination immediately after dosing: Cammack et 

al. (120) Developmental: 60 300 

(depletion of germinal epithelium and/or ↓seminiferous tubule diameter, ↓absolute testes weight, and ↑absolute liver weight)

10% Level: 106

1 SD Level: 125 

(testis weight)

Examination at 90 days of age:Developmental: 60 300 

(↓absolute testis weight)

10% Level: 190

1 SD Level: 105 

(testis weight)

Sprague-DawleyRat

Feed1.5, 10, 30, 100, 300, 1000, 7500, or 10,000 ppm(0.09– 0.12, 0.5 – 0.8, 1.4–2.4, 4.8–7.9, 14 –23, 

46–77, 392 –592, and 543– 775 

mg/kg bw/day)

Study authors:

Maternal: 46–77Development: 46–77

Study authors:

392–592

(body weight changes)

Study authors:

392 – 592 

(Pregnancy indices, litter data)

10% Level: 33–56 1 SD Level: 45–75 (F3 sperm/cauda)

The National 

Toxicology 

Program 

(114)

Expert Panel:

Maternal: 46–77Developmental: 3–5

Expert Panel:

392–592 

(↓body weight gain)

Expert Panel:

14 – 23 

(combined F1 and F2gross observations of small reproductive organs)

Sprague-DawleyRat

Gavage 500, and 1000 (Experiment 1) 0, or 0, 125, 250, and 500 mg/kg bw/day (Experiment 2) GD 7–18

Maternal: 500 Developmental: 500

1000 (↓maternal body weight)

1000(↑intrauterine mortality, ↓live fetuses/litter).

10% Level: 334 

1 SD Level: 490 

(↓live fetuses)

Shirota et al. 

(112)
Appendix II

II-105

Species and 

Strain Treatment

Effect Levels (mg/kg bw/day)

NOAEL Maternal LOAEL Developmental Reference LOAEL BMDLa

WistarRat

GavageGD 7–17 at 0, 300, or 750 mg/kg bw/day.

Maternal: ≥750Developmental: NA

NA 300 

(postnatal death, male anogenital distance, retained nipples, abnormal testis histology)

10% Level: 13 (postimplantation loss)1 SD Level: 179 (male anogenital distance)

Jarfelt et al. 

(117)

WistarRat

Gavage0, 300, or 750 mg/kg bw/dayGD 7–PND 17

Maternal: NDDevelopmental: 300

NDc 750 

(plasma hormone changes)

Not calculable from data in study report.

Borch et al. 

(115)

Gavage 0, 300, or 750 mg/kg bw/dayGD 7–PND 17

Maternal: NDDevelopmental: <300

ND 300 

(↑apoptosis in fetal tissue)

Not calculable from data in study report.

Borch et al. 

(118)

CD-1 Mouse Feed 

0, 17, 47, and 140 mg/kg bw/day during gestation, 0, 60, 172, and 493 mg/kg bw/day During lactation0, 16 –19, 48– 56, and 145 –171 mg/kg bw/day From weaning to 9 weeks of age in F1offspring.

Maternal: 140–493Developmental: 47–172 

NA 140–493 

(↓survival during lactation period)

Tanaka (133)

aSee the footnote to Table 20 for definitions and a discussion of the use of benchmark dose in this report.bNA: Not applicable; cND: Not Determined
II-106

Appendix II

Conclusions Based Only on Literature Appearing 

Since the First Expert Panel Report

There is insufficient evidence in humans that DEHP causes developmental toxicity when

exposure is prenatal. While there was one human study (108) judged to be useful, it was not 

sufficient to draw conclusions regarding developmental toxicity following prenatal exposure. The 

study found no significant association between maternal prenatal urinary MEHP and anogenital 

index in male offspring, and the interpretation of this novel index as applied to humans has not 

been established. 

There is insufficient evidence in humans that DEHP causes developmental toxicity when

exposure is during childhood. While there were two human studies judged to be useful, they 

were not sufficient to draw conclusions regarding developmental toxicity. One study (109) had 

very small sample size with no measurement of exposure. The other study (17) was limited in 

size and by the possibility of contamination by breast pump use.

There issufficient evidence that DEHP exposure in rats causes developmental toxicity with

dietary exposure during gestation and/or early postnatal life at 14 – 23 mg/kg bw/day as

manifested by small or absent male reproductive organs (114). There were multiple other 

studies supporting effects on the developing male reproductive tract at higher dose levels. The 

critical period for effects on the testes extends into the immediate postnatal period (120) with 

decreased Sertoli cell proliferation seen in male rats exposed by oral gavage to DEHP 100 

mg/kg/day on PND 3 (119). 

There is sufficient evidence that DEHP causes developmental toxicity with 21 days of IV

exposure starting at PND 3 – 5 at 300 mg/kg/day as manifested by decreased testes weight,

depletion of germinal epithelium, and decreased seminiferous tubule diameter (120). The 

reduced testicular weights persisted through at least 90 days of age. These findings are consistent 

with those observed after oral exposure. 

These data are assumed relevant to assessment of human risk.

NOTE: The definitions of the term sufficient and the terms assumed relevant, relevant, and not 

relevant are in the CERHR guidelines at http://cerhr.niehs.nih.gov/news/guidelines.html.
Appendix II

II-107

Conclusions from the original Expert Panel 

evaluation 

The original Expert Panel report on DEHP contained conclusions about developmental toxicity 

in Section 5. These conclusions have been extracted and reproduced below, with the section 

numbering as found in the original document. The references listed in the conclusion are listed, and 

the table to which the conclusions refer is reproduced, numbered Table 71 as in the original.

5.1.3

There were no studies located on the developmental toxicity of DEHP or its metabolites in 

humans.

5.1.3.1

Developmental toxicity findings were remarkably consistent. DEHP was found to produce malformations, as well as intrauterine death and developmental delay. The pattern of malformations seen 

in fetuses is consistent across studies. It included morphological abnormalities of the axial skeleton 

(including tail), cardiovascular system (heart and aortic arch), appendicular skeleton (missing 

limb bones, finger abnormalities), eye (including open eye), and neural tube (exencephaly).

In general, across studies there was not a strong relationship between the type and amount of 

maternal toxicity and developmental toxicity.

In addition to the studies of developmental toxicity with post-conception exposure discussed 

above, developmental toxicity was also manifested in reproductive toxicity studies . . .. The 

database as a whole identified CD-1 mice as the most sensitive species for DEHP developmental 

toxicity via the oral route. The critical papers are [149,150,168,186]. LOAELs and NOAELs for 

some relevant developmental toxicity studies are presented in Table 71. Developmental effects 

in reproduction studies are listed in Table 76 [See Section 4 of this report.] Studies that address 

developmental toxicity are consistent in identifying the lower effective range of oral exposure, 

taking into account differences in duration of treatment.

The DEHP database contains four rat studies conducted by a route other than oral: an IV study, two 

IP studies, and an inhalation study. These studies provide valuable information but do not contain 

enough data for separate route-specific hazard identification and NOAEL/LOAEL selection. 

The panel is not confident that the lowest dose has been established at which developmental 

toxicity (the development of the male reproductive system) occurs.

5.1.3.6

The database provides adequate information to identify DEHP as a developmental toxicant by 

the oral route and for identification of NOAELs and LOAELs for dose–response assessment. 
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Appendix II

The data are also sufficient to identify the metabolites (MEHP, 2-EH, 2-EHA) as developmental 

toxicants. However, there are not enough studies for independent hazard identification and 

dose –response assessment for the parenteral route. Because of the known role of intestinal 

lipase in DEHP metabolism, it is not possible to readily generalize dose–response assessment 

from the oral to intravenous route. Existing PBPK models do not include fetal compartments; 

and hence have limited use at present.

5.2 

As will be discussed below, there are sufficient data in rodents to conclude confidently that oral 

exposure to DEHP can cause reproductive and developmental toxicity in rats and mice. Further, 

an effect observed in rats involves adverse effects on the development, structure, and function 

of the male reproductive tract. Thus, for DEHP, the effects on reproduction and development 

are intertwined.

The developmental toxicity database contains well-conducted and reported studies, many available as full GLP study reports, and additional, more restricted studies that provide supplemental 

and supportive information. The database is somewhat limited in that it consists almost entirely 

of studies in rats and mice orally exposed during gestation where effects are seen by examining 

physical development of rodent pups just prior to birth (i.e. prenatal assessment). These studies 

indicate that a range of effects may occur, including malformations (tail malformations, axial 

and appendicular skeletal abnormalities, cardiovascular malformations, and neural tube closure 

defects), developmental delays, and intrauterine death. The NOAEL based on malformations 

in rodents was ~40 mg/kg bw/day and a NOAEL of 3.7 –14 mg/kg bw/day was identified for 

testicular development/effects in rodents. In contrast, functional reproductive endpoints that are 

evaluated through postnatal observation have not been adequately studied. This is a significant 

data limitation. There are a limited number of studies by the inhalation, dermal, and intravenous 

administration routes. It was noted that results are consistent across studies, taking into account 

doses, route, species, timing,

The examination of effects during the late gestational and neonatal periods is quite recent and 

incomplete. Despite the general belief among expert panel members that this represents a time 

of potentially high sensitivity to DEHP-induced disruption of the reproductive system, the 

dose –response relationships for reproductive effects following exposures in gestational versus 

postnatal ages are unknown. Low-dose studies examining sensitive endpoints following late 

gestational exposure are a critical data need.

There is a study that demonstrated the same spectrum of developmental toxicity (as seen in 

‘normal’ mice) in mice that were genetically incapable of expressing peroxisome proliferation 

due to lack of PPAR-alpha. 

DEHP data from rats and mice are assumed relevant to judging hazard to human reproduction 

and development; they are the standard mammalian test systems used.
Appendix II

II-109

References From the Original report cited above

[146] Price CJ, Tyl RW, Marr MC, Myers CB, Sadler BM, Kimmel CA. Reproduction and 

fertility evaluation of diethylhexyl phthalate (CAS No. 117-81-7) in CD-1 mice exposed during 

gestation. Research Triangle Park, NC: National Toxicology Program, 1988.

[149] Tyl RW, Price CJ, Marr MC, Kimmel CA. Developmental toxicity evaluation of 

dietary di(2-ethylhexyl)phthalate in Fischer 344 rats and CD-1 mice. Fundam Appl Toxicol 

1988;10:395– 412.

[150] Huntingdon HLS-A. Phthalic acid, di(2-ethylhexyl) ester (DEHP): study of embryo-foetal 

toxicity in the CD-1 mouse by oral gavage administration. Report no.: 95/EHM007/0705, 

1996.

[156] Price CJ, Tyl RW, Marr MC, Sadler BM, Kimmel CA. Reproduction and fertility evaluation of diethylhexyl phthalate (CAS No. 117-81-7) in Fischer 344 rats exposed during gestation 

NTP 86-309. Research Triangle Park, NC: National Toxicology Program, 1986.

[168] Lamb IV JC. Reproductive effects of four phthalic acid esters in the mouse. Toxicol Appl 

Pharmacol 1987;88:255 –69.

[185] Tyl R, Price CJ. Teratological evaluation of diethylhexylphthalate (CAS No. 117-81-7) in 

CD-1 mice. In: Jefferson AR, editor i.n National Center for Toxicological Research, 1984.

[186] Reel JR, Tyl RW, Lawton AD, Jamb JC. Diethylhexyl phthalate (DEHP): Reproduction 

and fertility assessment in CD-1 mice when administered in the feed. PB84-181734. 

Springfield, VA: available from: NTIS, Research Triangle Park: National Toxicology 

Program,1984.
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Appendix II

Table 71. Summary of DEHP Effects in Developmental Toxicity Studies 

with Oral Exposure

Protocol & Doses NOAEL 

(mg/kg bw/day)

LOAEL (mg/kg bw/day) Fetal Effects at Maternal Higher Doses Developmental 

Prenatal feeding study 

in CD-1 mice. 30/group 

received 0, 44, 91, 191, 

or 293 mg/kg bw/day on 

GD 0−17. Dams and pups 

examined in late gestation.

[149, 185]*

Maternal: 44

Developmental: 44

91 

Clinical signs

↓ Weight gain

91

↑Skeletal, 

visceral, and 

external malformations

↑ Skeletal, visceral, 

and external 

malformations

↑ Prenatal mortality

↓ Fetal weight

Prenatal gavage study 

in CD-1 mice. 14 /group 

received 0, 40, 200, or 

1,000 mg/kg bw/day on 

GD 6−15. Dams and pups 

examined in late gestation.

[150]*

Maternal: 200

Developmental: 40

1000 

↑ Liver weight

↓ Weight gain

200

↑ Visceral 

and external 

variations and 

malformations

↑ Skeletal, visceral, and 

external variations and 

malformations

↑ Prenatal mortality

Prenatal feeding study in 

Fischer 344 rats. 20/group 

received 0, 164, 313, or 573 

mg/kg bw/day on GD 0−20. 

Pups evaluated postnatally.

[156]*

Maternal: 164

Developmental: 164

313

↓ Food Intake

313

↑ Prenatal 

mortality

↑ Prenatal mortality

↓ Pup body weight on 

PND 1 only

Prenatal feeding study in 

CD-1 mice. 28/group received 0, 19, 48, or 95 mg/

kg bw/day from GD 0−17. 

Pups evaluated postnatally.

[146]*

Maternal: 95

Developmental: 48

No higher 

doses

95 

↑ Prenatal 

mortality

↓ Pup survival 

on pnd 4

No higher doses

*Doses calculated by study authors.
Appendix II

II-111

4.0 REPRODUCTIVE TOXICITY DATA

4.1 Human Data

Since the initial CERHR Expert Panel Report on DEHP, human studies have evaluated measures of male 

reproductive function and endometriosis in females in association with estimates of DEHP exposure.

Modigh et al. (152), supported by the Swedish Environmental Protection Agency and Swedish Work 

Environment Fund, evaluated time-to-pregnancy in the partners of men potentially exposed to DEHP. 

Men employed or the partners of women employed in 1 of 3 plants were invited to participate. Among 

the 284 men identified as eligible, 234 responded. The responders had produced 397 pregnancies. After 

excluding pregnancies for which information was unavailable, 326 pregnancies were available for 

analysis. Information on time-to-pregnancy was obtained in a telephone or written interview in which 

couples were asked how many months they had unprotected intercourse prior to achieving pregnancy. 

Pregnancies were counted if the couple was attempting to become pregnant or not attempting but not 

avoiding pregnancy. Only pregnancies ending in 1987 or later were counted. Information was accepted 

from either partner; the woman’s answer was used if there was disagreement. Exposure was estimated 

from employed subjects’ description of work tasks and measurements that had been made in each of 

the 3 workplaces during the general time period relevant for the pregnancies. Exposure categories were 

created as follows: unexposed pregnancies (n=182) were fathered by operators who were not exposed 

during the time leading up to pregnancy, by office staff, or unexposed male partners of female workers; 

low-exposure pregnancies (n=100) were fathered by men with estimated non-zero DEHP exposures 

<0.1 mg/m3; and high-exposure pregnancies (n=44) included 25 pregnancies fathered by men exposed 

to DEHP 0.1–<0.2 mg/m3, 15 pregnancies fathered by men exposed to DEHP 0.2–<0.5 mg/m3, and 

4 pregnancies fathered by men with DEHP exposures of 0.5 mg/m3 or higher. The highest estimated 

mean DEHP exposure level was 1.9 mg/m3. To account for possible effects at any time during the 70-day 

period of spermatogenesis, exposures were evaluated 1, 2, and 3 months prior to the month of attempted 

pregnancy. Fecundability ratios calculated from a Cox proportional hazards model were estimated using 

the unexposed pregnancies as the referent. A binomial regression model was used to control for potential 

confounders (father’s age, mother’s age, and length of time to recall). An additional analysis was conducted 

using only the first pregnancy from couples for which more than 1 pregnancy was available.

Median time-to-pregnancy was 3.0 months in the unexposed group, 2.25 months in the low-exposure 

group, and 2.0 months in the high-exposure group. The crude and adjusted fecundability ratios for 

the exposed pregnancies were all close to 1.0, and the 95% confidence intervals all overlapped unity. 

There was no significant effect of restricting the analysis to the first pregnancy of couples with more 

than 1 pregnancy or of excluding pregnancies conceived by employed women. Excluding couples with 

known fertility problems did not influence the findings. The results did not depend on whether exposure 

status was used for the month under consideration or lagged 1, 2, or 3 months. The authors concluded 

that there was no evidence of a DEHP-associated prolongation in time-to-pregnancy, although they 

recognized that there were few highly exposed men in their sample; the mean DEHP exposure level 

for men in the study was less than 0.5 mg/m3.

Strengths/Weaknesses: Time-to-pregnancy can be a sensitive marker of reproductive impairment. The use 

of unexposed men from the same workplaces as referents is a strength. Measurements of exposure were 

objective and independent of self-reports of work tasks and locations. The study considered numerous 
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Appendix II

potential confounders. Weaknesses include the small number of highly exposed men and measurement 

error involved in the retrospective assessment of paternal exposure. The use of only men who fathered 

pregnancies is a limitation of the retrospective time-to-pregnancy assessment. Other weaknesses include 

the low response rates and the inability to mask participants to exposure and outcome status.

Utility (Adequacy) for CERHR Evaluation Process: This paper is useful in the evaluation process.

Rozati et al. (153), support not indicated, measured phthalate esters in the seminal plasma of 21 men 

with unexplained infertility. The men were male partners in couples presenting for infertility evaluation 

[not otherwise defined]. All subjects had a sperm concentration <20 million/mL, rapidly progressive 

motility <25%, total progressive motility <50%, or <30% normal forms. Sperm concentration was 

assessed using a hemocytometer, and morphology was assessed after Papanicolaou staining. [Details

on motility evaluation were not provided.The number of semen samples per subject was not indicated.] Additional testing included eosin-nigrosin staining to determine vitality, hypo-osmotic swelling 

test, chromatin decondensation after treatment with SDS and EDTA, and chromatin susceptibility to 

acid denaturation, determined with acridine orange staining and fluorescent microscopy. Seminal fluid 

concentrations of phthalate esters [as a group] were assessed by HPLC, using a commercial phthalate 

esters mixture as a standard. [According to the manufacturer’s web site,this mixture contains 0.2%

each DEHP, di-n-octyl, dimethyl, diethyl, di-n-butyl, and benzyl butyl phthalate in hexane (http://

www.sigmaaldrich.com/cgi-bin/hsrun/Suite7/Suite/HAHTpage/Suite.HsSigma AdvancedSearch.

formAction,accessedApril 27,2005).]Comparison was made to seminal plasma phthalate concentrations 

in a control group of 32 men with evidence of conception and normal semen analysis [not otherwise

characterized] using Student t test. Correlation between seminal phthalate ester concentration and 

individual sperm test results was evaluated using linear regression analysis. [Regression terms were

not specified. Polychlorinated biphenyl concentration was also evaluated in seminal plasma. It is

not stated whether the regression analysis adjusted for polychlorinated biphenyl concentration.]

The mean±SD seminal plasma phthalate ester concentration in the infertile group was 2.03±0.214 

μg/mL, compared to 0.06±0.02 μg/mL in the control group (P<0.05). There was a significant inverse 

correlation between seminal phthalate ester concentration and normal sperm morphology (r=– 0.769, 

P<0.001) and a positive correlation between seminal phthalate ester concentration and the percent 

acid-denaturable sperm chromatin (r=0.855, P<0.001). There was no significant correlation between 

seminal phthalate ester concentration and ejaculate volume, sperm concentration, progressive motility, 

sperm vitality, sperm osmoregulation, or sperm nuclear chromatin decondensation.

The authors concluded that adverse effects on fertility of phthalate esters, which they called xenoestrogens, were consistent with published data on male reproductive toxicity of these compounds.

Strengths/Weaknesses: There was extensive reproductive assessment of cases, but the sample size was 

small, and there was very little information on the selection of controls for infertile cases. There was 

very limited assessment of possible confounders (mean age, urban/rural, fish consumption) and no 

evidence that exposure assessment was carried out blind to case/control status of participants.

Utility (Adequacy) for CERHR Evaluation Process: This report is of limited usefulness in the evaluation 

process.
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Duty et al. (154), supported by NIEHS, evaluated urinary MEHP and semen analysis parameters. 

Subjects included 168 men being evaluated in a clinic as part of a fertility evaluation. A questionnaire 

was used to obtain information on lifestyle factors. A single semen sample was produced by masturbation 

after instructions to abstain from ejaculation for 48 hours. Sperm concentration and percent motility 

were assessed using computer-assisted sperm analysis, and morphology was evaluated by light 

microscopy of air-dried smears after application of a commercial stain (Diff-Quik). A single spot urine 

was collected on the same day as the semen sample and analyzed using HPLC with tandem MS for 

MEHP and for monoethyl, monomethyl, mono-n-butyl, monobenzyl, mono-n-octyl, mono-isononyl, and 

monocyclohexyl phthalates. Urine phthalate concentrations were adjusted based on urine specific gravity 

and were dichotomized as high or low based on median values. Sperm parameters were dichotomized 

based on published norms. Abnormal sperm concentration was <20 million/mL, abnormal motility 

was <50% motile, and abnormal morphology was <4% normal forms. Mantel-Haenszel chi-squared 

test was used to assess the relationship between high/low phthalate concentration in the urine and 

normal/abnormal semen parameter. Multivariate logistic regression was used to adjust for smoking 

status, age, race, body mass index, and abstinence time.

The study population included 28 men (17%) with low sperm concentration, 74 men (44%) with <50% 

motility, and 77 men (46%) with >4% normal forms. There were 77 men (46%) who were normal in 

all 3 domains. There were no significant associations between abnormal semen parameters and MEHP 

urine concentration above or below the group median. [Associations were identified and explored

with respect to monobutyl, monobenzyl, and monomethyl phthalate, which are not discussed

here.] The authors did not present conclusions relative to MEHP. In this paper and in the following 

papers from this group, the authors discussed limitations of their methods:

Use of single spot urines. The authors indicated that because phthalates are rapidly eliminated, use 

of a single spot urine assumes steady state exposure from food and personal care products.

Adjustment of urine phthalate concentrations using specific gravity. The authors acknowledge 

that creatinine is often used to adjust urine concentrations, but argue that use of creatinine may 

not be appropriate for compounds that are not excreted through glomerular filtration. They 

further note the dependence of creatinine secretion on muscle mass, physical activity, time of 

day, diet, urine flow, and disease states.

Use of a fertility population. The authors expressed doubt that a fertility sample would include 

men that necessarily differ from men in the general population in their testicular response to 

phthalate exposure.

Strengths/Weaknesses: The detection of phthalate metabolites in urine eliminated contamination issues. 

There was good evaluation of confounders, but the use of a subfertile population is a limitation, and 

the use of only one semen sample per individual is also a limitation.

Utility (Adequacy) for CERHR Evaluation Process: This study is useful in the evaluation process.

Duty et al. (30), supported by NIEHS, evaluated urinary MEHP and sperm motion parameters by 

computer-assisted sperm analysis. Subjects were the male partners in couples presenting for fertility 

evaluation, without regard to whether the male had a fertility problem. Of the 259 men who agreed 

to participate, 234 provided a urine sample and a semen sample. [It is assumed, though not stated,

•

•

•
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Appendix II

thatsome of these subjects were also reported in Duty et al.(154).] Thirteen semen samples did not 

contain motile sperm, and 1 semen sample was not submitted for computer-assisted analysis, leaving 220 

subjects with motile sperm measurements and spot urine samples. After elimination of urine samples 

with specific gravities below 1.010 or above 1.030, 187 semen-urine pairs remained for evaluation. 

Urine samples were frozen for subsequent phthalate monoester analysis by HPLC and tandem MS. 

The phthalate monoesters included the monoethyl, monomethyl, mono-n-butyl, monobenzyl, monon-octyl, mono-isononyl, and monocyclohexyl, as well as MEHP. Urinary phthalate determinations 

were normalized using specific gravity. Multiple linear regression analysis was used to evaluate the 

association between tertile of normalized urinary MEHP concentration and sperm motion parameter. 

Covariates included in the model were smoking status, race, age, body mass index, and abstinence 

interval prior to collection of semen. 

Subjects had a mean±SD age of 36.3±5.6 years. Mean±SD sperm concentration was 115.6±99.2 

million/mL with 13.2% of samples having a sperm concentration <20 million/mL. Mean±SD percent 

sperm motility was 52.2±22.6% with 41.8% having <50% motile sperm. Mean±SD percent normal 

morphology was 7.4±4.6% with 22.3% having <4% normal forms. [The cut-offs representing the

norms cited in Duty et al. (154).] The authors stated that there was evidence of a dose-response 

relationship with respect to MEHP and straight-line velocity, curvilinear velocity, and linearity with 

P values for trends of 0.1 – 0.3. They further stated that use of quartiles instead of tertiles, use of 

phthalate concentration as a continuous parameter, and use of actual phthalate concentrations rather 

than concentrations adjusted for specific gravity produced results that were consistent with the initial 

analysis. [Data and analyses were not shown.]

The authors concluded that there was an overall pattern of decline in straight-line velocity, curvilinear 

velocity, and linearity, which was identified with mono-n-butyl and monobenzyl phthalate as well as MEHP. 

They postulated that the lack of statistical significance may have reflected the relatively small sample 

size. They indicated that if phthalates were associated with sperm motion abnormalities, their study may 

have under-ascertained the effect because immotile sperm did not give rise to motility parameters.

Strengths/Weaknesses: The detection of phthalate metabolites in urine eliminated contamination issues. 

There was good evaluation of confounders, but the use of a subfertile population is a weakness.This report 

appears to include the same subjects as Duty et al. (154) and cannot be considered independent.

Utility (Adequacy) for CERHR Evaluation Process: This study is useful in the evaluation process.

Duty et al. (155), supported by NIEHS, evaluated a possible association between urinary phthalate 

monoester concentrations and sperm DNA damage, assessed using the neutral comet assay [so named

because fragmented DNA streams away from the main cell body on electrophoresis, producing

a visual image that looks like a comet]. The subjects and samples were as described in Duty et al. 

(30), although the number of samples tested was lower (n=141). Semen samples were frozen prior 

to comet assay. Urinary phthalate monoester concentrations were adjusted for specific gravity and 

analyzed in quartiles using multiple linear regression adjusted for smoking status, race, age, body 

mass index, and abstinence interval prior to collection of semen. There were no significant associations between comet assay parameters and MEHP urinary concentrations. [Significant associations

were identified only for monoethyl phthalate.] Inclusion of urine samples that had been excluded 
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II-115

based on specific gravities below 1.010 or above 1.030 did not change the results. The authors did not 

express conclusions relative to MEHP.

Strengths/Weaknesses: The detection of phthalate metabolites in urine eliminated contamination issues. 

There was good evaluation of confounders, but the use of a subfertile population is a weakness.This report 

appears to include the same subjects as Duty et al. (154) and cannot be considered independent.

Utility (Adequacy) for CERHR Evaluation Process: This study is useful in the evaluation process.

Duty et al. (156), supported by NIEHS, evaluated the relationship between serum concentrations 

of testosterone, sex hormone-binding globulin, inhibin B, FSH, and LH and phthalate monoester 

concentrations in spot urine samples. The subjects included 295 men attending a clinic as part of a fertility 

evaluation. [It is not known how many of these men were also included in the previously discussed

studies from this group (30, 154, 155).] Blood, semen, and urine samples were collected. Serum was 

frozen until assayed using RIA (testosterone), enzyme immunometric assay (sex hormone-binding 

globulin), enzyme-linked immunosorbent assay (inhibin B), or microparticle enzyme immunoassay (LH, 

FSH). Urine concentrations of MEHP and monomethyl, monoethyl, mono-n-butyl, and monobenzyl 

phthalate were assayed using HPLC with tandem MS and were adjusted based on urine specific gravity. 

Spearman correlation coefficients were calculated in the exploratory analysis followed by multiple 

linear regression with adjustment for smoking status, age, race, body mass index, previous fertility 

evaluation, prior fathering of a pregnancy, season, and time of day.

In their primary analysis, using all urine samples, the authors identified a “negative non-significant 

association” between urine MEHP concentration and serum testosterone, with a change in serum 

testosterone of −0.47 nmol/mL (95% CI−1.03 to 0.10, P=0.10) for each quartile increase in MEHP 

concentration. In a secondary analysis, in which urine samples were excluded if they had a specific 

gravity <1.010 and >1.030, the association between MEHP concentration and testosterone was described 

as weaker (−0.42 ng/mL for each quartile increase; 95% CI−1.05 to 0.21; P=0.19). [Additional

associations were identified between urinary mono-n-butyl and monobenzyl phthalate and serum

concentrations of inhibin B and FSH.]

The authors did not draw conclusions with respect to MEHP. They indicated that they could not tell 

whether the associations they identified represented physiologically relevant changes or were the result 

of conducting multiple comparisons.

Strengths/Weaknesses: The detection of phthalate metabolites in urine eliminated contamination issues. 

There was good evaluation of confounders, but the use of a subfertile population is a weakness.This report 

appears to include the same subjects as Duty et al. (154) and cannot be considered independent.

Utility (Adequacy) for CERHR Evaluation Process: This study is useful in the evaluation process.

Jönsson et al.(85), supported by the Swedish Research Council, AFA Foundation, the Swedish Government Funding for Clinical Research, the Crafoordska Fund, the Ove Tulefjords Fund, the Foundation 

for Urological Research, and the Medical Faculty of Lund University, studied semen parameters and 

urinary phthalate monoester levels in 234 military recruits. The subjects were 18 –21 years old at the 
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Appendix II

time of examination. Combined testicular volume was estimated based on ultrasound measurements, 

semen was obtained by masturbation, and spot urine samples were collected for measurement of 

phthalic acid, MEHP, and monoethyl, monobutyl, and monobenzyl phthalate. The limit of detection for 

MEHP was 15 ng/mL [μg/L]. Seminal plasma was assayed for neutral α-glucosidase, zinc, prostatespecific antigen, and fructose. Blood samples were collected for determination of serum FSH, LH, sex 

hormone-binding globulin, testosterone, 17b-estradiol, and inhibin. Seminal sperm were assessed for 

concentration and motility, including computer-assisted parameters, and were subjected to the sperm 

chromatin structure assay. Subjects were categorized into quartiles by urine concentration of individual 

phthalate monoesters (uncorrected and creatinine-adjusted), and ratios with 95% confidence intervals 

were calculated for highest:lowest quartile groups. 

The median urinary MEHP concentration was below the limit of detection. The 75th and 95th percentile values were 5.1 and 12 ng/mL [μg/L], respectively. The maximum value was 25 ng/mL. There 

were no significant associations between highest versus lowest urinary MEHP quartile and any of the 

dependent variables. The authors found no evidence of interaction between phthalate metabolites and 

polychlorinated biphenyl (PCB)-153 on testicular function.

Strengths/Weaknesses: Population not selected based on fertility characteristics was a strength. However, because only 14% of men agreed to participate, they may not represent the source population. 

Although participants may have been more concerned with their fertility than non-participants, they 

were blind to their phthalate exposure, making it unlikely that participation was biased by exposure. 

Comprehensive and objective measurements of semen parameters (ultrasound assessment of testicular 

volume, computer assisted assessment of motility) were strengths. Assessment of confounding by 

abstinence time and smoking was a strength, but lack of assessment of confounding by age or BMI 

was a limitation.

Utility (Adequacy) for CERHR Evaluation Process: This study is useful in the evaluation process.

Cobellis et al. (157), support not indicated, measured DEHP and MEHP concentrations in the plasma 

and peritoneal fluid of 35 women identified by laparoscopy as having endometriosis. The operations 

were performed for ovarian cysts, chronic pelvic pain, or dysmenorrhea. A comparison group consisted 

of 24 age-matched controls without known reproductive disease [laparoscopy was not performed on

these women, and it is not known how many of them may also have had endometriosis]. Blood 

samples were collected from the women undergoing surgery either the day prior to the procedure or 

“immediately before anaesthesia for laparoscopy.” [The paper does not indicate how many, if any,

of the patients were receiving iv infusions at the time of sampling.The Expert Panel notes that

Section 1.2.4 of the original CERHR report containsinformation on the amount of DEHP that can

be transferred by medical infusions.] Blood samples were obtained from the age-matched controls 

at the same phases of the menstrual cycle as the surgical patients. Estimation of DEHP and MEHP 

concentration was by HPLC. The proportions of women in each group with detectable concentrations 

were compared using the Fisher exact test, and concentrations were compared using the Wilcoxon 

test. Correlation between stage of endometriosis (a semi-quantitative estimate of the extent of visible 

endometriosis implants) and DEHP/MEHP concentration was made by Spearman correlation coefficient. There was no difference in the proportion of women in either group with detectable DEHP or 

MEHP (91.4% of surgical patients compared to 92.6% of control women). The median concentration 
Appendix II

II-117

(interquartile range) of DEHP in the patients was 0.57 (0.06–1.23) μg/mL compared to a control value 

of 0.18 (0–0.44) g/mL (P=0.0047). The median concentration (interquartile range) of MEHP in the 

patients was 0.38 (0.1– 0.97) μg/mL compared to a control value of 0.58 (0.34–0.71) g/mL (P=0.12). 

There was no significant association between DEHP/MEHP concentration in plasma or peritoneal fluid 

and stage of endometriosis in women undergoing laparoscopy. The authors concluded that there could 

be a plausible causal relationship between DEHP exposure and endometriosis, but that, “further studies 

are needed in order to elucidate the mechanisms underlying the observed statistical association.”

Strengths/Weaknesses: Weaknesses include possible exposure of cases due to medical procedures, 

very limited information on the selection of controls for endometriosis cases, very limited evaluation 

of confounding, and small sample size.

Utility (Adequacy) for CERHR Evaluation Process: This study is not useful in the evaluation process.

Hauser et al. (158), supported by NIEHS, evaluated possible interactive effects of polychlorinated 

biphenyls and phthalates on sperm motility in male partners of couples seeking infertility evaluation. 

Phthalate exposure was estimated based on urinary monoester concentrations, corrected for specific 

gravity, and polychlorinated biphenyl exposure was estimated using measurements in blood samples. 

Both phthalate and polychlorinated biphenyl levels were dichotomized at the median as high or low, 

and sperm concentration was dichotomized as normal or abnormal based on World Health Organization 

criteria. Other sperm parameters were measured, but motility had the strongest association with phthalate 

monoesters and polychlorinated biphenyls, and only the motility data were presented. Multivariate logistic 

regression was used to explore interactions. A relative excess risk of the interaction was calculated as 

1 plus the relative risk (of low motility) associated with high values for both chemical classes less the 

relative risk associated with a high value for 1 chemical class and a low value for the other chemical 

class. Men with low values for both chemical classes were the reference group. This calculation was 

made for the individual phthalate monoesters, including MEHP and monobutyl, monobenzyl, monoethyl, 

and monomethyl phthalate, and for individual and grouped polychlorinated biphenyls.

There were 303 men with urinary phthalate monoester levels. The median unadjusted MEHP concentration was 6.6 ng/mL [μg/L], with a 95th percentile value of 112 ng/mL. Interactions were identified 

for monobutyl phthalate and different classes of polychlorinated biphenyls. There were no significant 

interactions between urinary MEHP and any of the polychlorinated biphenyls or groupings, either 

with regard to sperm motility or other sperm parameters.

The authors concluded that there were statistical interactions between some polychlorinated biphenyls and 

phthalates in relation to low sperm motility. They hypothesized that polychlorinated biphenyl metabolites 

could interfere with phthalate metabolism through inhibition of UDP-glucuronyl transferase.

Strengths/Weaknesses: This report contains additional analyses of the population in the Duty et al. 

studies, which were well-conducted and only suffered the limitation of being conducted on a subfertile 

population. The groupings of PCBs were made a priori based on structure-activity relationships and 

PCBs were expected to share metabolic pathways with phthalates. 

Utility (Adequacy) for CERHR Evaluation Process: This study is useful for the evaluation process.
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Appendix II

4.2 Experimental Animal Data

Since the initial CERHR Expert Panel Report on DEHP, studies in rodents have evaluated effects on 

ovarian follicles or their constituents. Additional studies in male rodents and marmosets have been 

performed to characterize testicular toxicity and to evaluate mechanisms of toxicity.

4.2.1 	 Female

Lovekamp and Davis(159), supported by NIEHS, evaluated the effects of MEHP on F344 rat granulosa 

cells in culture. Four-week-old animals were stimulated with diethylstilbestrol or with pregnant mare 

serum gonadotropin (PMSG) following which ovaries were removed and granulosa cells harvested. 

Cells were cultured in the presence of 500 nM testosterone and 200 ng/mL FSH. MEHP [purity not

specified] was added to cultures at concentrations of 0 –200 μM [0 –55.6 mg/L] in a comparison with 

other phthalate monoesters and with Wy-14,643, another peroxisome proliferator. After 48 hours, 

estradiol and progesterone were measured in media using commercial RIA kits. RNA was extracted 

from cells and RT-PCR performed to quantify mRNA for aromatase and for cholesterol side-chain 

cleavage enzyme. Aromatase protein was quantified by Western blot. Statistical comparisons were 

made using the Student t test or ANOVA, followed by least significant difference test.

A comparison with other phthalate monoesters showed a decrease in estradiol in the medium (controlled for protein content) with MEHP 100 or 200 μM [27.8 or 55.6 mg/L] but not with comparable 

molar concentrations of monomethyl, -ethyl, -propyl, -butyl, -pentyl, or -hexyl phthalate. Monopentyl 

phthalate was associated with a decrease in estradiol production at 400 μM. mRNA for aromatase 

was estimated in media after culture with MEHP 0, 25, 50, or 100 μM [0, 7.0, 13.9, or 27.8 mg/L]. 

Graphically, there appeared to be a concentration-dependent decrease in estradiol concentration and 

in aromatase mRNA; pair-wise comparisons with control were statistically significant for the 100 

μM concentration for estradiol and for the 50 and 100 μM concentrations for aromatase mRNA. The 

peroxisome proliferator Wy-14,643 also decreased estradiol and aromatase mRNA. Cholesterol sidechain cleavage enzyme was not altered by MEHP, suggesting specificity of the effect on aromatase 

mRNA. Aromatase protein was decreased by MEHP at concentrations of 100 and 200 μM [27.8 and

55.6 mg/L]. In a final experiment, granulosa cells were incubated for 48 hours with MEHP 0 or 200 

μM [0 or 56 mg/L] and 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP) added for the last 24 

hours. mRNA for aromatase and cholesterol side-chain cleavage enzyme and medium progesterone 

levels were increased by 8-Br-cAMP in the absence of MEHP. In the presence of MEHP, mRNA for 

aromatase and medium estradiol levels were suppressed, but there was no suppression of P450 sidechain cleavage enzyme mRNA or progesterone. The authors interpreted their results as consistent 

with transcriptional suppression of aromatase by MEHP independent of the FSH-cAMP pathway. 

They proposed a PPAR pathway as a candidate mechanism for MEHP suppression of granulosa cell 

steroidogenic function.

Strengths/Weaknesses: The strengths of this study include appropriate technical proficiency and appropriate study controls and statistics, as well as examination of a set of structurally diverse phthalates. 

The Panel has confidence in the veracity of these results. This study identified a probable main point 

of interference with steroid production, showed that it was most affected by MEHP and not other 

phthalates (thus identifying MEHP as the main concern), and proposed a possible mechanism by 

which this interference might occur (PPAR mediation). This in vitro study comes after several in vivo
Appendix II

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studies that demonstrated low-estradiol-related changes in female rats, so relevance is another strength. 

Weakness includes examining cells from only 1 species and using MEHP of uncertain purity.

Utility (Adequacy) for CERHR Evaluation Process: The data point towards a subcellular site of action. 

Their utility in risk assessment would come in helping to identify vulnerable species as those having 

these target enzymes. The in vitro data are presumed relevant for in vivo protection when circulating 

blood levels of MEHP (or even better, tissue levels) are known.

Lovekamp-Swan et al. (160), supported by NIEHS, evaluated the interaction of MEHP with PPAR 

pathways as a mechanism for modifying ovarian steroidogenic functions. Granulosa cells were harvested 

from 4-week-old Fisher rats 24 hours after injection of PMSG. Cells were cultured for 48 hours with 

500 nM testosterone and 200 ng/mL FSH with or without MEHP [purity not specified] 50 μM [13.9

mg/L]. Culture media were assayed for estradiol using a commercial RIA kit. RNA was extracted 

from cells and specific RT-PCR probes used to amplify mRNA for quantification. The mRNAs were 

chosen to reflect the activity of genes involved in the PPAR pathways, steroidogenesis, or phthalate 

toxicity. The mRNAs included aromatase, 17b-hydroxysteroid dehydrogenase IV (which metabolizes 

estradiol to estrone), cholesterol side-chain cleavage enzyme, the aryl hydrocarbon (Ah) receptor, 

cytochrome P450 1B1 (CYP1B1), epoxide hydrolase, and heart-fatty acid binding protein (H-FABP, 

which is associated with luteal transformation of granulosa cells). mRNA results were normalized to 

glyceraldehyde 3-phosphate dehydrogenase as an internal control. 

To test the hypothesis that MEHP inhibition of granulosa cell aromatase is mediated through different 

PPAR isoforms, the experiments included the coadministration with MEHP of troglitazone (a PPARγ

ligand), selective PPARα and γ agonists, a selective PPARγ antagonist, a selective retinoic acid X receptor 

(RXR) agonist (because a PPARγ:RXR heterodimer is believed to decrease aromatase), 9-cis-retinoic 

acid (which binds both RXR and the retinoic acid receptor, RAR), and 15-deoxy-Δ12,14-prostaglandin 

J2 (a PPAR activator). Statistical comparisons were made using the Student t test or ANOVA, followed 

by the least significant difference test. 

MEHP in culture reduced aromatase mRNA by >40% compared to control. The addition of a PPARγ

antagonist partially reversed the decrease in aromatase mRNA. PPARα and γ agonists reduced aromatase 

mRNA to an extent similar to that after treatment with MEHP. To test the hypothesis that MEHPactivated PPARγ plus activated RXR results in aromatase suppression mediated by the PPARγ:RXR 

heterodimer, cells were treated with MEHP plus RXR or RAR ligands. All treatments decreased 

aromatase, with significant additional suppression by MEHP when it was added with 9-cis-retinoic 

acid. To demonstrate that MEHP activity could be mediated through PPARα, MEHP treatment was 

shown to increase mRNA for 17b-hydroxysteroid dehydrogenase IV, which is inducible by PPARα. The 

addition of a PPARγ antagonist did not alter the MEHP induction of 17b-hydroxysteroid dehydrogenase 

IV, suggesting MEHP activation of the α-isoform of PPAR. Both MEHP and a selective PPARα

agonist, but not a selective PPARγ agonist, increased the expression of Ah receptor, CYP1B1, and 

epoxide hydrolase. Cholesterol side-chain cleavage enzyme was not induced by MEHP or by either 

PPAR isoform agonist. H-FABP was induced by MEHP and by each specific PPAR isoform agonist. 

The authors concluded that MEHP effects on granulosa cell gene expression, which would serve to 

decrease estrogen production, were mediated by both PPAR pathways.
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Appendix II

Strengths/Weaknesses: These data further dissect the molecular pathways by which MEHP reduces 

estradiol production in granulosa cells. The experiments were well-conceived and executed, used 

appropriate statistics, and the Expert Panel has confidence in the veracity of these studies. Possible 

weaknesses for the evaluation process include limited species evaluation, and relying on the presumption that these in vitro data report processes that are relevant and active in vivo.

Utility (Adequacy) for CERHR Evaluation Process: These in vitro data are adequate for the evaluation 

process and are presumed relevant for in vivo biology. An additional assumption implicit in these experiments is that immature stimulated rodent granulosa cells are appropriate models for human granulosa 

cells, and that the agonists and antagonists employed are specific for their intended targets. The data 

are uniquely valuable in describing a mechanism of toxicity that may be tested in other species, and 

that is presumed relevant for other species sharing a similar biochemistry.

Anas et al. (161), supported by the Japanese Ministry of Agriculture, Forestry, and Fisheries, evaluated 

the effect of MEHP on in vitro maturation of bovine oocytes. Cumulus-oocyte complexes were obtained 

by aspirating 2–5 mm follicles from slaughterhouse beef ovaries. Oocytes with unexpanded cumulus 

layers were selected for study. In experiment 1 (n=91–99 oocytes/group), cumulus–oocyte complexes 

were cultured for 24 hours with MEHP (90% purity) at 0, 10, 25, 50, 75, or 100 μM [0, 2.8, 7.0, 13.9,

20.9, or 27.8 mg/L], after which they were evaluated for cumulus expansion and for stage of oocyte 

maturation. [Maturation was assessed in ethanol-fixed orcein-stained oocytes using unspecified

criteria.] In experiment 2 (n=123–131 oocytes/group), denuded oocytes were cultured with MEHP 

at the same concentrations with the addition of a 5 μM [1.4 mg/L] concentration level, followed by 

assessment of oocyte maturation stage. In experiment 3, cumulus-oocyte complexes were cultured for 

24 hours with MEHP 0, 50, or 100 μM [0, 13.9, or 27.8 mg/L], following which some oocytes were 

evaluated for maturational stage. Other MEHP-treated oocyte cultures were continued in MEHP-free 

culture medium for an additional 24 hours or were continued at their original MEHP concentration (50 

or 100 μM) for an additional 24 hours. The treatment groups (n=124–135 oocytes/group) consisted 

of MEHP exposure for 24 hours, MEHP exposure for 24 hours followed by 24-hour “recovery,” and 

MEHP exposure for 48 hours. Statistical analysis was by ANOVA and Fisher protected least significance 

test. Experiments 1 and 2 showed a concentration-dependent decrease in progression through oocyte 

maturation stages, with a significant decrease in oocytes reaching metaphase-II beginning at MEHP 25 

μM [7.0 mg/L] for cumulus-oocyte complex culture and at 10 μM [2.8 mg/mL] for denuded oocytes 

(Figure 6). Cumulus expansion was not impaired in experiment 1 [suggesting no effect of MEHP on

granulosa cell division and differentiation]. In experiment 3, impairment of progression to metaphase 

II was seen with the initial 24-hour culture with MEHP, as expected. Culture in MEHP-free medium 

permitted the progression of oocyte maturation to metaphase II in 64.5–71.1% of oocytes, although 

the proportion of oocytes reaching metaphase II did not recover completely to control levels (83.2% at 

24 hours). The recovery of maturation ability suggested that the MEHP-associated decrease in oocytes 

reaching metaphase II was not likely to be due to nonspecific cytotoxic or lethal effects. When oocytes 

were cultured in MEHP 50 μM for 48 hours, the proportion reaching metaphase II was higher at 48 hours 

(41.6%) than at 24 hours (26.9%), suggesting that MEHP delayed maturation rather than preventing 

maturation altogether.

Strengths/Weaknesses: The strengths of this study include very large sample sizes, reasonable statistics, 

a useful study design that included recovery, and a novel approach. It would have been more valuable 
Appendix II

II-121

to have included additional measures of cytotoxicity and perhaps to have gone past simple description 

to the use of specific agonists or antagonists (see Lovekamp et al. (160), above).

Utility (Adequacy) for CERHR Evaluation Process: The data are adequate for the evaluation process and 

are presumed relevant for humans. They show a direct effect on the ovary, although not a unique effect. 

They add little to mechanistic understanding. They do, however, expand the list of affected species.

 Figure 6. Percent of Bovine Oocytes Reaching Metaphase II after 24-hour Culture with MEHP

0

20

40

60

80

O

o

c

yte

s

at M

eta

p

h

a

s

e II (%)

0 20 40 60 80 100

MEHP Concentration (μM)

B B

B

B B

B

J

J J

J

*

* *

*

* *

*

*

J J J

Cumulus-Oocyte Complexes

Denuded Oocytes

B

J

*Significant difference from 0 ppm group. Drawn from Anas et al. (161).

Sekiguchi et al. (162), supported by the Cooperative System for Supporting Priority Research in 

Japan Science and Technology Corporation, evaluated number of ova ovulated in response to 15 or 30 

international units (IU) equine chorionic gonadotropin as an assay of female reproductive toxicity in 

F344 rats. DEHP was used as an illustrative chemical in their system. The equine chorionic gonadotropin 

was injected intramuscularly (im) at 25 days of age. The animals were killed 72 hours later, and uteri 

and ovaries were removed and weighed. Ova were flushed from the oviducts with saline, denuded with 

hyaluronidase, and counted under a microscope. Because the maximum number of ova recovered in 

spontaneously ovulating rats was 11, superovulation was defined as the presence of more than 11 ova in 

the oviducts. To evaluate the effects of the test chemical, DEHP [purity not specified] 0 or 500 mg/kg 

bw/day in olive oil was given by sc injection daily from 24 to 27 days of age. There were 12 rats treated 

with 15 IU equine chorionic gonadotropin, 6 of which received DEHP, and 7 rats treated with 30 IU 

equine chorionic gonadotropin, 3 of which received DEHP. Ovulation was induced in 4 of the 6 rats that 

received 15 IU equine chorionic gonadotropin in the presence or absence of DEHP treatment. In rats 

given 30 IU equine chorionic gonadotropin, ovulation occurred in 1 of 3 rats given DEHP and 4 of 4 rats 

given the vehicle. This difference was described by the authors as not statistically significant, possibly 

due to the small number of animals [statistical methods not described; P=0.14, Fisher exact test by

CERHR]. None of the rats treated with DEHP demonstrated superovulation compared to 2 of 4 rats 

given 15 IU equine chorionic gonadotropin and 1 of 4 rats given 30 IU equine chorionic gonadotropin. 
II-122

Appendix II

The mean number of recovered ova was described by the authors as having been reduced by DEHP 

treatment (mean±SEM 12.7±7.75 [control] compared to 2.50±0.85 [DEHP] in animals given 15 IU 

equine chorionic gonadotropin and 8.00±4.69 [control] compared to 0.33±0.33 [DEHP] in animals given 

30 IU equine chorionic gonadotropin [P=0.22 and 0.23,ttest by CERHR]). The authors concluded that 

DEHP may have suppressed ovulation due to disrupted ovarian steroidogenesis and/or follicle growth.

Strengths/Weaknesses: The use of intact animals allows the assessment of an integrated physiologic 

response. This is overshadowed by the small sample sizes, the lack of appropriate statistics, and the 

fact that the studies did not move past simple description, and thus, add little that is new or of value 

to our understanding.

Utility (Adequacy) for CERHR Evaluation Process: These data are inadequate for the evaluation 

process, based on small sample size and inadequate statistics.

A 65-week feeding study in marmosets performed at Mitsubishi Chemical Safety Institute, Ltd. (92) 

contained information on ovarian and uterine weight and histology. Because the focus of this study 

was on testicular effects, the study is discussed in Section 4.2.2.2.

4.2.2 	 Male

Studies on the male reproductive effects of DEHP in rodents often employ young animals because 

these animals are more sensitive to the testicular effects of phthalates. Although toxicity produced in 

prepubertal animals can be considered developmental toxicity, these studies attempt to characterize 

testicular effects that are not unique to the developing gonad and are included in the reproductive 

toxicity section. There is a single study in which prepubertal boars were treated, and this study is 

considered in this section as well.

4.2.2.1 Cultured cells and tissues

Dees et al. (163), supported by NIEHS, reported the effects of MEHP on MA-10 cells in culture. The 

MA-10 cell is a mouse Leydig tumor cell that resembles the normal Leydig cell in ultrastructure and 

in steroid synthesis, except that it produces progesterone instead of testosterone. The cells were grown 

for 24 hours in the presence of MEHP [purity not specified] 0, 0.3, 1, 3, 10, 30, 100, 300, 1000, 

3000, and 10,000 μM [0, 0.08, 0.28, 0.84, 2.8, 8.4, 28, 84, 279, 838, and 2794 mg/L]. After MEHP 

exposure, cells were washed and incubated for 2.5 hours with human chorionic gonadotropin (hCG) 

50 ng/mL in the absence of MEHP. Media were assayed by RIA for progesterone. Cell protein was 

quantified, and cell viability was assessed by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl 

tetrazolium bromide (MTT). Electron microscopy was performed and cells photographed at 6000× for 

morphometric analysis based on nuclear and cytoplasmic volumes as a percent of total cell volume. 

Statistical analyses were performed by ANOVA with post hoc Tukey test. [The methods state that

cells were grown to confluence in 24-well plates, and that there were 2 experimental replicates.

A figure legend indicates n=16 from 5 different experiments for progesterone and protein levels

and n=9 from 3 different experiments for cell viability using MTT determination.]

Progesterone production was stimulated by hCG in cells not exposed to MEHP. Significant reductions 

in progesterone production occurred after incubation with MEHP at 3, 10, 3000, and 10,000 μM but not 

at the intervening concentrations. Viability and protein were decreased at 3000 and 10,000 μM. MEHP 
Appendix II

II-123

treatment was associated with an increase in cytoplasmic lipid droplets at MEHP concentrations of 1 μM 

and higher. Morphometric analysis did not show alterations in nuclear or cytoplasmic volumes up to an 

MEHP concentration of 1000 μM, the highest tested concentration at which viability was not affected. 

Abnormal mitochondria, including pale, swollen, and ring-form mitochondria and mitochondria with 

longitudinal or degenerating cristae, were seen at MEHP concentrations of 1 μM and higher. Rough 

endoplasmic reticulum was decreased at concentrations of 100 and 1000 μM. The authors concluded 

that these data were consistent with an MEHP effect on Leydig cell mitochondria and steroidogenesis, 

with consequent cholesterol accumulation resulting in an increase in lipid droplet accumulation. 

Strengths/Weaknesses: This report is useful for having performed an in-depth analysis of structural 

alterations in an immortalized cell line that produces steroids and for confirming previous reports. 

A weakness is that only an integrated biochemical measure was taken (progesterone secretion), and 

no other probes were used to explore the site or nature of the biochemical disruption or to confirm 

possible mechanisms for the observed changes (mitochondrial changes, increased lipid droplets). The 

authors could offer no convincing explanation for the peculiar dose-response curve.

Utility (Adequacy) for CERHR Evaluation Process: This paper is largely confirmatory of other studies. 

The model is modestly relevant (all immortalized cell lines have some significant biochemical changes 

that confer immortality, and thus separate them from the rest of life) providing little new knowledge.

Kang et al. (164), supported by the Korean Ministry of Environment, evaluated the effect of DEHP 

on gap junctional intercellular communication in cultured mouse Sertoli cells obtained from normal 

11–13-day-old mice. [Most of the text indicates TM4 cells, but the abstract, a figure legend, and

the discussion indicate TM5. The Expert Panel assumes based on the endpoints being studied

that the TM4 cell was used. The study authors present this paper as primarily an investigation

of altered gap-junctional communication as a possible mechanism of DEHP carcinogenicity but

discuss possible importance of gap-junctional communication for Sertoli–germ cell interactions.] A 

neutral-red uptake assay for cytotoxicity was performed using 24-hour exposures to DEHP concentrations 

of 0, 1, 10, 100, 250, 500 and 1000 μM [0, 0.39, 3.91, 39.1, 97.7, 195.3, and 390.6 mg/L], showing 

the highest concentration tested not to be cytotoxic. DNA content of the lysed cultured cells increased 

after a 3-day exposure to DEHP 500 μM [195.3 mg/L; the figure legend, however, says 100 μM

(39.1 mg/L)], interpreted as an indicator of cell proliferation. Gap junctional communication was 

evaluated using the scrape-loading dye transfer technique in which Lucifer yellow was added to the 

culture medium and scrape-loaded into the cells using a single pass of a razor blade across the cells, 

thus injuring some cells and allowing dye entry. This procedure was performed on a monolayer of 

cultured cells that had been exposed for 24 hours to DEHP 0, 100, or 500 μM [0, 39.1, or 195.3

mg/L]. The distance the dye traveled in 3 minutes, observed using fluorescence microscopy, was 

evaluated as decreased at both concentrations of DEHP compared with the control [data not shown;

amount of decrease not specified]. Morphologic nuclear apoptotic changes in response to 12 hours 

of serum deprivation were assessed using the stain Hoechst 33258. Cells pretreated with DEHP 500 

μM [195.3 mg/L] for 24 hours showed less nuclear staining than control cells, implying a reduction in 

apoptosis [data not shown]. Western blot analysis was used to quantify phosphorylated connexin-43, 

a gap-junction protein, and poly(ADP-ribose) polymerase (PARP), a substrate for caspase in the 

apoptosis pathway. Evaluation of phosphorylated connexin-43 protein was performed in lysed cells 

after exposure to DEHP 500 μM [195.3 mg/L] for 1, 4, or 9 hours. The DEHP exposure was described 
II-124

Appendix II

as reducing phosphorylated connexin-43 in a time-dependent manner [graph shown without error

bars or statistical analysis]. Evaluation of PARP cleavage products was performed in response to 

12 hours of serum deprivation in cells exposed to DEHP 500 μM [195.3 mg/L] for 9, 12, or 24 hours. 

Whereas control cells showed an increase in cleavage products with time, these products were largely 

absent in DEHP-exposed cells [graph shown without error bars or statistical analysis]. The study 

authors concluded that DEHP inhibited gap junctional communication, possibly associated with a 

decrease in phosphorylated connexin-43. A decrease in apoptosis associated with a decrease in PARP 

cleavage was believed possibly to be involved in the carcinogenicity of DEHP.

Strengths/Weaknesses: The weaknesses of this paper include numerous internal inconsistencies that 

make it difficult to ascertain what was actually done, a lack of statistical analysis, and uncertainty about 

experimental rationale and execution. Dye transfer experiments frequently require more than 3 minutes 

for the transfer of the dye to adjacent cells. The nuclear staining and early indicators of apoptosis are 

generally considered as issues separate from those relating to cell communication, so the combination 

here is perplexing. Sertoli cells are not targets of the tumorigenic activity of DEHP, so are a model of 

questionable relevance to the in vivo situation. The working dose most frequently used in this study 

was exceptionally high, and while perhaps useful as a biochemical tool, is of limited relevance. 

Utility (Adequacy) for CERHR Evaluation Process: This study is not useful for the evaluation process, 

as there is considerable uncertainty about what was done, whether statistical analysis was performed, 

and the relevance of the findings to the in vivo situation.

Suominen et al.(165), supported by the EU and the Academy of Finland, evaluated the effects of MEHP 

on segments of CD-1 mouse seminiferous tubules. Testes from 2 –3-month-old mice were decapsulated 

and microdissected to provide 1 and 2 mm tubule segments at stages III –V, VII –VIII, and IX–XI 

(determined by transillumination and confirmed in squash preparations). Tubule segments at stages 

IX–XI were incubated for 24 hours with MEHP [purity notspecified] at concentrations of 0, 0.01, 0.1, 

and 1.0 mM [0, 2.8, 28, and 279 mg/L] for 24 hours. Tritiated thymidine was added for the last 4 hours 

of incubation for estimation of DNA synthesis by thymidine incorporation. Graphic representation of 

the incorporated label at the end of the experiment suggested a concentration-related decrease in DNA 

synthesis, but none of the values were statistically different from the control by ANOVA. Incubation 

of 2 mm segments of tubules at stages III–V and VII –VIII with MEHP 0.1 mM [28 mg/L] also had 

no significant effect on thymidine incorporation. Stage IX–XI tubule segments were incubated for 

8 hours with MEHP at 0, 0.01, 0.1, and 1.0 mM [0, 2.8, 28, and 279 mg/L], following which squash 

preparations were evaluated for apoptosis using in situ 3’ end-labeling followed by light microscopy 

to count positive cells. A subsequent time-course experiment was performed in which stage IX–XI 

tubules segments were incubated with MEHP 0.1 mM [28 mg/L] for 4, 8, or 24 hours and evaluated 

for apoptosis by in situ 3’ end-labeling. Stage specificity of apoptosis induction was evaluated using 

segments of tubules at stages III–V and VII –VIII incubated with MEHP 0.1 mM [28 mg/L] for 8 

hours. Statistical evaluation used ANOVA with post hoc Dunnett test for dose-response experiments 

and t test for comparison of stage-specific results with one another. The number of apoptotic cells 

per tubule segment was approximately doubled [estimated from a graph] by MEHP 0.01 and 0.1 

mM [2.8 and 28 mg/L] compared to control but was not significantly altered by MEHP 1.0 mM [279

mg/L; lack of effect attributed by the study authors to a high incidence of early cell death at this

concentration]. In the time-course experiment, MEHP 0.1 mM [28 mg/L] significantly increased the 
Appendix II

II-125

number of apoptotic cells per tubule segment only at 8 hours. [The numeric value appeared to be

higher from a graph at 24 than at 4 or 8 hours, but the standard error was large, and statistical

significance was not identified.] In the stage-specificity experiment, only tubule segments at stages 

IX–XI showed an increase in number of apoptotic cells after MEHP exposure. The authors concluded 

that their finding of stage specificity in the mouse was “partly in agreement” with previous reports that 

stages XI –XIV and I– II were the most sensitive to phthalate testicular toxicity in the rat, associated 

with the reliance of these stages on FSH and with inhibition by phthalates of FSH binding.

Strengths/Weaknesses: One strength of these studies is that they were performed with technical competence and were not over-interpreted. No biochemical mediators of cell death or stage-specific biochemistry 

were evaluated in these studies, which is something uniquely available in this type of preparation. 

Utility (Adequacy) for CERHR Evaluation Process: These studies appear adequate for the evaluation 

process but add little that is new to our understanding of where and how DEHP/MEHP works in the 

testis.

Andriana et al. (166), supported by the Japanese Ministry of Health, Labor, and Welfare, evaluated 

morphologic MEHP-associated alterations in spermatogenic cells in Sprague-Dawley rat testis culture. 

Testes were harvested from an unspecified number of 20-day-old animals and 1 mm3 portions were 

cultured on filter paper. MEHP [purity not specified] was added to culture media at 0, 10–6, 10–3, 

1, or 100 μM [0, 0.00028, 0.28, 279, or 27,936 mg/L]. Testis fragments exposed at each concentration 

were evaluated at 1, 3, 6, and 9 hours. TUNEL staining using a commercial kit was applied to 5 μm 

paraformaldehyde-fixed paraffin-embedded sections with methyl green counterstaining. Apoptotic cells 

were counted in 25 randomly selected round tubules from 20 tissue cultures, expressed as a percentage, 

and compared using ANOVA. Transmission electron microscopy was used to characterize ultrastructural 

evidence of apoptosis and necrosis. Tissues for electron microscopy were fixed in 2.5% glutaraldehyde/0.05 

M cacodylate buffer, post-fixed in osmium tetroxide, and embedded in Araldite-M. [No quantitative

results were presented from the transmission electron microscopy portion ofthe study.] In the control 

cultures, apoptosis increased after 9 hours of culture with 2.8% of cells positive. In the MEHP-treated 

cultures, apoptosis increased in a time- and concentration-dependent manner, with a maximum of 14% 

of cells affected in the 100 μM culture at 9 hours (Figure 7). At the highest concentration, there was 

evidence of Sertoli cell apoptosis at 1 hour, characterized by the authors as partial lysis of the nuclear 

membrane by transmission electron microscopy. Partial lysis of spermatogenic cell nuclear membranes 

was noted at 1 hour at the 1 and 100 μM MEHP concentrations. The authors concluded that “even a low 

concentration of MEHP caused permanent changes in testicular tissue cultures of rats.”

Strengths/Weaknesses: One strength of this design was the novel exposure system, which allowed for 

normal cell-cell interactions to be maintained. Another unique aspect was the wide range of concentrations used. One weakness is the unspecified purity of the MEHP, and also the fact that all tubules 

were not affected equally by MEHP, so the selection of random tubules for assessment depended on 

truly random selection, which would almost certainly have diluted the effect of the MEHP. Another 

weakness is the uncertainty surrounding the use of electron microscopy-derived membrane structure 

to reach conclusions about cell death, and the fact that this finding is inconsistent with the published 

in vivo literature (i.e., Sertoli cell death is not reported after in vivo dosing and evaluation). The use 

of the word “permanent” when describing effects of a 9-hour exposure is questionable.
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Appendix II

Utility (Adequacy) for CERHR Evaluation Process: This study appears adequate for the evaluation 

process, although it produces no new insights into the site or mechanism of MEHP toxicity, confirming 

what was known previously.

Figure 7. Percent Apoptotic Cells in Rat Testicular Culture with MEHP

0

4

8

12

A

p

o

ptotic

C

ells (%)

0 3 6 9

Time in Culture with MEHP (hrs)

100 μM

1 μM

1 nM

Control

1 pM

Drawn from data in Andriana et al. (166). Standard error bars omitted for clarity. 

Statistical comparisons were not indicated in the original.

Andriana et al. (167), supported by the Japanese Ministry of Health, Labour, and Welfare, evaluated the effects of MEHP [purity not specified] on cultured goat testis. Testes were harvested from 

2-month-old Shiba goats, decapsulated, and cut into 1-mm3 pieces for culture on filter paper floated in 

Dulbecco’s Minimal Essential Medium. The medium contained antibiotics, DMSO, and ethanol. Some 

cultures included MEHP at 0.001, 1, or 100 nmol/mL [0.0028, 2.8, or 279 mg/L (nmol/mL=μM);

DMSO and ethanol concentrations, control culture conditions, number of animals, and number

of cultures were not specified]. Explants were harvested at 1, 3, 6, or 9 hours of culture and fixed in 

2.5% glutaraldehyde/0.05 M cacodylate buffer. Specimens were prepared for light microscopy using 

toluidine blue staining or transmission electron microscopy using uranyl acetate and lead citrate. 

Results were expressed qualitatively. Light microscopy showed a concentration-related increase in 

apoptotic or necrotic Sertoli and spermatogenic cells with vacuolization and sloughing of germ cells 

beginning at 3 hours “at each concentration.” Transmission electron microscopy showed infrequent 

distended mitochondria, abnormal nuclear vesicles, and ruptured mitochondrial membranes, which 

were apparent as early as after 1 hour of exposure to 1 μM MEHP [the results are not specific about

which cells were affected, but the figure legend and discussion suggest that the Sertoli cells

were affected first]. At 3 hours, vacuolization and abnormal vesicles were described as frequent, 

with damage in Sertoli and germ cells. At 6 hours, there were apoptotic spermatogonia. The lowest 

concentration (0.001 μM) produced marginated chromatin in Sertoli cells at 6 hours. The authors 

concluded that even the lowest MEHP concentration produced permanent testicular damage, and that 

their results support the Sertoli cell as the primary target of MEHP toxicity, with germ cell damage 

as a consequence of Sertoli cell alterations.
Appendix II

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Strengths/Weaknesses: A strength of this paper is that it adds another species to the list of those 

shown to be affected by DEHP/MEHP, and it uses methods shown to have worked for the more commonly employed rat model. It avoids having to expose the whole goat, which saves compound and 

avoids unknown toxicokinetic issues. Significant weaknesses are the uncertainty about control culture 

conditions, number of animals used, and the number of experiments, not to mention the uncertainty 

introduced by including the solvents DMSO and ethanol in the medium. Paucity of experimental detail 

limits confidence in the relevance of these data, and confidence is further decreased by the solvent 

issues because DMSO can significantly enhance the toxicity of lipophilic compounds in vitro. There 

is uncertainty about the relevance of the findings at the lower dose levels because these concentrations 

in vivo have not been previously associated with the reported effect.

Utility (Adequacy) for CERHR Evaluation Process: This study is of limited relevance and utility to 

the evaluation process, based on the uncertainty about the methods, solvents, and the doubt around 

the results at the lowest concentrations, and the assertions of “permanent” damage after short-term in 

vitro exposure and assessment. 

Awal et al.(168), supported by the Japan Society for Promotion of Sciences and the Japanese Ministry 

of Health, Labour, and Welfare, evaluated the effects of MEHP on guinea pig seminiferous tubule 

culture. Tubules harvested from 28-day-old animals were exposed to MEHP [purity not specified]

in corn oil at 0, 1, 10, and 100 μM [0, 279, 2794, or 27,936 mg/L] for 3, 6, or 9 hours. Cultured 

tissues were evaluated by light and transmission electron microscopy and by in situ TUNEL staining 

for apoptosis. Intact round seminiferous tubules (100 per dose group) were counted for apoptotic 

spermatogenic cells, expressed as a percent. Statistical analysis was by ANOVA with the Fisher least 

significant differences test. After 3 or more hours of MEHP exposure, germ cell detachment was evident 

by light microscopy and Sertoli cell vacuolation was identified by electron microscopy [effect levels

not given, but the figures show tubule sections from the 100 μM group, for which the response

was said to be maximal]. The number of apoptotic cells by TUNEL staining was said to increase 

with exposure level and time. [The data figure does not have significant differences marked; the

legend says that significance between treated and control cultures is recognized at P<0.05.] The 

authors concluded that “MEHP induces testicular toxicity in guinea pigs in vitro.”

Strengths/Weaknesses: It is unclear whether the animals were treated with MEHP before donating 

tissues to culture or the tissues were treated in vitro and exposed to MEHP dissolved in corn oil and 

added to the culture. There is sufficient confusion about the wording to make the method quite unclear, 

although much of the description seems to indicate the latter (corn oil was added to the cultures). 

Weaknesses of this study include this confusion, the unusual exposure paradigm in the absence of any 

determination of MEHP levels in the medium, uncertainty in the paper about which effects were seen 

at which concentrations, and the implausibility of finding these effects in cultured tissue fragments 

due to highly lipophilic compounds being dosed into an aqueous medium in corn oil. The only lightlevel micrographs use different magnifications for the control and treated cultures, making it difficult 

for the reader to closely assess the adequacy (i.e., normal structure) of the control cultures. The study 

confirms an affected cell type but adds little that is detectably new to our understanding.

Utility (Adequacy) for CERHR Evaluation Process: This study is inadequate for the evaluation process 

due to the confusion about the exposure and the fact that the data are redundant of previous literature. 
II-128

Appendix II

4.2.2.2 In Vivo Studies With or Without Ex Vivo Component

Richburg et al. (169), supported by NIEHS, NIH, and the Burroughs Wellcome Fund, evaluated the 

effect of MEHP on testicular apoptosis in gld mice, which have a nonfunctional form of fas-ligand 

(fasL). The study was performed to explore the involvement of the fasL, a mediator of cell-cell death, 

and hypothesized to mediate germ cell death after Sertoli cell damage, in the development of the DEHP 

lesion in mice. [The study also evaluated radiation-induced injury; only the MEHP results are

presented here.] Wild-type controls were C57BL/6 males at 28 days of age. Wild-type and gld mice 

were given MEHP (>94% purity) in corn oil at 0 or 1000 mg/kg as a single gavage treatment. The 

animals were killed 0, 3, 6, 12, or 24 hours later, and testes were harvested. One testis was fixed in 

10% neutral buffered formalin embedded in glycol methacrylate, sectioned, and stained with periodic 

acid-Schiff/hematoxylin for light microscopy. The other testis was frozen until analyzed for apoptosis 

using a commercial TUNEL staining kit. Apoptosis was evaluated (3 animals/group) using the number 

of tubules with 0 – 3 or >3 apoptotic germ cells. Homogenization-resistent testicular sperm heads 

were also counted. Statistical comparisons were made using ANOVA with Fisher least significant 

difference test. Baseline testicular parameters (n=15 gld and 17 wild-type mice at time 0) showed 

the gld mice to be 15% heavier and to have 6% higher testis weights than wild-type mice. There were 

17% more sperm heads/testis in the mutants, which also had 2.2 times the proportion of tubules with 

>3 apoptotic germ cells. After MEHP treatment, there was histologic evidence of injury (germ cell 

sloughing, increased tubule lumen, Sertoli vacuolation) in both gld and wild-type mice, but only the 

wild-type mice had histologic findings consistent with germ cell apoptosis (cell shrinkage, chromatin 

condensation). TUNEL labeling increased in wild-type mice 3 – 12 hours after MEHP treatment, 

with the proportion of tubules containing >3 apoptotic germ cells increasing 2.7-fold in that interval 

[estimated from a graph]. By contrast, there was only a 1.7-fold increase in tubules containing 

>3 apoptotic germ cells in the gld mice at 12 hours [estimated from a graph]. The proportion of 

tubules meeting these criteria was statistically higher in wild-type than mutant mice at 6, 12, and 24 

hours. The authors concluded that the insensitivity of gld mice to MEHP-induced germ cell apoptosis 

“further underscores the participation of the [f]as system in the regulation of germ cell apoptosis after 

MEHP-induced Sertoli cell injury.”

Strengths/Weaknesses: Strengths of this study are the technical competence with which it was performed and the creativity of the approach to address the experimental question. The data are internally 

consistent and support the proposed hypothesis. The study used MEHP as a tool to show the involvement of fasL in a lesion.

Utility (Adequacy) for CERHR Evaluation Process: These data are adequate for the evaluation process 

in that they appear to define the mechanism by which the germ cells die. These data were not intended 

to shed any light on the means by which MEHP affects Sertoli cells.

Ichimura et al.(170), support not indicated, evaluated the expression of fas, fasL, and caspase-3, which 

are 3 apoptosis-associated proteins, as well as TUNEL positivity and electrophoretic DNA laddering 

in the testes of DDY mice treated with DEHP. DEHP [purity not specified] in corn oil was given 

orally [gavage assumed] to 4-week-old male mice as a single dose of 0, 4, 40, 400, or 4000 mg/kg 

bw (n=3/dose group) [doses given in the original in mg/g and converted by CERHR to mg/kg by

multiplying × 1000]. Testes were removed 12 hours after the treatment. One testis per animal was fixed 

in paraformaldehyde and sectioned for immunohistochemistry for fas, fasL, and caspase-3. TUNEL 
Appendix II

II-129

labeling was evaluated in sections as an indicator of apoptosis. DNA extraction and electrophoresis 

was performed on homogenates of both testes from 3 other animals, with the density of electrophoretic 

bands estimated photographically using Adobe PhotoShop® software. FasL positivity was identified 

in a distribution consistent with Sertoli cell cytoplasmic processes and associated spermatocytes. Fas 

and caspase-3 co-localized in the middle to outer portion of the epithelium. [Results are described

for the group given DEHP 4000 mg/kg bw.] TUNEL-positive nuclei were clustered in the middle 

layer of the epithelium. The maximum number of positive nuclei per section in the 0, 4, 40, 400, 

and 4000 mg/kg bw groups were 3, 3, 5, 7, and 22, respectively. [Statistical comparisons were

not presented, and numbers of total cells/section were not given.] DNA laddering was identified 

in all dose groups, with band density estimated as 2.2 times the control in the high-dose group and 

1.1 times the control in the other dose groups. [Statistical comparisons were not presented.] The 

authors interpreted the numeric increase in number of TUNEL-positive nuclei per section as possibly 

indicating a dose-response relationship, concluding that exposure to DEHP at as little as 40 mg/kg bw 

might produce an increase in apoptosis in mouse testis. Further, they concluded that the 10% increase 

in electrophoretic band density in the lower dose groups meant that DEHP might have an effect on 

mouse testis after a dose as low as 4 mg/kg bw.

Strengths/Weaknesses: The strengths of this study include the technical co-localization approach and 

the number of doses of DEHP used. Weaknesses include the means of quantifying the density of bands 

after electrophoresis.

Utility (Adequacy) for CERHR Evaluation Process: This study is adequate for the evaluation process, 

although it does not add significantly to our understanding of the early events in the production of the 

testis lesion, nor was it intended to. The point of this study was to examine the co-localization of factors 

involved in cell death after treatment with a compound that would predictably induce that death. The 

changes at the lowest two doses, although detectable, are probably of little biological consequence.

Giammona et al. (171), supported by NIEHS, examined the role of MEHP in inducing apoptosis in 

pubertal rodent testicular germ cells and evaluated the role of fas-independent receptors in apoptosis. 

A single dose of 1000 mg/kg bw MEHP (> 97% purity) in corn oil was administered by gavage to 

5-week-old Sprague-Dawley rats, 28-day-old wild-type C57CL/6 mice, or 28-day-old gld mice. The gld

mice express a dysfunctional fasL protein, which cannot bind to the fas receptor to initiate apoptosis. 

A commercial TUNEL staining kit was used to identify apoptosis. Immunohistochemistry was used 

in sections of rat testis to identify fas and DR4, DR5, and DR6 receptors, which are fas-independent 

death receptors in the tumor necrosis factor (TNF) superfamily. Western blot was used to measure 

caspases, fas receptor, and DR receptors in homogenized mouse and rat testis. [Statistical analysis

methods were not described.] Following MEHP exposure, apoptosis was found to occur primarily in 

spermatocytes in both wild-type and gld mice. In wild-type mice, germ cell apoptosis was significantly 

increased from 6 to 48 hours following MEHP exposure. Apoptotic activity peaked between 12 and 24 

hours with ~5-fold increases compared to baseline levels. A significant ~2-fold increase in apoptosis 

compared to baseline levels was observed at 12 and 48 hours following MEHP exposure of gld mice. 

In both groups of mice, apoptotic activity returned to baseline levels by 96 hours following exposure. 

Western blot analyses revealed that fas expression significantly increased in wild-type mice (~3-fold) 

at 3 hours following MEHP exposure. There was no significant alteration in fas expression following 

MEHP exposure in gld mice. Expression of DR4, DR5, and DR6 proteins occurred in both wild-type and 
II-130

Appendix II

gld mice, but MEHP exposure did not increase expression in either strain. DR5 but not DR4 expression 

significantly increased in Sprague-Dawley rat testes (~1.5-fold) at 1.5 and 3 hours following MEHP 

exposure. Procaspase 8 cleavage products, downstream receptor-mediated signals of apoptotic pathways, 

were detected in testes of wild-type and gld mice, but expression was significantly increased only in gld

mice at 6 hours following MEHP exposure. Electrophoretic mobility shift assays demonstrated that DNA 

binding of NFkB, a receptor-mediated downstream signal possibly involved in cell death or survival, 

was generally reduced in wild-type mice but upregulated in gld mice following MEHP exposure. The 

study authors concluded that these findings demonstrate that germ-cell related death receptors and 

downstream signaling products appear to respond to MEHP-induced cell injury; it has not yet been 

determined if these factors are involved in testicular apoptosis following MEHP exposure.

Strengths/Weaknesses: The creative experimental approach and competence are both strengths of this 

paper. These studies used MEHP as a tool to explore the cell death mechanism in the rat and mouse 

testis and were effective in charting the expression of death-related genes in germ cells after exposure 

to a Sertoli cell toxicant.

Utility (Adequacy) for CERHR Evaluation Process: The data are considered adequate and useful for 

the evaluation process, mostly by way of identifying those factors involved in germ cell death. 

Dalgaard et al.(172), supported by the Danish Medical Research Council, evaluated the acute effects 

of MEHP on the testes of 28-day-old Wistar rats in an attempt to infer initial effects on the androgen 

receptor or intracellular levels of cyclic AMP. A single dose of MEHP [purity not specified] was 

given in corn oil by gavage at 0 or 400 mg/kg bw. Rats given corn oil were killed 2 hours later, and 

rats given MEHP were killed 3, 6, or 12 hours later (n=12 per time point). Testis sections were stained 

with hematoxylin and eosin for light microscopy or were immunostained for vimentin and androgen 

receptor. Testis sections were also TUNEL stained for determination of apoptosis. Portions of testis 

and ventral prostate were processed with RT-PCR for quantification of TRPM-2 gene message, and 

caspase-3 was estimated in testis samples based on its interaction with a colorimetric substrate. A 

commercial ligation-mediated PCR kit was used to detect nucleosomal ladders in testicular tissue, and 

TUNEL staining was performed using a commercial kit. Randomly selected tubules were evaluated 

for the presence of 0, 1–3, or >3 apoptotic germ cells/tubule. Statistical comparisons were made using 

ANOVA with post hoc Dunnett test. Light microscopy showed germ cell sloughing beginning 6 hours 

after MEHP treatment. Collapse of vimentin filaments in the Sertoli cell cytoplasm and perinuclear 

condensation were evident by immunostaining at 3 hours and persisted throughout the study. Androgen 

receptor staining was not altered by treatment at any time point. TRPM-2 expression was increased 

at 3 hours after MEHP treatment but returned to control levels at 6 and 12 hours. Caspase-3 activity 

was increased by treatment, although the authors noted that there was considerable caspase-3 activity 

in the control and that the 6-hour value was not statistically different from the control (Figure 8).

There were no detectable treatment effects on number of apoptotic cells per tubule or DNA laddering 

patterns. The authors concluded that the collapse and perinuclear condensation of vimentin fibers 

beginning at 3 hours was an early and sensitive MEHP-associated change, occurring at an exposure 

level 5 times lower than previously reported. The increase in caspase-3 was consistent with MEHP 

initiation of apoptosis but could not be confirmed by later apoptotic events, perhaps due to the large 

variability in TUNEL staining and DNA laddering among individuals.
Appendix II

II-131

Strengths/Weaknesses: This study attempted to investigate potential early events in the lesion produced 

by MEHP and repeated some previous findings. A weakness is the inability to explain the discordance 

between some of the early events the authors report and the absence of expected later events.

Utility (Adequacy) for CERHR Evaluation Process: The data are adequate for the evaluation process 

and eliminate some possible early events (changes in androgen receptor events or cAMP levels) as 

possible mediators of MEHP toxicity.

Figure 8. Testicular Caspase-3 Activity After Administration of a Single Oral Dose

 of MEHP to Wistar Rats 

J

J

0

200

400

600

800

1000

J

J

Control 3 6 9 12

Hours After MEHP Dosing

C

a

s

p

a

s

e-3

A

ctivity

(p

m

ol 4-nitro

a

nilin

e lib

e

rate

d/

m

g

p

rotein/h

r)

Drawn from Dalgaard et al. (172). Mean±SD, n=8. 

*Different from control, P<0.05 by ANOVA with post hoc Dunnett test.

Park et al. (173), supported by NIH, evaluated the role of zinc depletion as a causative factor in the 

testicular toxicity of DEHP in young Sprague-Dawley rats. Animals were treated beginning at 25 days 

of age. Dosing was by gavage with DEHP in corn oil at 0 (n=15) or 2000 (n=35) mg/kg bw daily 

for up to 14 days. Five control animals per time point were decapitated after 2, 7, or 14 days of treatment, and 5 DEHP-treated animals per time point were decapitated after 1, 2, 3, 5, 7, 10, and 14 days 

of treatment. Testes were removed and weighed, following which 1 testis was frozen for subsequent 

estimation of ZnT-1 mRNA by branched DNA signal amplification using a commercial kit. The other 

testis was fixed in neutral buffered formalin. One-half of the fixed testis was sectioned and stained with 

hematoxylin and eosin for histologic evaluation by light microscopy and for quantification of apoptosis 

using the TUNEL assay. An apoptotic index was calculated based on 1000 –1500 cells per animal, 

evaluated at 400× for the percentage showing apoptosis. The other half of the testis was digested with 

nitric acid for zinc determination by atomic absorption spectrophotometry. Comparisons of testicular 

ZnT-1 mRNA, zinc, and apoptotic index were made using ANOVA, pooling data from the 3 control 

time points, which did not differ from one another. Body weight comparisons, made using repeated 

measures ANOVA, showed a decrease in body weight gain after DEHP exposure beginning on the 

third day of treatment. Absolute testis weight was decreased in the DEHP group on treatment days 
II-132

Appendix II

7 and 14 to, respectively, 45 and 25% of control [estimated from a graph]. Relative testis weight 

was also significantly decreased by DEHP at these time points. DEHP treatment was associated with 

histologic evidence of apoptosis, which occurred early and was most marked by treatment day 3, and 

necrosis, which was most severe from treatment day 7. Testicular zinc did not change over the 14-day 

treatment period in control animals but decreased in DEHP-treated animals beginning on treatment 

day 3 and declining to 73% of the control level by treatment day 14 [estimated from a graph]. The 

testis level of ZnT-1 mRNA was not altered by DEHP until treatment day 14, when it was about 30% 

lower than the control level. The authors noted that the reduction in testicular zinc associated with 

DEHP treatment was consistent with other studies but concluded that this reduction was likely to be 

secondary to DEHP testicular toxicity rather than a cause of it. They noted that zinc depletion was 

not demonstrated prior to treatment day 3, whereas histologic abnormalities of the testis were evident 

within 1 day of initiating treatment. The maintenance of normal ZnT-1 mRNA until treatment day 14, 

well after the earliest demonstration of testicular toxicity, suggested that this transporter protein does 

not play a role in mediating DEHP testicular toxicity.

Strengths/Weaknesses: The technical approach and competence are two strengths of this paper. This 

study explored the loss of zinc as a causative factor that might be unique to the DEHP lesion. The 

authors clearly showed a separation between germ cell death and loss (early) and changes in the zinc 

levels and transporter expression, which happened after the initial germ cell changes. The authors 

marshal considerable previous data in support of their contention that zinc changes are probably 

secondary and do not mediate the germ cell death after DEHP exposure.

Utility (Adequacy) for CERHR Evaluation Process: This study is adequate for the evaluation process 

and is useful in providing a summary of, and some recent data on, zinc changes in the testis and their 

relationship to the DEHP-induced lesion. Collectively, the data suggest that zinc loss is not a causative 

factor in this lesion.

Kasahara et al. (174), supported by the Japanese Ministry of Education, Culture, Sports, Science, 

and Technology, evaluated the role of oxidative stress in DEHP-associated testicular toxicity in Wistar 

rats. In the first experiment, males at 4 –5 weeks of age were given DEHP [purity not specified]

in corn oil by gavage at 0, 1000, or 2000 mg/kg bw/day (n=8–15/group) for 7 days. Animals were 

killed, and body weight and weights of testis, liver, and kidneys were determined. Total glutathione, 

low-molecular-weight thiols, and ascorbic acid content of testis, liver, and kidney were determined, 

and in 5 animals/group, glutathione peroxidase and catalase were estimated in these tissues. Body 

and kidney weights were decreased by about 20% [estimated from a graph] by 2000 mg/kg bw/day 

DEHP. DEHP 1000 mg/kg bw/day increased liver weight by about 50% and decreased testis weight by 

about 20%, with an additional 50% decrease in testis weight at 2000 mg/kg bw/day [estimated from a

graph]. Total glutathione, low-molecular-weight thiols, and ascorbic acid were decreased in testis after 

treatment with DEHP 1000 mg/kg bw/day. Glutathione and low-molecular-weight thiols were increased, 

and ascorbic acid was unchanged in liver and kidney at both DEHP dose levels. These antioxidants 

were unchanged in plasma and brain of treated animals [data not shown]. Glutathione and catalase 

were increased in testis at both doses of DEHP and unchanged or decreased in liver and kidney.

Testicular cells were obtained from minced testis, and differential centrifugation was used to obtain 

predominantly spermatocytes. These cells were incubated with a chemiluminescent probe and digitonin, 
Appendix II

II-133

following which 5 mM NADH was added to initiate release of reactive oxygen species. The release of 

reactive oxygen species was measured as increased chemiluminescence that was reported to increase 

in a time-dependent manner in cells derived from animals treated with DEHP 2000 mg/kg bw/day. 

[The data graph is not marked with significant differences.] The increase in chemiluminescence 

was prevented by addition of superoxide dismutase, catalase, or sodium azide, suggesting to the authors 

that the superoxide radical and hydrogen peroxide were the reactive oxygen species responsible for the 

increased chemiluminescence. Reactive oxygen species generation was also evaluated in Sertoli cell 

and germ cell cultures obtained from 14-day-old rats. Cultures were grown in the presence of DEHP 

or MEHP [the MEHP was synthesized and HPLC-confirmed to be minimally contaminated] at 

concentrations of 0, 25, 50, 100, and 200 μM, using fluorescence of 2′,7′-dichlorofluorescein to estimate 

reactive oxygen species. Fluorescence in germ cells was increased by MEHP in a dose-dependent manner 

beginning at 50 μM [19.6 mg/L]. There was no increase in fluorescence at any concentration of DEHP 

in germ cells and no increase in fluorescence in Sertoli cells at any concentration of either chemical. 

Testicular sections were stained using the TUNEL method 12 hours after administration of DEHP 

2000 mg/kg bw [it is not stated whether these were animals from the first experiment or if this

experiment involved new animals given a single dose of DEHP]. TUNEL-positive cells were reported 

to be markedly increased in the testes of DEHP-treated animals, and TUNEL-positive cells showed 

chromatin condensation after propidium iodide staining, confirming apoptosis [no data were shown

for TUNEL staining or propodium iodide staining].

In a final experiment, mitochondria were isolated from fresh testes [age of animals not specified,

n=3 indicated in data figure] and incubated with DEHP or MEHP at concentrations of 0, 2, 10, 

or 50 μM for 5 minutes. Samples were centrifuged at 8000 g for 5 minutes, and supernatants were 

evaluated using SDS-PAGE and Western blotting for cytochrome c. The authors described an increase 

in cytochrome c release from mitochondria exposed to MEHP but not DEHP [the data figure is not

marked with significant differences].

The authors concluded that DEHP treatment of rats causes testicular atrophy through generation of 

reactive oxygen species and reduction in antioxidant enzymes. MEHP-associated apoptosis in germ 

cells was attributed, at least in part, to release of cytochrome c from mitochondria in response to 

oxidative stress. 

Strengths/Weaknesses: The conclusions from these extensive studies are minimally but measurably 

reduced by the uncertainties noted above. The variety of approaches used for these studies is a clear 

strength. The report is weakened by the use of whole-testis mitochondria, which is a problem, given that 

the testis is composed of DEHP-responsive cells and DEHP-resistant cells, at least as far as oxidative 

response is concerned. Of greater significance are the data indicating that germ cells were affected but 

Sertoli cells were not; this finding contradicts the existing gestalt in the field, supported by numerous 

histologic studies that show changes in Sertoli structure prior to germ cell death. The localization data, 

combined with the time course of apoptosis in vivo and active oxygen generation (Figures 7 and 4B, 

respectively, of the report), make it difficult or impossible to conclude that the oxidative damage was 

a cause of the germ cell death. Additionally, several of the time-dose points had a great deal of cell 

death occurring, making this separation impossible. Thus, it is likely that oxidation occurs as part of 

the cell death process in dying germ cells. 
II-134

Appendix II

Utility (Adequacy) for CERHR Evaluation Process: These data are adequate for the evaluation process, but 

the uncertainties over whether the oxidative damage or the cell death comes first, limit their mechanistic 

utility. The data showing the presence of oxidative damage are clear and believable; the uncertainties 

lie with the assertion that this damage produces rather than results from the germ cell death.

Ablake et al. (175), supported by the Japanese Ministry of Education, Science, Sports, and Culture, 

evaluated the effect of vitamins C and E in accelerating recovery from the testicular toxicity of DEHP 

in CD mice. Ten-week-old males were given 0 or 2% dietary DEHP (>98% purity) for 16 days, following which all animals were given untreated feed for an additional 50 days. A vitamin-supplemented 

group was given drinking water containing vitamin C 3.0 mg/mL and vitamin E powder 1.5 mg/mL 

during the 50-day recovery period. [Based on the reported estimated mean feed consumption of

5 g/mouse/day and mean fluid consumption of 5 mL/mouse/day, and using the starting mean

body weight of 35 g/mouse (estimated from a graph), CERHR calculated a mean DEHP intake

during the treatment period of 2857 mg/kg bw/day. During the recovery period,the mean intake

of vitamin C was calculated at 429 mg/kg bw/day and the mean intake of vitamin E at 214 mg/kg

bw/day.] Counting the first day of DEHP treatment as day 0, 4 – 7 animals/treatment group were 

killed on each of days 0, 3, 5, 7, 9, 12, 15, 25, 35, 45, 55, and 65. [The Expert Panel notes that time

points after day 15 did not include DEHP treatment and included supplemented vitamins C and

E.] Testes were harvested and samples prepared for light microscopy, electron microscopy, TUNEL 

staining, BrdU incorporation, or histochemical detection of lipid peroxidation. Light microscopy 

was performed on sections fixed in Bouin fluid, embedded in plastic, and stained with hematoxylin 

and eosin. Spermatogenic disturbance was graded in at least 100 tubule sections per mouse using a 

10-point scale, and the ranks were averaged to produce an overall score per mouse. Electron microscopy, 

TUNEL staining (using a commercial kit), BrdU incorporation, and detection of lipid peroxidation 

were performed on samples obtained on days 0, 7, and 15 (before, in the middle, and at the end of the 

DEHP treatment period). Statistical analysis was by ANOVA. 

Body weight decreased during the 16 days of DEHP exposure, recovering to control levels 10 days 

after DEHP was removed from the feed. Absolute testis weight increased on day 5 of DEHP treatment, 

and testes appeared grossly edematous. By day 12, testis weight was significantly decreased by DEHP 

treatment. Testis weight recovered toward control levels 20 days after the cessation of DEHP treatment, 

although at the end of the experiment on day 65, testis weight had not completely recovered. Testis 

weight was higher in the group given vitamin-supplementation than in the group not given vitamin 

supplementation during the recovery period [although pair-wise statistical comparisons of these 2

groups were not reported]. Relative testis weight showed a pattern similar to that of absolute testis 

weight. The mean histologic scores declined during the period of DEHP treatment from 10 to 2 [estimated

from a graph]. The scores returned toward control levels after cessation of DEHP treatments with the 

vitamin supplemented group reaching nearly control levels by day 45 and the unsupplemented group 

reaching nearly control levels by day 55. There were significant differences between supplemented 

and unsupplemented DEHP-treated animals in histologic score on experiment days 25, 35, and 45. 

Electron microscopy during the DEHP treatment period showed vacuoles and dark droplets in Sertoli 

cell cytoplasm in contrast to light microscopic findings, which apparently did not show Sertoli cell 

damage. Lipid peroxidation was prominent in the basal compartment of the seminiferous epithelium 

and surrounding interstitial tissues on day 15 of treatment. A few cells with TUNEL staining and BrdU 

staining were noted on day 15, but these evaluations appeared limited due to the hypocellularity of 
Appendix II

II-135

tubules in day 15 DEHP-treated animals. The authors concluded that the antioxidant effects of vitamins 

C and E accelerated the recovery of the testis from DEHP-induced injury.

Strengths/Weaknesses: The novel design of this study is a strength. This study really tests the ability 

of the testis to recover after DEHP-induced damage, and it is not clear that the DEHP damage is 

significantly different from any other toxicant-induced lesion. The findings are weakened by the lack of 

measurement of feed and water intake and the consequent inability to report actual DEHP or vitamin 

consumption, the lack of data on the levels of these two antioxidants in the mouse chow, and the lack 

of verification of water levels of these two vitamins (vitamin E is not inherently water-soluble, making 

this verification more important). The authors concluded that oxidative damage is the mechanism of the 

DEHP lesion, which is consistent with previous literature they cite, but cannot be a logical conclusion 

of this paper because they did not test for abrogation of the DEHP lesion by concurrent vitamin and 

DEHP administration. The DEHP-specific conclusion is also significantly limited by the fact that 

only a single toxicant was used. Had the authors employed another testicular toxicant that works by a 

presumably different mechanism, they might have seen a difference, in which case a DEHP-specific 

conclusion would be warranted. It would also have been very useful for the authors to have evaluated 

this finding in another species to address its extensibility.

Utility (Adequacy) for CERHR Evaluation Process: These data are adequate for the evaluation process, 

as the study was conducted credibly. The data are of use in considering treatments for recovery but 

are of limited use in determining mechanisms of DEHP toxicity.

Kijima et al.(176), support not indicated, administered a single oral dose of DEHP (99% purity) in corn 

oil [gavage assumed] to 6-week-old male Crj:CD(SD)IGS rats at dose levels of 0, 20, or 2000 mg/kg 

bw. This study was conducted to evaluate expression changes in many genes, looking for new leads on 

mechanism. Rats were killed 3, 6, 24, or 72 hours after dosing and testes were excised. There were 6 rats 

per dose group and time point. The right testis of each rat was fixed in 4% paraformaldehyde, embedded 

in paraffin, sectioned at 5 μm, and either stained with hematoxylin and eosin or used for TUNEL staining. 

The left testis was frozen at −80oC for subsequent RNA extraction. RNA extracts were pooled within 

dose and time groups and analyzed using a microarray consisting of 3060 cDNA probes. Altered regulation was defined as expression at less than half or more than twice the control level for the microarrays. 

Real-time PCR was used to evaluate mRNA expression for the apoptosis-related genes caspase-2, -3, -6, 

-8, -9, and -11, bcl-2, bax, fas, and fasL, normalized to b-actin. Statistical analysis was performed using 

the Student t test. There were no treatment-related histologic changes, but apoptotic cells were identified 

by TUNEL staining near the seminiferous tubule basement membrane in testes from rats treated with 

DEHP 2000 mg/kg bw. At 3 and 6 hours, the apoptosis involved spermatogonia, and at 24 hours the 

apoptosis also involved spermatocytes and spermatids. At 72 hours after exposure, only spermatogonia 

and spermatocytes were affected. The proportion of tubules with at least 3 apoptotic cells was increased 

significantly compared to control at 24 and 72 hours after treatment with DEHP 2000 mg/kg bw, with 

maximal response [almost 40%, estimated from a graph] at 24 hours. The microarray analysis patterns 

showed only “slight” overlap for the response to the 20 and 2000 mg/kg bw DEHP treatments. The genes 

up-regulated by DEHP 2000 mg/kg bw included genes involved in apoptosis, cell proliferation, metabolism, stress response, cell adhesion, immune response, DNA repair, and expression sequence tags. The 

RT-PCR results for apoptosis-related genes showed no effect of treatment with DEHP 20 mg/kg bw except 

for a transient increase in bcl-2 at 6 hours. Following treatment with DEHP 2000 mg/kg bw, significant 
II-136

Appendix II

increases in message were seen as early as 3 hours for capsase-2, caspase-3, caspase-8, caspase-9, bax, 

fas, and fasL. These increases persisted through the 24-hour time point and, for the caspases, through the 

72-hour time point. There was a significant decrease in bcl-2 message in the 2000 mg/kg bw group at 3, 

6, and 24 hours after the dose. The authors concluded that DEHP affected all stages of spermatogenesis 

with apoptosis peaking at 24 hours. They postulated that the decrease in identification of apoptosis at 72 

hours may have represented Sertoli cell phagocytosis of apoptotic spermatogenic cells.

Strengths/Weaknesses: The scientific rigor of the approach and the combined use of histology and 

focused gene expression methods are strengths. The RT-PCR experiments were not described in 

adequate detail, and there is an apparent lack of adequate controls. Other weaknesses include lack of 

replicates and insufficient and inappropriate statistical analyses.

Utility (Adequacy) for CERHR Evaluation Process: This study is adequate for the evaluation process, 

although it does not fundamentally add to our knowledge of the initiating mechanisms of DEHP 

damage. These data may help identify the subcellular pathways of cell death used by the testis after 

DEHP exposure.

Kim et al. (177), supported by the Japanese Ministry of Education, Science, Sports, Culture, and 

Technology, evaluated DEHP-associated changes in testosterone catabolism in 4-week-old male 

Wistar rats. Animals (6/group) were given DEHP orally [purity not specified; gavage assumed] in 

corn oil at 0, 100, or 1000 mg/kg bw/day for 5 days. Two weeks later, animals were killed and testes 

and livers were removed and weighed. The tissues were homogenized, and microsomal fractions were 

isolated by centrifugation. Microsomal protein was evaluated using SDS-PAGE and Western blot for 

CYP2C11 and CYP3A2. Blood was obtained for estimation of testosterone using a commercial RIA 

kit. The microsomal fraction was used for quantification of 17b-hydroxylation of androstenedione, 

6b-(CYP3A2) and 16α-hydroxylation (CYP2C11) of testosterone, and 3-hydroxylation (CYP3A2) 

and N-desmethylation (CYP2C11) of diazepam. HPLC was used to quantify the reaction products. 

5α-Reductase was quantified in the microsomal fraction [method specified by reference to a 1979

article]. Total RNA was extracted from testes, and RT- PCR was used to quantify aromatase (CYP19). 

Statistical testing was performed using ANOVA [post hoc testing was not discussed]. Results are 

summarized in Table 29. The authors proposed that the observed increase in 5α-reductase may have 

been a compensatory response to reduced testicular testosterone, and that the reduction in aromatase 

would be expected to result in decreased estrogen availability. The alterations in CYP isoforms were 

characterized by the authors as more sensitive markers of DEHP disruption of testicular steroid 

catabolism than testosterone synthesis from androstenedione.

Strengths/Weaknesses: The use of a recovery period after dosing ended complicates the interpretation 

of DEHP effects. The absence of histopathology renders the condition of the testis at the end of the 

experiment uncertain. The absence of measurements of the end products of each of these enzymatic 

reactions also reduces confidence in the biological importance of the findings. Not having a biological 

correlate or sequelae to these changes also reduces their value.

Utility (Adequacy) for CERHR Evaluation Process: The study is inadequate for the evaluation process. It 

adds slightly to our appreciation of testosterone catabolic pathways, although how and why the findings 

are important, particularly with the two-factored dosing scheme (dosing plus recovery), is not clear.
Appendix II

II-137

Table 29. Testosterone Metabolism after DEHP Treatment of 4 Week-old Rats

Parameter

DEHP dose group 

(mg/kg bw/day × 5 days)

100 1000

Testis weight ↔ ↓ 62%

Serum testosterone ↔ ↓ 34%

Androstenedione 17b-hydroxylation, testis ↔ ↔

5α-Reductase, testis a ↑ 21% ↑ 36%

5α-Reductase, liver ↔ ↔

6b-Hydroxylation of testosterone, testis a ↑1.2-fold ↑1.3-fold

16α-Hydroxylation of testosterone, testis a ↑1.2-fold ↑1.6 fold

3-Hydroxylation of diazepam, testis ↔ ↑1.4-fold

N-Desmethylation of diazepam, testis ↔ ↑ 4.6-fold

Microsomal CYP3A2, testis, Western blot ↔ ↑ 2.3-fold

Microsomal CYP2C11, testis, Western blot ↔ ↑ 2.6-fold

Aromatase mRNA expression, testis b ↓41% ↓53%

aEstimated from graph. 

b [Results in text state that aromatase was reduced by 59 and 47% compared to

control. Figure suggests that the results should have stated that aromatase was

reduced to 59 and 47% of control values.]

↑,↓ Statistically significant increase, decrease in the parameter; ↔ no significant 

change in the parameter.

From Kim et al. (177).

Kim et al.(178), supported by the Japanese Ministry of Education, Science, Sports, Culture, and Technology, performed a follow-up study to evaluate cytosolic phospholipase A2 activity in testes obtained 

from DEHP-treated rats. Four-week-old male Wistar rats were given DEHP [purity not specified] in 

corn oil at 0, 100, or 1000 mg/kg bw/day for 5 days. Testes were removed 2 weeks after the last treatment, and cytosol and microsomes were isolated by centrifugation of S-9 fractions. Phospholipase A2 

activity was measured using a commercial kit. Phospholipase A2, cyclooxygenase-2, 12-lipoxygenase, 

and CYP4A1 were evaluated using SDS-PAGE with chemiluminescence detection. Arachidonic acid 

was measured using reverse-phase HPLC. Statistical analysis was by Dunnett test. DEHP treatment 

did not alter rat body weight, although serum testosterone and testicular weight were decreased by 

treatment with DEHP 1000 mg/kg bw/day. Cytosolic phospholipase A2 was decreased to 62% of control 

values by DEHP 1000 mg/kg bw/day. Arachidonic acid concentrations were decreased 18% by DEHP 

100 mg/kg bw/day and 24% by DEHP 1000 mg/kg bw/day. The expression of lipo­oxygenase-12 and 

CYP4A1 was increased by DEHP [quantification not given], but cyclooxygenase-2 was not altered 

by treatment. The authors postulated that a DEHP-associated decrease in arachidonic acid may be 

at least partly responsible for a decrease in testosterone synthesis. The decrease in arachidonic acid 

may be due to a decrease in formation by phospholipase A2 and a decrease in metabolism by lipooxygenase-12 and CYP4A1. The increase in CYP4A1 may have been due to PPARα activation by 

DEHP (not measured in this study) and would be expected to increase lipid oxidation. Lipoxygenase 

activity would also be expected to generate superoxide.
II-138

Appendix II

Strengths/Weaknesses: This study used the same confounding dosing-plus-recovery schedule did in the 

previous study (177). However, the evaluation of active fatty acids is a significant plus, as it broadens 

our appreciation of biological sequelae of DEHP exposure. The dosing sequence renders the results 

difficult to interpret.

Utility (Adequacy) for CERHR Evaluation Process: The dosing and recovery schedule make this study 

difficult to interpret and prevent it from being useful in the evaluation process. The focus on phospholipase A2 and arachidonic acid pathways may be a potentially important contribution to mechanistic 

understanding of DEHP toxicity but is not interpretable at this time based on the treatment schedule.

Akingbemi et al. (111), supported by NIEHS, evaluated the effect of DEHP on Leydig cell function 

in male Long-Evans rats exposed during pregnancy, lactation, or post-weaning stages (pregnancy and 

lactation results are presented in Section 3.2.1.1). Prepubertal rats were exposed to DEHP (>99% purity) 

on PND 21 –34, 35–48, or 21 –48, and young adult rats were exposed on PND 62 –89. DEHP was 

administered by gavage in corn oil at 0, 1, 10, 100, or 200 mg/kg bw/day (n=10/dose group). Animals 

were decapitated within 24 hours of the final dose, and trunk blood was collected for measurement of 

LH and testosterone by RIA. Testes and seminal vesicles were weighed, and testicular interstitial fluid 

was collected for measurement of fluid testosterone by RIA. Testicular histology was evaluated. Leydig 

cell cultures were prepared from testes by Percoll density gradient preceded in 49- and 90-day-old rats 

by centrifugal elutriation. The resulting Leydig cell preparations were 95 – 97% pure, as evaluated by 

staining for 17-hydroxysteroid dehydrogenase. Cultures were evaluated after 3 hours incubation with 

and without a maximally stimulating concentration of ovine LH. Testosterone was measured in the 

medium. The activity of different enzymes in the testosterone biosynthesis pathway were evaluated 

by incubating Leydig cells for 3 hours with saturating concentrations of substrate for the enzyme of 

interest: 22R-hydroxycholesterol for P450-cholesterol side-chain cleavage enzyme, pregnenolone 

for 3b-hydroxysteroid dehydrogenase, progesterone for P450-17α hydroxylase/17,20-lyase, and 

androstenedione for 17b-hydroxysteroid dehydrogenase. The steroid products were measured by HPLC 

with ultraviolet detection. Statistical analysis was by ANOVA and Duncan multiple range test.

There were no treatment-related effects on body weight gain or feed consumption. Treatment of prepubertal rats on PND 21–34 or PND 35–48 did not produce alterations of serum LH or testosterone, but 

longer treatment (on PND 21–48) produced a dose-related increase in serum LH and testosterone and in 

interstitial fluid testosterone that was statistically significant at a DEHP dose level of 10 mg/kg bw/day 

(Figure 9). Leydig cells isolated from rats after DEHP treatment on PND 21–34 showed a decrease in 

basal and LH-stimulated testosterone production in vitro (LOAEL 100 mg/kg bw/day). Leydig cells 

isolated from rats after DEHP treatment on PND 35–48 displayed decreased basal and LH-stimulated 

testosterone production in vitro (LOAEL 10 mg/kg bw/day) and decreased activities of enzymes in 

the testosterone synthesis pathway. All tested enzyme activities were affected, with the most sensitive 

being 17b-hydroxysteroid dehydrogenase (reduced 74% at 10 mg/kg bw/day compared to control; other 

enzyme activities were significantly reduced at DEHP dose levels of 100 or 200 mg/kg bw/day). Leydig 

cells isolated from prepubertal rats that had been treated on PND 21–48 showed an increase in basal 

and LH-stimulated testosterone production with a LOAEL of 10 mg/kg bw/day. There was no effect 

of DEHP treatment in adults at any tested dose on serum testosterone or LH or on in vitro Leydig cell 

steroidogenesis [data not shown]. There were no effects of any treatment on testicular histology.
Appendix II

II-139

Figure 9. Effect of DEHP Treatment in Male Rats on PND 21 –48.

B

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LOAEL=10 mg/kg bw/day by pair-wise testing (drawn from data presented by Akingbemi et al. (111)).

The authors postulated that the decrease in in vitro steroidogenesis after treatment of rats on PND 

35–48 without a decrease in evidence of in vivo testosterone synthesis may have represented enzyme 

inhibition that was not great enough to alter in vivo serum testosterone levels; i.e., the animals could 

compensate for the treatment effect on synthesis. The authors pointed out that the substrates were 

provided for the Leydig cell culture experiments in higher amounts than may typically be available 

in vivo. The authors believed that the increased testosterone and LH serum levels and the increase in 

in vitro testosterone biosynthetic ability of Leydig cells after 28 days of DEHP treatment represented 

a compensatory mechanism, with a disruption of the negative feedback mechanism that typically 

maintains homeostatic testosterone levels. They identified 1 mg/kg bw/day as a NOAEL in their 

experiments and 10 mg/kg bw/day as a LOAEL.

Strengths/Weaknesses: This study benefits from intelligent study design, technical expertise, and 

appropriate dosing intervals and quantities. Some of the groups have fewer animals than might be 

optimal. Appropriate statistics is a strength.

Utility (Adequacy) for CERHR Evaluation Process: This study is adequate and useful for the evaluation 

process because it helps identify a vulnerable window of exposure and during that more-vulnerable 

period, does a good job of parsing the dose-response curve. The study effectively separates inherent 

effects from compensatory change by the smart use of ex vivo cultures and identifies critical enzymatic 

steps in steroidogenesis.

Akingbemi et al.(179), supported by NIEHS, evaluated Leydig cell hyperplasia and steroid production 

in response to DEHP treatment of male Long-Evans rats as a follow-up to the previous study (111) 

in which treatments were given on PND 21 – 48. Three experiments were described in the current 

paper, with comparisons performed using ANOVA and post hoc Dunnett multiple range test. In the 

first experiment, animals were gavaged on PND 21 to PND 90 or to PND 120 with DEHP [purity
II-140

Appendix II

not specified] in corn oil at 0, 10, or 100 mg/kg bw/day (n=10/group). These treatments caused no 

significant change in body weight or paired testis weight at 120 days of age. At the end of the treatment periods, serum LH and testosterone were measured by RIA, and Leydig cells were harvested 

by collagenase digestion and Percoll density centrifugation [methods obtained from the earlier

paper (111)]. The Leydig cells were cultured for 3 hours with and without maximally stimulating 

concentrations of ovine LH. Medium testosterone was measured by RIA and normalized to cell number. 

In the animals treated until PND 90, serum LH and testosterone were increased in both DEHP dose 

groups compared to controls, and testosterone production in Leydig cell culture was significantly 

reduced, both with and without added LH. On PND 120, the increase in serum LH and testosterone 

and the decrease in basal and stimulated testosterone production in culture were identified only in the 

animals exposed to DEHP 100 mg/kg bw/day. The study authors believed the results were consistent 

with Leydig cell hyperplasia because serum testosterone was increased in the face of a reduction in 

testosterone production per Leydig cell.

In the second experiment, the possibility of Leydig cell hyperplasia was evaluated using markers 

of cell division (PCNA, cyclins D3 and G1, and p53, estimated by RT-PCR of steady-state mRNA 

levels in Leydig cells), incorporation of tritiated thymidine in cultured Leydig cells, and counting of 

Leydig cells in 10 sections/testis. The treatment regimens were the same as in the first experiment. 

mRNA levels were increased in both DEHP exposure groups for PCNA and both cyclins and in the 

high-dose group for p53. Leydig cell numbers were also increased in both DEHP dose groups. [Data

for these outcomes were only described for the animalstreated until PND 90.] Tritiated thymidine 

incorporation was increased in both DEHP exposure groups in animals treated until PND 120. [Data

not described or shown for animals treated until PND 90.]

The third experiment evaluated the hypothesis that a DEHP-associated increase in LH induced estrogen 

synthesis. Male rats were gavaged with DEHP in corn oil at 0, 10, or 100 mg/kg bw/day from PND 21 

to PND 48 or 90 at which time serum estradiol was measured by RIA. Leydig cells were cultured for 

3 hours with or without exogenous LH, and estradiol in the medium was measured and normalized 

for cell number. Aromatase mRNA was measured in Leydig cells. Serum estradiol was increased on 

PND 48 in rats in both DEHP exposure groups. Basal estradiol production in cultured Leydig cells 

was increased in the 100 mg/kg bw group, and LH-stimulated estradiol production was increased in 

both DEHP dose groups on PND 48. Aromatase mRNA was also increased in Leydig cells from both 

DEHP-treated groups. On PND 90, there was no significant difference between control and DEHPtreated animals in serum estradiol level, in basal or LH-stimulated estradiol production in culture, or 

in aromatase mRNA. MEHP but not DEHP was detectable in serum by HPLC on PND 48 and 90, 

suggesting that MEHP mediated the testicular effects of DEHP treatment. Serum MEHP was more 

than 6-fold lower [estimated from a graph] on PND 90 than on PND 48.

The authors concluded that DEHP increases Leydig cell populations associated with chronically 

increased LH and testosterone levels, and that a decrease in testosterone and increase in estradiol 

synthesis (per cell) were consistent with induction of aromatase activity in Leydig cells. They further 

proposed that the increase in estradiol and LH could act together to increase cyclin proteins and thereby 

increase Leydig cell numbers. They concluded that the Leydig cell hyperplasia identified in their study 

might have implications in the production of testicular neoplasms, and that chronic exposure to DEHP 

had anti-androgenic and estrogenic activity.
Appendix II

II-141

Strengths/Weaknesses: The multiple technical approaches and good design of these studies are buttressed by the sequential hypothesis-testing approach to produce an excellent paper. This report extends 

our knowledge about the hormonal perturbations to include hyper-estrogenization, and deepens our 

appreciation of the complexity of this syndrome.

Utility (Adequacy) for CERHR Evaluation Process: The data are adequate for the evaluation process 

and increase the useable data-set by adding estrogen-induced effects to the spectrum of sequelae. The 

use of 100 mg/kg bw/day is not meant to plumb a NOAEL but to employ an effective dose level. This 

paper suggests that elevated levels of estradiol might be present in some of these animal models and 

human populations and should be considered in further evaluations.

Rasoulpour and Boekelheide (180), support not indicated, evaluated MEHP activation of the transcription factor NF-κB, which is a mediator of the Fas signaling pathway. Fischer 344 rats were treated 

when 28 days old with single gavage doses of MEHP [purity not given] in corn oil at 0 or 1000 mg/kg 

bw. Testes were excised and processed 1, 3, 6, and 12 hours after treatment (n=3 or 4 rats/treatment/time 

point). Sections were immunostained for the NF-κB subunits p50, p65, c-Rel, and Rel-B. TUNEL 

staining was performed on additional sections and seminiferous tubules evaluated for the presence of 

0, 1 – 3, or >3 apoptotic cells/tubule section. Nuclear protein was extracted from homogenized testes 

and used to assess NF-κB-binding by electrophoretic mobility shift assay. Statistical analysis was by 

ANOVA with Bonferroni correction.

Within 1 hour of MEHP treatment, p50 staining increased in spermatocytes. Distribution of the p65 

subunit (Sertoli cells, Leydig cells, spermatogonia, and spermatids) was not altered by MEHP treatment 

at 1 hour, but at 3 hours some spermatocytes showed staining and Sertoli cell staining was intense. 

At 6 hours, all germ cells were positive for p50, p65, and c-Rel, and at 12 hours the interstitium was 

also involved. Rel-B staining was not influenced by treatment at any time point. TUNEL staining was 

decreased by MEHP at 6 hours post-treatment, but was increased at 12 hours with 37.4% of tubules 

showing >3 apoptotic cells compared to 4.1% of control tubules. Increased NF-κB-binding activity 

was shown by electrophoretic mobility shift, which peaked 1 hour after treatment, with p50 and c-Rel 

identified as the main subunits activated.

The authors considered the changes in NF-κB subunit localization in spermatocytes to be consistent 

with the sensitivity of these cells to MEHP-induced damage. They hypothesized that NF-κB through 

its anti-apoptotic effects may play a protective role in the MEHP-exposed testis, with a decrease in 

apoptosis at 6 hours that subsequently is overwhelmed. 

Strengths/Weaknesses: This paper examined the testicular response of NF-κB to phthalate exposure 

with a focus on examining activation of this system as part of the injury response. Strengths include 

appropriate techniques for assessing a whole-testis response, including use of immunohistochemistry 

and electrophoretic mobility shift assay. The biological relevance of these proteins and their involvement in cell death is a strength as are the many time points examined. Weaknesses include the use of 

a single high dose of exposure and the uncertainty of how these changes relate to cell death since all 

cells showed the changes whether or not they were killed by phthalates.

Utility (Adequacy) for CERHR Evaluation Process: This paper may add to the mechanistic under-
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Appendix II

standing of the response to phthalate treatment but does not contribute to the evaluation of risk in the 

CERHR process.

Awal et al. (181), supported by the Japan Society for the Promotion of Sciences and the Japanese 

Ministry of Health, Labor, and Welfare, evaluated the testicular effects of MEHP given to pre-pubertal 

guinea pigs. The animals were treated at 5 weeks of age with a single gavage dose of MEHP [purity

not given] in corn oil at 0 or 2000 mg/kg bw. Animals were killed 3, 6, or 9 hours after treatment 

for evaluation of testes by light and electron microscopy. [The number of animals per group was

not explicitly stated. If group sizes were equal, there were 2 guinea pigs/treatment/time point.]

For light microscopy, testes were dehydrated in ethanol, cleared in xylene, and embedded in paraffin. 

Sections were stained with hematoxylin and eosin or with hematoxylin and periodic acid Schiff stain. 

TUNEL staining was used for evaluation of apoptotic spermatogenic cells, expressed as a percentage 

of total spermatogenic cells, and analyzed with ANOVA and Fisher least significant difference test.

At 3 hours post-dosing, testicular morphology was described as normal by light microscopy, although 

the authors noted sloughed spermatogenic cells in the efferent ductules. There was “progressive detachment” of spermatogenic cells between 3 and 9 hours [not otherwise quantified]. Electron microscopy 

showed degenerative vacuolated Sertoli cells [time course not described]. TUNEL staining showed a 

time-dependent increase in the percentage of apoptotic spermatogenic cells beginning at 3 hours and 

reaching a value of ~9% at 9 hours [estimated from a graph; control value ~0.7%].

The authors concluded that MEHP causes testicular toxicity in guinea pigs manifested as an increase 

in germ cell apoptosis mediated by direct effects on the apoptotic pathway or through altered Sertoli 

cell function.

Strengths/Weaknesses: This paper essentially replicates work from the early 1980s by re-demonstrating 

the known sensitivity of the guinea pig to phthalate exposure, and shows that germ cells rapidly die by 

apoptosis after high-dose exposure. The low number of animals and high dose level are weaknesses.

Utility (Adequacy) for CERHR Evaluation Process: Because of the single high dose used and because 

these studies have been done previously and incorporated into the first evaluation of DEHP, this paper 

adds nothing to the current evaluation.

Ljungvall et al.(182), supported by the Swedish Environmental Protection Agency, treated pre-pubertal 

male pigs with DEHP (99.6% pure) 50 mg/kg bw/dose, estradiol benzoate 0.25 mg/kg bw/dose, or 

peanut oil vehicle. Treatments were given twice/week im for 5 weeks, beginning 1 week after weaning, 

at 6 weeks of age [DEHP 50 mg/kg bw/dose twice/week would be 100 mg/kg bw/week or about 14

mg/kg bw/day; the Expert Panel notes that this calculated daily dose after twice-weekly injection

may not be comparable to a similar oral daily dose.The authorsjustified the im route asthe most

practical route for treating young pigs.] Evaluations were planned for 5 days after the last treatment (the 

immediately evaluated group) and for 4.5 months after the last treatment period (the delayed-evaluation 

group). The immediately evaluated group consisted of 1 male pig from each of 4 litters assigned to each 

treatment (4 male pigs/treatment group). The delayed-evaluation group consisted of 5 male pigs from 

4 litters assigned to DEHP, an additional 5 male pigs from 4 litters assigned to the vehicle control, and 

3 male pigs from 3 litters assigned to estradiol benzoate. In the immediately evaluated animals, blood 
Appendix II

II-143

was sampled weekly during treatment for plasma testosterone, 17b-estradiol, and LH by RIA. Five 

days after the last treatment, immediately evaluated animals were necropsied and relative testicular 

weight was recorded. In the delayed-evaluation group, blood was collected for the plasma testosterone, 

17b-estradiol, and LH before the first treatment, after the last treatment, and at the time of slaughter 

at 7.5 months of age. Necropsy was performed, and relative testicular and seminal vesicle weights 

were recorded. Hematoxylin and eosin-stained testis sections were evaluated by light microscopy, and 

immunohistochemistry for vimentin was performed. Digitized images of testis sections were evaluated for 

Leydig cell area and tubule diameter. Leydig cell area relative to tubule area, the proportion of testicular 

morphologic abnormalities, and the proportion of tubules in Stage VIII were compared between groups 

with the Wilcoxon-Mann-Whitney test. ANOVA was used for other comparisons.

In the immediately evaluated group, there were no significant differences in plasma testosterone concentration in DEHP- or estradiol benzoate-treated animals compared to control. At 7.5 months of age, DEHP 

treatment was associated with an increase in plasma testosterone, and estradiol benzoate treatment was 

associated with a decrease in plasma testosterone compared to control. There were no DEHP-related 

effects on plasma estradiol. Plasma LH was described by the authors as tending to be higher after 2 weeks 

of DEHP treatment (P=0.08) in the immediately evaluated group. In the delayed-evaluation group, LH 

was lower in the DEHP group prior to treatment compared to the control group. There were no significant 

effects of DEHP treatment on relative weight of the testis or seminal vesicles. There were no significant 

differences in testis morphology in the DEHP-treated animals, although Leydig cell area relative to 

tubule area was increased by DEHP to 0.45±0.026 (SEM) from a control value of 0.35±0.033. The 

authors noted a “weak indication that spermatogenesis was affected by DEHP” based on an increase in 

proportion of Stage VIII tubules (P=0.1). They indicated that although they had characterized DEHP 

toxicokinetics in male pigs after oral administration (183), they did not have data on metabolism or 

kinetics after im administration in male pigs. They noted that in rats, parenterally administered DEHP 

is metabolized to MEHP. The authors concluded that DEHP administered before puberty in male pigs 

increased plasma testosterone and testosterone-producing cells 4.5 months after the last exposure.

Strengths/Weaknesses: The variety of appropriate measures and time points and the examination of a 

new species make this a useful and relevant study. The im injection route complicates the toxico­kinetic 

profile somewhat but does not fundamentally detract from the utility of this study. The low numbers of 

animals used, while logistically justifiable, complicates the interpretation and reduces our confidence 

in the veracity of the data. The twice-weekly administration, in the absence of blood level measures 

of MEHP, also weakens the direct comparison of these data to other daily dosed lab animal data.

Utility (Adequacy) for CERHR Evaluation Process: This study is adequate and useful for the evaluation 

process in showing Leydig cell hypertrophy in young swine (in parallel with that shown in rats in other 

studies). The serum hormone changes previously documented in rats do not appear as consistently 

in these swine, perhaps because of the low numbers and twice-weekly dosing schedule. These data 

are sufficient to show that twice-weekly dosing with 50 mg/kg to young swine can cause Leydig cell 

hypertrophy 4.5 months after the end of exposure.

Ljungvall et al. (184), supported by the Swedish Environmental Protection Agency, treated postweaning male piglets with DEHP and evaluated post-pubertal sexual function. The pigs were Swedish 

Yorkshire×Swedish Landrace crossbreeds. Two male siblings in each of 10 litters were selected 
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Appendix II

to receive DEHP (99.5% pure) 300 mg/kg bw/dose or water. Treatments were by oral gavage and 

were given 3 times/week for 4 weeks beginning at weaning (3 weeks of age). During the exposure 

period, blood samples were taken weekly for measurement of testosterone, 17b-estradiol, and LH. 

Mating behavior was evaluated with an artificial sow when males were 6 – 9 months of age. Mating 

trials occurred twice/week for 14 weeks. Two pigs died of unrelated illnesses, and their siblings were 

removed from the trial, resulting in 8 sibling pairs for the mating evaluations. At 9 months of age, 

the male pigs had iv catheters placed while under anesthesia, following which the GnRH agonist 

buserelin was administered. Blood samples were drawn periodically from the catheters from 1 hour 

before administration of buserelin until 12 hours thereafter, and the response of LH and testosterone 

to the GnRH agonist was determined. Statistical analysis included paired t-tests, chi-squared, and the 

Wilcoxon nonparametric test.

During the second week of exposure to DEHP, control animals showed ~44% reduction in plasma LH 

[estimated from a graph] compared to pretreatment and week 1 values. DEHP-treated animals did 

not demonstrate the week 2 LH decrease. There were no other differences between DEHP-treated and 

control pigs in plasma LH, testosterone, or 17b-estradiol during the treatment period. The response 

of plasma LH and testosterone to buserelin treatment was similar in DEHP-treated and control pigs 

except for 1 time point of 36 (45 minutes), when plasma LH was lower in DEHP-treated animals 

than in controls. Sexual behavior, evaluated by number of trials to first mount, number of trials to 

first ejaculation, time to first mount, proportion of trials with mount, and proportion of trials with 

ejaculation, did not differ by treatment group. 

The authors concluded that decreased LH response to GnRH stimulation occurred, although the significance of the finding was unknown. The hormonal effects of DEHP in young pigs were described 

as “minor,” and the lack of effect on mating behavior was considered possibly due to species differences between rats and pigs. [The Expert Panel disagrees with the authors’ conclusions that the

decreased LH at 45 minutes was meaningful, treatment-related, or represented a “lasting effect

on the hypothalamus-pituitary-gonadal axis.” Given that only 1 (the 45 min) time point out of

36 was significant and there was no supporting trend for depressed LH at other time points, this

single change appears to be spurious. In addition, if each time point was analyzed with α=0.05,

approximately 2 false positives out of the 36 evaluations would be expected.This single findings

falls within the expected false positive rate.]

Strengths/Weaknesses: Strengths include the use of another species, although no rationale was given 

for this particular species, and multiple timepoints for analysis. There was excellent statistical design 

and control for litter and sibling effect. A split-litter design was used, balancing littermates across the 

treatment and control groups. In addition, since the piglets and not the sows were dosed post-weaning, the individual piglet was the appropriate unit of analysis. Post-weaning dosing also diminished 

maternal and litter influence. The sample size was sufficient given the use of repeated measures and 

the inclusion of an animal factor in the statistical designs to account for repeated measure on the 

same animal. A robust evaluation of LH and testosterone was conducted following GnRH stimulation, 

utilizing repetitive blood sampling at short intervals. Weaknesses include the single dose level used. 

This dose did not produce significant adverse effects. It is not clear if the pig is a more or less relevant 

species (than the rat) for evaluating effects on human reproductive hormones and sexual behavior 

following exposure to DEHP. 
Appendix II

II-145

Utility (Adequacy) for CERHR Evaluation Process: These data show that exposure to DEHP at 300 

mg/kg, 3 times per week, between 3 and 7 weeks of age, did not produce adverse effects on LH, 

testosterone, or mating behavior of male pigs. Because only a single dose level was used, the data 

cannot be used in models to estimate NOAELs/LOAELs. It is not clear that the conclusion of the 

authors that different species (rats versus pigs) may respond differently to perinatal DEHP exposure 

is supported by their findings. Additionally, statements in the Discussion (“noted lower concentrations 

of LH in the DEHP-treated animals after GnRH stimulation.”) appear to contradict the data in Figure 

2 of the paper. These data are of limited utility in the evaluation process.

4.2.3 	 Male and Female

Kim et al. (185), support not indicated, evaluated the effects of DEHP (99% pure) on gonad development and serum vitellogenin in Japanese medaka. DEHP was dissolved in acetone and added to 

aquarium water at up to 250 μL/L; controls received acetone. Serum vitellogenin was assessed after 

exposure for 5 days to DEHP-containing water at 0, 10, 50, and 100 μg/L, n=10 fish/concentration 

[both males and females; ratio not indicated]. Serum was evaluated using SDS-PAGE, and a band 

at 200 kDa was taken to represent vitellogenin based on the finding that 17b-estradiol produced a 

prominent band in this location. The heaviness of the band on the gel was evaluated with the naked 

eye to compare the effects of treatments on vitellogenin content of serum, with the finding that DEHP 

exposure appeared to decrease the heaviness of the band in female fish [the text says that this effect

occurred at 1 μg/L, which was not listed in the Methods as 1 of the concentrations used in this

experiment]. A chronic experiment was performed using DEHP concentrations of 0, 1, 10, and 50 

μg/L with exposure from 1 or 2 days after hatching until 3 months of age. Serum vitellogenin was 

evaluated in 1 male and 1 female per exposure level. The remaining fish were fixed in neutral buffered 

formalin, weighed, and measured; gonads were removed for weighing and histologic evaluation. After 

the chronic exposure, vitellogenin was again described by visual inspection as reduced in females 

[effect level not given]. Mean weight and length were not altered by treatment. Relative testicular 

weight and testicular histology were not altered by treatment, but relative ovarian weight was reduced 

at DEHP concentrations of 10 and 50 μg/L [to 33 and 38% of control values]. Histological evaluation revealed impaired maturation of oocytes, with the achievement of yolk deposition in 54% of 

control females, 37% of females exposed to DEHP 1 mg/L, no females exposed to DEHP 10 μg/L, 

and in 22% of females exposed to DEHP 50 μg/L. The authors concluded that the decrease in serum 

vitellogenin and the impairment of oocyte maturation associated with DEHP exposure were consistent 

with an anti-estrogenic effect of DEHP. [Actual concentrations of DEHP were noted to be 88% of

target at 0 hours and 57% of target at 72 hours. The authors calculated that their protocol of

changing the DEHP solutions every 48– 72 hours would have resulted in exposure to 73 – 78%

of the nominal concentrations as a time-weighted average.]

Strengths/Weaknesses: The strengths of this paper include the measurement of DEHP in the aquarium 

water. The paper is weakened both by the fact that vitellogenin was not determined by Western blotting 

or some other method of positive identification and by the extremely low numbers of animals used to 

collect the vitellogenin data after “chronic” exposure (1/group). A repeated t-test was not the correct 

statistic to use to compare multiple dose groups to controls. Consequently, it is difficult to believe the 

significance values for the relative gonadal weights, especially given that the mean and SD for the controls 

are the same approximate size (making a reduction by approximately 67% in the middle-dose group 
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Appendix II

of dubious significance). The bar graph representation of the oocyte progression data was useful. The 

non-dose-related changes increase our uncertainty of the veracity and replicability of these findings.

Utility (Adequacy) for CERHR Evaluation Process: This paper is adequate to show that water-based 

exposure to ~7 and ~35 μg/L DEHP was effective in reducing ovarian development, although the 

actual degree of this reduction is as difficult to ascertain. The relevance to mammalian reproductive 

processes of these data is uncertain.

Mitsubishi Chemical Safety Institute,Ltd.(92), in an unpublished report, described a 65-week oraldose toxicity study of DEHP in marmosets. [Data from this report also appeared in an abstract

(186).] The study was sponsored by the Japan Plasticizer Industry Association. DEHP (99.6% purity) 

was administered by gavage in corn oil to juvenile marmosets beginning at 90–115 days of age at dose 

levels of 0, 100, 500, and 2500 mg/kg bw/day (n=9 males and 6 females/dose group). The rationale 

for the age at onset of treatment was to start treatment as early “as technically possible.” The treatment 

period was designed to extend to the age of sexual maturation at about 18 months. Wasting and death 

occurred in 1–3 males/group but was not treatment related, and these animals were replaced. Blood was 

drawn every 13 weeks for hematology, chemistry, and determination by RIA of testosterone, estradiol, 

triiodothyronine, and thyroxine. Animals were killed 1 day after the final DEHP dose. Six males and 

all 6 females in each dose group underwent gonad removal. The left testis and epididymis were fixed 

in Bouin fluid for light microscopy. The right testis and epididymis were frozen for sperm counting, 

and a portion of the frozen right testis was used for determination of zinc, sorbitol dehydrogenase, 

γ-glutamyl transpeptidase, total glutathione, glutathione-S-transferase, and glutathione peroxidase. 

Left ovaries were frozen for histochemistry for 3b-hydroxysteroid dehydrogenase determination. 

Livers from both sexes were analyzed for enzyme levels or activities. An additional 3 males per dose 

group were perfused with glutaraldehyde, and testes were used for 3b-hydroxysteroid dehydrogenase 

histochemistry and for electron microscopy. Data were analyzed using ANOVA with post hoc Dunnett 

or Scheffé test or Kruskal-Wallis test with post hoc Dunnett rank-sum or Scheffé test.

In analyzing their data, the study investigators excluded certain animals from inclusion in summary 

tables because they were considered to be “growing.” [Although exclusion was reportedly related

to low body weight, this criterion appears to have been applied somewhat arbitrarily without a

clearly stated rationale.Although it is recognized thatsexually immature animals may reasonably

be excluded from the summary analysis of certain parameters (i.e., sperm counts), the lack of a

clear rationale makesthe interpretation and independent assessment of the conclusions difficult.]

According to the summary tables reported, there were no treatment-related alterations in hematology, 

blood chemistry, or blood hormones. Body weights were not affected by treatment. Organ weights were 

not affected except for ovarian and uterine weights, which were significantly increased at DEHP dose 

levels of 500 and (for ovaries) 2500 mg/kg bw/day (Table 30). These increases in ovarian and uterine 

weight were associated with elevations, relative to controls, in serum 17b-estradiol at DEHP dose 

levels of 500 and 2500 mg/kg bw/day [suggesting an accelerated onset of puberty with exposure]. 

Mean serum testosterone levels were highly variable, but the data suggested the possibility of a delay 

in the onset of puberty with increasing DEHP dose. Testicular enzymes were not altered by treatment, 

although there was a 16 – 21% decrease in testicular zinc at the 100 and 500 mg/kg bw/day dose levels. 

There were no alterations in sperm counts; however, 1 animal in each of the DEHP groups was omitted 

from the analysis as having exceptional values due to these animals being diagnosed as “growing.” 
Appendix II

II-147

Hepatic cytochrome P450 content and testosterone 6b-hydroxylation were significantly increased in 

females at DEHP 500 mg/kg bw/day, but hepatic peroxidase enzymes were not altered by treatment. 

There were no gonadal histopathologic findings by light or electron microscopy that were attributable 

to DEHP treatment, although small growing animals had testicular findings consistent with immaturity. 

Degenerative testis changes similar to those described elsewhere for rodents were not identified in 

marmosets. Immunohistochemistry findings were not altered by treatment. 

Table 30. Ovary and Uterine Weight Findings in a Marmoset 65-Week DEHP Feeding Study

Organ Weightb Dose Level (mg/kg bw/day) Benchmark Dose a (mg/kg bw/day)

100 500 2500 BMD10 BMDL10 BMD1 SD BMDL1 SD

Ovary

Absolute 100 180* 169* 507 259 2063 1196

Relative 106 167* 162* 572 303 1999 1173

Uterine

Absolute 106 188* 168 562 258 2545 1356

Relative 100 167* 150 677 296 2759 1374

aCalculated by CERHR. See Table 22 for definitions and a discussion of the use of benchmark dose in this report.

bData presented as percent of control. 

*Significantly different from control, P<0.05. From Mitsubishi Chemical Safety Institute, Ltd. (92).

The authors concluded that in spite of demonstration of absorption of the compound, as manifested 

by adaptive hepatic changes, DEHP in marmosets did not exert testicular toxicity at a dose level as 

high as 2500 mg/kg bw/day. The authors suggested, based on an accompanying pharmacokinetic study 

(reviewed in Section 2.1), that the most likely explanation for the lack of testicular toxicity of DEHP 

in marmosets as opposed to rodents was the limited accumulation of DEHP and its metabolites in the 

marmoset testis. [The Expert Panel noted the paper by Li et al.(187),which evaluatesthe marmoset

as a model for reproductive studies. Li et al. described the relatively high free levels of steroids

in marmosets, which result in a generalized end-organ steroid insensitivity syndrome and differ

from steroid levels in rats, humans, and other Old World primates (to which humans are most

closely related).This insensitivity would be unremarkable except in the context of a toxicant the

mechanism of action of which involves reduced steroid levels and downstream effects flowing from

the absence of that steroid. An animal model that does not need gonadal steroids to the degree

that humans do would not be an optimal model for this situation. In addition, Li et al. noted

the short gut transit time (4 –8 hours) and the propensity to diarrhea, which would combine to

limit absorption and keep circulating levels of an orally dosed compound low.Another factor is

that common marmosets require high levels of dietary vitamin C in their diet, and vitamin C is

protective against the testicular effects of DEHP in rats and mice (175, 188).In addition,the lack

of LH (and concomitant use of chorionic gonadotropin to fulfill the general functions of LH) is

another, arguably less serious, difference between marmosets and humans.]

Strengths/Weaknesses: Strengths of this study are the use of a primate species, the length of exposure, 

the determination of DEHP and MEHP levels in numerous tissues, and the numerous endpoints 

relevant to the reproductive system. Limitations include concerns about the husbandry practices, 

because 1–3 animals per group had to be replaced during the treatment period due to “wasting;” the 

marked variability within groups for some of the endpoints (for example, serum testosterone); failure to 
II-148

Appendix II

collect all appropriate data (for example, testis weights in the Group 2 animals); a somewhat arbitrary 

exclusion of certain animals from consideration in summary tables because of apparent immaturity; 

and a general lack of transparency in the study design and the intended use of the animals. Additional 

weaknesses are those of marmosets as noted above, which significantly limit our reliance on this species 

as a surrogate for humans.

Utility (Adequacy) for CERHR Evaluation Process: These data are useful for the evaluation process 

and suggest that marmosets, exposed to DEHP at up to 2500 mg/kg bw/day from ~3 months of 

age until ~18 months of age (sexual maturity), had no discernable effect on the testis, either at the 

ultrastructural, histochemical, or gross functional (sperm count) level, although there are significant 

limitations to the confidence that can be placed on these male data. In addition, it is important to note 

apparently unique features of endocrine responsivity in the developing male marmoset that limit the 

relevance of these data to the evaluation process. The data are adequate to show an effect on ovary and 

uterine weight in these young adults at necropsy at administered doses of 500 and (for ovary) 2500 

mg/kg bw/day associated with increases in serum 17b-estradiol and suggestive of an earlier onset of 

puberty in the 2 high-dose groups relative to the control and low-dose groups.

Schilling et al. (151), sponsored by the European Council for Plasticizers and Intermediates (a sector 

group of CEFIC, the European Chemical Industry Council), performed a 2-generation reproductive 

toxicity study of DEHP in Wistar rats. DEHP (99.7% pure) was administered in feed at 0, 1000, 3000, 

and 9000 ppm, resulting in estimated DEHP intakes of 0, 113, 340, and 1088 mg/kg bw/day. F0 male 

and female animals were 36–38 days old when they were placed on test (n=25/group). Animals were 

mated at least 73 days after the beginning of treatment. Females littered and raised their own pups. 

On PND 4, litters were standardized to 8 (4 males and 4 females where possible). Pups were weaned 

on PND 21. Treated feed was provided throughout the gestation and lactation period, and pups were 

weaned to the diet assigned to their parents. During the rearing period, 25 males and 25 females per 

dose group (1/sex/litter where possible) were assigned to be F1 parents. Mating (non-sibling) occurred 

at least 75 days after assignment. F2 litters were standardized on PND 4 and weaned on PND 21. On 

PND 28, 10 F2 offspring/sex/dose group were evaluated by functional observation battery and motor 

activity testing, and an additional 10 F2 offspring/sex/dose group were evaluated in a water maze test 

of learning and memory. Estrous cycle was monitored daily for all F0 and F1 females for at least 3 

weeks prior to mating. Cauda epididymal sperm were assessed for motility, morphology, and head 

count in F0 and F1 males at necropsy. Sperm head count was also performed in testes. Reproducing 

females were killed after weaning, and uteri were stained in 10% ammonium sulfide for calculation of 

implantation sites, used in the calculation of postimplantation loss. Male pups were evaluated on PND 

12 and 13 for the presence of nipples and areolae. Age and weight at vaginal opening and preputial 

separation were assessed in pups. One pup/sex/litter was killed on or after PND 21, and brain, spleen, 

thymus, liver, kidneys, testes, ovaries, and uterus (with oviducts and cervix) were weighed. Gross 

necropsy was performed on pups culled on PND 4 and on those pups not selected as parents for the 

next generation or for neurobehavioral testing (on PND 21). Histologic examination was performed on 

reproductive organs and other selected organs, and follicle counts were performed in ovaries. Statistical 

comparisons were made using the Dunnett test for means and the Fisher exact test for proportions or 

the Kruskal-Wallis test with Wilcoxon test for proportions of affected pups/litter, pup organ weights, 

and several neurobehavioral endpoints. Selected results are given in Table 31. 
Appendix II

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Table 31. Results of 2-Generation Study of DEHP in Wistar Rats

Parameter Dose in feed (ppm) Benchmark dose (ppm) a

1000 3000 9000 BMD10 BMDL10 BMD1 SD BMDL1 SD

F0 Feed Consumption

During pregnancy ↔ ↔ ↔

Laction day 1–4 ↔ ↔ ↓ 18% 5433 4016 7244 5257

Laction day 4 –7 ↔ ↔ ↓ 21% 6886 5015 8578 5792

Laction day 7 –14 ↔ ↔ ↓ 33% 6928 4465 6986 4577

Other F0 Parameters

F0 Body weight gain during 

Pregnancy ↔ ↔ ↓ 11% 8849 7167 8965 7894

F1 Body weight on lactation day 21 ↔ ↔ ↓ 14% 8741 7225 8547 6170

F0 Males with confirmed mating ↔ ↔ ↔

F0 Males with confirmed fertility ↔ ↔ “↓”12%b

F0 Sperm parameters ↔ ↔ ↔

F0 Females with stillborn pups ↔ ↔ ↑ 4-fold 6414 250

F0 Litter size (F1 pups/litter) ↔ ↔ ↔

F1 Pups Surviving (pup basis)

PND 0 – 4 ↔ ↓ 4% ↓ 7% 11,399 8541

PND 4– 21 ↔ ↔ ↔

Other F1 Parameters

Live F1 pups/litter on PND 4, 7, 14, 

and 21 ↔ ↔ ↔

Sex ratio F1 pups ↔ ↔ ↔

Postimplantation loss per F0 female ↔ ↔ ↑ 2.1-fold 7850 813 9070 7659

F1 Pup Body Weight, Male

PND 1 ↔ ↔ ↓ 6% 9274 9034 9139 8389

PND 4 ↔ ↔ ↔

PND 7 ↔ ↔ ↓ 6% 8780 5470 8919 6431

PND 14 ↔ ↔ ↓ 26% 5448 3903 5516 3463

PND 21 ↔ ↔ ↓ 31% 4661 3005 4615 2875

F1 Pup Body Weight, Female

PND 1 ↔ ↔ ↔

PND 4 ↔ ↔ ↔

PND 7 ↔ ↔ ↓ 16% 7643 4684 7777 5202

PND 14 ↔ ↔ ↓ 27% 5460 3818 5224 3451

PND 21 ↔ ↔ ↓ 31% 4733 3386 4583 3084

F1 PND 1 Anogenital Distance

Male ↔ ↔ ↓ 14% 6943 5417 5514 4242

Female ↔ ↔ ↔
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Appendix II

Parameter Dose in feed (ppm) Benchmark dose (ppm) a

1000 3000 9000 BMD10 BMDL10 BMD1 SD BMDL1 SD

Other F1 Genital Parameters

F1 Males with nipples/areolae per 

litter ↔ ↔ ↑ 38-fold 6238 2222

F1 Days to vaginal opening ↔ ↔ ↑ 12% 7921 6534 5407 3631

F1 Days to preputial separation ↔ ↔ ↑ 19%c 5780 4325 3986 2592

F1 PND 21 Absolute Organ Weights (sexes combined; male and female changes were similar)

Brain ↔ ↔ ↓ 7% 10,964 9118 6484 4136

Thymus ↔ ↓ 12% ↓ 39% 2506 2056 3443 2728

Spleen ↓ 15% ↓ 13% ↓ 57% 2446 1265 3962 2578

Liver ↔ ↑ 17% ↔ 1271 805 2713 1817

Kidney ↔ ↔ ↓ 33% 5946 4458 8487 5214

Testis ↔ ↔ ↓ 37% 5357 2995 6122 3863

Ovary ↔ ↔ ↓ 32% 8472 2265 9013 6273

Uterus ↔ ↔ ↓ 22% 8537 2859 9235 8276

F1 PND 21 Relative Organ Weights (sexes combined; male and female changes were similar)

Brain ↔ ↔ ↑ 38% 5292 3104 5274 3095

Thymus ↔ ↔ ↓ 12% 2467 1541 9534 9379

Spleen ↓ 11% ↓ 8% ↓ 38% 3450 2224 4300 2801

Liver ↑ 8% ↑ 22% ↑ 30% 1138 911 1184 956

Kidney ↔ ↑ 5% ↔

Testis ↔ ↔ ↔

Ovary ↔ ↔ ↔

Uterus ↔ ↔ ↔

F1 Feed Consumption

GD 0 – 7 ↔ ↑ 5% ↓ 7% 9107 8803 8893 7787

GD 7–14 ↔ ↔ ↓ 9% 8989 8218 8782 7034

GD 14–20 ↔ ↔ ↔

Laction day 1– 4 ↔ ↔ ↓ 32% 4587 3079 6266 4328

Laction day 4 –7 ↔ ↔ ↓ 31% 4540 2715 5159 3040

Laction day 7 –14 ↔ ↓ 12% ↓ 44% 2556 1848 3352 2193

Other F1 Parameters

Body weight gain during pregnancy ↔ ↔ ↓ 15% 6015 3920 7748 5283

Body weight on lactation day 21 ↔ ↔ ↓ 21% 8539 5480 8501 5238

Males with confirmed mating ↔ ↔ ↔

Males with confirmed fertility ↔ ↔ “↓” 24%b

Sperm count (testis, epididymis) ↔ ↔ ↔

Percent abnormal sperm ↔ ↔ ↑ 27% 3061 1174 25,588 9256

Percent motile sperm ↓ 2% ↔ ↔

Females with stillborn pups ↔ ↑ 3-fold “↑” 2.8-fold

Litter size (F2 pups/litter) ↔ ↔ ↓ 19% 5790 3137 9657 6697
Appendix II

II-151

Parameter Dose in feed (ppm) Benchmark dose (ppm) a

1000 3000 9000 BMD10 BMDL10 BMD1 SD BMDL1 SD

F2 Pups Surviving (Pup Basis)

PND 0 – 4 ↔ ↓ 15% ↓ 13% 2325 2045

PND 4– 21 ↔ ↔ ↔

Other F2 Parameters

Live F2 pups/litter on PND 4,7,14,21 ↔ ↔ ↔

Sex ratio F2 pups ↔ ↔ ↔

Postimplantation loss per F1 female ↔ ↔ ↔

F2 Pup Body Weight, Male

PND 1 ↔ ↔ ↔

PND 4 ↔ ↔ ↔

PND 7 ↔ ↔ ↓ 11% 7703 2940 9299 6072

PND 14 ↔ ↔ ↓ 29% 4204 2940 3827 2521

PND 21 ↔ ↔ ↓ 35% 4085 2790 4142 2787

F2 Pup Body Weight, Female

PND 1 ↔ ↔ ↔

PND 4 ↔ ↔ ↔

PND 7 ↔ ↔ ↓ 11% 7894 5078 10,689 6703

PND 14 ↔ ↓ 8% ↓ 21% 3691 2724 3356 2476

PND 21 ↔ ↔ ↓ 33% 4523 3094 3652 2318

F2 PND 1 Anogenital Distance

Male ↔ ↓ 9% ↓ 10% 8810 6204 6597 3981

Female ↔ ↔ ↔

F2 Males with nipples/areolae per litter ↔

↑ 45-

fold ↑ 54-fold 1610 Failedd

F2 PND 21 Absolute Organ Weights (sexes combined; male and female changes were similar)

Brain ↔ ↔ ↓ 7% 11,077 9201 5432 3686

Thymus ↔ ↔ ↓ 39% 2872 1957 4185 2963

Spleen ↔ ↔ ↓ 53% 4059 2262 5555 3828

Liver ↑ 14% ↑ 20% ↔ ↓ 1618 1096 2557 1480

Kidney ↔ ↔ ↓ 30% 6134 4155 6591 4700

Testis ↔ ↔ ↓ 38% 4063 2447 4905 3252

Ovary ↔ ↔ ↓ 26% 3157 2015 9484 6354

Uterus ↔ ↔ ↔

F2 PND 21 Relative Organ Weights (sexes combined; male and female changes were similar)

Brain ↔ ↔ ↑ 39% 4598 3379 4307 3000

Thymus ↔ ↔ ↓ 12% 7302 4633 9860 6359

Spleen ↔ ↔ ↓ 32% 5423 3424 6230 4311

Liver ↑ 12% ↑ 24% ↑ 33% 939 636 1637 1149

Kidney ↑ 6% ↑ 6% ↔

Testis ↔ ↔ ↓ 10% 9393 6427 8853 5832
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Appendix II

Parameter Dose in feed (ppm) Benchmark dose (ppm) a

1000 3000 9000 BMD10 BMDL10 BMD1 SD BMDL1 SD

Ovary ↔ ↔ ↔

Uterus ↔ ↔ ↑ 28% 8469 3304 8913 6381

From Schilling et al. (151).

↑ ,↓ ,↔ Increase, decrease, or no change in parameter compared to 0 ppm group by statistical testing. “↓” , “↑” 

refers to study author conclusion of a difference in the absence of statistical confirmation.

aCalculated by CERHR. See Table 22 for definitions and a discussion of the use of benchmark dose in this report. 

Benchmark dose was calculated only when a treatment effect was demonstrated by the study authors’ analysis.

bHistologic changes in testes and/or epididymal sperm led the study authors to conclude that there was a treatment-related reduction in fertility in this dose group although statistical significance was not shown.

cTwo males with hypospadias, penile hypoplasia, and cleft prepuce.

dBMDL calculation could not be performed by EPA program using Hill model, which gave the best fit.

In general, the high-dose level (9000 ppm) was associated with a decrease in feed consumption and 

weight gain at several intervals during the study. Clinical signs leading to unscheduled sacrifice were 

present in 1 high-dose F0 female. Six high-dose adult F1 females died or were killed in moribund 

condition during the pregnancy or lactation periods; 3 of these females were found to have liver necrosis. 

F2 pups in the high-dose group were smaller and gained less weight from birth through the period of 

assessment of functional observation battery and water maze testing. Grip strength was reduced in 

males, and hind-limb splay was reduced in both sexes in the high-dose group. There were no other 

treatment-related findings in the functional observation battery or in the water maze. Differential 

ovarian follicle counts of F0 and F1 adults showed a deficit in growing follicles and corpora lutea in 

the high-dose group [expressed per follicle].

The authors concluded that reproductive performance and fertility were affected at the 9000 ppm dose 

level with a NOAEL of 3000 ppm, and that developmental toxicity was noted at 3000 and 9000 ppm, 

with an increase in stillbirth, an increase in PND 0–4 pup mortality, retardation of F2 pup body weight 

gain, reduced male anogenital distance, and increased retained nipples/areolae in males. A delay in sexual 

maturation was also noted in F1 male and female offspring at 9000 ppm. The NOAEL for developmental 

toxicity was considered by the study authors to be 1000 ppm. The alterations in pup organ weights 

were noted, but the changes in spleen and thymus weight were assessed as causally related to the body 

weight alterations and not as primary effects of DEHP. The alterations in liver weight were considered 

likely to be due to peroxisome proliferation and were not representative of developmental toxicity. The 

NOAEL for systemic toxicity was considered by the study authors to be 1000 ppm. [The Panel noted

that focal tubular atrophy, the most subtle manifestation of the phthalate effect, was noted in the

F0 animals in 0, 1, 3, and 6 males, and in the F1 generation, in 2, 7, 4, and 13 male rats.The Panel

considers the increases in affected animals to be treatment-related at all doses,so that the lowest

effective dose was 1000 ppm (~100 mg/kg bw/day).These effects are consistent with other reports

in the literature and show a reasonable dose-response that becomes more severe in gestationally

exposed animals. The lowest BMD10 for reproductive toxicity was 2325 ppm (BMDL10: 2045

ppm) for F2 pup survival on PND 0–4. Based on the dose regression provided by the authors, the

corresponding DEHP intake is 263 mg/kg bw/day (BMDL10: 231 mg/kg bw/day).]

Strengths/Weaknesses: Strengths of this study include the completeness and widely accepted rigor (i.e., to 

GLP standards) with which it was performed, the number of animals, and the number of endpoints.
Appendix II

II-153

Utility (Adequacy) for CERHR Evaluation Process: This study was useful for the evaluation process 

and showed a LOAEL of 1000 ppm (~100 mg/kg bw/day) based on testicular histopathology in both 

generations. That this is a conclusion of the Expert Panel and not the authors is a cause for concern 

and limits the confidence that this conclusion can bear.

Tanaka et al. (137), support not indicated, gave DEHP (>97% purity) to CD-1 mice in the diet from 5 

weeks of age in the F0 generation to 9 weeks of age in the F1 generation. A single dietary dose level of 

0.03% was used, with control animals receiving untreated basal feed (n=20/sex/treatment group). At 

9 weeks of age, 10 DEHP-treated females were paired with DEHP-treated males, 10 DEHP-treated 

females were paired with control males, 10 control females were paired with DEHP-treated males, 

and 10 control females were paired with control males. The females’ diet was available to males 

during the 5-day cohabitation phase. Females reared their own unadjusted litters, which were weaned 

at 4 weeks of age. One female and male from each litter were retained and fed their dam’s diet until 

9 weeks of age. Statistical analyses were performed using ANOVA or Kruskal-Wallis test, followed 

by Bonferroni multiple comparison test. Proportions were evaluated using chi-squared or Fisher test. 

Based on measured feed consumption, mean DEHP intake by treated males [rounded by CERHR]

was 46 mg/kg bw/day. Treated females received 53 – 57 mg/kg bw/day during the preconception 

period, ~43 mg/kg bw/day during mating, 46 –49 mg/kg bw/day during gestation, and 154 – 171 

mg/kg bw/day during lactation. DEHP had no effect on feed consumption or dam body weight. There 

were no significant treatment effects on the number of pregnant females, number of litters, number 

of offspring, average litter size or weight, or sex ratio. The authors concluded that “DEHP caused 

few significant adverse effects on reproductive or neurobehavioral parameters.” [Neurobehavioral

testing of the F1 offspring, the focus of the study, was discussed in Section 3.2.2.]

Strengths/Weaknesses: The crossover design of this study is a strength, as are the statistical evaluations 

and the presentation of the data. The use of a single dose level weakens the study for use in assessing 

reproductive risk, which admittedly was not the major focus of the study.

Utility (Adequacy) for CERHR Evaluation Process: The data are sufficient to conclude that DEHP 

dosing between 46 and 154 mg /kg bw/day was insufficient to materially change any reproductive 

parameter measured in this study in mice. 

The National Toxicology Program (114) sponsored a multigeneration continuous breeding study in 

rats with the intent of evaluating whether responses seen at very low doses might be different from, 

or forerunners of, responses seen at higher doses. Task 1 of the study was conducted to determine the 

doses used in subsequent tasks. Sprague-Dawley rats (13 weeks old; 8/sex/group) were given feed 

containing 0, 5000, or 10,000 ppm DEHP from 7 days prior to mating through the cohabitation period, 

which extended until necropsy. [The time period of cohabitation was not specified.] DEHP intake 

was estimated by the study authors at 0, 321.42, and 643.95 mg/kg bw/day. In litters born during the 

cohabitation period, anogenital distance was measured on PND 1, and growth and mortality were 

monitored through PND 21. Signs of systemic toxicity included decreased feed and water intake 

during lactation in females from the 5000 and 10,000 ppm groups and a decrease in body weight gain 

in females from the 10,000 ppm group. Reproductive effects included a decrease in uterus, cervix, 

and vagina weights in PND 21 pups from the 5000 and 10,000 ppm groups. At 10,000 ppm, ratio of 

anogenital distance to pup weight was increased in female pups, and pup weights were decreased on 

PND 4 and 21. 
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Appendix II

In task 2 of the study, 17 Sprague-Dawley rats/sex/group were randomly assigned to groups and fed 

diets containing 1.5 (control group exposed to background DEHP levels in feed), 10, 30, 100, 300, 

1000, or 7500 ppm DEHP (99.8% pure) from the first day of the study until the day of necropsy. Due 

to a lack of reproductive effects in the first litter produced, the study was repeated with 2 additional 

doses, 1.5 (control) and 10,000 ppm. [It is not clear why the authors concluded there were no

reproductive effects in the 7500 ppm group when several significant effects were observed in the

first litter, as discussed below.] Ranges of DEHP intake in the F0, F1, and F2 animals were estimated 

at 0.09– 0.12, 0.47 – 0.78, 1.4 – 2.4, 4.8–7.9, 14 –23, 46 –77, 392 –592, and 543 –775 mg/kg bw/day. 

At about 5 weeks of age, F0 rats were fed the DEHP-containing diets for 6 weeks prior to mating 

and were then cohabitated for 9 weeks. Concentrations of dosing solutions were verified. The first 

2 litters delivered during the cohabitation period (F1a and F1b) were counted, weighed, assessed for 

anogenital distance, and then discarded. The third litter (F1c) was raised by the dam until weaning on 

PND 21 [designation for day of birth not specified]. Following weaning of pups, vaginal cytology 

was monitored in F0 females for 14 days. After completion of crossover studies described below, at 

least 10 F0 rats sex/group were necropsied. Sperm analyses were conducted, and organs were collected 

for histopathological evaluation. Ovaries were preserved in Bouin fluid. Testes and epididymides were 

preserved in 2% paraformaldehyde/3% glutaraldehyde. F1 pups were counted, weighed, and examined 

for anogenital distance and nipple retention during the lactation period. On PND 16, 1 female per 

litter was evaluated for vaginal opening, and a second was selected for F1 mating. One male per 

litter was selected for mating, and 4 or 5 males per litter were evaluated for testicular descent and 

preputial separation; both groups of rats were necropsied. At weaning (PND 21), pups were given diets 

containing the same DEHP concentrations as their parents. On PND 81, the F1 rats chosen for mating 

(17/sex/group) were randomly assigned to breeding pairs (preferably non-sibling) and cohabited for 9 

weeks. The study conducted in F0 parents and F1 offspring was repeated in F1 parents and F2 offspring, 

except that the third F3 litter born (F3c) did not undergo the continuous-breeding protocol. Selected 

F3c males were necropsied on PND 63 –64 and selected females on PND 60 –74. Statistical analyses 

included Jonckheere test to determine if data should be analyzed by Shirley or Dunn test. Shirley test 

was used to evaluate data that consistently increased or decreased according to dose. Dunn test was 

used to evaluate data with severe departures from monotonicity. Additional statistical analyses included 

Wilcoxon, Cochran-Armitage, and chi-squared tests. 

Some systemic effects were consistent across all generations. During numerous time periods of the 

study and especially at necropsy, body weight gains were decreased in rats from the 7500 and 10,000 

ppm groups. Dam body weights during delivery and lactation were decreased by 8– 20% in the F0

10,000 ppm group. Increases and decreases in feed intake were observed at most dose levels. In the 

F0 7500 and 10,000 ppm groups, feed intake was decreased during lactation. The liver was identified 

as a target of toxicity, with increases in liver weight and hepatocellular hypertrophy observed at dose 

levels ≥1000 ppm. Changes in organ weights and lesions were also observed in kidney at ≥7500 ppm 

and adrenal gland at 10,000 ppm.

Reproductive toxicity findings in all generations of rats are summarized in Table 32. 
Appendix II

II-155

Table 32. Results of Continuous Breeding Multigeneration Study of DEHP in Sprague-Dawley Rats

Parameter Dose in feed (ppm) Benchmark dose (ppm) a

10 30 100 300 1000 7500 10,000 BMD10 BMDL10 BMD1 SD BMDL1 SD

F0 FemalesCumulative days to deliver ↔ ↔ ↔ ↔ ↔ ↔ ↑ litter 1Estrous cycle effects ↔ ↔ ↔ ↔ ↔ ↔ ↔F0 MalesSpermatid/testis ↑16.5% ↔ ↔ ↔ ↔ ↔ ↓31%Sperm velocity ↔ ↔ ↔ ↔ ↔ ↔ ↓11%Body weight at necropsy ↔ ↔ ↔ ↔ ↔ ↔ ↓6%Absolute organ weight

Cauda epididymis ↔ ↔ ↔ ↔ ↔ ↔ ↓19%Epididymis ↔ ↔ ↔ ↔ ↔ ↔ ↓16%Testis ↔ ↔ ↔ ↔ ↔ ↔ ↓23%

No. with small right testis 0 0 0 0 0 0 2/10F0 MatingsLive pups/litter (F1a) ↔ ↔ ↔ ↔ ↔ ↓18% ↓21% 7008 3237 6771 2849Live males/litter (F1a) ↔ ↔ ↔ ↔ ↔ ↓20% ↓26% 3802 3303 2650 2227Proportion liveborn pups ↔ ↔ ↔ ↔ ↔ ↔ ↓4%Pup body weight ↔ ↔ ↔ ↔ ↔ ↔ ↓9%

Adjusted for litter size ↔ ↔ ↔ ↔ ↔ ↔ ↓10%

Pup anogenital distance

Male F1a ↔ ↔ ↔ ↔ ↔ ↓7% ↓14% 10,566 7631 3411 2382Male F1b ↔ ↔ ↔ ↔ ↔ ↓8% ↓15% 8408 7465 2874 2127Female, relative to bw ↔ ↔ ↔ ↔ ↔ ↔ ↑10 –17%

Male pup retained nipples ↔ ↔ ↔ ↔ ↔ ↔ ↔Pup weights PND 1–21 

Male ↔ ↔ ↔ ↔ ↔ ↔ ↓30%Female ↔ ↔ ↔ ↔ ↔ ↔ ↓30%

Pup survival, PND 4 –21 ↔ ↔ ↔ ↔ ↔ ↔ ↔
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Appendix II

Parameter Dose in feed (ppm) Benchmark dose (ppm) a

10 30 100 300 1000 7500 10,000 BMD10 BMDL10 BMD1 SD BMDL1 SD

F1 Males and femalesAge at vaginal opening ↔ ↔ ↔ ↔ ↔ ↑3 days ↑8 daysAge at preputial separation ↔ ↔ ↔ ↔ ↔ ↑4 days ↑11 daysAge at testes descent ↔ ↔ ↔ ↔ ↔ ↑3 days ↑6 daysPregnancy index, first 2 litters (control 76 – 100)

100 94 100 94 94 71 0

76 88 88 81 82 59 0

Cumulative days to litter ↔ ↔ ↔ ↔ ↔ ↔ no littersF1 FemalesEstrous cycle effects ↔ ↔ ↔ ↔ ↔ ↔ ↑0.4 daysBody weight at necropsy ↔ ↔ ↔ ↔ ↔ ↔ ↓19%Relative uterine weight ↔ ↔ ↔ ↔ ↔ ↔ ↑36%Relative ovarian weight ↔ ↔ ↔ ↔ ↔ ↔ ↑35%F1 Breeder malesBody weight at necropsy ↔ ↔ ↔ ↔ ↔ ↔ ↓16%Absolute organ weight 

Cauda epididymis ↔ ↔ ↔ ↔ ↔ ↓37% ↓62% 6532 3576 5970 2224Epididymis ↔ ↔ ↔ ↔ ↔ ↓35% ↓55% 6764 3896 6184 2226Testis ↔ ↔ ↔ ↔ ↔ ↓51% ↓80% 6577 1693 5940 511Seminal vesicles ↔ ↔ ↔ ↔ ↔ ↔ ↓29%Ventral prostate ↔ ↔ ↔ ↔ ↔ ↓28% ↔ 7056 3062 6998 2725Dorsolateral prostate ↔ ↔ ↔ ↔ ↔ ↔ ↓29%

No. with small testes 0 0 0 0 0 8/10 10/10No. with small epididymides 0 0 0 0 0 2/10 0F1 Non-breeder malesBody weight at necropsy ↔ ↔ ↔ ↔ ↔ ↓9% ↓21% 6752 6408 1634 1229Absolute organ weight

Epididymis ↔ ↔ ↔ ↔ ↔ ↓20% ↓54% 6845 3349 5787 797Seminal vesicles ↔ ↔ ↑9% ↔ ↔ ↔ ↔
Appendix II

II-157

Parameter Dose in feed (ppm) Benchmark dose (ppm) a

10 30 100 300 1000 7500 10,000 BMD10 BMDL10 BMD1 SD BMDL1 SD

Cauda epididymis ↔ ↔ ↔ ↔ ↔ ↓20% ↓44% 6802 4188 5669 1450Testis ↔ ↔ ↔ ↔ ↔ ↓34% ↓80% 2945 2584 1221 946

Relative organ weight

Epididymis ↔ ↔ ↔ ↔ ↔ ↔ ↓42% 6474 5814 3088 1965Testis ↔ ↔ ↔ ↔ ↔ ↓28% ↓75% 3895 3253 1903 1312Cauda epididymis ↔ ↔ ↔ ↔ ↔ ↔ ↓33.8%

No. with small:

Testes 0 0 0 3/45 0 9/30 21/21Epididymides 0 0 0 2/45 0 0 21/21Seminal vesicles 0 0 0 2/45 0 0 1/21Ventral prostates 0 0 0 0 3/43 1/30 1/21Caudae epididymides 0 0 0 1/45 0 0 21/21

Spermatid/testis ↔ ↔ - ↔ - ↓69% ↓100% 5834 1329 5534 916Sperm/cauda ↔ ↔ ↔ ↔ ↔ ↓61% ↓99.8% 6771 2865 6663 2472Epididymal sperm density ↔ ↔ ↔ ↔ ↔ ↔ ↓99.6% 6389 2819 6181 2310F1 MatingsLive F2 pups/litter ↔ ↔ ↔ ↔ ↔ ↔Live pup weight ↔ ↔ ↔ ↔ ↔ ↓10% 7496 7119 5996 1762Adjusted for litter size ↔ ↔ ↔ ↔ ↔ ↓11% 7245 6708 2123 1247Male F2a anogenital distance ↔ ↔ ↔ ↔ ↔ ↓13% 5749 4582 1093 963Male F2c anogenital distance ↔ ↔ ↔ ↔ ↔ ↓18% 5375 3284 3702 2517Male pup weight PND 1 ↔ ↔ ↔ ↔ ↔ ↓13% 7192 4135 6788 2649Male pup weight PND 4 ↔ ↔ ↔ ↔ ↔ ↓22% 6960 2715 6362 2365Female pup weight PND 1 ↔ ↔ ↔ ↔ ↔ ↓17% 5801 3505 6669 2036Female pup weight PND 21 ↔ ↔ ↔ ↔ ↔ ↓28% 5097 2144 4638 2209Male pup survival PND 1 –21 ↔ ↔ ↔ ↔ ↔ ↓20% 7004 2992 7082 3504Combined survival PND 1– 21 ↔ ↔ ↔ ↔ ↔ ↓19% 7293 3057 6924 3130
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Appendix II

Parameter Dose in feed (ppm) Benchmark dose (ppm) a

10 30 100 300 1000 7500 10,000 BMD10 BMDL10 BMD1 SD BMDL1 SD

F2 Males and females Age at vaginal opening ↔ ↔ ↔ ↔ ↔ ↑6 daysPreputial separation (days) ↑1.9 ↑1.0 ↑1.4 ↑0.7 ↑6.5Testes descent (days) ↔ ↑1.4 ↑0.8 ↑0.4 ↑3.4Pregnancy index, 1st 2 litters (control 100)

100 100 100 100 100 53(↓)100 100 100 100 94 47(↓)

Cumulative days to deliver ↔ ↔ ↔ ↔ ↔ ↔No. cycling rats ↔ ↔ ↔ ↓29% ↔ ↔No. litters/pair ↔ ↔ ↔ ↔ ↔ ↓F2 Males (breeders) Body weights at necropsy ↔ ↔ ↔ ↔ ↔ ↓14% 7346 4638 7003 3233Absolute organ weight

Epididymis ↔ ↔ ↔ ↔ ↔ ↓36% 6593 1846 4490 678Seminal vesicles ↔ ↔ ↔ ↔ ↔ ↓24% 2916 2009 2209 1705Testis ↔ ↓10% ↔ ↔ ↔ ↓60% 5874 1329 2408 1300Cauda epididymis ↔ ↔ ↔ ↔ ↔ ↓63% 6493 1641 6711 2432

Relative testis weight ↔ ↔ ↔ ↔ ↔ ↓53% 2334 1224 1815 1065No. with small:

Testes 0 0 0 0 0 8/10Epididymides 0 0 0 0 0 8/10Caudae epididymides 0 0 0 0 0 8/10

F2 Males (non-breeders)Body weight at necropsy ↔ ↔ ↔ ↔ ↔ ↓14.5% 7190 4446 6289 3153Absolute organ weight

Epididymis ↔ ↔ ↔ ↔ ↔ ↓27% 4350 2269 2574 1270Testis ↔ ↔ ↔ ↔ ↔ ↓49% 1788 1362 987 758Cauda epididymis ↔ ↔ ↔ ↔ ↔ ↓32% 2532 2005 1132 895
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II-159

Parameter Dose in feed (ppm) Benchmark dose (ppm) a

10 30 100 300 1000 7500 10,000 BMD10 BMDL10 BMD1 SD BMDL1 SD

Relative organ weight

Testis ↔ ↔ ↔ ↔ ↔ ↓40% 1948 1470 1132 1003Cauda epididymis ↔ ↔ ↔ ↔ ↔ ↓20% 4187 2714 1718 1472

No. with small:

Testes 0 0 0 1/21 1/25 11/20Epididymides 0 0 0 1/21 1/25 7/20Caudae epididymides 0 0 0 1/21 1/25 6/21

Spermatid/testis ↔ ↔ ↔ ↔ ↔ ↓74% 2885 1092 2043 1161Sperm/cauda ↔ ↔ ↔ ↔ ↔ ↓72% 1795 935 1374 854Epididymal sperm density ↔ ↔ ↔ ↔ ↔ ↓64% 1158 897 1830 1542Percent motile sperm ↔ ↔ ↔ ↔ ↔ ↓25% 3825 2038 554 412F2 Matings Live F3 pups/litter ↔ ↔ ↔ ↔ ↔ ↔Live pup weight ↔ ↔ ↔ ↔ ↔ ↔Male anogenital distance, F3a ↔ ↔ ↔ ↔ ↔ ↓13% 7322 4638 6846 2661

Pup weight (PND 1–21) ↔ ↔ ↔ ↔ ↔ ↔

Pup survival (PND 1 – 21) ↔ ↔ ↔ ↔ ↔ ↔

F3 FemalesAge at vaginal opening ↔ ↔ ↔ ↔ ↔ ↑6F3 MalesRetained nipples ↔ ↔ ↔ ↔ ↔ ↑11%Age at preputial separation ↔ ↔ ↔ ↔ ↔ ↑5.1Age of testes descent ↔ ↔ ↔ ↔ ↔ ↑2.5Body weights at necropsy ↔ ↔ ↔ ↔ ↔ ↔Absolute organ weights

Dorsolateral prostate ↔ ↔ ↔ ↔ ↔ ↓41% 1926 1401 2204 1840Testis ↔ ↔ ↔ ↔ ↔ ↓45% 1960 1399 1060 851
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Appendix II

Parameter Dose in feed (ppm) Benchmark dose (ppm) a

10 30 100 300 1000 7500 10,000 BMD10 BMDL10 BMD1 SD BMDL1 SD

Relative organ weights

Testis ↔ ↔ ↔ ↔ ↔ ↓48% 1870 1307 1330 1097Epididymis ↔ ↔ ↔ ↔ ↔ ↓35% 3223 1625 2332 1285Spermatid/testis ↔ ↔ ↔ ↔ ↔ ↓79% 1159 782 1100 695Sperm/cauda ↔ ↔ ↔ ↔ ↔ ↓95% 787 728 1188 970Epididymal sperm density ↔ ↔ ↔ ↔ ↔ ↓94% 797 750 1108 933

aCalculated by CERHR. See Table 22 for definitions and a discussion of the use of benchmark dose in this report. The 10,000 ppm dose group was not 

included in the benchmark dose calculations because it used a different control than the other dose groups. Benchmark dose was calculated only when there was a significant treatment effect according to the study authors’ analysis.

↑ ,↓ ,↔ Increase, decrease, or no change in parameter compared to 0 ppm group by statistical testing.From NTP (114).
Appendix II

II-161

In the F0 generation, there were no effects on fertility or estrous cycles. The only reproductive effects 

observed in the F0 parents occurred at 10,000 ppm and included decreases in sperm counts and 

velocity, reductions in testis and epididymis weights, and increased numbers of rats with small 

testes. Histopathological findings associated with small testes included minimal-to-marked atrophy 

of seminiferous tubules characterized by loss of germ cells. The lowest dose level producing doserelated effects in F1 offspring was 7500 ppm, and those effects included decreases in number of live 

pups/litter, reduced male anogenital distance, and delays in vaginal opening, preputial separation, 

and age of testicular descent. Additional effects noted in the F1 offspring from the 10,000 ppm group 

included decreased live pup weight at birth and during the lactation period and increased ratio of 

female anogenital distance to body weight. 

In contrast to findings in the F0 generation, fertility was compromised in the F1 rats from the 10,000 

ppm group, which did not produce any viable litters. Other reproductive effects observed in F1 parents 

were similar to those observed in F0 parents but usually occurred at lower dose levels. In the nonmating F1 adult males of the 300 ppm group, there was a small increase in the number of animals (3 

of 45) with small testes and/or epididymides. The effects were not observed at the next higher dose 

(1000 ppm), but small testes were observed in 10 of 30 males of the 7500 ppm non-mating group. 

Small testes and epididymides were observed in 21 of 21 animals of the 10,000 ppm non-mating 

group. A small percentage (3 – 7%) of non-mating rats treated with ≥1000 ppm had small ventral 

prostates. In rats that were mated, the only decreases in reproductive organ size occurred in testes at 

7500 and 10,000 ppm (8 of 10 and 10 of 10 affected at each dose) and epididymides at 7500 ppm (2 

of 10). Histopathological findings observed in all animals of the 7500 and 10,000 ppm groups were 

consistent with those observed in the F0 generation and included minimal-to-marked seminiferous 

tubule atrophy and occasional sperm release failure. Minimal seminiferous tubule atrophy was observed 

in 1 of 10 males in the 100 and 300 ppm groups. Reductions in numerous reproductive organ weights 

were observed in mating and non-mating F1 males treated with ≥7500 ppm. Additional reproductive 

effects observed in F1 rats were reduced sperm counts at 7500 ppm and higher and increased uterus 

and ovary weights at 10,000 ppm. Estrous cycle length was slightly increased at 10,000 ppm. In the 

F2 pups, delays in preputial separation and testicular descent occurred at every dose level above the 

control. [In no other generation did delays in preputial separation and testicular descent occur

at such low doses, but the study authors did not offer any explanations for this observation.The

Expert Panel believes these findings are consistent with a problem with the control group in that

generation.] All other effects occurred in F2 pups of the 7500 ppm group and included delayed vaginal 

opening and reductions in live pup weight at birth and during the lactation period, male anogenital 

distance, and survival during the lactation period. 

No F2 adult rats from the 10,000 ppm group were available due to complete infertility in F1 rats of that 

group. In F2 adult rats, a decreased number of cycling females observed in the 300 ppm group was not 

observed at any other dose level. In non-mating males, rats with small testes and epididymides were 

observed at ≥300 ppm (1 of 21), 1000 ppm (1 of 25), and 7500 ppm (7 –11 of 20). However, in males 

that were mated, small epididymides and testes were only observed at the 7500 ppm dose level (8 of 

10). Seminiferous tubule atrophy was observed in 10 of 10 males of the 7500 ppm group. All other 

reproductive effects in F2 adults occurred at 7500 ppm and included decreases in pregnancy index, the 

number of litters per pair, male reproductive organ weights, sperm counts, and sperm motility. In F3

pups, a decrease in postnatal survival of females was observed only on PND 7 in the 300 ppm group 
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Appendix II

but was not observed on any other day or dose level. All other effects in F3 pups occurred at 7500 ppm 

and included decreases in male anogenital distance, delayed vaginal opening, preputial separation, 

and testicular descent, and an increase in male pups with nipples. F3 pups were the only generation 

of rats to experience an increase in males with nipples. At necropsy of adult F3 rats, effects were only 

observed at the 7500 ppm dose level and included reduced sperm counts and weights of dorsolateral 

prostate, testis, and epididymis.

The study authors discussed the relevancy of small male reproductive organ sizes observed in both 

F1 and F2 rats of the 300 ppm groups. They noted that although incidences were low, the effects were 

consistent with phthalate-induced toxicity. The incidence of small testes and epididymides exceeded 

historical control data from the laboratory. Therefore, the study authors considered the effects as 

potentially treatment-related. However, the study authors concluded that the overall significance of the 

effects could not be determined due to lack of histopathological data and lack of adverse reproductive 

effects at 300 and 1000 ppm. 

Two crossover breeding studies were conducted. The first was conducted to investigate the lack of 

litters in F1 rats of the 10,000 ppm group. The second was conducted to investigate the decrease in 

F3 pup body weight in the 7500 ppm group. The studies were conducted by mating the control and 

high-dose rats of each sex (n=17/sex/group) with naïve animals for 7 days or until a vaginal plug was 

detected. Pups were counted, weighed, assessed for anogenital distance, and discarded. Implantation 

sites were examined in naïve females. The crossover studies (Table 33) demonstrated that pregnancy 

and fertility indices were reduced in males of the 7500 ppm and 10,000 ppm groups. Implantation 

sites were reduced in the naïve rats mated with DEHP-treated males. A decrease in pup weight and 

male anogenital distance was seen in offspring born to females treated with 7500 and 10,000 ppm 

DEHP and mated to naïve males. 

Table 33. Reproductive Crossover Breeding Study

Parameter Dam

×

Sire

F1 DEHP treatment 

Groups

F2 DEHP Treatment 

Groups

10,000 ppm 

×

naïve

Naïve 

×

10,000 ppm

7500 ppm 

×

naïve

Naïve 

×

7500 ppm

Pregnancy index ↑ ↓ (0 Pregnant) ↔ ↓

Fertility index ↑ ↓ (0 Fertile) ↔ ↓

Mating index ↔ ↓ (0 Mated) ↔ ↔

Implantation sites Not Reported ↓97.9% Not Reported ↓54.5%

Adjusted live male pup weight ↓13.6% NA ↓8.2 – 12.3% ↔

Male pup anogenital distance ↓16.9% NA ↓11.5% ↔

Female anogenital distance/bw ↑17.6% NA ↔ ↔

Live female pups/litter ↔ NA ↔ ↓31.0%

↑ ,↓ ,↔ Increase, decrease, or no change in parameter

NA=Not Applicable

From NTP (114).
Appendix II

II-163

The study authors concluded, “The findings obtained in this study indicate that DEHP is clearly a 

reproductive and developmental toxicant at 7500 and 10,000 ppm based upon changes in fertility and 

pregnancy indices, litter data, sperm parameters, sexual development, and/or histopathological changes 

in testes.” Intake at 7500 ppm was estimated at 392–592 mg/kg bw/day, and intake at 10,000 ppm was 

estimated at 543 –775 mg/kg bw/day. [The lowest BMD10 was 787 ppm based on F3 sperm/cauda.

Extrapolating from the authors’ estimates of intakes at 1000 mg/kg bw/day, this intake level

is 36 – 61 mg/kg bw/day. The lowest BMD1 SD is 554 ppm based on F2 percent motile sperm.

Extrapolating from the authors’ estimates of intakes at 300 ppm, this intake level is 9 –15 mg/kg

bw/day.The Expert Panel notes that Sertoli cell vacuolation, which was the endpoint driving the

LOAEL in the original CERHR Expert Panel evaluation of DEHP, was not increased by DEHP

treatment in this study.]

Strengths/Weaknesses: Clearly, a major strength of this study is the number of doses evaluated. The 

relatively small group sizes were compensated by the unusually high numbers of groups and the 

very low doses used. An additional strength is the fact that more offspring were evaluated early for 

alterations in the development of the reproductive system; a weakness might be that not all animals 

were so evaluated. The quality of the histology is another strength. The lack of vacuoles is perplexing, 

but not lethal to the study. 

Utility (Adequacy) for CERHR Evaluation Process: These data are adequate for the evaluation process 

and show that 10,000 and 7500 ppm are clearly toxic to the developing reproductive system in rats. 

The Expert Panel considers 300 ppm and 1000 ppm to represent the tail of the dose-response curve in 

this study, based on the incidence of testicular abnormalities, which would put the NOAEL for these 

developmental effects at 100 ppm, in the 3 –5 mg/kg bw/day range.

4.2.4 	 Abstracts

CERHR retrieved several abstracts reporting reproductive toxicity associated with DEHP exposure. 

Although information from abstracts is not considered by the Expert Panel in reaching final conclusions, 

the abstracts are briefly summarized for the sake of completeness. 

Boekelheide et al. (189) presented an abstract on the use of mutant mice to investigate the MEHP 

mechanism of testicular toxicity. Knockout mice deficient in fasL, p53, or both were protected against 

testicular toxicity after treatment with MEHP. The authors concluded that the apoptotic response of 

mouse testis to MEHP is at least partially dependent on fasL and p53. [The Expert Panel notes

that the results for fasL-deficient mice reported in this abstract appear similar to the results in

Richburg et al. (169), discussed above, from the same laboratory.]

Noriega et al.(190) treated male Sprague-Dawley rats on PND 23–43 or 44 with DEHP 0, 100, 300, or 

900 mg/kg bw/day [route notspecified]. There were reductions in the 300 and 900 mg/kg bw/day groups 

in testis, seminal vesicle, Cowper gland, levator ani, and bulbocavernosus muscle weights compared to 

the controls. Testicular testosterone (basal and stimulated) was reduced in these 2 groups, and serum 

testosterone was reduced in the 900 mg/kg bw/day group. There was a reduction in the proportion of 

males with complete preputial separation in the 300 and 900 mg/kg bw/day groups compared to the 

controls. Female rats dosed from PND 23 concurrently with males showed no difference in time to 

vaginal opening in the 900 mg/kg bw/day group compared to the corn-oil control group.
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Nakajima et al.(191) gave wild-type and PPARα-null mice DEHP in the diet for 4 weeks at exposure 

levels of 0 or 0.05%, following which the animals were mated within dose and genetic-strain groups. 

Two generations (F1 and F2), produced without direct exposure to DEHP, showed decreases in number 

of pups born and in pups surviving to 16 weeks of age. Only wild-type mice were affected. In another 

experiment, dietary DEHP exposures at 0, 0.01, and 0.05% were used, producing an increase in 

resorptions and a decrease in newborn survival among wild-type mice born to treated animals. There 

were no alterations in parental reproductive organs. 

Kang and Lee (192) treated male Sprague-Dawley rats orally for 4 weeks with phthalate diesters at 

500 mg/kg bw/day or phthalate monoesters at 250 mg/kg bw/day. Animals were evaluated for body 

weight, testicular and epididymal weights, epididymal sperm count, and sperm motion parameters. 

There were no significant effects of treatment on body or reproductive organ weight. The order 

(strongest to weakest) for effects on sperm motility for the diesters was DEHP>DnOP>DEP>DUP

>DIDP>BBP, and for the monoesters was MBuP>MEP>MEHP. [Abbreviations were not defined

but are assumed to be: DnOP=di-n-octyl phthalate; DEP=diethyl phthalate; DUP=diundecyl

phthalate; DIDP=diisodecyl phthalate; BBP=butyl benzyl phthalate; MBuP= monobutyl

phthalate; MEP=monoethyl phthalate.]

Jayes and Davis(193) cultured granulosa cells from PMSG-stimulated immature rats. Medium containing FSH and testosterone was treated with MEHP at 0, 0.001, 0.01, 0.1, 1, 2, or 5 μM. Estradiol was 

assayed in media at 51 hours, and aromatase was measured in cells exposed to 0, 1, or 5 μM MEHP. 

Estradiol and aromatase were decreased by MEHP at 1 or 5 μM compared to controls.

4.4 Summary of Reproductive Toxicity Data

4.4.1 	 Human Data

There are 7 useful studies of adult male exposure to MEHP and various reproductive endpoints. The 

studies are consistent in that they do not identify any significant associations between MEHP and 

adverse semen parameters, hormone levels, time-to-pregnancy, or infertility diagnosis. However, in 2 

of these studies, there were suggestions of associations between MEHP and decreased sperm velocity 

and serum testosterone. 

Modigh et al. (152) evaluated time-to-pregnancy in the partners of men potentially exposed to DEHP. 

Exposure was estimated from employed subjects’ description of work tasks and classified as unexposed 

pregnancies (n=182, fathered by operators who were not exposed), low-exposure pregnancies (n=100, 

fathered by men with estimated non-zero DEHP exposures <0.1 mg/m3), and high-exposure pregnancies 

(n=44, by men exposed to DEHP>0.1 mg/m3). There was no association found between time-topregnancy and exposure group (median time-to-pregnancy was 3.0 months in the unexposed group, 

2.25 months in the low-exposure group, and 2.0 months in the high-exposure group). The crude and 

adjusted fecundability ratios for the exposed pregnancies were all close to 1.0, and the 95% confidence 

intervals all overlapped unity. The Expert Panel noted that the exposures were not assessed using biologic 

markers, and misclassification of exposure was possible, biasing the results toward the null.

Duty et al.(154) evaluated urinary MEHP and semen analysis parameters in 168 men being evaluated 

in a clinic as part of a fertility evaluation. A single semen sample and single spot urine were collected 
Appendix II

II-165

on the same day. There were no significant associations between abnormal semen parameters and 

MEHP urine concentration above or below the group median. 

Duty et al. (30) evaluated urinary MEHP and sperm motion parameters in 187 male partners of 

couples presenting for fertility evaluation. A single spot urine was collected on the same day as the 

semen sample. Multiple linear regression analysis was used to evaluate the association between tertile 

of normalized urinary MEHP concentration and sperm motion parameters. There were no significant 

relationships between MEHP and sperm motion parameters, although there was a suggestion of an 

association with straight line velocity (P=0.08). 

Duty et al. (155) evaluated the association between urinary phthalate monoester concentrations and 

sperm DNA damage, assessed using the neutral comet assay. The 141 subjects were a subset of those 

described in Duty et al. (30). Urinary phthalate monoester concentrations were adjusted for specific 

gravity and analyzed in quartiles using multiple linear regression adjusted for smoking status, race, 

age, body mass index, and abstinence interval prior to collection of semen. There were no associations 

between comet assay parameters and MEHP urinary concentrations. 

Duty et al. (156) evaluated the relationship between serum concentrations of testosterone, sex hormone-binding globulin, inhibin B, FSH, and LH and phthalate monoester concentrations in spot urine 

samples. The subjects included 295 men attending a clinic as part of a fertility evaluation. No significant 

associations between MEHP and hormone levels were reported although there was a suggestion of a 

negative association between MEHP and serum testosterone (P=0.10).

Hauser et al. (158) evaluated possible interactive effects of polychlorinated biphenyls and phthalate 

on sperm motility in male partners of couples seeking infertility evaluation. Both phthalate and polychlorinated biphenyl levels were dichotomized at the median as high or low, and sperm concentration 

was dichotomized as normal or abnormal based on World Health Organization criteria. There were no 

significant interactions between urinary MEHP and any of the polychlorinated biphenyls or groupings, 

either with regard to sperm motility or other sperm parameters.

The Duty et al. (30, 154-156) and Hauser (158) papers represented an overlap of subjects in a special 

population of men presenting for fertility evaluation. The studies generally had good evaluation of and 

control for potential confounders. Although the studies found no statistically significant effects for MEHP 

with sperm abnormalities (concentration, motility and normal forms), sperm motion, sperm DNA damage, 

or hormone levels, there were suggestions of dose-response with reduced sperm motility measures (P=0.08) 

and marginal significance for the association of MEHP with reduced serum testosterone (P=0.10). 

Jönsson et al. (85) studied semen parameters and urinary phthalate monoester levels in 234 military 

recruits aged 18–21 years. Combined testicular volume was estimated based on ultrasound measurements, semen was obtained by masturbation, and spot urine samples were collected for measurement 

of MEHP. Seminal sperm were assessed for concentration and motility, including computer-assisted 

parameters, and were subjected to the sperm chromatin structure assay. Subjects were categorized 

into quartiles by urine concentration of individual phthalate monoesters (uncorrected and creatinineadjusted), and ratios with 95% confidence intervals were calculated for highest:lowest quartile groups. 

There were no significant associations between highest versus lowest urinary MEHP quartile and any 
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Appendix II

of the dependent variables. They also found no evidence of interaction between phthalate metabolites 

and PCB-153 on testicular function.

4.4.2 	 Experimental Animal Data

4.4.2.1 Female

Mitsubishi Chemical Safety Institute, Ltd. (92), in an unpublished report, described a 65-week 

oral-dose toxicity study of DEHP in marmosets. DEHP was administered by gavage in corn oil to 

juvenile marmosets beginning at 90–115 days of age until 18 months of age (young adulthood) at dose 

levels of 0, 100, 500, and 2500 mg/kg bw/day. Both males and females were assessed with a battery 

of in-life hormonal assays and with histopathology at necropsy. In the female marmosets, DEHP at 

500 and 2500 mg/kg bw/day was associated with early increases in serum 17b-estradiol consistent 

with an early onset of puberty, manifested as a significant increase in ovarian and uterine weights at 

necropsy in these groups.

4.4.2.2 Male

Male reproductive toxicity data in experimental animals are summarized in Table 34. 

At dose levels of 10 and 100 mg/kg bw/day, Akingbemi et al. (111, 179) showed reduced Leydig cell 

testosterone production ex vivo, increased serum LH, testosterone, and 17b-estradiol, and Leydig cell 

hyperplasia. The authors concluded, and the Expert Panel concurs, that DEHP increases Leydig cell 

populations associated with chronically increased LH and testosterone levels, and that a decrease in 

testosterone and increase in estradiol synthesis (per cell) was consistent with induction of aromatase 

activity in Leydig cells. The authors identified 1 mg/kg bw/day as a NOAEL and 10 mg/kg bw/day 

as a LOAEL.

In another study, 10-week-old male mice were given 0 or 2% dietary DEHP for 16 days following which 

all animals were given untreated feed for an additional 50 days with vitamin supplementation (175).

[CERHR calculated a mean DEHP intake during the treatment period of 2857 mg/kg bw/day.]

Groups of animals were killed during and after the end of exposure and assessed for testicular effects 

by weight and histology. By day 12 of exposure, testis weights were decreased and recovered towards 

control levels 20 days after cessation of exposure. Similarly, testis histopathology abnormalities were 

seen during treatment and recovered after the end of exposure. Groups of animals were killed during 

and after the end of exposure and assessed for testicular effects by weight and histology. By day 12 of 

exposure, testis weights were decreased and recovered towards control levels 20 days after cessation 

of exposure. Similarly, testis histopathology abnormalities were seen during treatment and recovered 

after the end of exposure.
Appendix II

II-167

Table 34. Summary of Male Reproductive Toxicity Data from Studies in Rats and Mice

Species and Dosing Most Sensitive Outcome Effect Levels 

(mg/kg bw/day) Reference

Single Dose-Level Studies (excluding control)

Mice, C57Bl6 wild-type and gld 

mutant

MEHP 1000 mg/kg bw×1 by 

gavage

TUNEL labeling greater in wildtype than fasL-deficient mutant 

mice 1000 Richburg et al. 

(169)

Mice, C57Bl6 wild-type and gld 

mutant

MEHP 1000 mg/kg bw×1 by 

gavage

Apoptotic response decreased in 

fasL-deficient mutant compared to 

wild-type mice 1000 Giammona et 

al. (171)

Mice, CD

DEHP 2% in the diet for 16 

days

Decreased testis weight, abnormal 

histopathology findings [2857] Ablake et al. 

(175)

Rat, Sprague-Dawley

MEHP 1000 mg/kg bw×1 by 

gavage

Increased expression of death 

receptors 1000 Giammona et 

al. (171)

Rat, Wistar

MEHP 400 mg/kg bw×1 by 

gavage

Germ cell sloughing, collapse of 

Sertoli cell vimentin filaments 3 

hours after treatment

400 Dalgaard et al. 

(172)

Rat, Sprague-Dawley

DEHP 2 mg/rat/day 

[19,000–29,000 mg/kg bw/

day]×1–14 days

Decreased body and testis weight, 

testis histologic evidence of apoptosis beginning treatment day 3 [19,000–29,000] Park et al. 

(173)

Multiple Dose Levels

Rat, Wistar

DEHP 0, 1000, or 2000 mg/kg 

bw/day by gavage×7 days 

Decreased total glutathione, low 

molecular weight thiols, and 

ascorbic acid in testis

LOAEL 1000 Kasahara et al. 

(174)

Rat, Wistar

DEHP 0, 100, or 1000 mg/kg 

bw/day by gavage×5 days

Decreased aromatase and 

increased CYP2C11 and CYP3A2 

testosterone hydroxylation in 

testis

LOAEL 100 a Kim et al. 

(177)

Rat, Long-Evans

DEHP 0, 1, 10, 100, or 200 

mg/kg bw/day by gavage×14 or 

28 days

Decreased 17α-hydroxylase in 

testis, altered ex vivo Leydig cell 

testosterone synthesis

LOAEL 10

NOAEL 1

Akingbemi et 

al. (111)

Rat, Long-Evans

DEHP 0, 10, or 100 mg/kg bw/

day by gavage×70 or 100 days

Increased serum LH and testosterone, decreased ex vivo Leydig cell 

testosterone synthesis (PND 90)

LOAEL 10 Akingbemi et 

al. (179)

aThe use of a recovery period in the dosing schedule prevented the Expert Panel from being able to interpret the 

results.
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Appendix II

4.4.2.3 Male and Female

Reproductive toxicity data in male and female rats are summarized in Table 35.

Schilling et al. (151) performed a 2-generation reproductive study of DEHP in Wistar rats. DEHP 

was administered in feed at 0, 1000, 3000, and 9000 ppm, resulting in estimated DEHP intakes of 0, 

113, 340, and 1088 mg/kg bw/day. The high-dose level (9000 ppm) was associated with a decrease in 

feed consumption and weight gain at several intervals during the study. An extensive evaluation was 

conducted, including a functional observation battery, water maze, estrous cyclicity, cauda epididymal 

sperm evaluation, testicular spermatid head counts, presence of nipples and areolae in male pups, 

and day and weight at vaginal opening and preputial separation. F2 pups in the high-dose group were 

smaller and gained less weight from birth through the assessment of functional observation battery 

and water maze testing. Grip strength was reduced in males and hind-limb splay was reduced in both 

sexes in the high-dose group. Differential ovarian follicle counts of F0 and F1 adults showed a deficit 

in growing follicles and corpora lutea in the high-dose group. [The Expert Panel noted dose-related

increasesin the number of animalsshowing focal tubular atrophy in both generations, beginning

at the lowest dose (1000 ppm, ~113 mg/kg bw/day). The LOAEL for reproductive toxicity was

considered by the Expert Panel to be 1000 ppm (the lowest dose in the study), which was also

the NOAEL for systemic toxicity.]

Mitsubishi Chemical Safety Institute,Ltd.(92), in an unpublished report, described a 65-week oraldose toxicity study of DEHP in marmosets. DEHP was administered by gavage in corn oil to juvenile 

marmosets beginning at 90 –115 days of age until 18 months of age (young adulthood) at dose levels 

of 0, 100, 500, and 2500 mg/kg bw/day. Both males and females were assessed with a battery of in-life 

hormonal assays and with histopathology at necropsy. Although there were significant limitations in the 

study and the reporting of findings, the results suggest little effect of DEHP exposure at 2500 mg/kg 

bw/day in marmosets on testicular structure and function. Mean serum testosterone levels were highly 

variable, but the data suggested the possibility of a delay in the onset of puberty in male marmosets 

with increasing DEHP dose. The authors suggested, based on an accompanying pharmacokinetic 

study (reviewed in Section 2.1), that the most likely explanation for the lack of testicular toxicity of 

DEHP in marmosets as opposed to rodents was the limited accumulation of DEHP and its metabolites 

in the marmoset testis. This limited accumulation may in part be a result of the short gut transit time 

and propensity to diarrhea. In addition, the male marmoset has an apparent relative end-organ steroid 

resistance compared to humans. [Because of the limited blood levels achieved in this study, differences between marmosets and humans in terms of their steroid resistance, and the difficulties

in ascertaining the exact disposition of animals and inclusion of animals in the final data set, the

male data from the Mitsubishi study are of limited ultimate use in the evaluation process.]

The NTP (114) sponsored a multigeneration continuous-breeding study in rats. Sprague-Dawley rats 

were fed diets containing 1.5 (control group exposed to background DEHP levels), 10, 30, 100, 300, 

1000, or 7500 ppm DEHP from the first day of the study until the day of necropsy. Due to a described 

lack of reproductive effects in the first litter produced, the study was repeated with 2 additional doses, 

1.5 (control) and 10,000 ppm. Ranges of DEHP intake in the F0, F1, and F2 animals were estimated 

at 0.09–0.12, 0.47–0.78, 1.4 – 2.4, 4.8 – 7.9, 14– 23, 46–77, 392– 592, and 543 –775 mg/kg bw/day. 

At about 5 weeks of age, F0 rats were fed the DEHP-containing diets for 6 weeks prior to mating and 

were then cohabitated for 9 weeks. The first 2 litters delivered during the cohabitation period (F1a 
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and F1b) were counted, weighed, assessed for anogenital distance, and discarded. The third litter (F1c) 

was raised by the dam. Following weaning of pups, vaginal cytology was monitored in F0 females 

for 14 days. After completion of crossover studies described below, at least 10 F0 rats sex/group were 

necropsied. Sperm analyses were conducted, and organs were collected for histopathological evaluation. F1 pups were counted, weighed, and examined for anogenital distance and nipple retention 

during the lactation period. On PND 16, 1 female per litter was evaluated for vaginal opening, and 

a second was selected for F1 mating. One male per litter was selected for mating, and 4 or 5 males 

per litter were evaluated for testicular descent and preputial separation. At weaning on PND 21, pups 

were given diets containing the same DEHP concentrations as their parents. On PND 81, the F1 rats 

chosen for mating (17/sex/group) were randomly assigned to breeding pairs (preferably non-sibling) 

and cohabited for 9 weeks. The study conducted in F0 parents and F1 offspring was repeated in F1 

parents and F2 offspring, except that no F3 offspring were mated. Selected F3c males were necropsied 

on PND 63– 64 and selected females on PND 60–74.

The liver was identified as a target of toxicity with increases in liver weight and hepatocellular hypertrophy observed at dose levels ≥1000 ppm. Changes in organ weights and lesions were also observed 

in the kidney at ≥7500 ppm and the adrenal gland at 10,000 ppm.

Reproductive effects observed in the F0 parents occurred only at 10,000 ppm and included decreases in 

sperm counts and velocity, reductions in testis and epididymis weights, and increased numbers of rats 

with small testes. The lowest dose level producing dose-related effects in F1 offspring was 7500 ppm, 

and those effects included decreases in number of live pups/litter, reduced male anogenital distance, 

and delays in vaginal opening, preputial separation, and age at testicular descent. 

F1 rats from the 10,000 ppm group produced no viable litters. At and above 7500 ppm, rats had reduced 

sperm counts, seminiferous tubular atrophy, and delayed preputial separation and testicular descent. 

In the F3 offspring, reproductive toxicity was noted in numerous endpoints at 7500 ppm but not at 

lower dose levels. Effects included seminiferous tubule atrophy and decreases in pregnancy index, the 

number of litters per pair, male reproductive organ weights, sperm counts, and sperm motility. 

[The Expert Panel carefully considered the finding of small reproductive organ sizes by gross

observations in non-mating F1 and F2 rats. The combined F1 and F2 data were reviewed to

determine the occurrence of these alterations on a per animal and per litter basis across the

dose range, as shown inTable 23 in Section 3.2.1.1. Based on the incidence of small reproductive

organ size at necropsy, the Expert Panel considered 300 ppm (about 14 – 23 mg/kg bw/day) to be

an effect level, giving a NOAEL of 100 ppm, about 3– 5 mg/kg bw/day. The Expert Panel notes

that Sertoli cell vacuolation, which wasthe endpoint driving the LOAEL in the original CERHR

Expert Panel evaluation of DEHP, was not increased by DEHP treatment in this study.]
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Appendix II

Table 35. Summary of Reproductive Toxicity Studies Involving Male and Female Rats

Species and Dosing Most Sensitive Outcome LOAEL, ppm NOAEL, ppm Lowest BMDLa, ppm Reference

Rat, WistarDEHP in feed 0, 1000, 3000, or 9000 ppm × 2 generationsDEHP intakes estimated as 0, 113, 340, or 1088 mg/kg bw/day)

↑Abnormal sperm↓ Male fertility↓ Testis size↓ Litter size and viabilityDeficit in growing follicles and corpora lutea

Study authors:

9000 [~1088 mg/kgbw/day]

Expert Panel:

1000 [~100 mg/kgbw/day], based on testicular histopathology

Study authors:

3000 [~340 mg/kgbw/day]

Expert Panel:

Not applicable

10% level:

2045 ppm [~231mg/kg bw/day]based on F2 pup 

survival PND 0 – 4

Schilling et 

al. (151)

Rat, Sprague-DawleyDEHP in feed at 1.5, 10, 30, 100, 300, 1000, 7500, or 10,000 ppm (the 10,000 ppm dose was run separately with its own 1.5-ppm control). Rats were treated over 3 generations using a continuousbreeding protocol for up to 3 litters/pair. DEHP intake was estimated at 0.09– 0.12 and 0.47 – 0.78 for the 2 controls, 0.09 – 0.12, 0.47–0.78, 1.4– 2.4, 4.8 –7.9, 14–23, 46–77, 392 –592, and 543–775 mg/kg bw/day

Fertility and pregnancy indices, litter data, sperm parameters, sexual development, and histopathological changes in testes

Study authors:

2 mg/kg bw/day; occasional effects at 300 ppm were considered possibly treatment-related.)

Expert Panel:

300 ppm (14–23 

mg/kg bw/day) 

Study authors:

1000 ppm (46 –77 

mg/kg bw/day)

Expert Panel:

100 ppm (3 – 5 

mg/kg bw/day)

10% level:

728 [33 –56 mg/kgbw/day] based on F3 sperm/cauda

1 SD level:

412 [19 –32 mg/kgbw/day] based on F2 percent motile sperm

National 

Toxicology 

Program 

(114)

aCalculated by CERHR. See Table 22 for definitions and a discussion of the use of benchmark dose in this report.↑ ,↓  Increase or decrease in parameter
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Conclusions Based Only on Literature Appearing Since 

the First Expert Panel Report

There is insufficient evidence in humans that DEHP causes male or female reproductive

toxicity.There were 7 human studies judged to be useful for evaluating male reproductive toxicity. 

One study addressed occupational exposure and 6 addressed non-occupational exposure. Five 

of the 6 studies were conducted in the same population of men seeking evaluation for infertility, 

which may limit the studies’ generalizability. There were suggestions of associations between 

specific male reproductive parameters and urinary MEHP but none reached a conventional level 

of statistical significance (P<0.05). In the one study of an occupationally exposed group of 

men, no association was found with time-to-pregnancy. This study was limited by small sample 

size and indirect exposure estimates. There were no studies judged to be useful for evaluating 

female reproductive toxicity. 

There is sufficient evidence in female rats to conclude that DEHP causes reproductive

toxicity (decreased numbers of corpora lutea and growing follicles) with dietary exposure

at 1088 mg/kg bw/day for multiple generations (151).

There issufficient evidence in female marmosetsto conclude that DEHP causes reproductive

toxicity (increased ovary weight and uterine weight) when exposure is by oral gavage at

500 mg/kg bw/day for ~15 months in the peripubertal period (92). The Expert Panel found 

these data consistent with precocious puberty in the 2 highest dose DEHP-exposed groups (500 

and 2500 mg/kg bw/day).

There issufficient evidence in male ratsto conclude that DEHP causes reproductive toxicity

when exposure is by oral gavage or in feed at 10– 113 mg/kg bw/day for exposures that

included gestational and/or peripubertal periods. The critical effects are small reproductive 

organ size (14 –23 mg/kg bw/day (114)), focal tubular atrophy (113 mg/kg bw/day (151)), and 

Leydig cell hyperplasia and altered reproductive hormones (10 and 100 mg/kg bw/day (111, 

179)). The Expert Panel found the data suggestive of male reproductive toxicity caused by 

changes in the gonadal hormonal response as well as direct effects on Sertoli cells. 

There is sufficient evidence to conclude that DEHP causes reproductive toxicity in adult

male mice at dietary exposure levels of 2857 mg/kg bw/day as manifested by decreased

testis weight and histopathologic alterations (175).

The experimental animal data are assumed relevant for consideration of human risk. 

Note: The definitions of the term sufficient and the terms assumed relevant, relevant, and not 

relevant are in the CERHR guidelines at http://cerhr.niehs.nih.gov/news/guidelines.html.
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Appendix II

Conclusions from the original Expert Panel 

evaluation

The original Expert Panel report on DEHP contained conclusions in Section 5. These conclusions 

have been extracted and reproduced below, with the section numbering as found in the original 

document. The references listed in the conclusion are listed, and the table to which the conclusions 

refer is reproduced, numbered 76 as in the original.

5.1.4 Reproductive Toxicity

There are no data on the reproductive toxicity of DEHP or its major metabolites in humans.

5.1.4.1.1 Females

 There are data that indicate that oral exposure to DEHP can affect reproductive processes in rats 

and mice. Data presented in [168] clearly show adverse functional effects at a dietary dose of 

425 mg/kg bw/day where complete infertility was observed, although the design did not allow 

conclusions as to whether males, females, or both sexes contributed to reduced number of pups 

and pup viability. Only the data from [219] come close to evaluating broadly the effects on the 

female tract, and this study examined only a high dose. Davis et al. [219] showed clear effects on 

estradiol synthesis and ovulation in rats at 2000 mg/kg bw/day. No histopathological structural 

changes were seen in the uterus or vagina. Further, there are no studies that have evaluated adult 

female reproductive structure and function after prenatal exposure. Current data are not adequate 

to confidently ascribe a NOAEL or LOAEL for female reproduction (Table 76).

5.1.4.1.2 Males 

The oral exposure studies of Lamb [168] and Schilling et al. [169] are sufficient to conclude 

that DEHP is a reproductive toxicant in male rats and mice. In the Lamb study [168], only the 

control and high-dose F0 mice were necropsied; thus, it is not known if the reduced fertility at 

~141 mg/kg bw/day (the middle dose) is partly male-mediated resulting from testicular damage. 

Schilling et al. [169] reported effects (androgen mediated and testicular lesions) at 1060 mg/kg 

bw/day in rats, and no effects (i.e. a NOAEL) at ~ 339 mg/kg bw/day. The study of Lamb 

[168] has a NOAEL of ~14 mg/kg bw/day (Table 76)... it is clear from the existing data that 

testicular pathology and reduced sperm numbers are consistent effects. The data are sufficient 

to conclude that DEHP is a reproductive toxicant in male rats, mice, ferrets, and guinea pigs 

when administered orally. There is greater uncertainty when determining lowest-adverse and 

no-adverse effect levels of exposure. The two studies [75,169] that used peripubertal dosing 

(believed to be the most sensitive period for causing adverse effects) and evaluated the rats when 

at, or close to, maturity (believed to be the most sensitive period for observing adverse effects) 

present markedly different NOAELs. The expert panel could not confidently reconcile these 

differences (~339 versus 3.7 mg/kg bw/day). Confidence in results observed at a given dose 

in [169] is eroded slightly by the small group size. The study of Poon et al. [75] was thorough 

in its design and execution, including verification of dose. While there were 20 animals per 

dose group, only 10 were males; the study design did not incorporate measures of reproductive 
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function. The authors were clear in asserting that “...the mild Sertoli cell vacuolation at 500 

ppm (~38 mg/kg bw/day) should be considered an adverse effect.” The expert panel finds there 

is a reasonable basis for such a conclusion (i.e. a LOAEL). The NOAEL from this study is 

therefore 3.7 mg/kg bw/day. When comparing the NOAELs from [75,168], 3.7 mg/kg bw/day 

versus 14 mg/kg bw/day, it is reasonable to conclude that these values are indistinguishable 

given the wide dose spacing and inherent biological variability in the endpoints. It is the panel’s 

view that the existing data support a NOAEL within the range of 3.7−14 mg/kg bw/day for oral 

exposure in rats.
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Appendix II

References from the original report cited above

[75] Poon R, Lecavalier P, Mueller R, Valli VE, Procter BG, Chu I. Subchronic oral toxicity 

of di-n-octyl phthalate and di(2-ethylhexyl) phthalate in the rat. Food Chem Toxicol 1997; 

35:225–39.

[168] Lamb IV JC. Reproductive effects of four phthalic acid esters in the mouse. Toxicol Appl 

Pharmacol 1987;88:255 –69. 

[169] Schilling K, Deckardt K, Gembardt C, Hildebrand B. Di-2-ethylhexyl phthalate – twogeneration reproduction toxicity range-finding study in Wistar rats, continuous dietary 

administration Laboratory Project ID: 15R0491/997096: BASF Aktiengesellschaft, 1999. 

[186] Reel JR, Tyl RW, Lawton AD, Jamb JC. Diethylhexyl phthalate (DEHP): Reproduction 

and fertility assessment in CD-1 mice when administered in the feed. PB84-181734. Springfield, 

VA: available from: NTIS, Research Triangle Park: National Toxicology Program, 1984.

[219] Davis BJ, Maronpot RR, Heindel JJ. Di-(2-ethylhexyl)phthalate suppresses estradiol 

and ovulation in cycling rats. Toxicol Appl Pharmacol 1994; 128:216 –23.
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Table 76. Summary of DEHP Reproductive Toxicity Studies with Oral Exposure

Protocol & Study NOAEL (mg /kg bw/day) LOAEL and Effects (mg/kg bw/day) Reproductive Effects at Higher Doses Reproductive Systemic

Continuous breeding and crossover mating study 11-week- old CD-1 mice 20 pairs of mice Fed diets with DEHP (0, 14, 141, and 425 mg/kg/day) for 7 days prior to mating and during a continuous 98-day mating period. (168, 186)

Reproductive:

14

Systemic:

Not known since lower dose groups not examined

141Reduced fertility↓ Live pups per litter

425↑ Liver weight

Complete infertility in females and reduced fertility in malesSeminiferous tubule damage with adverse effects on sperm numbers, motility, and morphology ↓ Male reproductive organ 

weights

Dose-setting two-generation dietary study Wistar rats. 10 pairs per group Received 0, 110, 339, or 1,060 mg/kg/day for 70 days prior to mating through gestation and lactation. (169)

Reproductive:

339

Systemic:

None

1,060↓ Anogenital distance in F2 males↓ Ovary weight in F0↓ Testes and epididymis weight in F1Testicular lesions↑ Nipple development in F1 males Spermatocyte loss in F1↓ Postnatal survival in F1 pups and 

prenatal survival in F1 and F2 pups

110↑ Liver weight 

in F0 females

No higher doses

90 Day repeat-dose dietary studySprague-Dawley rats. 10 rats/sex/group (4−6 weeks old) Received doses of:

M: 0, 0.4, 3.7, 38, 375F: 0, 0.4, 4.2, 42, 419

(75)

Reproductive:

37 (M)

Systemic:

37−42 (M and F)

38 (M)Mild Sertoli cell vacuolation

38(M)−42(F)

Liver enzyme effects

Atrophy of seminiferous tubules, loss of spermatogenesis, and Sertoli cell vacuolation
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Appendix II

Protocol & Study NOAEL (mg /kg bw/day) LOAEL and Effects (mg/kg bw/day) Reproductive Effects at Higher Doses Reproductive Systemic

1−12 day repeat-dose gavage study Female Sprague-Dawley rats. 6−10 rats per group (60−70 days old) received doses of 0 or 2,000 mg/kg/day. (219)

Reproductive:

Not known since only one dose administered 

Systemic:

Not examined 

2,000↓ Estradiol levels and suppression of 

ovulation

Not examined No higher doses
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5.0 SUMMARY, CONCLUSIONS, AND CRITICAL DATA NEEDS

5.1 Developmental and Reproductive Toxicity

5.1.1 	 Developmental Toxicity (Update)

One human study was considered useful for evaluating the association between maternal prenatal 

urinary MEHP excretion and anogenital index in male offspring. No significant association was found. 

Two human studies that were considered useful for evaluating DEHP exposure and development during 

childhood suffered from small sample sizes and problems in accurate exposure characterization. The 

data are insufficient to evaluate the prenatal or childhood effects of DEHP exposure in humans. 

Developmental toxicity has been assessed in rats following exposure to DEHP during gestation and 

postnatally to sexual maturation. Most of the relevant rat studies focused on effects on male offspring. 

Data from a multigeneration study indicated that dietary exposures in the range of 14 – 23 mg/kg 

bw/day result in small and/or absent components of the male urogenital tract. At higher levels of both 

dietary and gavage exposure, effects on in utero survival, reduced anogenital distances, undescended 

testes, retained nipples/areolae, incomplete preputial separation, and disruption in spermatogenesis 

were evident in postnatal animals. Based on two studies that exposed neonatal male rats to DEHP by 

oral gavage, the critical period for effects on the testis extends into the immediate postnatal period. In 

one of the studies, decreased Sertoli cell proliferation was seen in male rats exposed by oral gavage 

to DEHP 100 mg/kg bw/day on PND 3. In the other study, neonatal rats were exposed by the iv

route starting on PND 3 – 5 and continuing for 21 days. Exposures to 300 mg/kg bw/day and higher 

resulted in decreased testis weight, depletion of germinal epithelium, and decreased seminiferous 

tubule diameter. The reduced testis weights persisted through at least 90 days of age. The NOAEL for 

this study was 60 mg/kg bw/day. The findings from the iv route were similar to those observed after 

oral exposure to the same dose levels. 

These data are sufficient to conclude that DEHP is a developmental toxicant in rats by the dietary, oral 

gavage, and iv routes of administration at the indicated dose levels. These animal data are assumed 

relevant to the assessment of human risk. 

5.1.2. Reproductive Toxicity (Update)

Several human studies have examined the relationship between adult male exposure to MEHP and 

various reproductive endpoints. While there were suggestions of associations between MEHP and 

decreased sperm velocity and serum testosterone, no significant associations were found between 

MEHP exposure and adverse semen parameters, hormone levels, time-to-pregnancy, or infertility 

diagnosis. There were no studies of adult female exposure that were judged to be useful. The data are 

insufficient to evaluate the reproductive effects of DEHP exposure in humans.

There are data to indicate that oral exposure to DEHP can affect reproductive processes in female 

marmosets. The Mitsubishi study found an increase in serum 17b-estradiol at weeks 52 and 65, and 

an increase in ovary and uterine weights at necropsy in the 500 and 2500 mg/kg bw/day groups (92). 

There were no other new studies on female reproductive function. 

The oral exposure studies of Akingbemi et al. (111, 179), Schilling et al. (151), and the NTP (114) 
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Appendix II

are sufficient to conclude that DEHP is a reproductive toxicant in male rats at the indicated dose 

levels. All of those data are assumed relevant. Despite some internal inconsistencies in the Akingbemi 

studies, the data provide a plausible explanation for the Leydig cell hyperplasia observed in other 

studies, with a LOAEL of 10 mg/kg bw/day and a NOAEL of 1 mg/kg bw/day. These data are assumed 

relevant for human reproduction. The Schilling et al. data show an effect of DEHP exposure (increased 

seminiferous epithelium vacuolation) at ~113 mg/kg bw/day in male rats in a multigeneration study. 

The NTP study found an increase in grossly small male reproductive tissues at necropsy in multiple 

generations, which was first seen at 300 ppm in feed (~14– 23 mg/kg bw/day) and which increased in 

a dose-related manner at higher doses. These effects are in the range of effects observed by Poon et al. 

(194) and cited in the previous Expert Panel Report on DEHP, which found seminiferous epithelium 

vacuolation at 500 ppm (~38 mg/kg bw/day) in a multigeneration study in rats. Despite a less-thanthorough evaluation of all the animals born into the NTP study, the NOAEL in that study was 4.8–7.9 

mg/kg bw/day, which is also below the LOAEL in Akingbemi et al. The convergence of data from the 

NTP study, Akingbemi, and Poon around the 10 –30 mg/kg bw/day range gives added confidence that 

this is the range of the lowest effective dose level. It is the panel’s view that the existing data support 

a NOAEL between 1 and 10 mg/kg bw/day for oral DEHP exposure in rats.

5.2 Human Exposure

The information in this report is an update of the first Expert Panel Report on DEHP, and the reader 

is referred to the Summary of Exposure in Section 5.1 of that report for background and context for 

the following remarks.

DEHP is ubiquitous in the environment. Humans can be exposed to DEHP through many routes 

including ingestion (food, infant formula, and breast milk), contact with contaminated household dust 

and consumer products (cosmetics and toys), inhalation, and through medical procedures. The largest 

source of general population exposure to DEHP is dietary. Food surveys show a range of DEHP content 

with fatty foods, including dairy, fish, meat, and oils, containing the most.

DEHP is currently the primary phthalate plasticizer used in PVC-containing medical devices. Medical 

exposures can be iv, oral, and inhalational, and the exposures can be either DEHP alone or mixtures 

of DEHP and MEHP.

Exposures to DEHP can be estimated using probabilistic calculations from measurements in environmental matrices or dose reconstruction from urinary metabolite measurements. Probabilistic estimates can 

accurately estimate exposures if all routes/pathways of exposure are accounted for and the environmental 

matrices in these pathways are well-characterized. Dose reconstruction from urinary measurements 

can also accurately estimate exposures if the toxicokinetics are well-defined and reasonably stable. 

For both exposure estimate methods, uncertainties exist. For example, for probabilistic estimates, 

other pathways of exposure may contribute significantly such as medical exposures, occupational 

exposures, some indoor air exposures and potentially exposure from mouthing of DEHP-containing 

objects. Similarly, dose reconstruction is limited because the toxicokinetics vary within and among 

persons and the proportion of any given metabolite attributable to the total fraction excreted may 

vary within and between persons as well. In addition, a steady state excretion is assumed whereas the 

urinary measurement may have captured either a peak or background exposure. The limitations listed 

above for both dose estimation methods lead to uncertainties in exposure estimates; however, both the 
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probabilistic and dose reconstruction approaches agree within an order of magnitude suggesting that 

both methods are appropriate for estimating dose ranges with a reasonable degree of certainty.

For the purposes of comparison in this report, the Expert Panel relied primarily on dose reconstruction from urinary metabolites to estimate DEHP daily intake. The Expert Panel used the full range of 

reported urinary MEHP concentrations in determining exposure ranges; however, the literature provided 

2 separate approaches for dose reconstruction that differ primarily based upon the excretion fraction of 

MEHP. Both metabolite excretion fraction estimates were based upon limited data, and at this time, it 

is not possible to determine which excretion fraction estimate is most accurate. Thus, the Expert Panel 

chose to include both dose reconstruction approaches in determining a range of exposure for the US 

population and selected subpopulations. In determining exposure ranges from dose reconstruction, 

the Expert Panel used the median or geometric mean concentration of MEHP and the 95th percentile 

of the distribution of MEHP in each study to estimate the exposure range (Table 36).

Table 36. Estimated DEHP Dose Ranges for Selected US Population Groups

Population Group Estimated dose range

 (μg/kg bw/day)

General populationa

20+ years 1 – 30

12–19 years 1 – 25

6–11 years 1 – 30

<6 years Unknown c

Medical exposures, neonates b 130–6000

Lower range estimate is median or geometric mean values estimated using the 

excretion fraction used by David (37). Upper range estimate is 95th percentile 

using the excretion fraction used by Koch (31, 36), which gives higher values.

aBased upon NHANES 2001–2002 data (n=2782) (27).

bBased upon Calafat 2004 (n=6) (5).

cUS population-level urinary excretion data not available; the previous Expert 

Panel concluded that the level could be several-fold higher than in the general 

adult population.

The general population dose estimates based upon the most recent US urinary MEHP data are similar 

to those contained in the previous report. In addition, recently published probabilistic estimates for 

general population doses agree well with the ranges presented in Table 36, providing additional support 

for these estimates. However, dose estimates from medical procedures in neonates using both dose 

reconstruction and probabilistic estimates are highly variable, depending largely upon the medical 

treatments given and the duration of the treatments. Recent dose estimates are shown in Table 37.

Pregnant and lactating women represent a population of special concern because of the potential 

impact of their exposures on the fetus and nursing infant. For example, data suggest that metabolites 

cross the placenta and enter breast milk in free form. In addition, women undergoing certain medical 

procedures during pregnancy or lactation may increase their exposures significantly above the general 

population level, thus potentially resulting in higher exposures to the fetus and nursing infant. Another 

potential source of infant exposure is from expressed and stored breast milk that has been contaminated 

by DEHP-containing breast pumps.
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Appendix II

Table 37. Dose Estimates for DEHP/MEHP Exposures from Medical Procedures in Neonates

Dose Estimation 

Method

Estimate

(μg/kg bw/day) Reference

Dose reconstruction 130–6000a Calafat et al. (5)

Dose reconstruction 1–170 a,b Green et al. (63)

Probabilistic 7000 Loff et al. (56)

Probabilistic 2800 FDA (2)

Probabilistic 800– 2000 Kambia et al. (59)

aEstimated by Expert Panel according to the methods used in 

Table 36.

bUpper bound is 75th percentile.

5.3 Overall Conclusions

The Expert Panel noted that DEHP and some other phthalates have been shown to act through the same 

mode of action and to induce similar effects in exposed animals. The combined effects of multiple 

phthalate exposures have implications for exposure and risk assessment. The conclusions in this report 

assume exposure only to DEHP.

5.3.1. 	General Adult Population

First Report:

DEHP conversion to active MEHP involves intestinal lipases that appear to be at significantly greater 

levels in rodents than in primates; adult rodents require 1 –2 orders of magnitude more DEHP than is 

required in juvenile rodents to produce testicular effects; and adult marmosets (primates) showed no 

testis effects when exposed to DEHP at oral doses (2500 mg/kg bw/day) for 13 weeks, conditions that 

produce testicular toxicity in juvenile rodents. Based on these data, the panel has minimal concern 

that ambient human exposures adversely affect adult human reproduction. This level of concern is not 

appreciably altered for adults medically exposed to DEHP or MEHP.

Update:

Data not considered in the earlier report demonstrated that humans have ~2–3-fold lower levels of 

intestinal lipases than ferrets and rats. New information suggests that marmosets may be less susceptible 

to hormonal disruption, a key feature of DEHP toxicity, than most other species, including rats and 

humans. In the absence of significant new adult rodent toxicity data, the previous Lamb et al. (195) 

data remain informative: a LOAEL of 425 mg/kg bw/day, orally in the diet. Based on these data, the 

panel has minimal concern that general population exposures adversely affect adult human reproduction. In addition, there have been some, albeit insufficient, human studies addressing reproductive 

effects under general population exposures, and these do not increase our level of concern. This level 

of concern is not appreciably altered for adults medically exposed to DEHP or MEHP. This conclusion 

concurs with the conclusion of the first DEHP Expert Panel.

5.3.2 	 Healthy Infants and Toddlers

First Report: 

DEHP produces testicular toxicity at lower doses in juvenile rodents than in adults; the reproductive system 

is (and specifically, the Sertoli cells are) still in proliferative mode until puberty, and reproductive system 
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development has been shown to be sensitive to MEHP in rodents; intestinal lipase activity is found at 

adult levels in babies older than 6 months of age. All of these points increase the level of concern. While 

adult marmosets showed no testicular toxicity at doses that produced toxicity in adult rats, no data are 

available for infant primates. If healthy human infant/toddler exposure is several-fold higher than adults, 

the panel has concern that exposure may adversely affect male reproductive tract development.

Update: 

There are 2 new studies on the effects of DEHP following postnatal only exposure of rats. DEHP 

was shown to induce testicular toxicity following oral exposure of neonates, and the susceptibility of 

the testes of the young animal was confirmed in these studies to be greater than that for adults. In the 

first study (120), the BMDL10 for the effect on testicular weight was 77 mg/kg bw/day (there was no 

NOAEL in this study; the lowest dose was 300 mg/kg bw/day). In the second study (119), a NOAEL 

of 20 mg/kg bw/day was found for effects on Sertoli cell proliferation. 

There are no data for exposure levels of healthy infants under the age of one year. Exposure of children 

aged 1–6 years has been estimated to be up to several-fold higher than the general population exposure 

estimate of 1–30 μg/kg bw/day. There are indications that younger children have elevated ratios of the 

metabolites of MEHP in their urine compared to older individuals, suggesting differential internal exposure 

to the metabolites of DEHP. The toxicity of these metabolites has not been studied in this age group. 

If the level of exposure is at the high end of the estimated range, the Panel has some concern that 

exposure to DEHP can adversely impact reproductive development in male children older than 1 year. 

The Expert Panel has concern that DEHP exposure can adversely affect reproductive development in 

infants less than 1 year old because of their greater susceptibility and uncertainties regarding exposure. 

This conclusion is a refinement of the first Expert Panel’s conclusion in distinguishing concern by age 

group within the infant-toddler category. 

5.3.3 	 Critically Ill Infants

First Report: 

Documented parenteral medical exposure to DEHP of critically ill infants can exceed general population exposures by several orders of magnitude; parenteral exposures to DEHP involving blood and 

blood products include concurrent exposure to MEHP; the most sensitive process (reproductive system 

development) is still occurring; human parenteral exposures can approach the rat parenteral NOAEL. 

On the other hand, concern is lowered by the fact that there is less conversion of DEHP to MEHP by 

the parenteral route of exposure, although the exact degree of reduction is not known. It is not known 

if primate Sertoli cells are more, equally, or less sensitive than rodent Sertoli cells to the effects of 

MEHP. The available reproductive and developmental toxicity data and the limited but suggestive human 

exposure data indicate that exposures of intensively-treated infants/children can approach toxic doses in 

rodents, which causes the panel serious concern that exposure may adversely affect male reproductive 

tract development. The panel recognizes that benefits of medical procedures can outweigh any risks.

Update: 

New information on the mechanism of DEHP-induced developmental toxicity focuses on gonadal 

endocrine effects. Recent additional human studies confirm our previous assumptions and concerns 

that intensively medically treated infants are exposed to doses that are toxic in rodents. The Panel has 
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serious concerns that such exposures may adversely affect male reproductive tract development and 

function. The Panel believes that the benefits of medical procedures can be significant but that minimizing 

exposure to DEHP should be a goal. This conclusion concurs with that of the first Expert Panel.

5.3.4 	 Pregnancy and Lactation

First report: 

In utero development is a life stage of particular vulnerability; exposures may be on the order of 3 – 30 

μg/kg bw/day; the most relevant rodent data suggest a NOAEL for testis/developmental effects of 3.7 –14 

mg/kg bw/day; DEHP produces malformations in rodents, with a NOAEL of ∼40 mg/kg bw/day; even 

time-limited exposures are effective at producing irreversible effects; the active toxicant MEHP passes 

into breast milk and crosses the placenta. On the other hand, absorption from the primate gut appears 

to be less effective than from the rodent gut, which reduces the level of concern for oral exposure. 

Given that oral exposure is <30 μg/kg bw/day for humans and toxic effects are seen in rodents at >3 

mg/kg bw/day in rodents, even in the face of significant species differences in absorption, the panel 

has concern that ambient oral DEHP exposures to pregnant or lactating women may adversely affect 

the development of their offspring.

Update: 

Since the last report, 4 useful studies have been conducted involving exposure of rats to DEHP during 

gestation, 2 of which are multigenerational. Based upon a constellation of effects on the developing 

male reproductive tract and accessory sex organs seen across studies, a mode of action involving antiandrogenicity has been confirmed. Numerous other mechanistic studies support these observations. 

The lowest LOAEL identified for effects on testicular tract development was 14 – 23 mg/kg bw/day, 

with a NOAEL of 5 –8 mg/kg bw/day (114). This level was determined by the Expert Panel based 

upon an overall assessment of response across generations. Estimates of exposure to DEHP for the 

adult human population range from 1 to 30 μg/kg bw/day. 

Based upon the projected level of exposure and the toxicity observed in the offspring of treated rats, the 

Expert Panel has some concern for the effects of DEHP on male offspring of humans exposed to general 

population levels during pregnancy. This reduction in level of concern from that of the first Expert Panel 

is due to greater confidence in population exposure levels and greater confidence in the effect level in 

experimental animals. The Expert Panel notes that it has concern for possible effects on male fetuses of 

women undergoing certain medical treatments where additional exposure to DEHP could occur. Lactation 

concerns are expressed in Section 5.3.2., above, with reference to children under 1 year of age.

5.4 Critical Data Needs

1. First report: 

Identification and follow-up studies of human populations (e.g., premature infants) who were heavily 

exposed to DEHP. This would directly address the issue of whether there are functional effects in 

the most heavily and simultaneously the most vulnerable, human population. This would consist 

primarily of follow-up evaluation of reproductive system development and function.

Update: 

There is one study by Rais-Bahrami et al. (109) that evaluated sexual development and serum 

hormone levels in adolescents that had been subjected to ECMO treatment. However, the panel 
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found this study insufficient to draw conclusions regarding human developmental toxicity. This 

data need continues.

2. First report: 

Obtain better medical exposure data. Common clinical research designs with unified analysis 

approaches across centers, as are often used in the large group cooperative studies of cancer 

therapy, would be one approach to acquiring better data. Potential toxicity from medical exposures 

could also be evaluated using the multi-center model to study DEHP/MEHP exposed neonates and 

adults longitudinally over decades to capture the reproductive, developmental, and other outcomes 

of concern based upon animal toxicity studies. Finally, discussions with the manufacturers of the 

medical devices used in these procedures would be helpful to determine whether and how much 

the formulations of PVC blood bags, ECMO circuits, hemodialysis machines, and other medical 

devices that contain DEHP or MEHP have changed over time.

Update: 

There are additional data on DEHP release from medical devices (e.g., 55, 56, 59, 61). The 

panel also identified 3 studies (5, 63, 84) that better characterized medical exposures in infants 

undergoing intensive medical procedures. In these studies, DEHP urinary metabolite levels were 

measured, thus providing information on DEHP exposures through multiple routes. Studies with 

larger numbers of subjects relating the nature of the procedures to exposure levels using measures 

of internal dose with multiple metabolites are needed. 

3. Significance of perinatal exposure:

3.1 First report: 

Dose-response of male and female reproductive tract malformations. There is a need to gather 

dose-response data across a wider range of lower exposures in dam and pups in order to correlate blood levels of MEHP with reproductive effects.

Update: 

An NTP continuous breeding study (114) examined male and female reproductive organs in 

rats fed DEHP in the diet at concentrations of 10 – 10,000 ppm (0.47 –775 mg/kg bw/day). 

The doses address the range of estimated exposures in infants undergoing intensive medical 

procedures. The dose range included 3 dose levels below the LOAEL (300 ppm) at which 

developmental toxicity was observed. Information on MEHP blood levels and reproductive 

effects remains limited.

3.2 First report: 

Relevant animal model (in utero reproductive tract maturation) in guinea pig or non-human 

primate to correlate dose with effects, if any, and compare these doses with those of rodents 

where adverse effects do occur.

Update: 

There are no new studies further addressing prenatal developmental toxicity in a non-rodent 

species. This is still a data need. A pre/postnatal study in a cynomolgus monkey would be 

most useful.
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3.3 First report: 

Timing, PPAR, metabolism.

Update: 

There are no new studies examining the effects of timing of exposure on developmental toxicity. 

There are no new studies examining the possible role of PPAR on developmental toxicity. The 

Expert Panel considers PPAR to be a less critical data need given studies showing that the 

mode of developmental toxicity appears to be independent of this class of receptors. There are 

3 studies that examined metabolism and/or toxicokinetics in pregnant animals. Two studies 

were in rats (86, 94) and one in marmosets (92).

4. First report: 

Extension of PBPK model to

Pregnancy, because this is the human group thought to be most at risk.

Species (marmoset/human), as humans are the species of interest. The marmoset data provide 

a positive control to show that the PBPK model works as advertised.

ADME, specifically phase l and phase II metabolism extended across species, and into pregnant 

humans.

In order to acquire better data on primate/human toxicokinetics, including immature animals 

and humans, there is a need for a fetal compartment in the PBPK model and rate constants for 

fetuses and newborns for absorption, metabolism, and excretion.

Model DEHP/MEHP dose for iv exposure. This is another route of exposure for a great many 

people who may have reduced capability to clear the compound.

Update: 

There are no new relevant publications on PBPK modeling and this remains one of the most 

important data needs. As noted above, the most desirable study would include the cynomolgus 

monkey.

Additional data needs identified by the current Expert Panel include:

In vitro and in vivo metabolic data including information across ages and species on lipase, 

cytochrome P450, glucuronyl transferase, and dehydrogenase enzyme kinetics. There is a 

critical lack of human in vitro data.

Gene expression and enzyme induction studies on DEHP and its metabolites, particularly in 

DEHP target tissues and human cell types to better understand dose-response relationships.

Comparison of Cmax and AUC measurements of exposure to better understand the appropriate 

dose metric for toxicity extrapolation.

Human studies evaluating endocrine-mediated effects in males with consideration of confounding effects and with larger sample size.

Additional exposure and toxicokinetic information on pregnant women, infants, and children 

1–6 years of age. This information should include exposure in breast milk, including potential 

contamination from breast pumps.

Investigation of mode/mechanism of action.

Effects of mixtures of phthalates including questions of additivity and interference.

•

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•

•

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166.	Andriana, B. B., Tay, T. W.,Tachiwana, T., Sato,T., Ishii, M., Awal, M. A., Kanai,Y., Kurohmaru,

M. and Hayashi, Y. Effects of mono(2-ethylhexyl) phthalate (MEHP) on testes in rats in vitro.

Okajimas Folia Anat Jpn 2004; 80: 127-36.

167.	Andriana, B. B., Tay, T. W., Maki, I., Awal, M. A., Kanai,Y., Kurohmaru, M. and Hayashi,Y. An

ultrastructural study on cytotoxic effects of mono(2-ethylhexyl) phthalate (MEHP) on testes in

Shiba goat in vitro. J Vet Sci 2004; 5: 235-40.

168.	Awal, M.A., Kurohmaru, M., Ishii, M.,Andriana, B. B., Kanai,Y. and Hayashi,Y. Mono-(2-ethylhexyl) phthalate (MEHP) inducesspermatogenic cell apoptosisin guinea pig testes at prepubertal

stage in vitro. Int J Toxicol 2004; 23: 349-355.

169.	Richburg, J. H., Nanez, A., Williams, L. R., Embree, M. E. and Boekelheide, K. Sensitivity

of testicular germ cells to toxicant-induced apoptosis in gld mice that express a nonfunctional

form of Fas ligand. Endocrinology 2000; 141: 787-93.

170.	Ichimura,T., Kawamura, M. and Mitani,A. Co-localized expression of FasL, Fas, Caspase-3 and

apoptotic DNA fragmentation in mouse testis after oral exposure to di(2-ethylhexyl)phthalate.

Toxicology 2003; 194: 35-42.

171.	Giammona, C. J., Sawhney, P., Chandrasekaran, Y. and Richburg, J. H. Death receptor response

in rodent testis after mono-(2-ethylhexyl) phthalate exposure. Toxicol Appl Pharmacol 2002;

185: 119-27.

172.	Dalgaard, M., Nellemann, C., Lam, H. R., Sorensen, I. K. and Ladefoged, O.The acute effects of

mono(2-ethylhexyl)phthalate (MEHP) on testes of prepubertal Wistar rats. Toxicol Lett 2001;

122: 69-79.

173.	Park, J. D., Habeebu, S. S. and Klaassen, C. D. Testicular toxicity of di-(2-ethylhexyl)phthalate

in young Sprague-Dawley rats. Toxicology 2002; 171: 105-15.

174.	Kasahara, E., Sato, E. F., Miyoshi, M., Konaka, R., Hiramoto, K., Sasaki, J., Tokuda, M.,

Nakano, Y. and Inoue, M. Role of oxidative stress in germ cell apoptosis induced by di(2-ethylhexyl)phthalate. Biochem J 2002; 365: 849-56.

175.	Ablake, M., Itoh, M., Terayama, H., Hayashi, S., Shoji, S., Naito, M., Takahashi, K., Suna, S.

and Jitsunari, F. Di-(2-ethylhexyl) phthalate induces severe aspermatogenesis in mice, however,

subsequent antioxidant vitamins supplementation accelerates regeneration of the seminiferous

epithelium. Int J Androl 2004; 27: 274-81.

176.	Kijima, K., Toyosawa, K., Yasuba, M., Matsuoka, N., Adachi, T., Komiyama, M. and Mori, C.
II-200

Appendix II

Gene expression analysis of the rat testis after treatment with di(2-ethylhexyl) phthalate using

cDNA microarray and real-time RT-PCR. Toxicol Appl Pharmacol 2004; 200: 103-10.

177.	Kim, H. S., Saito, K., Ishizuka, M., Kazusaka, A. and Fujita, S. Short period exposure to di-

(2-ethylhexyl) phthalate regulates testosterone metabolism in testis of prepubertal rats. Arch

Toxicol 2003; 77: 446-51.

178.	Kim, H. S., Ishizuka, M., Kazusaka, A. and Fujita, S. Alterations of activities of cytosolic

phospho­lipase A2 and arachidonic acid-metabolizing enzymes in di-(2-ethylhexyl)phthalateinduced testicular atrophy. J Vet Med Sci 2004; 66: 1119-24.

179.	Akingbemi, B. T., Ge, R., Klinefelter, G. R., Zirkin, B. R. and Hardy, M. P. Phthalate-induced

Leydig cell hyperplasia is associated with multiple endocrine disturbances. Proc Natl Acad Sci

U S A 2004; 101: 775-80.

180.	Rasoulpour, R. J. and Boekelheide, K. NF-kappaB is activated in the rat testis following exposure to mono-(2-ethylhexyl) phthalate. Biol Reprod 2005; 72: 479-86.

181.	Awal, M. A., Kurohmaru, M., Andriana, B. B., Kanai, Y. and Hayashi, Y. Mono-(2-ethylhexyl)

phthalate (MEHP) induces testicular alterations in male guinea pigs at prepubertal stage. Tissue

Cell 2005; 37: 167-75.

182.	Ljungvall, K., Karlsson, P., Hultén, F., Madej,A., Norrgren, L., Einarsson, S.,Rodriguez-Martinez,

H. and Magnusson, U. Delayed effects on plasma concentration of testosterone and testicular

morphology by intramuscular low-dose di(2-ethylhexyl)phthalate or oestradiol benzoate in the

prepubertal boar. Theriogenology 2005;64:1170-84.

183.	Ljungvall, K., Tienpont, B., David, F., Magnusson, U. and Torneke, K. Kinetics of orally-administered di(2-ethylhexyl)phthalate and its metabolite mono(ethylhexyl)phthalate, in male pigs.

Arch Toxicol 2004; 78: 384-389.

184.	Ljungvall, K., Spjuth, L., Hulten, F., Einarsson, S., Rodriguez-Martinez, H., Andersson, K. and

Magnusson, U. Early post-natal exposure to low dose oral di(2ethylhexyl) phthalate affects the

peripheral LH-concentration in plasma, but does not affect mating behavior in the post-pubertal

boar. Reprod Toxicol 2006;21:160-6.

185.	Kim, E. J., Kim, J. W. and Lee, S. K. Inhibition of oocyte development in Japanese medaka

(Oryzias latipes) exposed to di-2-ethylhexyl phthalate. Environ Int 2002; 28: 359-65.

186.	Tomonari,Y., Kurata,Y., Kawasuso, T., David, R. M., Gans, G. and Katoh, M. Testicular toxicity

study of di(2-ethylhexyl) phthalate (DEHP) in juvenile common marmoset. Toxicologist 2003;

72: 385.

187.	Li,L.-H.,Donald,J.M. andGolub,M.S.Reviewon testicular development,structure,function, and

regulation in common marmoset. Birth Defects Res B Dev Reprod Toxicol 2005; 74: 450-469.
Appendix II

II-201

188.	Ishihara, M., Itoh, M., Miyamoto, K., Suna, S., Takeuchi, Y., Takenaka, I. and Jitsunari, F.

Spermato­genic disturbance induced by di-(2-ethylhexyl) phthalate is significantly prevented by

treatment with antioxidant vitamins in the rat. Int J Androl 2000; 23: 85-94.

189.	Boekelheide, K., Embree, M. E., Schoenfeld, H. A., Rasoulpour, R. J. and Gray, D. A. Hypothesis testing in male reproductive toxicology using mutant mice. Toxicologist 2002; 66: 382-3.

190.	Noriega, N. C., Furr, J., Lambright, C., Wilson, V. S. and Gray, L. E. Peripubertal DEHP Exposure Inhibits Androgen Sensitive Tissue Development And Delays Puberty In Male SpragueDawley Rats. Biol Reprod 2004; 166.

191.	Nakajima, T., Hata,Y., Ito,Y., Omura, M., Sone, H., Toyama, C., Gonzalez, F. J. and Aoyama, T.

Di(2-Ethylhexyl)Phthalate May Reduce Mouse Fertility Via Peroxisome Proliferator-Activated

Receptor alpha (PPARalpha). Toxicol Appl Pharmacol 2004; 197: 231.

192.	Kang, S. C. and Lee, B. M. Comparative evaluation of phthalates for sperm motility and male

fertility in Sprague-Dawley rats. Birth Defects Res Part A Clin Mol Teratol 2004; 70: 310.

193.	Jayes, F. L. and Davis, B. J. Granulosa Cell Function Is Impaired By Environmentally Relevant

Levels Of Mono-(2-Ethylhexyl) Phthalate (MEHP). Biol Reprod 2004; 166.

194.	Poon, R., Lecavalier, P., Mueller, R., Valli, V. E., Procter, B. G. and Chu, I. Subchronic oral toxicity

of di-n-octyl phthalate and di(2-ethylhexyl) phthalate in the rat. Food Chem Toxicol 1997; 35:

225-239.

195.	Lamb, J. C. I., Chapin, R. E., Teague, J., Lawton, A. D. and Reel, J. R. Reproductive effects of

four phthalic acid esters in the mouse. Toxicol Appl Pharmacol 1987; 88: 255-269.
Appendix III

Public Comments

ON di(2-ethylhexyl) phthalate


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ON THE EXPERT PANEL UPDATE
Dr. Michael B. Shelby, Director

Center for Evaluation of Risks to Human Reproduction

NIEHS

P.O. Box 12233, MD EG-32

Research Triangle Park, NC 27709

February 2, 2006

Re: NTP-CERHR expert panel update on the reproductive and developmental toxicity of di(2-

ethylhexyl)phthalate – November 2005

Dear Dr. Shelby:

On behalf of Health Care Without Harm (HCWH), we submit these comments on the NTPCERHR expert panel update on the reproductive and developmental toxicity of di(2-ethylhexylphthalate, issued November 2005.

Health Care Without Harm agrees with the expert panel's conclusions that:

- DEHP is a reproductive and development toxicant in animal studies that are relevant to 

humans;

- Health care delivery can be a significant source of DEHP exposure;

- Levels of DEHP exposure in sick infants receiving medical care are of serious concern; and 

- Because DEHP crosses the placenta, exposures in pregnant women receiving medical

treatments are also of concern.

We also emphasize that the expert panel’s experiences with the unpublished Mitsubishi and 

BASF (Schilling) studies confirm the importance of carefully reviewing study design, raw data,

data handling, data interpretation, and authors’ conclusions if such studies will continue to be

used by future CERHR and NTP panels. As the Expert Panel noted, for unjustified reasons the

authors of the marmoset study removed from data analysis specific animals that apparently 

showed adverse impacts of DEHP exposure. Moreover, the study authors failed to examine the

histopathology of the testis of each animal as required in their study design. The Expert Panel

also concluded that the BASF/Schilling study showed histopathologic impacts of DEHP in the

testes of animals in all treatment groups and that no NOAEL was identified in this study. The

authors of the original paper provided no justification for ignoring those pathologic changes in 

the lowest dose treatment group of animals. Appendix III
Detailed comments follow and are tracked in the PDF document attached. (Our comments are in 

page order.)

Page iii: In the introductory section to the Panel’s conclusions, the report indicates that there was

not consensus around reducing the concern level for pregnant women. We agree with some of

the panel members, that the level of concern for pregnant women identified in the 2000 expert

panel report should NOT be reduced. It is our position that pregnant women’s exposure remains

a concern, as stated, because:

1) MEHP passes the placenta in free form where it may not be detoxified by the fetus, 2)

exposure throughout pregnancy is not necessary to cause damage in animal models, and 3)

current exposure estimates in women of child bearing age do not distinguish peak or

episodic exposures from average exposures.

Page 4, Table 5: It does not look like the data are expressed as percentages but rather as

microgms/kg/day. Which is correct? The later reference on page 26 that “more than 90% of the

intake is from food” is not consistent with Table 5. Table 5 indicates that less than 90% intake

for infants is from food.

Page 11, Figure 2: It is not clear what the “x” axis represents on the graph.

Page 26 – The statement that estimates the percentage intake of DEHP from food is not accurate

for infants (See comment for page 4, Table 5 above).

Page 27: The report does not indicate what the expert panel thinks about the validity of David's

argument and Koch's response. Is there any other research to support David’s case? The expert

panel report should explicitly note that David has a conflict of interest inasmuch as he has

financial ties to the phthalate manufacturing industry.

Page 55: In reference to the Rais Bahrami, et al. study, the panel identifies the utility of the study 

as minimally significant due to the small sample size and the lack of a comparison group. A

major limitation of the study is the lack of measurements of phthalate exposure. The panel report

should explicitly note this important limitation of the study.

Page 98, Table 28: In reference to the developmental LOAEL, the panel's conclusion for the

LOAEL differs from the author's conclusion, which is apparently quoted in the Table. It is

important to note that the Table reflects the authors’ conclusion and does not reflect the panel’s

conclusion, especially so that the subsequent panel discussion of Schilling in Section 4 does not

appear to be in contradiction to what is noted here.

Page 143: In reference to the discussion of the Schilling study, the note in the “utility” section 

states that the study was useful in the evaluation process, showing a LOAEL of 1000 ppm (~100

mg/kg bw/day). This was the lowest dose tested. The panel, however, goes on to say that since

this is a conclusion of the Expert Panel and not the authors’, it is a cause for concern and limits

the confidence that this conclusion can bear. We disagree that the panel’s conclusion bears

Appendix III
limited confidence. A review of the original paper clearly shows histological impacts that the

authors simply ignored when deriving their conclusion. Adverse impacts of DEHP exposure are

discernable at every dose tested.

Page 169: In the section updating the science on reproductive toxicity, the report should make it

clear that in the Schilling study ~113 mg/kg/day was the lowest dose tested.

Page 171: In reference to the Schilling study, the “update” states, “Data not considered in the

earlier report demonstrated that humans have ~2–3-fold lower levels of intestinal lipases than 

ferrets and rats.” We are unable to identify a reference for this in the draft report. The summary 

of Ito et al. (page 41) discusses rodents and marmosets, but not humans. The 2001 FDA “Safety

Assessment of DEHP Released from Medical Devices” states (page 31):

“Consequently, individuals with high rates of lipase activity and/or low rates of

glucuronidation activity could be at higher risk of DEHP-induced adverse effects than the

rest of the population. Polymorphisms in genes coding for pancreatic (Hegele et al.,

2001) and hepatic (Cohen et al., 1999) lipase in humans are known to exist and these

polymorphisms can result in lipase deficiency. Low lipase activity would be expected to 

exert a protective effect in these individuals with regard to DEHP-mediated effects.

Conversely, pancreatic lipase activity is increased by heparin administered to patients on

hemodialysis (Montalto et al., 1997) and plasma lipoprotein lipase activity is increased by 

erythropoietin (Goto et al., 1999), which is also administered to patients on hemodialysis.

Increased lipase activity would facilitate the conversion of DEHP to its active metabolite.

Smoking is also known to increase lipase activity (Kong et al., 2001) and DEHP itself

induces lipase activity in rodents (Mocchiutti and Bernal, 1997). Consequently, some

individuals in the DEHP-exposed population can convert DEHP to MEHP more

efficiently than others. This variability is evidenced, to some extent, by the variability in 

the rate at which intestinal mucosal cell preparations obtained from two humans

hydrolyzed a number of di-n-alkyl phthalates (Lake et al., 1977). The metabolic rates

between these two individuals differed by around 3- to 6-fold. Presumably, the degree of

variability would increase with a larger sample size.”

We also note that ref 96 in the Expert Panel report discusses differences in phthalate hydrolysis

among non-human primates.

It is also important to keep in mind the absorption and serum levels of DEHP metabolites

reported by Koch after he ingested labeled DEHP. Levels were comparable to those in

marmosets given far higher doses. This suggests that there may be differences between 

humans and marmosets with respect to intestinal or pancreatic lipase levels and marmosets

should not be assumed to accurately reflect levels in humans without empirical data

supporting that conclusion. Moreover, intestinal and pancreatic lipase is inducible and in 

vitro assays will not accurately portray in vivo circumstances.

Page 175: In the section identifying additional data needs, we also suggest noting a need for

human in vivo data. Enzymes are inducible and vary with diet and co-exposure to xenobiotics Appendix III
capable of enzyme induction. These variables may influence individual susceptibility to DEHP

exposure.

Thank you for considering our comments. We look forward to seeing the CERHR/NTP

monograph on DEHP.

Sincerely,

Ted Schettler, MD, MPH

Science and Environmental Health Network

(978) 462-4092

Anna Gilmore Hall, RN

Executive Director, Health Care Without Harm

(703) 505-3239

Julie Silas, JD

Co-Chair, Safer Materials Workgroup, Health Care Without Harm

(510) 594-8270

Appendix III
February 2, 2006

Dear Dr. Shelby:

B. Braun Medical Inc., a global leader in safety-minded and environmentally-sound hospital 

products, commends the findings of the expert panel convened by the National Toxicology

Program (NTP), Center for the Evaluation of Risks to Human Reproduction (CERHR). We 

share the panel’s concerns regarding the potential impact of DEHP on reproductive 

development in critically ill infants, and in women pregnant with male fetuses who are 

undergoing medical treatments where high exposure to DEHP could occur. B. Braun 

applauds the NTP/CERHR for convening this second independent panel of experts to review

and update its previous report.

At B. Braun, our commitment to patient and environmental safety is evident in our products,

which provide clinicians the ability to administer their infusion therapy needs with devices that

are manufactured with DEHP- and PVC-free alternatives. B. Braun encourages all efforts to 

reduce and, wherever possible, eliminate actual or potential toxic substances such as DEHP 

in all medical products.

Today, B. Braun is the only full-line manufacturer of basic IV solutions in PVC-free and 

DEHP-free containers, and a leader in patient-safety technology and policy. The two major 

critical-care products B. Braun markets – EXCEL® and PAB® IV Containers – are 

manufactured with nontoxic, biologically inert copolymers of ethylene and propylene, and 

provide the most drug-compatible IV containers available.

In 2003, B. Braun recommended that all medical product companies minimize DEHP use.

B. Braun urged the U.S. Food and Drug Administration to draft and support a regulation to 

mandate labeling for all medical devices plasticized with DEHP, allowing clinicians to make 

informed decisions when using products containing DEHP in clinical practice.

Until there are revised national standards for DEHP leachate levels or requirements to label 

products that leach DEHP, healthcare professionals will need to evaluate the data and make 

their own determination of the risks involved. As these caregivers consider available options,

and the possible impact on patients’ long-term health, B. Braun will continue to be at the 

forefront of evaluating and producing products that are DEHP-free.

Sincerely,

Marcus Schabacker, MD, PhD

Corporate Vice President

Research and Development and Medical and Clinical Affairs

B. Braun Medical Inc.
February 3, 2006 

VIA EMAIL: shelby@niehs.nih.gov 

Dr. Michael D. Shelby 

CERHR Director 

CERHR 

79 T.W. Alexander Drive 

Building 4401, Room 103 

Research Triangle Park, NC 27709. 

Dear Dr. Shelby: 

The American Chemistry Council Phthalate Esters Panel (PE Panel) is submitting the 

attached comments on the November 2005 Expert Panel Update on the Reproductive and Developmental 

Toxicity of Di(2-ethylhexyl) Phthalate (Final Update) to assist the NTP-CERHR in its review of the 

reproductive and developmental toxicity of di(2-ethylhexyl) phthalate (DEHP) and in response to NTP’s 

request for comments on the Final Update. 70 Fed. Reg. 69567 (Nov. 16, 2005). The PE Panel includes 

the major domestic manufacturers of phthalate esters and some users. 

The PE Panel appreciates the Expert Panel’s work in preparing the Final Update and 

believes that the Final Update, in general, provides a reasonable summary of the new information that has 

become available on DEHP since the first Expert Panel review in 2000. The PE Panel believes that the 

Final Update suffers from several shortcomings due to some aspects of the process by which the Final 

Update was produced, and that some of the scientific conclusions in the Final Update are not supported by 

the scientific data for DEHP. In particular, the PE Panel has obtained opinions from two experts in 

marmoset toxicology, which indicate the Final Report understates the value of recent marmoset data for 

evaluating human male testicular development, while it overstates the value of the marmoset data for 

evaluating human female reproductive development. 

For the reasons discussed in the PE Panel’s comments on the August 2005 Draft Update, 

and the comments presented here, the PE Panel believes that the available information for DEHP supports 

a conclusion that the overall concern for risk to human reproduction from DEHP exposure is minimal. 

If you have any questions, or if you need any further information, please call Marian K. 

Stanley, Senior Director and Manager of the Phthalate Esters Panel, at (703) 741-5623, email her at 

marian_stanley@americanchemistry.com, or write her at the address below. 

Sincerely yours, 

Hasmukh C. Shah 

Acting Vice President, CHEMSTAR 

Attachments

 Responsible Care®

1300 Wilson Boulevard, Arlington, VA 22209 ♦ Tel 703-741-5000 ♦ Fax 703-741-6000 ♦ 

http://www.americanchemistry.com

Appendix III
Comments of The 

American Chemistry Council Phthalate Esters Panel 

On the Final NTP-CERHR Expert Panel Update On The 

Reproductive And Developmental Toxicity Of Di(2-Ethylhexyl) Phthalate 

February 3, 2005 

 

TABLE OF CONTENTS

Page

INTRODUCTION AND SUMMARY............................................................................................1 

I. THE FINAL UPDATE CONTAINS SCIENTIFIC CONCLUSIONS THAT 

WERE NOT FULLY DELIBERATED OR SUBJECT TO PUBLIC COMMENT ...........4 

A. Stakeholders Were Not Given Adequate Time to Analyze and Comment 

on Late-Arising Information....................................................................................4 

B. The Update Report Does Not Adequately Reflect the Complete Weight of 

Evidence for DEHP, Because the Expert Panel Reviewed Post-2000 Data 

in Isolation from Other DEHP Data.........................................................................6 

C. Several Significant Issues Were Raised for the First Time in the Final 

Minutes of the Public Meeting, and Thus Were Not Fully Deliberated, but 

Were Nonetheless Included in the Final Update......................................................6 

D. NTP-CERHR Should Be More Flexible In Allowing Audience 

Participation .............................................................................................................8 

II. THE MARMOSET IS A VALUABLE MODEL FOR MALE HUMAN 

REPRODUCTIVE TOXICITY ...........................................................................................9 

A. The Review Relied Upon by the Expert Panel, Li et al. (2005), Exhibits an 

Unwarranted Bias Against the Marmoset as a Model for Male 

Reproductive Toxicity .............................................................................................9 

B. The Marmoset Has Similarities to Human Reproductive Biology that 

Make it a Valuable Model......................................................................................10 

C. The Mitsubishi Study Provides Valuable Information Regarding the 

Sensitivity of Primates to DEHP............................................................................12 

D. Toxicokinetic Data Support the Lower Sensitivity of Primates to the 

Effects of DEHP ....................................................................................................13 

III. COMMENTS ON SECTION I: USE AND HUMAN EXPOSURE.................................14 

A. The Final Update Could Have Used Available Biomonitoring Data to 

Show That DEHP Exposures to Children Ages 12-18 Months Are Not 

Several Fold Higher than Adults............................................................................14 

B. The Final Update Erroneously Indicates that Cosmetics and Breast Milk 

Pumps Are Significant Sources of Human Exposure to DEHP.............................15 Appendix III
ii

IV. COMMENTS ON SECTION 3: DEVELOPMENTAL TOXICITY DATA ....................16 

A. The Data Reviewed in the Final Update Do Not Support an Oral 

Developmental NOAEL of 3-5 mg/kg/day............................................................16 

V. SECTION 4: REPRODUCTIVE TOXICITY DATA .......................................................18 

A. The Data From the Mitsubishi Study Showing Increased Uterine and 

Ovarian Weights in Female Marmosets Do Not Support the Conclusion 

that DEHP Exposure Resulted in Precocious Puberty in Marmosets....................18 

B. The Studies by Akingbemi et al. Reviewed in the Final Update Are 

Flawed and Do Not Support a NOEL of 1 mg/kg/day...........................................19 

CONCLUSION..............................................................................................................................22 

REFERENCES ..............................................................................................................................22 

ATTACHMENTS..........................................................................................................................25 

Appendix III
INTRODUCTION AND SUMMARY 

The American Chemistry Council Phthalate Esters Panel (PE Panel) submits these 

comments on the November 2005 Expert Panel Update on the Reproductive and Developmental 

Toxicity of Di(2-ethylhexyl) Phthalate (Final Update) to assist the NTP-CERHR in its review of 

the reproductive and developmental toxicity of di(2-ethylhexyl) phthalate (DEHP) and in 

response to NTP’s request for comments on the Final Update. 70 Fed. Reg. 69567 (Nov. 16, 

2005).1

 The PE Panel includes the major domestic manufacturers of phthalate esters and some 

users.2

The PE Panel believes that the Final Update, in general, provides a good summary 

of the new information that has become available on DEHP since the first Expert Panel review in 

1999-2000. However, the PE Panel also believes that the Final Update suffers from several 

shortcomings that NTP-CERHR should take into account while reviewing the reproductive and 

developmental toxicity of DEHP. The following comments and suggestions are intended to 

assist NTP-CERHR in its review and use of the Final Update and to enhance the robustness of 

NTP-CERHR’s conclusions. Attached to these comments are expert opinions by Professor 

Stefan Schlatt and Dr. Suzette Tardif, both scientists with extensive experience in the use of 

marmosets for toxicology. Those opinions provide perspectives that differ from those of the 

Final Update and which should be seriously considered by the NTP-CERHR. 

These comments make the following points: 

• The Expert Panel review process, while well-executed in many respects, suffered in 

several critical aspects, such that the Final Update contains some scientific conclusions 

which have not been fully deliberated or subjected to public comment. Key process 

concerns include: 

o The Expert Panel relied heavily on a study that was published on the last day of the 

public comment period, and there was no prior public notice that the study would 

even be discussed at the public meeting. The study was highly important to the 

deliberations, because it questioned the suitability of marmoset studies for assessing 

potential human health hazards, and was used by the Expert Panel as a primary 

rationale for largely disregarding a key DEHP study in marmosets that showed no 

male reproductive effects following very high exposure. Stakeholders were given 

inadequate opportunity to provide scientific comment on this important publication. 

As a consequence, the Expert Panel appeared to adopt the critical positions in this 

publication with no meaningful reflection on other information supporting the use of 

marmosets as an experimental model for testicular toxicity. The PE Panel is 

providing with these comments the opinions of two experts with extensive experience 

in marmoset research. These opinions rebut many of the hasty conclusions adopted 

during the Expert Panel deliberations. 

 

1

 The Final Update is available at 

http://cerhr.niehs.nih.gov/chemicals/dehp/DEHP__Report_final.pdf. 

2

 The Panel members are: BASF Corporation, Eastman Chemical Company, ExxonMobil 

Chemical Company, Ferro Corporation and Teknor Apex Inc. 

1

Appendix III
o The Update was conducted in a manner that prevented reaching conclusions based on 

an integrated view of the entire database for DEHP. For example, the Expert Panel 

discussed the marmoset data in the Mitsubishi (2003) study virtually independent of 

several other studies which also indicate primate insensitivity to testicular effects 

from DEHP. As a consequence, the Expert Panel’s conclusions regarding concern for 

male reproductive toxicity were unduly influenced by its unfavorable view of the 

Mitsubishi marmoset study, and insufficiently influenced by the total weight of the 

evidence from all primate studies. 

o Several significant scientific issues were raised by the Expert Panel for the first time 

in the final minutes of the public meeting, and thus were not fully deliberated, but 

were nonetheless included in the Final Update. This, combined with the inability of 

the public to comment on these issues at the meeting, led to several hastily- and 

scantily-considered conclusions being adopted as the consensus opinion of the Expert 

Panel. 

• The PE Panel believes that these procedural flaws compromised the ability of the Expert 

Panel to render an objective and thoughtful opinion as to the reproductive toxicity of 

DEHP. The PE Panel makes recommendations to enhance the Expert Panel review 

process in the future, including: 

o Being more cautious about relying on information that becomes available at the last 

minute, and where the information is significant to the deliberations, adjusting the 

process to allow adequate scientific input from stakeholders, including allowing 

additional comments and expert opinions after the close of the public meeting for 

consideration by the Expert Panel, and even reconvening the Expert Panel where 

necessary; and 

o Being more flexible about accepting stakeholder scientific input during the Expert 

Panel deliberations, as critical issues arise. 

• The marmoset, despite some unique aspects of its biology, is a valuable model for male 

human reproductive toxicity – particularly for the evaluation of developmental and 

toxicological aspects of the testis and spermatogenesis. 

o The review of the Mitsubishi study relied upon by the Expert Panel exhibits an 

unwarranted bias against the marmoset as a model, focusing on negative information 

and ignoring data that indicate the marmoset is a good model. 

o The marmoset has several similarities to human reproductive biology, in particular 

Sertoli cell development – a primary concern of the Expert Panel – that makes it an 

excellent model for male reproductive toxicity. 

o There is no evidence that the generalized steroid hormone resistance in the marmoset 

applies to sex steroids. 

2

Appendix III
o As discussed in previous comments to NTP-CERHR, the marmosets’ requirement of 

dietary Vitamin C and lack of luteinizing hormone are not significant disadvantages 

of the marmoset model. 

o Additional criticisms of the marmoset model have been addressed in other comments, 

which are provided as an attachment to these comments. 

• The Mitsubishi marmoset study, while presenting some concerns about the health of the 

animals, nonetheless provides valuable information regarding the sensitivity of primates 

to DEHP. The study provides strong evidence that DEHP had no major effect on 

testicular development even after long-term DEHP exposure at very high concentrations 

that would have profound adverse effects on rodent testes. 

• In its comments on the August 2005 Draft Update, the PE Panel summarized an extensive 

body of literature indicating that differences between rodents and primates in the 

absorption, distribution, metabolism and excretion (ADME) of DEHP can explain in 

large part the lower sensitivity of primates to the reproductive effects of DEHP. Because 

most, but not all, of the primate ADME data came from the marmoset, the Expert Panel 

apparently chose to disregard these data due to perceived significant limitations with the 

Mitsubishi study. At the very least, the ADME data should provide the Expert Panel with 

sufficient information to acknowledge that the use of rat data for human risk assessment 

is likely to be very conservative (i.e., health protective). 

• While the PE Panel applauds the Expert Panel for considering the PE Panel’s 

recommendation in its comments on the Draft Update to calculate exposures based on 

available biomonitoring data, the Final Update also could have used available 

biomonitoring data to show that DEHP exposures to children ages 12-18 months are not

several fold higher than adults. 

• The Final Update erroneously indicated that cosmetics and breast milk pumps are 

significant sources of human exposure to DEHP. To the contrary, the data on DEHP in 

cosmetics and pumped breast milk indicate that DEHP exposures from these sources are 

minute or non-existent. 

• The data reviewed in the final update do not support an oral developmental NOAEL as 

low as 3-5 mg/kg/day. The Expert Panel failed to include a significant number of 

individuals (F1 and F2 breeding males and F2 non-breeding males) in the overall number 

of individuals examined in the NTP multi-generation study (NTP 2004), which caused 

the Expert Panel to significantly overstate the incidence rate of “small” organs and, in 

turn, significantly overstate the magnitude of the effect. In addition, no laboratory 

historical control data were made available for review in these studies, which makes it 

difficult to adequately evaluate the statistical significance of the very low reported 

incidence rate of treatment effects. 

• The data from the Mitsubishi study showing increased uterine and ovarian weights in 

female marmosets do not support the conclusion that DEHP exposure resulted in 

precocious puberty in marmosets. The increased uterine and ovarian weights correlate to 

3

Appendix III
body weights which were higher in the higher dose females, versus lower body weights 

in controls and low dose females (probably the result of an unhealthy marmoset colony). 

Those higher body weights likely reflected healthier animals that were able to reach 

sexual maturity, with its accompanying increase in ovarian weight, rather than an effect 

of DEHP. 

• The studies by Akingbemi et al. (2001; 2004) reviewed in the Final Update are flawed 

and do not support a NOEL of 1 mg/kg/day. The studies: 1) rely on single-point 

measures of serum hormones to support the report of abnormal changes in testosterone 

production in response to DEHP exposure; and 2) fail to account for differences in 

Leydig cell density between treatments and controls, which leads to the authors analyzing 

only a subset of the Leydig cells present in the treatment group. 

Based on the complete database for DEHP, for the reasons discussed in the PE 

Panel’s comments on the August 2005 Draft Update and the comments presented here, the PE 

Panel believes that the available information for DEHP supports a conclusion that the overall 

concern for risk to human reproduction from DEHP exposure is minimal. 

I. THE FINAL UPDATE CONTAINS SCIENTIFIC CONCLUSIONS THAT WERE 

NOT FULLY DELIBERATED OR SUBJECT TO PUBLIC COMMENT 

The NTP-CERHR intended that the Expert Panel’s review “provide objective and 

scientifically thorough assessments of the scientific evidence that adverse reproductive/ 

developmental health effects may be associated with [DEHP] exposures.”3

 Toward that end, 

CERHR solicited scientific information on DEHP and nominations for the Expert Panel (see 70 

Fed. Reg. 6024 (Feb. 4, 2005)), provided a draft of the Report for public comment, and held a 

public meeting for the Expert Panel deliberations. Given the considerable amount of information 

that had become available after the first Expert Panel Report was published in October 2000,4

the PE Panel agrees that an “objective and scientifically thorough” update is needed. However, 

we believe that in several respects the Final Update falls short of the mark, and that the 

shortcomings reflect at least in part inadequacies in the assessment process. 

A. Stakeholders Were Not Given Adequate Time to Analyze and Comment on LateArising Information 

Public comments on the Draft DEHP Update were due September 28, 2005.5

 At 

the public meeting on October 11-12, 2005, the Expert Panel relied heavily on a paper by Li et 

al. (2005) that only became available as an online preprint on September 28 – the day public 

comments were due. Even assuming the PE Panel became aware of that paper the day it was 

released, it clearly could not have commented on the paper within the comment deadline. Input 

 

3

 Final Update Preface, page ii. 

4

 NTP-CERHR Expert Panel Report on Di(2-ethylhexyl) Phthalate, October 2000. Available at: 

http://cerhr.niehs.nih.gov/chemicals/dehp/DEHP-final.pdf. 

5

 See 70 Fed. Reg. 43870 (Jul. 29, 2005). 

4

Appendix III
is now being provided with these comments, but it is too late to be reflected in the Expert Panel’s 

deliberations. 

The Li et al. paper critiqued the suitability of the marmoset as a human 

reproductive model. The Li paper was critical to the Expert Panel’s deliberations, because it 

raised doubts about reliance on a key study for DEHP in marmosets that showed no effects on 

male reproductive development, even at very high doses (Mitsubishi, 2003). The PE Panel was 

not made aware that the Expert Panel would be discussing the Li et al. paper and relying on it to 

critique the DEHP study until the second day of the public meeting. When the lack of 

opportunity for public comment was pointed out, an opportunity for brief oral comment to the 

Expert Panel was provided, but this clearly was an unsatisfactory situation. Stakeholders had a 

very limited amount of time to review and comment on the study, and the Expert Panel had 

extremely limited time in which to consider those undoubtedly incomplete comments. 

As detailed in Section II.A below, a critique of Li et al. by an expert in marmoset 

reproductive biology shows that the Li et al. paper overstates the case against using marmosets as 

a model, and fails to acknowledge factors that make marmosets good models for human testes 

development. Without the benefit of that input from a scientist who has done extensive research 

in marmosets and is expert in marmoset reproductive biology, the Expert Panel adopted the 

positions in Li et al. with little or no discussion. The net result, as explained more fully later in 

these comments, is that the Expert Panel received a one-sided and overly-critical view of the 

scientific value of DEHP marmoset studies, and inappropriately discounted the results of those 

studies. 

The PE Panel realizes that its more robust comments on the Li et al. review will 

now be considered by the NTP-CERHR as its prepares its final brief on DEHP. However, the 

Expert Panel report, as a consensus document of an independent group of experts, carries great 

weight. Further, until the NTP-CERHR brief is public, the Expert Panel report is the “last word” 

on DEHP reproductive toxicity. Therefore, it is important that the Expert Panel report reflect 

truly fair and robust consideration of all key studies cited in its report. That did not happen in the 

case of the paper by Li et al., because the process did not allow it to occur. 

There were other instances of the Expert Panel considering last minute 

information for which the public had no meaningful opportunity for comment. These include a 

summary of raw data from a key multi-generation study (see Section IV.A) and speculation on 

the potential contribution to exposure from use of breast milk pumps (see Section III.B). 

In the future, the PE Panel strongly recommends that NTP-CERHR exercise 

caution in using or relying on last minute information, and that it adjust its process so that 

stakeholders have adequate opportunity to provide comment and scientific input concerning the 

late-arising information, including statements by independent experts where appropriate. For 

highly significant information, NTP-CERHR should allow a post-public meeting comment 

period with an opportunity for the Expert Panel to review and consider those comments. In some 

cases, it may be necessary to reconvene the Expert Panel to entertain other expert scientific input 

relevant to the new information. Otherwise, there is a risk that the Expert Panel report will 

appear up-to-date but in fact be the product of hasty and premature conclusions, as the PE Panel 

believes occurred in this case. 

5

Appendix III
B. The Update Report Does Not Adequately Reflect the Complete Weight of 

Evidence for DEHP, Because the Expert Panel Reviewed Post-2000 Data in 

Isolation from Other DEHP Data 

The PE Panel considers that a major failing of the update process was that the 

post-2000 data was reviewed almost in complete isolation from the pre-existing database for 

DEHP. Thus, the Update Report conclusions essentially reflect a view of DEHP based on the 

post-2000 set of data, rather than on the complete weight of evidence for DEHP. 

A prime example of the imbalance created by this approach is the Expert Panel’s 

consideration of and conclusions relating to primate data. In drawing its conclusions about the 

significance of primate data for assessing potential risks of DEHP, the Expert Panel focused 

only on the recent Mitsubishi (2003) marmoset study and its limitations. However, as discussed 

in Section II.C, below, several pre-2001 studies in both old world and new world primates show 

that primates are generally much less sensitive to testicular effects from DEHP exposure than are 

rodents. Consideration of these data in addition to the Mitsubishi study might well have 

modified the Expert Panel’s conclusions. To complete a comprehensive review, the Expert 

Panel should have considered the post-2000 data as an integrated package with previous data, 

both while drafting the Final Update and during the public meeting. However, there was no 

mechanism in the review process for doing so. For its final brief of DEHP, NTP-CERHR should 

itself consider the full body of data for DEHP. The PE Panel also urges that the process for 

future updates to provide a mechanism for integrating previous and update information. 

C. Several Significant Issues Were Raised for the First Time in the Final Minutes of 

the Public Meeting, and Thus Were Not Fully Deliberated, but Were Nonetheless 

Included in the Final Update 

Several significant issues were raised in the final minutes of the public meeting 

and, despite not being fully deliberated, were included in the Final Update and represented to be 

the Expert Panel’s consensus opinion. One such issue, as pointed out in an October 12, 2005 

letter from the PE Panel to NTP-CERHR,6

 concerns the first paragraph of Section 5.3 (Overall 

Conclusions) of the Final Update. This paragraph states that “[t]he combined effects of multiple 

phthalate exposures have implications for exposure and risk assessment.” 7

 There were 

absolutely no deliberations concerning additive effects of DEHP and any other chemical by the 

Expert Panel, and no opportunity for public comment on this paragraph, which was written at the 

very end of the meeting. As stated in the PE Panel’s letter, the only study available on this issue, 

Foster et al. (2002), found no additive effect from a combination of DBP and DEHP. Foster et 

al. concluded “[t]his study did not indicate an additivity of response or an interaction of the two 

phthalates in combination. Aggregation of risk of these doses would not be appropriate.”8

 Thus, 

the paragraph suggesting a concern for additivity of effects is presented as the consensus of the 

Expert Panel after receiving no deliberation or opportunity for comment, and in direct opposition 

 

6

 Letter from Marian K. Stanley, Manager PE Panel, to Dr. Michael Shelby, CERHR Director, Re: 

DEHP Update Expert Panel Report, dated October 12, 2005. 

7

 Final Update at page 171. 

8

 Id.

6

Appendix III
to the only available evidence about additivity. For these reasons, this paragraph should not have 

been included in the Final Update. 

In addition, the Final Update concludes that “[t]here is sufficient evidence in 

female marmosets to conclude that DEHP causes reproductive toxicity (increased ovary weight 

and uterine weight) when exposure is by oral gavage at 500 mg/kg bw/day for ~15 months in the 

peripubertal period . . . .”9

 The Final Report interprets this observation by stating that “[t]he 

Expert Panel found these data consistent with precocious puberty . . . .”10 However, this 

conclusion was not the result of any true deliberation by the Expert Panel. In the closing minutes 

of the public meeting, a comment was made that the observation of increase in ovarian weight 

should be accompanied by a statement about the implications of that observation. A single panel 

member, almost off-handedly, asserted that the data were indicative of precocious puberty. This 

interpretation was accepted by the Expert Panel with no discussion and no opportunity for public 

comment. For this assertion, which has very significant implications for concern about potential 

DEHP effects,11 there was absolutely no deliberation by the Expert Panel or opportunity for 

public comment about the interpretation of the data. In fact, as discussed in Section V.A, below, 

the increased ovarian and uterine weights may simply correlate with increased female body 

weights, and therefore represent no adverse effect at all. Yet as a result of there being no 

deliberation or opportunity for public comment, the conclusion that DEHP causes precocious 

puberty in marmosets is now represented as the consensus opinion of the Expert Panel. 

As a final example, Table 23 of the Final Update, which was compiled at the last 

minute with no real deliberation or quality checking, arguably contains errors in the total 

numbers of animals observed at each dose (see Section IV.A, below). These errors likely 

affected the Panel’s interpretation of the data, and consequently its conclusions regarding the 

toxicity of DEHP. 

Due to the lack of any deliberation or opportunity for public comment, the PE 

Panel believes it is highly inappropriate that the above statements are included in the Final 

Update as the Expert Panel’s consensus opinion. In denying the PE Panel’s request that the 

paragraph suggesting additivity of effects be removed from the Final Update, NTP-CERHR 

disclaimed that “the conclusions reached by CERHR expert panels are their own” and “should 

not be construed to represent the views of the National Toxicology Program.”12 Despite this 

disclaimer, it is likely that the Final Update will nonetheless be pointed to by interested parties as 

the consensus view of an ostensibly objective “eleven-member panel of government and non-

 

9

 Final Update at page 163. 

10 Id.

11 Given current debate about endocrine disruption, “precocious puberty” is a highly charged term. 

12 Letter from Dr. Michael D. Shelby, Director CERHR, to Marian K. Stanley, Manager PE Panel, 

dated October 28, 2005. NTP-CERHR also stated that “because the expert panel report is not a 

government document, the NTP is unable to [remove the paragraph].” Id. It seems somewhat 

disingenuous, however, to suggest that NTP-CERHR is powerless to correct a documented flaw 

in the report. If nothing else, NTP-CERHR could have shared the PE Panel’s letter with the 

Expert Panel to receive direction on whether to remove the offending paragraph. 

7

Appendix III
government scientists.”13 Consequently, it is not sufficient merely to state that the conclusions 

of the Expert Panel are their own, and not those of NTP-CERHR. NTP-CERHR should take a 

hard look at its Expert Panel process and implement safeguards to ensure that the “consensus 

opinions” put forth in panel reports are indeed the fully-deliberated consensus view of the panel, 

and not last-minute, hastily accepted but substantively important add-ons. 

The need for such a deliberative period is illustrated by the note included in the 

Preface to the Final Update which states that “[w]hile the expert panel reached consensus on all 

conclusions during the panel meeting, following the meeting . . . three panel members 

reconsidered their position on one conclusion. Upon reconsideration, they did not concur with 

[the conclusion arrived at the public meeting].”14 The mere existence of this note shows that a 

hastily-arrived-at “consensus” is really no consensus at all. The Final Update should reflect a 

true consensus, in which case there would be no need of a note such as this. Allowing an 

additional period of reflection and comment would help ensure that panel reports reflect a true 

consensus of the expert panel. 

D. NTP-CERHR Should Be More Flexible In Allowing Audience Participation 

The negative impact of the process flaws identified above might have been 

partially mitigated had the public meeting been run in a similar manner as the first phthalate 

Expert Panel meetings in 1999 and 2000. At those meetings, members of the public were 

allowed to raise their hands during the meeting to comment and provide clarification on various 

scientific issues or specific data. These public comments were presented in a respectful and 

orderly fashion, by highly qualified scientists, and contributed to, rather than detracted from, the 

Expert Panel’s deliberations. Indeed, the value of that scientific input was acknowledged by 

many participants in the public meetings. In the 2005 meeting, however, public comments 

appeared to be more discouraged than encouraged. They were limited to 15 minute presentations 

and there was no opportunity to comment during the Expert Panel’s discussion.15 Without 

adequate opportunity for the public to comment during the deliberations, there was no effective 

way to address or even identify many of the issues described above. Indeed, there was 

effectively little or no way to address issues that had not first surfaced in the draft Expert Panel 

report. 

 The issues that are addressed by an NTP-CERHR Expert Panel frequently are 

cutting-edge scientific issues that benefit from robust discussion. That discussion, of course, is 

centered around the Expert Panel, which hopefully will have the requisite expertise and be free 

of conflict or bias. However, industry scientists or other stakeholder scientists often will have 

important points to contribute, and may in some cases be able to offer the perspective of 

expertise that is lacking on the Expert Panel. The PE Panel believes NTP-CERHR should not 

 

13 See Final Update at page ii. 

14 Id. at page iii. 

15 Comments could be given to the CERHR director to be passed on to the Expert Panel chair, but 

this was very ineffective. It is not clear that all comments were given to the chair, or that all 

comments were presented to the full Expert Panel, and even when comments were passed on, 

they often “lost something in the translation.” 

8

Appendix III
manage the deliberations in a way that shuts out that input, but instead should encourage 

participation in a reasonable and respectful manner, as occurred with the initial phthalate Expert 

Panel assessments. 

If NTP-CERHR’s goal truly is an “objective and scientifically thorough 

assessment,” then NTP-CERHR should be flexible in the way it manages the public meetings, 

and give scientists in the audience some credit for being able to exercise discretion in offering 

scientific input during the deliberations. An overly rigid approach can only serve to deny Expert 

Panel members access to relevant scientific input. A more flexible approach can only enhance 

the quality and objectivity of the final work product. 

II. THE MARMOSET IS A VALUABLE MODEL FOR MALE HUMAN 

REPRODUCTIVE TOXICITY 

The Final Update lists several aspects of marmoset biology that differ from 

human biology and, based largely on a review of marmoset reproductive biology by Li et al. 

(2005), ultimately concludes that these differences “significantly limit our reliance on this 

species as a surrogate for humans.”16 While the marmoset, like all animal models, is not a 

perfect model for all aspects of human toxicity, the PE Panel believes strongly that marmoset is a 

good model for male reproductive development. In particular, the marmoset is a particularly 

good model for the reproductive endpoints investigated in the Mitsubishi (2003) study. 

To assess the usefulness of the marmoset as a model for human reproductive 

toxicity, the PE Panel engaged Professor Stefan Schlatt to render his opinion on the value of the 

marmoset model. Dr. Schlatt is an Assistant Professor in the Department of Cell Biology and 

Physiology at the University of Pittsburgh School of Medicine, and is an expert in mammalian 

reproductive biology. Dr. Schlatt has more than 13 years of experience researching reproductive 

function and endocrine activities in animal models, including rats, hamsters, and various 

nonhuman primates, including marmosets, and has authored more than 50 refereed articles on 

various aspects of this field. In Dr. Schlatt’s opinion (provided as Attachment 1 to these 

comments), “there is no doubt that the marmoset is a useful model to explore developmental and 

toxicological aspects of the testis,” and “many similarities in regard to testicular organization, 

general developmental pattern and hormonal regulation render the marmoset a much more useful 

model when compared to rodents.”17

A. The Review Relied Upon by the Expert Panel, Li et al. (2005), Exhibits an 

Unwarranted Bias Against the Marmoset as a Model for Male Reproductive 

Toxicity 

In discussing the validity of the marmoset model, the Final Update relies heavily 

on the review by Li et al. (2005), which points out several differences between marmoset and 

human reproductive biology. This review, however, ignores information or data that indicate the 

marmoset is a good model, and focuses on negative information. As stated by Dr. Schlatt, the Li 

 

16 Id. at page 141. 

17 Opinion of Dr. Schlatt, Attachment 1, at page 4. 

9

Appendix III
et al. “interpretation of the suitability and validity of the [marmoset] model carries an unjustified 

negative bias.”18 As discussed above, the merits of the Li et al. criticisms were not sufficiently 

evaluated by the Expert Panel, due to the timing of its publishing versus the public comment 

period. 

B. The Marmoset Has Similarities to Human Reproductive Biology that Make it a 

Valuable Model 

As discussed in Dr. Schlatt’s opinion (Attachment 1), several features of 

marmoset reproductive biology make it a valuable model for human reproductive toxicity. 

1. The Similarity of Human and Marmoset Sertoli Cell Development Make 

the Marmoset a Good Model for Studying Effects on Germ Cell 

Development – a Primary Concern of the Expert Panel 

One of the key concerns of the Expert Panel was the effect of DEHP exposure on 

Sertoli cells (vacuolation and reduced proliferation) in developing animals. This concern is 

based on data from rats, in which Sertoli cell effects are seen at relatively low oral doses. 

However, the data in Mitsubishi (2003) indicate that extremely high oral doses of DEHP, up to 

2500 mg/kg/day, had no effects on marmoset testes even at the cellular level. Because of this 

disparity in effects, the value of the marmoset as a model of human reproduction becomes an 

essential question. 

As stated by Dr. Schlatt, there are “striking similarities of organization of the 

marmoset and human spermatogenic epithelium (both species have a multistage organization of 

spermatogenic stages per tubular crossection),” and “quite similar mechanisms of germ cell 

development and clonal expansion of germ cells in marmosets and man.” 19 Also, while Li et al. 

point out an unusual uniformity of Sertoli cell morphology throughout the marmoset 

spermatogenic cycle, Dr. Schlatt explains that it is not yet known whether humans and 

marmosets differ with respect to this observation, and that “it appears likely that the human and 

marmoset Sertoli cell show a high degree of similarity in this respect”.20 Dr. Schlatt concludes: 

With choice of the correct timepoints [the marmoset] should be 

highly useful and informative for exploring the effects on Sertoli 

cell differentiation, testicular growth and effects of FSH on the 

testis. The striking similarities to man make it an excellent model 

for studying effects on germ cell development, the organization of 

the seminiferous epithelium and changes to the kinetics of 

spermatogenesis.21

 

18 Id.

19 Opinion of Dr. Schlatt, Attachment 1, at page 2. 

20 Id.

21 Id. at page 4. 

10

Appendix III
Thus, similarities in biology indicate that the marmoset is a good model for 

determining the potential effects of DEHP on human Sertoli cells, and available data indicate 

that the marmoset is far less sensitive to oral DEHP exposure than rodents. Despite concerns 

with the health of the animals in the Mitsubishi study (see discussion in Section II.C below), the 

extreme insensitivity of marmosets to oral DEHP exposure, at the very least, provides 

information that puts the rodent model into perspective as being much more sensitive to 

testicular effects of DEHP than primates. Therefore, the marmoset data should not have been 

completely disregarded for purposes of evaluating male reproductive toxicity. 

2. There Is No Evidence that the Generalized Steroid Hormone Resistance 

in the Marmoset Mentioned by Li et al. Applies to Sex Steroids 

Based primarily on the review of Li et al. (2005), the Expert Panel concludes that 

marmosets have a general end-organ steroid resistance relative to humans, as indicated by their 

high serum levels of steroids, which limits their value as a human reproductive model. However, 

as explained by Dr. Schlatt (see Attachment 1), while a general insensitivity has been proposed 

for the mineralocorticoid and glucocorticoid hormones, it is unknown whether such steroid 

resistance exists for the gonadal (sex) steroids. Marmosets, but not mice, have sex hormone 

binding globulin that separates the serum testosterone into active free (biologically active) and 

bound (biologically inactive) fractions. Due to the binding affinities of these proteins, 

marmosets appear to have high levels of unbound testosterone in the circulation which is similar 

to their unusually high levels of glucocorticoids. However, these higher testosterone levels may 

have little or no biological significance since the kinetics of testosterone-receptor binding and 

post-receptor binding events is unknown. Therefore, high levels of serum testosterone in 

marmosets are not necessarily indicative of sex steroid resistance. As stated by Dr. Schlatt, “in 

the absence of solid data on sex steroids it appears poorly justified and premature to transfer the 

conclusion of high sex steroid resistance from the glucocorticoid and mineralocorticoid system to 

the sex steroid system.”22 

3. The Marmoset’s Requirement of Dietary Vitamin C and Lack of 

Luteinizing Hormone Are Not Significant Disadvantages of the 

Marmoset Model 

The Final Update, citing the review of Li et al. (2005), mentions, as additional 

disadvantages of the marmoset model, the potentially protective action of Vitamin C in the 

marmosets’ diet and the marmosets’ lack of Luteinizing Hormone (LH). Each of these concerns 

was addressed in the PE Panel’s April 2005 submission to NTP-CERHR23 and the Vitamin C 

issue again in the PE Panel’s September 2005 Comments on the Draft Update,24 and has been 

 

22 Id. at page 3. 

23 Recent Information on Exposure to and Toxicology of Di(2-ethylhexyl) Phthalate (DEHP), 

American Chemistry Council Phthalate Esters Panel, submitted to NTP-CERHR April 21, 2005. 

(April 2005 Comments.) 

24 See Section IV.B. of: Comments of The American Chemistry Council Phthalate Esters Panel On 

the Draft NTP-CERHR Expert Panel Update On The Reproductive And Developmental Toxicity 

11

Appendix III
shown to be of no substantial importance in assessing the validity of the marmoset model. As 

discussed in the PE Panel’s previous submissions, the marmosets’ dietary Vitamin C requirement 

should not be of concern because: 1) the levels of Vitamin C used in the Mitsubishi study are not 

high relative to the marmoset’s requirements, and 2) based on the available science, it is not clear 

that Vitamin C affords any protection to primates from DEHP exposure. Moreover, if the level 

of Vitamin C in the marmosets’ diet in the Mitsubishi study in fact provided the degree of 

protection necessary to be responsible for the observed lack of effects, then the level of Vitamin 

C in the average human diet would be protective of any likely exposure to DEHP. In other 

words, the Vitamin C levels in the marmoset diet in the Mitsubishi study were similar to normal 

levels in the human diet and, consequently, whether Vitamin C had a protective effect in this 

study is not directly relevant to a risk assessment. 

As for the marmoset’s lack of LH, the data cited in the PE Panel’s earlier 

submission show that this difference in the hormone that initiates testosterone synthesis between 

the common marmoset and humans does not provide a sufficient basis for rejecting the marmoset 

as a model for human testicular development and function.25 This opinion is shared by Dr. 

Schlatt who states: “Despite . . . the exchange of LH by CG the marmoset shows many 

similarities to man. The function and regulation of FSH and CG and their feedback mechanisms 

resemble other primates.”26 Even with this hormonal difference, Dr. Schlatt concludes that the 

marmoset is a valuable model, particularly for the investigation of effects of DEHP exposure on 

Sertoli cell proliferation, which is regulated primarily by Follicle Stimulating Hormone in 

marmosets and other primates, but not rodents. 

4. Other Criticisms in the Li at al. Review Are Insufficient to Invalidate Use 

of the Marmoset Model to Evaluate Potential Effects of DEHP on 

Human Reproductive Development 

The Li et al. review includes some additional criticisms of the marmoset model, 

not explicitly discussed in the Final Update. Both these and some of the foregoing criticisms 

have been addressed in the context of comments by the PE Panel to the California Office of 

Environmental Health Hazard Assessment (OEHHA). A copy of those comments is provided as 

Attachment 2. As NTP-CERHR prepares the final brief for DEHP, to the extent it considers the 

Li et al. review paper, it also should consider Section I of the comments to OEHHA. 

C. The Mitsubishi Study Provides Valuable Information Regarding the Sensitivity of 

Primates to DEHP 

The Final Update notes concern about the body weights of some of the animals in 

the Mitsubishi marmoset study, as does Dr. Tardif in her independent review (See Attachment 

3). However, as pointed out in the opinion of Dr. Schlatt, notwithstanding these concerns, the 

Mitsubishi study nonetheless provides “strong evidence that DEHP had no major effect on 

 

Of Di(2-Ethylhexyl) Phthalate, Submitted to NTP-CERHR September 28, 2005. (Comments on 

Draft Update.) 

25 See Section II.B.2.d.2. of April 2005 submission. 

26 Opinion of Dr. Schlatt, Attachment 1, at page 3. 

12

Appendix III
testicular development even after very long and intense DEHP exposure.”27 Dr. Schlatt 

concludes that the Mitsubishi data “should be carefully and critically considered for evaluating 

the risk of gonadotoxic effects in humans after exposure to DEHP.” 

Indeed, the Mitsubishi data should not be completely disregarded with respect to 

implications for the degree of concern for male reproductive effects in humans. The 

shortcomings of the study might limit its usefulness on a quantitative level, but it still provides 

important qualitative information about the relative toxicity of DEHP to primates versus rodents, 

specifically that very high concentrations of DEHP that would have profound adverse effects on 

rodent testes had no adverse effects on the testes of marmosets. Moreover, despite it being 

considered essentially in isolation in the Final Update, the Mitsubishi study results are supported 

by several other primate studies. In other studies of both old world and new world primates, no 

testicular effects have been observed at doses up to 2500 mg/kg/day (Pugh et al., 2000; Kurata et 

al., 1998; Rhodes et al., 1986; Short et al., 1987). Combined with the Mitsubishi study, the clear 

conclusion is that primates are much less sensitive than rodents to the effects of DEHP. Even if 

primates are not used to establish the NOAEL for male reproductive effects, the PE Panel 

strongly believes that these data should be used to modify the degree of concern for effects in 

human testicular development from DEHP. 

Due to the limitations in the process discussed above, the Expert Panel essentially 

ignored these older studies and instead focused only on the Mitsubishi data and its limitations. 

The Panel should consider the Mitsubishi data in light of these other primate studies, which 

together provide substantial evidence that primates are less sensitive to DEHP than rodents. The 

Expert Panel’s lack of consideration of Mitsubishi in the context of this additional primate data 

might have been avoided had the Public Meeting allowed for public comments beyond the 15 

minute presentations allowed. 

In summary, substantial evidence, not all of which appears to have been 

adequately considered by the Expert Panel, indicates that marmosets are a valuable model for the 

investigation of human reproductive toxicity. Like any other non-human model, the marmoset is 

useful for some comparisons but not others. In this case, the parameters measured in the 

Mitsubishi study, particularly the lack of Sertoli cell effects, were those for which a marmoset 

model would be appropriate, and in fact superior to the rodent model. The use of these data 

should not be precluded by the fact that the marmoset may not be a good model in other respects 

not germane to the issues at hand. 

D. Toxicokinetic Data Support the Lower Sensitivity of Primates to the Effects of 

DEHP 

In Section II of its comments on the August 2005 Draft Update, the PE Panel 

summarized an extensive body of literature indicating that differences between rodents and 

primates in the absorption, distribution, metabolism and excretion (ADME) of DEHP can explain 

in large part the lower sensitivity of primates to the reproductive effects of DEHP. The ADME 

data provide a consistent explanation both among mammals (i.e., rodents vs. primates) and 

within primates (i.e., marmoset vs. cynomolgus monkey) as to why primates absorb less of the 

 

27 Id. at page 6 (emphasis in original). 

13

Appendix III
toxicologically relevant metabolite (MEHP) and exhibit lower MEHP / DEHP levels in the blood 

and target tissues than rodents. The Expert Panel does acknowledge some of the ADME data 

that have appeared since the first Expert Panel review in 2000 but in its conclusions seems 

unwilling to make the connection between lower systemic doses of MEHP in primates and the 

lower sensitivity of primates to DEHP toxicity. Because most, but not all, of the primate ADME 

data came from the marmoset, the Expert Panel apparently chose to disregard these data due to 

perceived significant limitations with the Mitsubishi study. At the very least, however, the 

ADME data should provide the Expert Panel with sufficient information to acknowledge that the 

use of rat data for human risk assessment is likely to be very conservative (i.e., health 

protective). 

III. COMMENTS ON SECTION I: USE AND HUMAN EXPOSURE 

A. The Final Update Could Have Used Available Biomonitoring Data to Show That 

DEHP Exposures to Children Ages 12-18 Months Are Not Several Fold Higher 

than Adults 

The PE Panel applauds the Expert Panel for considering the PE Panel’s 

recommendation in its comments on the Draft Update to calculate exposures based on available 

biomonitoring data. It was the PE Panel’s recommendation that “[t]he section. . .on human 

exposures to DEHP could be significantly improved by including conversions of urinary 

metabolite levels to estimates of environmental exposure,” and that “[t]he CDC biomonitoring 

data are the most comprehensive and accurate estimates available of exposures of the U.S. 

population, including children, to DEHP.”28 Using this approach, the PE Panel developed 

exposure estimates from the biomonitoring data that were consistent with probabilistic estimates 

based on sources of exposure. Although the Expert Panel articulated the uncertainties associated 

with each method, the PE Panel agrees that combining, or at least comparing, the two methods, 

as done in the Final Update, provides the best overall approach. 

However, the Expert Panel did not extend the process of exposure estimation 

from biomonitoring data to children ages 12-18 months (Brock et al., 2002), a subpopulation that 

was considered at potentially greater risk. Instead, the Expert Panel indicated that children 1-6 

years old could have exposures several-fold higher than the population estimates based on the 

study by Koch et al. (2004) of German children ages 2.5-6.5, which showed higher levels of 

excreted phthalate per gram creatinine than adults in the same household.29 However, the 

Expert Panel could instead have used the same approach of calculating exposures on the Koch et 

al. study, and combined it with the study by Brock et al., to show that exposures of this age 

bracket are in fact not several fold higher than adults (see Table 1 below). The PE Panel urges 

NTP-CERHR to take this approach in assessing DEHP exposures for its final brief. 

 

28 Comments on Draft Update at page 7. 

29 As stated in the PE Panel’s September 2005 comments on the Draft Update, there are significant 

differences in creatinine excretion between adults and children – children generally excreting half 

the level of creatinine excreted by adults). 

14

Appendix III
Table 1: DEHP Exposures to Children Calculated from Biomonitoring Data in Koch et al. 

(2004) and Brock et al. (2002). 

Source 

Mean/median DEHP 

metabolite/g creatinine 

Exposure estimate 

(μg/kg/d) 

Koch et al. 55.8 (5-OH-MEHP)a 5.52 

Brock et al. 26.14 (MEHP)b 2.57 

a = calculated using the method of David (2000) and the excretion ratio of Koch et al. (2004). 

b = calculated using the method of David (2000) and the excretion ratio of Anderson et al. (2001). 

B. The Final Update Erroneously Indicates that Cosmetics and Breast Milk Pumps 

Are Significant Sources of Human Exposure to DEHP 

Section 1.0 of the Final Update states: “Phthalates are used in a variety of 

products including . . . perfumes, hairsprays, and cosmetics . . . ”, implying that DEHP is so 

used.30 The Final Update conclusions also imply that cosmetics are a significant source of 

human exposure to DEHP.31 However, information in the Final Update indicates that DEHP use 

in cosmetics and personal products is extremely limited or non-existent. As discussed in Section 

1.1.1, a survey of 42 perfumes, 8 deodorants, 21 nail polishes, and 31 hair care products 

marketed in Korea found DEHP in only 2 of the perfumes and 2 of the nail polishes, and none of 

the deodorants or hair products (Koo and Lee, 2004). The maximum level of DEHP detected 

was 25 mg/L (25 ppm). Similarly, a 2002 Environmental Working Group report found only 3 

products out of 72 tested that contained DEHP, again with a maximum concentration of 25 

ppm.32 That is a concentration of only 0.0025%. It is unlikely that DEHP would have 

functionality and therefore be intentionally added to a formulation at such a low level. More 

likely, the DEHP was a trace contaminant in the formulation or was an artifact of laboratory 

contamination. In fact, the Cosmetic, Toiletry and Fragrance Association has indicated that no 

cosmetic products currently manufactured in the US contain DEHP. 

In an exceedingly large number of places, often particularly prominent places 

such as boxed language, the Final Update speculates that breast milk expressed into breast 

pumps may be a source of DEHP exposure, and uses concern about the potential for such 

exposure as a basis for asserting uncertainty about exposures of infants to DEHP. See pages 7, 

33-34, 54, 55, 93, 97, 171, and 175 of the Final Update. Yet the Final Update cites not one 

analytical result demonstrating that breast milk pumps contribute any DEHP to breast milk, 

much less significant amounts that would warrant the great deal of attention given to this 

 

30 Final Update at 2. 

31 See Final Update at page 169, which states: “DEHP is ubiquitous in the environment. Humans 

can be exposed to DEHP through many routes including ingestion (food, infant formula, and 

breast milk), contact with contaminated household dust and consumer products (cosmetics and 

toys). . . .” 

32 Environmental Working Group (2002). Not too pretty: Phthalates, beauty products and the FDA., 

page 2. Available at: http://www.ewg.org/reports_content/nottoopretty/nottoopretty_final.pdf

15

Appendix III
speculative source. In fact, the available data cited in the Final Update suggests breast milk 

pumps do not affect DEHP levels in breast milk.33 

The PE Panel’s knowledge of breast pumps indicates that it is highly unlikely 

breast milk pumps would be a significant contributor to DEHP exposures. To the PE Panel’s 

knowledge, no milk container portion of any pump is made of vinyl that would contain DEHP 

(or any other phthalate), and therefore there would be no DEHP available to migrate into the 

milk during storage. Some breast milk pumps have flexible vinyl tubing that may contain 

DEHP; however, the tubing is used to pull air away from the container (creating the vacuum that 

pumps the milk); the milk does not come into contact with the tubing. The cup that is placed 

against the breast might be made of flexible vinyl that might contain DEHP, and it is possible 

some breast milk might come into contact with the cup, but any such contact would be fleeting 

and would allow for very little migration of DEHP into the milk.34

For these reasons, the rather extreme concern about potential infant DEHP 

exposures to DEHP from breast milk pumps is spurious. The PE Panel requests that, in its final 

brief, the NTP-CERHR clarify the potential role of breast milk pumps in DEHP exposures. To 

the extent that the Expert Panel expressed concern due to uncertainty over infant exposures 

because of the speculative contribution of breast milk pumps, NTP-CERHR should express a 

lower level of concern. 

IV. COMMENTS ON SECTION 3: DEVELOPMENTAL TOXICITY DATA 

A. The Data Reviewed in the Final Update Do Not Support an Oral Developmental 

NOAEL of 3-5 mg/kg/day 

The first CERHR Expert Panel Report did not identify a firm NOAEL for 

developmental toxicity on the developing male reproductive tract.35 In the Final Update, the 

Expert Panel cited the multigeneration continuous breeding study conducted by NTP (2004) as 

providing a developmental NOAEL of 3-5 mg/kg/day. This NOAEL is based on the incidence 

of gross observations of small reproductive organs (with no change in organ weights) observed 

in a few animals in the 14-23 mg/kg/day and the 46-77 mg/kg/day groups. In deriving this 

NOAEL, the Expert Panel stated that it evaluated the combined incidence of small male 

 

33 “The authors tested milk samples in 1 common Danish pump system and found no effect on 

phthalate monoester levels.” Final Update at 7. “Women who used a breast pump in Denmark 

had significantly higher levels of monoethyl and monobutyl phthalate. Breast pump-associated 

levels of other phthalates were not significantly different . . . .” Final Update at 55. 

34 Like the Li et al. paper, the concern about breast milk pumps is an example of information newly 

raised at the Expert Panel meeting for which there was no adequate opportunity for public 

comment (see Section I.A, above). The PE Panel attempted to provide an explanation such as 

given herein to the Expert Panel, but it is not clear that the explanation was conveyed to the 

Expert Panel Chair, much less the rest of the Expert Panel, a failing which could have been 

overcome by a more flexible approach to audience participation (see Section I.D., above). 

35 The Expert Panel stated “The Panel is not confident that the lowest dose has been established at 

which developmental toxicity (the development of the male reproductive system) occurs.” First 

Expert Panel Report at 88. 

16

Appendix III
reproductive organs in the F1 and F2 non-breeding males.36 However, the total number of 

examined organs listed in Table 23 of the Final Update does not reflect this, as the table contains 

only the number the F1 non-breeding males examined and omits the number of F2 non-breeding 

males. 

Counting the F2 non-breeding males, the total combined number or organs 

examined should have been 59, 61, 64, 61, 66, 68, 50 for the 1.5, 10, 30, 100, 300, 1000, and 

7500 ppm dose levels, respectively. In addition, the PE Panel believes that the number of 

breeding males in the F1 and F2 generations should also be included in the combined incidence. 

While the breeding males were older than non-breeding males at the time of sacrifice, the effect 

of small reproductive organs, if present, would not be expected to disappear over this time 

period. Including the breeding males would increase the total number of each group by 20. By 

not including in the total number of examined organs the number of F2 non-breeding males and 

breeding males, the Expert Panel significantly overstates the incidence rate of small organs, 

which in turn significantly overstates the magnitude of the effect. The PE Panel provides below 

what it believes to be the correct version of Table 23. 

Table 23. Reproductive Organ Abnormalities in Combined F1 + F2 Non-breeding Males in NTP 

Multigeneration Study 

 DEHP dose level, ppm in feed 

 __________(number of organs examined)___ 

1.5 10 30 100 300 1000 7500 

Organ (79) (81) (84) (81) (86) (88) (70) 

Testis 0 0 0 0 4 3 21 

Epididymis 0 0 0 0 3 3 7 

Seminal vesicles 0 1 0 0 2 0 0 

Prostate 0 0 0 0 0 4 1 

Any reproductive organ 0 1(1)* 0 0 5(4)* 7(5)* 22(14)*

*Data expressed as number of animals (litters) affected. From NTP (114) 

In addition, although the NTP study reported that the finding of small 

reproductive organs in the 300 and 1000 ppm groups was at a significantly higher incidence rate 

than laboratory historical control data, the historical data were not available for review. The PE 

Panel questions whether sufficient historical control data for small reproductive organs have 

been evaluated to ascertain whether the limited incidence of small organs seen in the NTP study 

can be definitively determined to be treatment related. For the low incidence of small organs 

observed in the treatment groups to be statistically significant, the control incidence of this effect 

would have to be zero, or very close to zero. As mentioned in the PE Panel’s comments to the 

Draft Update, historical data from contract laboratories indicate that there is a 2-3% incidence of 

testicular atrophy at necropsy in control populations of sexually mature Sprague-Dawley rats 

from Charles River Laboratories. In the absence of reduced organ weight (individual or group 

mean) or evidence of lowered reproductive success, which cannot be assessed because the effects 

were only reported for non-mating males, the small organs reported at 1000 and 300 ppm should 

 

36 Final Update at 66 and 94. 

17

Appendix III
not be considered toxicologically significant. Therefore, the PE Panel believes it is necessary to 

evaluate historical control data for this strain of rat to evaluate the statistical significance of the 

incidence of the small reproductive organs. Without such information, the PE Panel questions 

the validity of making the determination of a NOAEL in this study based on this finding. 

Moreover, at the Expert Panel public meeting, Dr. Robert Chapin, the Expert 

Panel member who most closely reviewed the NTP data, stated that the 300 ppm (about 14-23 

mg/kg/day) LOAEL was at the “very tail end of the response”37 and that the Expert Panel was 

“flying along in the weeds” at this level. This raises the issue of whether the effects reported in 

the NTP study at the 300 ppm LOAEL were in fact treatment related, or were non-treatment 

related noise. In other words, if 14 mg/kg/day is the LOAEL, then the NOAEL is likely much 

closer to this value than the value chosen by the Expert Panel, the lowest tested dose of 100 ppm 

(about 3-5 mg/kg/day). 

Thus, the PE Panel believes that the NTP (2004) data as reviewed by the Expert 

Panel do not support a NOAEL as low as 3-5 mg/kg/day. For the foregoing reasons, the PE 

Panel believes NTP-CERHR should consider 14-23 mg/kg/day as a NOAEL, or at the least 

should find the NOAEL to be near that level (e.g., 12 mg/kg/day). 

V. SECTION 4: REPRODUCTIVE TOXICITY DATA 

A. The Data From the Mitsubishi Study Showing Increased Uterine and Ovarian 

Weights in Female Marmosets Do Not Support the Conclusion that DEHP 

Exposure Resulted in Precocious Puberty in Marmosets 

After rejecting the Mitsubishi (2003) data for assessing male reproductive 

toxicity, due in part to concerns about the reliability of the study, the Expert Panel used data 

from that same study to conclude that increased uterine and ovarian weights in female marmosets 

were “consistent with precocious puberty in the 2 highest dose DEHP-exposed groups (500 and 

2500 mg/kg bw/day).”38 However, a review of the Mitsubishi study by Dr. Suzette Tardif, the 

Associate Director of the Southwest National Primate Research Center and an expert in female 

marmoset reproductive biology with over twenty years experience raising primates, determined 

that these data are inconclusive and do not support the conclusion that DEHP exposure causes 

precocious puberty in female marmosets. 

Dr. Tardif’s analysis of Mitsubishi (2003) (see Attachment 3 to these comments) 

shows that the increased uterine and ovarian weights correlate to body weights that were higher 

in the higher dose females, versus lower body weights in controls and low dose females. As Dr. 

Tardif explains, the females in the Mitsubishi study, particularly the controls and the low dose 

group, were of extremely low weight for their age at the end of the study (about 17 months), 

compared to a healthy marmoset colony. Dr. Tardif attributed this to the study procedures, 

which involved daily gavage for many weeks in a row and resulted in many “basically unhealthy 

 

37 This comment is acknowledged in the Final Update, which states at page 151: “The Expert Panel 

considers 300 ppm and 1000 ppm to represent the tail of the dose-response curve in this study....” 

38 Final Update at page 163. 

18

Appendix III
animals with impaired growth.”39 While there was no significant difference in average weight 

across the treatment groups, there was a trend for the females in the two highest dose groups to 

have the highest body weights, and these were the groups which also had increased uterine and 

ovarian weights. Specifically, only three out of six subjects in the control and low dose groups 

had a 17-month-old body weight that Dr. Tardif would consider suitable for research (275 g), 

five of six and six of six of the animals in the higher dose groups would have been suitable. 

 Moreover, based on measured estradiol concentrations, many of the animals in 

fact appeared to be pre-pubescent throughout the length of the study, which was terminated when 

the animals were at an age at which all should have been adults. The failure of many individuals 

to enter puberty was correlated with the abnormally low body weights. 

Dr. Tardif found that the higher ovarian weights in the higher dose groups were 

generally associated with higher body weights and the occurrence of ovulation and corpus 

luteum formation that accompanies sexual maturity. In Dr. Tardif’s opinion, the higher ovarian 

weights seen in the highest dose group were simply due to the higher weight group having more 

normal weight animals which were more likely to have ovulated. Normal ovarian function in 

marmosets includes the development and maintenance of a large, steroidogenic interstitial gland, 

and the persistent presence of this gland, along with the cyclical presence of corpus lutea, leads 

to the heavier, sexually mature females having higher ovarian weights. Thus, the higher ovarian 

and uterine weights in the two high dose groups is an artifact of those groups having higher 

weight females, and is not an effect of DEHP dose. Accordingly, the PE Panel disagrees with 

the Expert Panel’s conclusion that the increased female ovarian and uterine weights in the 

Mitsubishi study are indicative of precocious puberty.40

B. The Studies by Akingbemi et al. Reviewed in the Final Update Are Flawed and 

Do Not Support a NOEL of 1 mg/kg/day 

The Expert Panel concluded that the results from two Akingbemi et al. (2001; 

2004) studies reporting a NOEL of 1 mg/kg/day based on data obtained from purified Leydig 

cells were adequate for the evaluation process.41 The effects reported by Akingbemi et al. at the 

LOEL (10 mg/kg/day) were abnormal changes in testosterone production and altered Leydig cell 

proliferation in the testes of prepubertal rats. However, because these studies suffer from a 

methodological deficiency – they rely on single-point measures of serum LH and testosterone – 

the PE Panel has significant concerns regarding the suitability of these studies for deriving the 

NOEL. 

 

39 Opinion of Dr. Tardif, Attachment 2, at page 1. 

40 An equally plausible interpretation of the data is that high doses of DEHP enhance the health of 

females, thus enabling high dose females to mature in the usual time frame, with resulting 

increases in ovarian weights over the less healthy controls and lose dose females. The PE Panel 

dose not advance this hypothesis – it would be far too speculative a conclusion based on this one 

study. The PE Panel believes that the assertion of precocious puberty is equally speculative. 

41 Final Update at 168. 

19

Appendix III
Several papers and texts (Culler, 1998; Levine and Duffy, 1988; Creasy, 1999) 

provide excellent descriptions as to why single point measures of isolated or combined 

gonadotropin and gonadosteroid measurements do not allow for identifying toxicity from 

chemical exposures. The methodological problem arises from the fact that pulsatile releases of 

gonadotropin releasing hormone (GnRH) control LH release and pulsatile LH levels control 

testosterone production and release from Leydig cells. These pulsatile releases are superimposed 

on a circadian rhythm pattern for the secretion of these releasing factors and hormones. If 

multiple samples are not collected and analyzed from each animal over several hours during the 

peak phase of the circadian cycle, then the pulsatile releases may be missed. To ensure accurate 

comparisons among individuals, care must be taken to collect samples from all of the animals at 

precisely the same time relative to the circadian pattern described above. 

The Akingbemi et al. studies do not meet these criteria. The Materials and 

Methods Sections in both Akingbemi et al. papers simply state that the blood was taken within 

24 hours of the last DEHP exposure; there is no mention of any attempt on the part of the authors 

to control for the time of day (circadian pattern) between treatment groups. The lack of control 

for these circadian patterns and pulsatile releases of these factors and hormones is a critical flaw 

in the study design. For example, if all the control animals were sacrificed first, followed by the 

low dose, mid dose, and high dose animals, the investigators would be introducing a systematic 

sampling bias to their measurements because the animals may have been in different stages of 

the circadian pattern and have been experiencing varied pulsatile release of these 

factors/hormones. 

Another significant flaw in the Akingbemi et al. studies lies in the method by 

which the authors isolated the Leydig cells from these animals and how they interpreted the data 

from endpoints derived from these cells. Leydig cells in immature rats are relatively quiescent 

and small and will elute/migrate through a Percoll gradient with a different density than mature 

Leydig cells. As the authors describe in the 2001 paper, PND 21 (progenitor) Leydig cells 

elute/migrate within the Percoll gradient at a band representing a density of 1.062 – 1.070 g/ml. 

PND 35 control rats have immature Leydig cells which elute/migrate through the same gel at a 

band representing a density of 1.070 to 1.088 g/ml and the Leydig cells obtained from PND 49 

and 90 (mature) animals have densities greater than 1.070 g/ml. In addition, the authors used an 

enzyme critical for testosterone biosynthesis, 3β hydroxysteroid dehydrogenase (3β-HSD), as a 

biomarker for whether these cells were able to produce testosterone. PND 21 Leydig cells stain 

lightly or not at all for 3β-HSD indicating that they are quiescent in terms of testosterone 

biosynthesis. The Leydig cells from PND 35 and greater stain intensely for 3β-HSD indicating 

that testosterone biosynthesis can occur in those cells. Therefore, in control rat testes, as the 

Leydig cells mature and gain the ability to synthesize testosterone, their cellular density (a 

product of cell mass and size) changes and, correspondingly, the distance they elute/migrate 

within the Percoll gradient changes. 

Typically, one of the first alterations noted in cells undergoing abnormal cell 

division (e.g., hyperplasia) or reacting to a toxic insult, is a change in cell mass and size. These 

changes, which are well described in textbooks of toxicological pathology, are changes that 

pathologists look for microscopically. However, measuring changes in cell mass or size (i.e., 

density) is very difficult using histological techniques, as a three dimensional object (the cell) is 

20

Appendix III
only represented in two dimensions under a microscope. Consequently, it is not difficult to miss 

changes in cell density using microscopic histological techniques. 

In mature rat testes, Leydig cells make up a significant portion of the total weight, 

about 20 – 25% (Creasy, 1999), and any change in Leydig cell number should cause a change in 

that percentage. However, in the Akingbemi et al. papers, the authors note a lack of change in 

either testes weight or size. If there was Leydig cell hyperplasia (as proposed by these authors), 

but no corresponding increase in testicular mass, then it stands to reason that the Leydig cells 

present are of different size, shape, and density than those found in control testes. The authors, 

therefore, have made a fundamental error in these experiments; they assumed that the Leydig 

cells from the treated animals would have the same density as those from the control animals. 

Because they assumed that the control and treated animals would have the same Leydig cell 

density, the authors only collected the Leydig cells that migrated within the band of the Percoll 

gradient that matched the control Leydig cells’ migration. They did not consider that the Leydig 

cells from the treated animals may have a different density and therefore would migrate into a 

different band within the Percoll gradient. 

As a consequence, all of the reported measures from the isolated fraction of 

Leydig cells simply represent the remaining Leydig cells within the treated testes that have the 

same density as those of control Leydig cell populations at that developmental stage. The 

authors did not collect other density bands within the Percoll gradient and examine whether the 

Leydig cells within those bands could also synthesize testosterone or have other characteristics 

of mature Leydig cells. As described above, cells that are hyperplasic or that have undergone 

toxic insult can have a different density. These cells were never detected or collected in these 

experiments. 

Therefore, all the endpoints in Akingbemi et al. that make use of isolated Leydig 

cell preparations, (the ability of the Leydig cells to produce testosterone at either a basal level or 

in response to LH stimulation, the indicators of cell cycle stage used to investigate hyperplasia, 

the counting of the cells, aromatase levels, etc.) are flawed due to the assumption made by the 

investigators that the Leydig cells from the DEHP treated animals would migrate/elute in the 

same density band as those from the control animals. 

In addition to the above flaws, at least one member of the Expert Panel questioned 

the reliability of the data in the Akingbemi et al. studies at the Public Meeting, stating that the 

data were “messy and perplexing” and that “I would never ask this group to do toxicological 

work again.” However, rather than finding these data unsuitable for its review, the Expert Panel 

instead cherry-picked results from the studies which suggested DEHP toxicity. This is yet 

another example of procedural inequities in the review process described above, and is 

unacceptable in a review process that is supposed to be objective and deliberative. 

Because of the serious flaws in the Akingbemi et al. studies described above, the 

PE Panel does not agree with the Expert Panel that these studies “are sufficient to conclude that 

DEHP is a reproductive toxicant in male rats at the indicated dose levels.”42

 

42 Id.

21

Appendix III
CONCLUSION 

For the reasons stated herein, the PE Panel has significant concerns regarding 

both the process by which the Final Update was produced and some of the scientific conclusions 

present in the Final Update. The PE Panel believes that because of significant procedural flaws, 

the Final Update does not adequately represent an objective and fully-deliberated consensus 

opinion. In addition, the PE Panel believes that the Expert Panel incorrectly disregarded 

scientific data which show that very high concentrations of DEHP that would cause profound 

adverse effects in rodent testes had no adverse effects on the testes of marmosets. The marmoset 

is a useful model for human male reproductive toxicity and the Mitsubishi study, while not 

perfect, provides valuable information about the toxicity of DEHP to primates, with importance 

implications for the degree of concern for human health. In addition, several aspects of the 

Expert Panel’s review of reproductive and developmental toxicity do not, in fact, support the low 

NOAELs chosen, and there is less uncertainty about infant exposures than indicated by the Final 

Report. The PE Panel requests that the NTP-CERHR keep these points in mind as it considers 

the conclusions of the Expert Panel in the Final Update. 

22

Appendix III
REFERENCES 

Akingbemi, B. T., Youker, R. T., Sottas, C. M., Ge, R., Katz, E., Klinefelter, G. R., 

Zirkin, B. R. and Hardy, M. P. (2001). Modulation of rat Leydig cell steroidogenic function by 

di(2-ethylhexyl)phthalate. Biol Reprod; 65: 1252-9. 

Akingbemi, B. T., Ge, R., Klinefelter, G. R., Zirkin, B. R. and Hardy, M. P. (2004). Phthalate 

induced Leydig cell hyperplasia is associated with multiple endocrine disturbances. Proc 

Natl Acad Sci USA; 101: 775-80. 

Anderson, W., Castle, L., Scotter, M., Massey, R., Springall, C. (2001). A biomarker approach to 

measuring human dietary exposure to certain phthalate diesters. Food Additives & Contaminants 

18(12):1068-174. 

Brock, J. W., Caudill, S. P., Silva, M. J., Needham, L. L. and Hilborn, E. D. (2002). Phthalate 

monoesters levels in the urine of young children. Bull Environ Contam Toxicol; 68: 

309-14. 

Creasy, D.M. (1999). Chapter 16 “Hormonal Mechanisms in Male Reproductive Tract Toxicity” 

pgs. 355-406 in Endocrine and Hormonal Toxicology, John Wiley and Sons, Publishers. 

Culler, M.D. (1998). Chapter VIII “Circulating Hormones” pgs. 75-95 in An Evaluation and 

Interpretation of Reproductive Endpoints for Human Health Risk Assessment. HESI ILSI 

Publication. 

David, R.M. (2000). Exposure to phthalate esters. Environ Health Perspect 108:A440. Available 

at: http://ehp.niehs.nih.gov/docs/2000/108-10/correspondence.html 

Foster, P. M., Turner, K. J. and Barlow, N. J. (2002). Antiandrogenic effects of a phthalate 

combination on in utero male reproductive development in the Sprague-Dawley rat: additivity of 

response? Toxicologist, 66:233. 

Koch, H. M., Drexler, H. and Angerer, J. (2004). Internal exposure of nursery-school children 

and their parents and teachers to di(2-ethylhexyl)phthalate (DEHP). Int J Hyg Environ Health, 

207:15-22. 

Koo, H. J. and Lee, B. M. (2004). Estimated exposure to phthalates in cosmetics and risk 

assessment. J Toxicol Environ Health A; 67: 1901-14. 

Kurata, Y., Kidachi, F., Yokoyama, M., Toyota, N., Tsuchitani, M., and Katoh, M. (1998). 

Subchronic toxicity of di(2-ethylhexyl)phthalate in common marmosets lack of hepatic 

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42:49-56. 

23

Appendix III
Levine, J.E. and Duffy, M.T. (1988). “Simultaneous measurement of luteinizing hormone (LH)-

releasing hormone, LH, and follicle-stimulating hormone release in intact and short-term castrate 

rats“ Endocrinology 122(5):2211-2221. 

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450-469. 

Mitsubishi-Chemical-Safety-Institute. (2003). Sixty-five week repeated oral dose toxicity study 

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Chemical Safety Institute. 

National Toxicology Program (2004). Diethylhexylphthalate: Multigenerational reproductive 

assessment by continuous breeding when administered to Sprague-Dawley rats in the diet. 

Research Triangle Park NC: National Toxicology Program.. 

Pugh, G Jr., Isenberg, J.S., Kamendulis, L.M., Ackley, D.C., Clare, L.J., Brown, R., Lington, 

A.W., Smith, J.H., Klaunig, J.E. (2000). Effects of di-isononyl phthalate, di-2-ethylhexyl 

phthalate, and clofibrate in cynomolgus monkeys. Toxicol Sci 56:181-188. 

Rhodes, C., Orton, T.C., Pratt, I.S., Batten, P.L., Bratt, H., Jackson, S.J., Elcombe, C.R. (1986). 

Comparative pharmacokinetics and subacute toxicity of di(2-ethylhexyl) phthalate (DEHP) in 

rats and marmosets : extrapolation of effects in rodents to man. Environ Health Perspect. 65:299-

308. 

Short, R.D., Robinson, E.C., Lington, A.W., Chin, A.E. (1987). Metabolic and peroxisome 

proliferation studies with di(2-ethylhexyl)phthalate in rats and monkeys. Toxicol Ind Health

3:185-194 

24

Appendix III
ATTACHMENTS 

1. Curriculum Vitae and Opinion of Dr. Stefan Schlatt entitled: Evaluation of the marmoset 

(Callithrix jacchus) as a model for reproductive toxicity. 

2. Comments of the Phthalate Esters Panel of the American Chemistry Council on Notice of 

Intent to List Chemicals, submitted to the California Office of Environmental Health Hazard 

Assessment, May 24, 2005. 

3. Curriculum Vitae and Opinion of Dr. Suzette Tardif entitled: Findings regarding female 

reproductive physiology from the Mitsubishi Study #B000496, “Sixty-five week repeated oral 

dose toxicity study of DEHP in juvenile common marmosets.” 

 DC\832136.1 

25

Appendix III
Evaluation of the marmoset (Callithrix jacchus) as a model for reproductive toxicity 

Author: Stefan Schlatt, Ph.D. 

e-mail: schlatt@pitt.edu

Date: January 30, 2006 

1) Information on the Author 

I obtained a PhD from the University Münster, Germany and was awarded a number of postgraduate 

fellowships including a Welcome Trust Training Fellowship in Reproductive Research and a 

Heisenberg-Fellowship which is the most prestigeous German Senior Research Fellowship granted by 

the Deutsche Forschungsgemeinschaft. At present, I am an Assistant Professor at the University of 

Pittsburgh School of Medicine, PA, USA. In recent years I have been employed and trained at several 

Centers of Excellence in Reproductive Sciences including the Institute of Reproductive Medicine of the 

University Münster, Germany; the Institute of Reproduction and Development of Monash University, 

Clayton, Australia and the Center for Animal Transgenesis and Germ Cell Research of the University 

of Pennsylvania, Kennett Square, PA, USA. I have explored a wide spectrum of research in 

reproductive biology including such diverse topics as the photoperiod-dependent regulation of female 

and male reproductive organs in Djungarian hamsters as well as preclinical research using macaque and 

marmoset monkeys to analyse the role of inhibin in the testis, the effects of hormones on prepubertal 

testis development and the development of male contraceptive regimens. I and my colleagues 

established several milestones for the development of germ cell transplantation as a clinical tool 

performing the first successful germ cell transplant into a testis of non-human primates and men. 

Recently, a novel strategy to use testicular xenografting as a tool to generate fertile sperm was 

described and applied on marmosets, macaques and men. We were also able to describe xenografting 

as a new tool for exploration of toxic effects during testicular development in rhesus monkeys. 

This document has been compiled in response to a request by the American Chemistry Council 

Phthalate Esters Panel. I have been asked to critique a recent review by Li et al. (2005) and to render 

my opinion of the general value of the marmoset as a model for human testis development and its use 

for risk assessments of gonadotoxic exposures in the male. In addition I was asked to express my 

opinion on the validity and relevance of the Mitsubishi study. 

1

Appendix III
2) Introduction 

The marmoset has been intensely used for many years as a non-human primate model to describe the 

physiology of testicular development and function. In contrast to rodent animal models, this small new 

world primate shows many similarities with old world primates and man in regard to the general 

developmental pattern. Like human and old world primates the marmoset enters three periods of 

development with an active postnatal phase of the hypothalamus-pituitary-gonadal axis, a prepubertal 

period of quiescence and a re-awakening of the hormonal axis and final differentiation of the male 

reproductive system during puberty. This pattern is quite different from that in mice and rats which do 

not show a marked prepubertal period of no or slow gonadal growth and differentiation. Since the 

marmoset is easy to maintain and to reproduce and – as a primate - appears to be evolutionarily close to 

man, it has been considered a valuable and clinically relevant animal model to analyze toxic effects on 

the developing male reproductive system. However, the marmoset also exhibits some interesting and 

unique features in regard to hormonal regulation of its reproductive functions. A detailed review by Li 

et al. (2005) that summarizes similarities and differences concludes that interpretation of experimental 

findings on the testicular effects in marmosets should be made with caution. The present short report 

revisits the pro- and contra-arguments of using the marmoset as a model for testicular developmental 

toxicity and concludes that the marmoset presents a useful and valid model to explore several aspects 

of testicular development. I have also been asked to critically review an unpublished study from the 

Mitsubishi Chemical Safety Institute using the marmoset as a model to explore the testicular toxicity of 

DEHP. As discussed below, I conclude that the results of this study should be carefully and critically 

considered for the evaluation of risks associated with exposure to DEHP. 

3) The value of the marmoset as a model for male testis development and evaluation of bias in Li 

review 

3.1 Physiological characteristics of the male marmoset endocrine and reproductive systems 

The recent review by Li et al. (2005) is a comprehensive review of the testicular development and 

function in the marmoset. The authors present the current knowledge on embryonic testicular 

development and describe the changes occurring during the postnatal period (2-3 weeks) and the 

pubertal period (6-12 months). They show the striking similarities of organization of the marmoset and 

human spermatogenic epithelium (both species have a multistage organization of spermatogenic stages 

per tubular crossection). Although nine stages of the seminiferous epithelial wave have been 

distinguished originally, many authors have adapted the human staging system for the marmoset 

indicating quite similar mechanisms of germ cell development and clonal expansion of germ cells in 

marmosets and man. This organizational similarity has prompted scientists to promote the marmoset as 

a “suitable model for studies relevant to human testicular function” (Millar et al., 2000). 

Li et al. (2005) point out an unusual uniformity of Sertoli cell morphology throughout the marmoset 

spermatogenic cycle. In rodents and macaques Sertoli cells show specific morphological features 

related to specific stages of spermatogenesis. However, it is not clear how stage-specific changes of 

Sertoli cells can be identified in seminiferous tubules of men and marmosets which show a mixed 

pattern of spermatogenic stages. Although there are insufficient human data on Sertoli cell morphology 

to know whether such uniformity occurs in humans, it appears likely that the human and marmoset 

Sertoli cell show a high degree of similarity in this respect. 

2

Appendix III
Li et al. (2005) quote studies reporting a high efficiency of marmoset spermatogenesis although this 

finding appears slightly controversial in the literature. They indicate that FSH is the prime regulator of 

Sertoli cell proliferation which correlates well with the role of FSH in other primates but differs from 

its less significant role in rodents. 

One of the most interesting marmoset specific adaptations is the absence of the LH hormone. In the 

marmoset and other new world monkeys a CG type hormone is expressed in the pituitary and is 

responsible for regulation of steroid secretion in the ovary and the testis. This change is accompanied 

by a deletion of the LH receptor which lost exon 10 and can therefore not bind to LH, but has high 

affinity for CG. Despite of the exchange of LH by CG the marmoset shows many similarities to man. 

The function and regulation of FSH and CG and their feedback mechanisms resemble other primates. It 

is quite interesting to note that the LH receptor deletion in the marmoset mimics the situation in a 

hypogonadal patient (Gromoll et al., 2000). The availability of the marmoset as a model opened a new 

research field to explore the molecular mechanisms of hormone specificity of the LH receptor (Gromoll 

et al., 2003). 

Li et al. (2005) propose a generalized steroid hormone resistance in the marmoset. This phenomenon is 

indeed quite striking and has been proposed for the mineralocorticoid and glucocorticoid hormones. 

However, whether or not this resistance also exists for gonadal steroids is poorly understood. Although 

some evidence exists that the response to sex steroids is rather poor, these observations should be 

balanced with the facts that many aspects of the marmoset in regard to sex steroids are strikingly 

similar to humans and differ from those in the rodent. 

The authors mention that there is little information on marmoset testosterone synthesis and testosterone 

metabolism. The marmoset, but not the mouse, has sex hormone binding globulin separating the serum 

testosterone into active free and bound fractions. Due to affinities of these proteins the marmoset 

appears to have high levels of unbound testosterone in the circulation which is similar to the unusually 

high levels of glucocorticoids. However, in the absence of solid data on sex steroids it appears poorly 

justified and premature to transfer the conclusion of high sex steroid resistance from the glucocorticoid 

and mineralocorticoid system to the sex steroid system. 

Some other curiosities exist which – in my opinion- do not impact the use of the marmoset as a model. 

Intact male marmosets respond with positive feedback to estradiol. Such a response is usually observed 

in orchidectomized rats or macaques and many homosexual men. The development of the testis relies 

on the presence of FSH and CG. A delay in testicular development does not have serious 

consequences, similar to other primates. Our own studies using xenografting as a model system showed 

the expected outcome that mouse LH can not stimulate the androgen-dependent differentiation of 

grafted marmoset tissue due to its deleted LH-receptor (Schlatt et al., 2002). Co-grafting with hamster 

tissue was not sufficient to induce testicular development (Wistuba et al., 2004). Li et al (2005) 

interpreted our data as indicative of unique factors responsible for marmoset spermatogenesis. 

However, the more likely explanation is that CG is needed to stimulate organogenesis of the immature 

testicular fragments and that the testosterone supplied by the hamster graft is insufficient. This 

explanation is supported by very recent data showing that autografted marmoset testes can develop 

through puberty (Wistuba et al., 2005), an indication that their hormonal system can drive 

spermatogenesis in autografted tissue. 

3

Appendix III
In conclusion, the review by Li et al. (2005) presents a useful resource for any scientist interested in the 

marmoset model. However, the authors’ interpretation of the suitability and validity of the model 

carries an unjustified negative bias. 

3.2 Personal judgement of the value of the marmoset as research model for assessment of male 

reproductive toxicity. 

 In my opinion there is no doubt that the marmoset is a useful model to explore developmental and 

toxicological aspects of the testis. Certainly, old world primates appear to be favorable as they 

maintained a closer physiological relation to humans. However, many similarities in regard to testicular 

organization, general developmental pattern and hormonal regulation render the marmoset a much more 

useful model when compared to rodents. The marmoset therefore has its valid place when it comes to 

the analysis of toxic effects on testicular development. With choice of the correct timepoints this 

species should be highly useful and informative for exploring the effects on Sertoli cell differentiation, 

testicular growth and effects of FSH on the testis. The striking similarities to man make it an excellent 

model for studying effects on germ cell development, the organization of the seminiferous epithelium 

and changes to the kinetics of spermatogenesis. Due to its unusual gonadotropic and sex steroid 

regulation it is certainly an interesting biological model for exploring steroid feedback and the 

functions of the CG-molecule with less relevance for humans in these specific aspects. 

4) The value of the Mitsubishi study to determine male reproductive toxicity 

4.1 Validity of the results of the Mitsubishi study in respect to pitfalls and shortcomings 

This study is a unique effort to explore the effects of DEHP on testicular development in a non-human 

primate model. In the oral dose toxicity part of the study marmoset monkeys of 90-110 days of age 

were exposed to three doses of DEHP (100, 500, 2500 mg/kg) and vehicle for 65 weeks. The monkeys 

received a daily oral dose in corn oil by gavage. The size of the male treatment groups finishing the 

exposure was 8 (vehicle), 9 (100mg/kg), 10 (500 mg/kg) and 9 (2500 mg/kg). 3 animals in each group 

were subjected to perfusion allowing electron microscopic analysis of the testes. At the time of sacrifice 

an impressive number of endpoints were analyzed: 10 hematological parameters, 21 blood chemical 

parameters, levels of testosterone, estradiol and T3/T4 as endocrine parameters, body weights and 

organ weights of pituitary, thyroids, liver, pancreas, spleen, kidneys, testes, prostate, seminal vesicles 

and epididymides. Histological analysis of all organs was performed and additional histochemical 

(3betaHSD) and ultrastructural analysis was performed in the testes. Testicular homogenates were used 

to determine counts of spermatids (heads per g testicular tissue) and 6 testicular enzymes were analyzed 

biochemically. The activities of nine hepatic enzymes were determined in liver homogenates. 

The results of the study reveal no statistical changes in clinical parameters and body weights, 

hematology, blood chemistry, hormone levels and testicular and hepatic enzymes for the male monkey 

groups. In each DEHP treatment group and in the control group one monkey showed low testis and 

reproductive organ weights. The authors report that this usually correlated with low body weight and 

that these three monkeys were therefore considered to be immature. This implies that they consider 

each other monkey in the study to be fully adult at the time of sacrifice. However, for reasons discussed 

below, this may not be the case. When summing up the age at the start of the study and the duration of 

the study, the time point of sacrifice was around 20 months of age which corresponds to the late phase 

4

Appendix III
marmosets go through puberty. It is characteristic for this species that the variability to sexually mature 

is extremely wide (12-20 months). Therefore, a subgroup of four (or 5, see below) late pubertal 

monkeys among 36 monkeys is not unusual at this age. 

The apparent occurrence of different developmental stages could have been avoided by delaying the 

time point of sacrifice by several months. While the period of exposure was wisely selected and 

covered the full period of testis development after weaning, the period of sacrifice means that both 

adult and late pubertal monkeys were analyzed. The exclusion of some of the treated animals from 

sperm count analysis may be an indication that poor definition of developmental stages at the time of 

analysis may have influenced the outcome of the study and might have induced high variability in the 

data. 

I have some questions in regard to the interpretation of testicular histology. Photographs 1 and 2 show 

the histological patterns of qualitatively and quantitatively normal testes of fully adult marmosets. If 

these testicular crossections are – as indicated - representative of the control and high dose DEHP 

groups, these micrographs clearly reveal that the exposure to DEHP had no impact on testis 

development and spermatogenic induction in marmosets. Photographs 3 and 6 depict a rather unusual 

pattern of testicular development for a late pubertal monkey and are potentially showing some testicular 

damage. The degree of vacuolization is quite high and the number of germ cells is rather low for the 

late stage of pubertal testicular development. It is therefore difficult to determine whether animals 

10208 (Fig. 6) and 10204 (Fig. 3) are showing an immature or damaged testicular phenotype. 

Surprisingly an underdeveloped testis as shown for Mk 10208 has not been reported elsewhere in the 

study. It might be useful to send all testicular histological samples for a blinded re-analysis to a scientist 

who has abundant expertise in primate testicular development. Together with the results on testis 

weight and reproductive organ weights this might enable analysis of subtle effects and confirmation 

whether the proposed “growing” monkeys are indeed immature or whether their testes are damaged. 

There was a significant loss of monkeys during the study. About 15% of the experimental animals died 

during the exposure period and were replaced. Marmosets are highly sensitive and show high mortality 

rates when exposed to poor hygiene conditions or poor diet. The main reason for this high death rate 

(also known as wasting syndrome) is intestinal infection. Marmosets have a short gut transit time, 

easily and frequently develop diarrhea, and eventually develop colitis leading to a rapid loss of body 

weight and death. The animal husbandry conditions in the study which exposed isolated single 

monkeys to a solid chow without supplementation of additional fruit or additional protein components 

were suboptimal to maintain the animals in good health. These monkeys live in social family groups 

and isolation might have been an additional stressor throughout the experiment. In addition, the effect 

of daily exposure to approximately 0.2-0.5 ml of corn oil must have had additional negative effects on 

their digestive tract. The amendments and deviations from the study protocol as outlined in the report 

(see 4.4.3: addition of milk powder to the diet and 5.4: discontinuation of oral exposure in 6.6% of 

applications) show that the animal care staff and veterinarians experienced serious problems in respect 

to the health and the nutritional status of the monkeys. 

Given these factors, it is possible that the lack of statistical significance in this study might be due to 

high inter-animal variations in many of the analyzed parameters. Reasons for the high variation could 

be the different developmental status of the animals at the time of sacrifice, poor health of some of the 

monkeys, and/or a high individual variability known for marmosets. 

5

Appendix III
4.2 Personal judgement of the value of the Mitsubishi study for risk assessment 

Despite of the above described shortcomings this study presents strong evidence that DEHP had no 

major effect on testicular development even after very long and intense DEHP exposure. The study 

analyzed many different parameters of which hardly any showed a significant change. Unfortunately, 

the poor design of the study and suboptimal animal husbandry render this study not fully conclusive. 

The oral dose toxicity study has generated a unique and wide set of data in a primate species which – as 

outlined above – has been used and should be considered in the future as a valuable model for the study 

of testicular development. Although the marmoset has an unusual androgen-CG feedback loop, the 

most striking effect of DEHP in other animal models is related to direct effects on Sertoli cells during 

testicular growth. In regard to this parameter the marmoset should be considered an excellent animal 

model. The analysis of the histological testicular parameters in the Mitsubishi study are therefore valid 

and impressively confirm a minor – if any – negative effect of DEHP exposure on Sertoli cell 

development and induction of spermatogenesis. 

In my opinion the Mitsubishi study can therefore not be fully neglected and should be carefully and 

critically considered for evaluating the risk of gonadotoxic effects in humans after exposure to DEHP. 

Yours sincerely, 

(Stefan Schlatt, Ph.D.) 

(5) References 

Gromoll J, Eiholzer U, Nieschlag E, Simoni M. Male hypogonadism caused by homozygous deletion of exon 10 of the 

luteinizing hormone (LH) receptor: differential action of human chorionic gonadotropin and LH. J Clin Endocrinol Metab. 

2000; 85:2281-6. 

Gromoll J, Wistuba J, Terwort N, Godmann M, Muller T, Simoni M. A new subclass of the luteinizing hormone/chorionic 

gonadotropin receptor lacking exon 10 messenger RNA in the New World monkey (Platyrrhini) lineage. Biol Reprod. 2003; 

69:75-80. 

Li LH, Donald JM, Golub MS. Review on testicular development, structure, function, and regulation in common marmoset. 

Birth Defects Res B Dev Reprod Toxicol. 2005 74:450-69. 

Millar MR, Sharpe RM, Weinbauer GF, Fraser HM, Saunders PT. Marmoset spermatogenesis: organizational similarities to 

the human. Int J Androl. 2000 23:266-77. 

Schlatt S, Kim S, Gosden R. Spermatogenesis and steroidogenesis in mouse, hamster and monkey testicular tissue after 

cryopreservation and grafting. Reproduction 2002; 124: 323-329. 

Wistuba J, Mundry M, Luetjens CM, Schlatt S. Cografting of hamster (Phodopus sungorus) and marmoset (Callithrix 

jacchus) testicular tissues into nude mice does not overcome blockade of early spermatogenic differentiation in primate 

grafts. Biol Reprod. 2004; 71:2087-91. 

Wistuba J, Luetjens CM, Wesselmann R, Nieschlag E, Simoni M, Schlatt S. Meiosis in Autologous Ectopic Transplants of 

Immature Testicular Tissue Grafted to Callithrix jacchus. Biol Reprod. 2005 Dec 21; [Epub ahead of print] 

6

Appendix III
 Responsible Care®

1300 Wilson Boulevard, Arlington, VA 22209 ♦ Tel 703-741-5000 ♦ Fax 703-741-6000 ♦ 

http://www.americanchemistry.com

May 24, 2005 

Via Facsimile: (916) 323-8803 

Via Email: coshita@oehha.ca.gov 

Ms. Cynthia Oshita 

Office of Environmental Health Hazard Assessment 

P.O. Box 4010 

Sacramento, CA 95812-4010 

Dear Ms. Oshita: 

The American Chemistry Council Phthalate Esters Panel (Panel) submits these comments 

in response to the California Office of Environmental Health Hazard Assessment (OEHHA) 

Notice of Intent to List Chemicals of March 4, 2005. The Panel includes the major U.S. 

producers and some processors of phthalate esters. These comments pertain to the four 

phthalates for which OEHHA provided notice that it intends to list as chemicals known to the 

state to cause reproductive toxicity under the Authoritative Bodies Mechanism of Proposition 65 

– dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), di-n-hexyl phthalate (DnHP), and 

diisodecyl phthalate (DIDP). These comments will show that these four phthalates fail to meet 

the listing criteria of Proposition 65. 

The Panel renews in whole the comments it submitted to OEHHA on August 26, 2004. 

These comments reiterate and expand upon those earlier comments, and address some of 

OEHHA’s responses to those comments. These comments make the following points: 1) The 

marmoset is a scientifically sound model for investigating potential effects of phthalates on 

human reproduction, as pointed out in the Panel’s previous comments, and OEHHA’s responses 

to those comments are inadequate to disqualify the marmoset as such a model; 2) the marmoset 

data comprise scientifically valid data, not considered by NTP-CERHR, which establish that the 

phthalates do not meet the criteria for identification “as causing reproductive toxicity;” 3) NTPCERHR found minimal to negligible risk of human reproductive toxicity and, therefore, the 

Proposition 65 listing criteria are not met for these phthalates; and 4) OEHHA has no statutory 

mandate to list chemicals such as these phthalates, which pose no significant risk to public 

health, and the Panel believes it would be poor public policy to list such low-risk chemicals 

under Proposition 65. 

Appendix III
Ms. Cynthia Oshita 

May 24, 2005 

Page 2 

For the reasons given in these comments, and in its earlier comments, the Panel believes 

that DBP, BBP, DnHP and DIDP do not meet the criteria for listing under Proposition 65 

pursuant to the authoritative bodies mechanism, and that OEHHA should not list these four 

phthalates under Proposition 65. 

If you have any questions, please call Marian K. Stanley, Manager of the Phthalate Esters 

Panel, at (703) 741-5623 or email her at Marian_Stanley@americanchemistry.com. 

Sincerely yours, 

Courtney M. Price 

Vice President, CHEMSTAR 

Enclosure 

Appendix III
Before the 

California Environmental Protection Agency 

Office of Environmental Health Hazard Assessment 

COMMENTS OF THE 

PHTHALATE ESTERS PANEL OF THE AMERICAN CHEMISTRY COUNCIL 

ON NOTICE OF INTENT TO LIST CHEMICALS 

 

 ) 

Notice of Intent to List Chemicals ) 

California Regulatory Notice Register 05, No. 9-Z, ) 

pp. 289-290 (March 4, 2005) ) 

) 

 

Courtney M. Price 

 Vice President 

 CHEMSTAR 

Dell E. Perelman, Esq. 

 Vice President and 

 General Counsel 

Marian K. Stanley 

 Manager, Phthalate Esters Panel 

Karyn M. Schmidt, Esq. 

 Counsel, CHEMSTAR 

Of Counsel: 

William K. Rawson, Esq. 

Ann Claassen, Esq. 

Latham & Watkins LLP 

555 Eleventh Street N.W. 

Suite 1000 

Washington, D.C. 20004 

May 24, 2005 

AMERICAN CHEMISTRY COUNCIL 

1300 Wilson Blvd. 

Arlington, VA 22209 

(703) 741-5000

Appendix III
EXECUTIVE SUMMARY

The American Chemistry Council Phthalate Esters Panel (Panel) submits these comments 

in response to the California Office of Environmental Health Hazard Assessment (OEHHA) 

Notice of Intent to List Chemicals of March 4, 2005 (California Regulatory Notice Register 05, 

No. 9-Z, pp. 289-290 (March 4, 2005)). The Panel includes the major U.S. producers and some 

processors of phthalate esters. These comments reiterate and expand upon the Panel’s comments 

of August 26, 2004, which were submitted in response to OEHHA’s May 28, 2004 Request for 

Information on Chemicals Under Consideration for Possible Listing via the Authoritative Bodies 

Mechanism. In addition, these comments address portions of OEHHA’s response to those earlier 

comments, contained in a March 1, 2005 letter from Dr. George V. Alexeeff, OEHHA Deputy 

Director of Scientific Affairs, to Ms. Courtney M. Price, Vice President of CHEMSTAR. These 

comments pertain to the four phthalates for which OEHHA has provided notice that it intends to 

list as chemicals known to the state to cause reproductive toxicity under the Authoritative Bodies 

Mechanism of Proposition 65 – dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), di-nhexyl phthalate (DnHP), and diisodecyl phthalate (DIDP). For the reasons presented in both the 

August 2004 comments and these comments, the Panel strongly believes that these phthalates 

should not be listed under Proposition 65. These comments renew in whole the comments 

submitted by the Panel in August 2004, and make the following points: 

• OEHHA’s responses to the Panel’s earlier comments that new marmoset data strongly 

suggest the effects observed in rodents are not relevant to humans are not sufficient to 

invalidate the marmoset as a model for investigating the potential effects of phthalates on 

human reproduction. In general, a primate is considered to be a more relevant species 

than rats for human risk assessment, since humans are themselves primates. 

• OEHHA’s stated basis for listing these phthalates under the Authoritative Bodies 

Mechanism is the monographs published in 2003 by the National Toxicology Program 

Center for the Evaluation of Risks to Human Reproduction (NTP-CERHR). OEHHA 

acknowledged in its response that the marmoset data were “relevant for DBP, BBP, 

DnHP, and DIDP” and thus considered by OEHHA to be “‘new data’ that were not 

considered by the authoritative body.” Because the marmoset data were not considered 

by the authoritative body (NTP-CERHR), and those data clearly establish that the 

association between adverse reproductive effects in humans and the phthalates is not 

“biologically plausible,” these four phthalates fail to meet the listing criteria of 

Proposition 65. 

• In its representation of NTP-CERHR’s conclusions, OEHHA fails to acknowledge that 

NTP-CERHR found minimal to negligible risk of human reproductive toxicity for these 

phthalates. Because NTP-CERHR did not clearly conclude that the phthalates cause 

reproductive toxicity in humans, the Proposition 65 listing criteria are not met for these 

four phthalates. 

• OEHHA has no statutory mandate to list chemicals such as these phthalates, which pose 

no significant risk to public health. Therefore, listing these phthalates under Proposition 

i 

Appendix III
65 makes little sense from a public policy perspective, as it would likely to lead to public 

and regulatory concern about these substances that is not warranted in light of the data for 

them. Proposition 65 listing also is likely to lead to reformulation of products away from 

the listed phthalates, and toward other chemicals about which less reproductive toxicity 

information may be known. The Panel strongly believes that sound public policy would 

avoid promoting such consequences through Proposition 65 listing, where human 

exposures to substances – such as DBP, BBP DnHP and DIDP – have been shown to 

pose a very low risk of reproductive or developmental toxicity.

The Panel believes that DBP, BBP, DnHP and DIDP do not meet the criteria for listing 

under Proposition 65 pursuant to the authoritative bodies mechanism, and that in any event 

would be poor public policy to list such low-risk chemicals. Therefore, OEHHA should not list 

these four phthalates under Proposition 65.

ii 

Appendix III
TABLE OF CONTENTS

EXECUTIVE SUMMARY ............................................................................................................. i

INTRODUCTION ...........................................................................................................................1 

I. OEHHA’S RESPONSES TO THE PANEL’S EARLIER COMMENTS ARE 

NOT SUFFICIENT TO INVALIDATE THE MARMOSET AS A SUITABLE 

MODEL OF HUMAN REPRODUCTIVE PHYSIOLOGY ...............................................2 

A. The Features of Marmoset Reproductive Physiology Listed by OEHHA in 

its Response Letter Are Insufficient to Invalidate the Marmoset as a Model 

for Investigating the Potential Effects of Phthalates on Human 

Reproduction............................................................................................................2 

1. Marmoset Sertoli Cell Morphological Uniformity Does Not 

Invalidate the Marmoset as a Model for Human Reproductive 

Toxicity........................................................................................................3 

2. The Marmoset’s Pituitary Production of Chorionic Gonadotropin, 

Rather Than Luteinizing Hormone Does Not Invalidate the 

Marmoset as a Model for Human Reproductive Toxicity ...........................4 

3. The Inability to Co-Transplant Marmoset and Hamster Testicular 

Tissue into Nude Mice Does Not Invalidate the Marmoset as a 

Model for Human Reproductive Toxicity....................................................4 

4. Marmoset Twin Germ Cells Likely Are Not Chimeric, and 

Therefore Marmoset Chimerism Does Not Invalidate the 

Marmoset as a Model for Human Reproductive Toxicity ...........................5 

B. The Conclusions of Zuhkle and Weinbauer Regarding the Use of 

Marmosets as a Model for Human Reproductive Toxicity are Incorrect, 

and Possibly Biased .................................................................................................5 

C. Vitamin C Levels in the Marmoset Do Not Negate the Relevance of the 

Marmoset Study to Human Risk Assessment..........................................................6 

D. Based on Pharmacokinetic Differences, Marmosets Are Less Susceptible 

Than Rodents to Developmental Toxicity from Phthalate Exposure ....................10 

II. THE MARMOSET DATA COMPRISE SCIENTIFICALLY VALID DATA, 

NOT CONSIDERED BY NTP-CERHR, WHICH ESTABLISH THAT THE 

PHTHALATES DO NOT MEET THE CRITERIA FOR LISTING UNDER 

PROPOSITION 65.............................................................................................................10 

III. NTP-CERHR FOUND MINIMAL TO NEGLIGIBLE RISK OF HUMAN 

REPRODUCTIVE TOXICITY FOR THESE PHTHALATES; THEREFORE 

THE PROPOSITION 65 LISTING CRITERIA ARE NOT MET ....................................13 

i 

Appendix III
IV. OEHHA HAS NO STATUTORY MANDATE TO LIST CHEMICALS SUCH 

AS THESE PHTHALATES, WHICH POSE NO SIGNIFICANT RISK TO 

HUMAN HEALTH; THEREFORE, LISTING OF THESE PHTHALATES 

MAKES LITTLE SENSE FROM A PUBLIC POLICY PERSPECTIVE........................16 

CONCLUSION..............................................................................................................................17 

ii 

Appendix III
INTRODUCTION

The American Chemistry Council Phthalate Esters Panel (Panel) submits these comments 

in response to the California Office of Environmental Health Hazard Assessment (OEHHA) 

Notice of Intent to List Chemicals of March 4, 2005.1

 The Panel includes the major U.S. 

producers and some processors of phthalate esters.2

 These comments pertain to the four 

phthalates that OEHHA has stated it intends to list as chemicals known to the state to cause 

reproductive toxicity under the Authoritative Bodies Mechanism of Proposition 653

 – dibutyl 

phthalate (DBP), butyl benzyl phthalate (BBP), di-n-hexyl phthalate (DnHP), and diisodecyl 

phthalate (DIDP). OEHHA states that the basis for these listings is the monographs published in 

2003 by the National Toxicology Program Center for the Evaluation of Risks to Human 

Reproduction (NTP-CERHR, 2003a,b,c,d). 

At the outset, the Panel reaffirms in whole its August 26, 2004 comments, which were 

submitted in response to OEHHA’s May 28, 2004 Request for Information on Chemicals Under 

Consideration for Possible Listing via the Authoritative Bodies Mechanism.4

 In addition, these 

comments reiterate and expand upon the Panel’s earlier comments, and address portions of 

OEHHA’s response to those earlier comments, which are contained in a March 1, 2005 letter 

from Dr. George V. Alexeeff, OEHHA Deputy Director of Scientific Affairs, to Ms. Courtney 

M. Price, Vice President of CHEMSTAR. 

Part I of these comments addresses OEHHA’s responses to the Panel’s previous 

submission of marmoset data (not considered by NTP-CERHR), which strongly suggest that the 

effects of phthalates observed in rodents are not relevant to humans. In its response, OEHHA 

listed four features of marmoset reproductive physiology, based upon which it concluded that the 

marmoset is not a suitable model for evaluating the potential reproductive effects of phthalates 

on humans. Because these features have not been shown to be relevant to the mechanism by 

which phthalates affect reproduction in the species most sensitive to phthalate perturbation (i.e. 

rodents), and do not appear to interfere with other phthalate effects known to manifest in 

marmosets (e.g., liver enzyme induction), they do not invalidate the marmoset as a model for 

investigating the potential effects of phthalates on human reproduction. The Panel continues to 

believe that the marmoset data strongly suggest that phthalates do not pose a reproductive 

toxicity hazard for humans. 

OEHHA acknowledges in its response that the marmoset data were “relevant for DBP, 

BBP, DnHP, and DIDP” and thus considered by OEHHA to be “‘new data’ that were not 

considered by the authoritative body.” Part II of these comments explains that, because the 

marmoset data were not considered by the authoritative body (NTP-CERHR), and those data 

 

1

 California Regulatory Notice Register 05, No. 9-Z, pp. 289-290 (March 4, 2005); 

http://www.oehha.ca.gov/prop65/CRNR_notices/admin_listing/intent_to_list/noilpkg21.html. 

2

 The Panel’s members include BASF Corporation, Eastman Chemical Corporation, ExxonMobil 

Chemical Company, Ferro Corporation, and Teknor Apex Company. 

3

 See CAL. HEALTH & SAFETY CODE § 25249.8(b); CAL. CODE REGS. tit. 22, § 12306. 

4

 http://www.oehha.ca.gov/prop65/CRNR_notices/admin_listing/requests_info/dcallin21.html#get. 

1

Appendix III
clearly establish that the association between adverse reproductive effects in humans and the 

phthalates is not “biologically plausible,” these four phthalates fail to meet the listing criteria of 

Proposition 65. 

Part III of these comments points out that, even assuming that the rodent data cited by 

NTP-CERHR and relied upon by OEHHA are relevant to humans, NTP-CERHR generally found 

that these phthalates posed minimal to negligible risk of reproductive or developmental effects in 

humans. Nevertheless, in its representation of NTP-CERHR’s conclusions, OEHHA fails to 

acknowledge qualifying language, the result of which is an overstatement of NTP-CERHR’s 

findings of phthalate reproductive toxicity. Because NTP-CERHR did not clearly conclude that 

the phthalates cause reproductive toxicity, the Proposition 65 listing criteria are not met for these 

four phthalates. 

Part IV of these comments makes the point that OEHHA has no statutory mandate to list 

chemicals such as these phthalates, which pose no significant risk to public health. Therefore, 

the Panel believes that listing these phthalates under Proposition 65 makes little sense from a 

public policy perspective as it would likely to lead to public and regulatory concern about these 

substances that is not warranted in light of the data for them, and would also likely lead to 

reformulation of products away from the listed phthalates, and toward other chemicals about 

which less reproductive toxicity information may be known. 

For the reasons given in these comments, and in its August 2004 comments, the Panel 

believes that DBP, BBP, DnHP and DIDP do not meet the criteria for listing under Proposition 

65 pursuant to the authoritative bodies mechanism, and that in any event would be poor public 

policy to list such low-risk chemicals. Therefore, OEHHA should not list these four phthalates 

under Proposition 65. 

I. OEHHA’S RESPONSES TO THE PANEL’S EARLIER COMMENTS ARE NOT 

SUFFICIENT TO INVALIDATE THE MARMOSET AS A SUITABLE MODEL 

OF HUMAN REPRODUCTIVE PHYSIOLOGY 

A. The Features of Marmoset Reproductive Physiology Listed by OEHHA in its 

Response Letter Are Insufficient to Invalidate the Marmoset as a Model for 

Investigating the Potential Effects of Phthalates on Human Reproduction 

In its August 26, 2004 comments, the Panel stressed that a recent study on marmosets 

(MCSI, 2003; Tomonari, 2004), which had not been evaluated by NTP-CERHR, strongly 

suggests that DEHP and other phthalates do not pose a reproductive toxicity hazard for humans. 

This study demonstrated that daily administration of 2500 mg DEHP/kg/day from weaning 

through maturity did not affect male reproductive tract development in the marmoset. In its 

response to this comment, OEHHA stated: 

OEHHA agrees with [the Panel] that these data are relevant for DBP, BBP, 

DnHP, and DIDP. . .and thus considers this study as “new data” that were not 

considered by the authoritative body. 

However, OEHHA then listed four features of marmoset male reproductive physiology, based 

upon which it concluded that the marmoset is not a suitable species for investigating potential 

2

Appendix III
reproductive effects of DEHP (and by implication other phthalates) in humans. The comments 

below demonstrate that these four features of marmoset reproductive physiology do not 

invalidate the marmoset as a model for investigating potential human reproductive effects of 

phthalates. 

Initially, it is reasonable to presume that unique features of any non-human animal model 

can be cited that might affect the comparison of that model to humans. For example: 

• rhesus macaque males display seasonal variation in gonadal function and testosterone 

production that is not mirrored in humans, marmosets or rats; 

• macaques exhibit very low levels of inhibin B neonatally, relative to rats and humans; 

and 

• rats and macaques show a segmental/radial distribution of stages of spermatogenesis 

within the testis, while humans and marmoset exhibit a semi-helical organization 

(Sharpe, et al., 2000). 

Because all animal models will be different from humans in some respects, merely pointing out 

those differences does not provide justification sufficient to reject a particular model. Rather, it 

is critical to determine whether the differences between the animal model and humans are 

pertinent to the specific comparison, which in turn depends on whether the mechanism that 

produces the effects of a given compound is relevant to the particular species differences 

described. Absent strong evidence to the contrary, a primate is considered to be a more relevant 

species than rats for human risk assessment, since humans are themselves primates. 

With this in mind, what follows is an examination of the four features of marmoset 

reproductive physiology cited by OEHHA in its response to the Panel’s comments, and an 

analysis of whether the differences between marmoset and human male reproductive physiology 

are pertinent to a determination of whether the marmoset is a valid model of human male 

reproduction. This analysis shows that there is no evidence that the features of marmoset 

reproductive physiology listed in OEHHA’s response letter are related to the mechanism by 

which DEHP, or other phthalates, affect male reproduction. This analysis (and that of Section 

I.B.) is based on a review of relevant research by Dr. Suzette Tardif, Associate Director of the 

Southwest Foundation for Biomedical Research, who is a leading authority on marmoset 

reproduction.5

1. Marmoset Sertoli Cell Morphological Uniformity Does Not Invalidate 

the Marmoset as a Model for Human Reproductive Toxicity 

OEHHA’s Response 1 to Comment 1: “There is no morphological variation in the 

spermatogenic epithelium . . . [indicating that] marmosets are totally different from most 

other mammals studied, including rodents and humans.” 

 

5

 Dr. Tardif’s comments reflect her opinions and are not to be interpreted as necessarily the 

opinions of the Southwest Foundation for Biomedical Research. 

3

Appendix III
This statement appears to be based on the conclusions of Rune, et al. (1992) that there 

was no indication of “a dependent relationship between the spermatogenic stages and Sertoli cell 

morphology” and that “[a]s far as this finding is concerned, marmoset Sertoli cells differ from 

those of other species.” While Sertoli cells appear to be an initial target of DEHP (and MEHP) 

in rodents, there is no evidence that this difference (Sertoli cell morphological uniformity) is in 

any way related to the mechanism by which DEHP affects the Sertoli cells. In fact, despite this 

difference, Rune, et al. conclude that, because marmoset Sertoli cells appear and behave 

similarly in vitro and in vivo, “the adult marmoset monkey could provide a primate model for 

mature Sertoli cells in culture, since there is [also] a close similarity to human adult Sertoli cells 

in vitro and in vivo.” 

2. The Marmoset’s Pituitary Production of Chorionic Gonadotropin, 

Rather Than Luteinizing Hormone Does Not Invalidate the Marmoset 

as a Model for Human Reproductive Toxicity 

OEHHA’s Response 2 to Comment 1: [T]he pituitary of common marmosets does not 

produce [luteinizing hormone] LH. Instead, it produces chorionic gonadotropin (CG), 

which is only produced in the placenta of humans or rodents. . . .” 

The evidence does support the conclusion that the primary luteotrophic gonadotropin 

produced by the marmoset pituitary is CG, and not LH. However, this distinction may be 

inconsequential, as both CG and LH bind to the same receptors and CG essentially acts like LH 

in tissues such as the luteal cells of the ovary. Whether the difference in molecular structure of 

the pituitary gonadotropin is significant is unknown, and will depend upon the phthalates’ not yet 

established mechanism of action. On the other hand, it is clear that the basic hypothalamicpituitary-gonad control mechanisms present in other primates are also present in marmosets. For 

example, release of pituitary gonadotropins and, subsequently, of testosterone production in male 

marmosets are affected by GnRH analogues in a fashion identical to that of other primates, 

including humans (e.g., Prince, et al., 1998). 

3. The Inability to Co-Transplant Marmoset and Hamster Testicular 

Tissue into Nude Mice Does Not Invalidate the Marmoset as a Model 

for Human Reproductive Toxicity 

OEHHA’s Response 3 to Comment 1: “Recent studies using transplanting techniques 

have shown that the conditions needed for initiation of spermatogenesis in the marmoset 

are remarkably different from those present in other mammals.”

This comment appears to be based on the work of Wistuba et al. (2004) in which the 

authors co-grafted marmoset and hamster testicular tissue into nude mice, but were unable to get 

marmoset spermatogenesis to proceed beyond the spermatogonial stage. The failure of the 

grafting techniques described by Wistuba et al. is likely related to Response No. 2 above, i.e., the 

difference in gonadotropin structure in New World monkeys, such as marmosets, compared to 

Old World monkeys and rodents. Witsuba et al. acknowledge that although they attempted to 

circumvent this difference by administering human CG to the host hamsters, “it might be that the 

exogenous administration was not sufficient to achieve a microenvironment in the mouse 

recipient that mimics the situation in the marmoset.” This hypothesis would best be tested by 

4

Appendix III
attempting a similar grafting with testicular tissue from another New World monkey, such as a 

squirrel monkey, since all New World monkeys are thought to share the same LH receptor 

changes that likely drive the difference in pituitary gonadotropin. Such an experiment has not 

yet been attempted, and until is has been, no valid basis exists to conclude that the failed 

transplant experiments of Wistuba et al. indicate that marmosets are not a suitable human 

reproductive model. 

4. Marmoset Twin Germ Cells Likely Are Not Chimeric, and Therefore 

Marmoset Chimerism Does Not Invalidate the Marmoset as a Model 

for Human Reproductive Toxicity 

OEHHA’s Response 4 to Comment 1: “XX germ cells have been reported from the testes 

of male marmosets with a female twin . . . [t]he chimeric feature of [marmoset] twins is 

rare in most mammals including rodents and humans. . . .”

The chimeric nature of marmoset twin germ cells cited by OEHHA appears to be based 

on two older studies, Benirschke and Brownhill (1963) and Hampton (1973). However, the 

results of these older studies have not been replicated and OEHHA acknowledged in its response 

letter that the occurrence of germ cell chimeras in marmosets has been questioned (e.g., Ford and 

Evans, 1977) and that more study is needed to clarify this issue. Indeed, most investigators 

working with marmosets today agree that it is highly unlikely that germ cell chimerism occurs in 

marmosets (Gengozian et al., 1980; Ford and Evans, 1997). Moreover, given the presence of 

hematopoietic cells (which are known to be chimeric) in most marmoset tissues, it is difficult to 

definitively demonstrate that chimerism occurs in germ cells as opposed to supporting 

hematopoietic cells within the gonad. Consequently, this purported difference between 

marmosets and other mammals is unverified and should not be relied upon to conclude that 

marmosets are not a viable model for human reproduction studies. 

B. The Conclusions of Zuhkle and Weinbauer Regarding the Use of Marmosets 

as a Model for Human Reproductive Toxicity are Incorrect, and Possibly 

Biased 

OEHHA’s Response: “Because of the fundamental differences in the testis between 

common marmosets and humans, it has recently been suggested that ‘the use of this 

animal model cannot be recommended for reproductive toxicology assessment’ (Zuhkle 

and Weinbauer, 2003).”

Most of the points raised in Zuhkle and Weinbauer (2003) are the same as those 

enumerated by OEHHA in Responses 1 – 4 above. Some of the additional points raised by the 

authors (i.e., that high interindividual fluctuations of steroid hormone levels makes monitoring of 

ovarian cycle based upon serum concentrations difficult or not feasible; and that marmosets 

require a complex diet in captivity) are simply incorrect. In relation to the ability to monitor 

ovarian cyclicity based on serum hormone concentrations, numerous studies have used 

circulating estradiol and progesterone concentrations to track ovarian cyclicity, so this is not an 

issue. 

5

Appendix III
Moreover, the U.S. Food and Drug Administration (FDA), one of the five authoritative 

bodies specifically identified in Proposition 65 for the purposes of identifying chemicals as 

causing reproductive toxicity,6

 has proposed that the marmoset is an appropriate model for 

human health assessment, and may be a more appropriate model than the rat for evaluation of 

reproductive toxicity hazard to humans. For example, the FDA Safety Assessment of DEHP 

states that: 

Spermatogenesis in the marmoset is organizationally similar to the process that 

occurs in humans, with regard to length of the spermatogenic cycle, duration of 

spermatogenesis, and number of mitotic divisions (Millar et al., 2000; Weinbauer 

et al., 2001). Consequently, the marmoset has been described as an appropriate 

model for experimental studies of human spermatogenesis. By analogy, it can be 

assumed that DEHP-induced effects on this process seen in marmosets would be 

applicable for humans. 

(FDA, 2001, p. 35.) 

Finally, there is a possibility of bias of Zuhkle and Weinbauer in favor of the cynomolgus 

macaque as a model for reproductive toxicology, given that both authors work for Covance, a 

company that sells cynomolgus macaques (but not marmosets) to pharmaceutical firms and 

biomedical institutions. 

C. Vitamin C Levels in the Marmoset Do Not Negate the Relevance of the 

Marmoset Study to Human Risk Assessment 

OEHHA’s Response: “[V]itamins C and E are protective against the testicular effects of 

DEHP in rats or mice (Ishihara et al., 2000; Ablake et al., 2004). Common marmosets 

require high levels of dietary vitamin C so regular diets for this species usually contain 

high levels of vitamin C supplements (e.g., MCSI, 2003). Serum levels of vitamin C in 

common marmosets are markedly higher (2.56 mg/100ml in average; Flurer and Zucker, 

1987; 1989) than most other mammals (0.63 mg/100ml in average in humans; Hampl et 

al., 2004), suggesting a possibility of reduced sensitivity to DEHP in this species.”

As indicated by the above response, OEHHA is concerned that the lack of observed 

effects of DEHP on marmoset reproduction (e.g., MCSI, 2003; Tomonari et al., 2004) may be 

due to the protective action of high doses of vitamin C, rather than a difference in the effects of 

DEHP between rodents and primates. These concerns, however, are not well founded because: 

1) the levels of vitamin C used in Tomonari et al. (2004) are not high relative to the marmoset’s 

requirements and 2) based on the available science (discussed below), it is not clear that vitamin 

C affords any protection to primates from DEHP exposure. Moreover, if the level of vitamin C 

in the marmosets’ diet in Tomonari et al. in fact provided the degree of protection necessary to 

be responsible for the observed lack of effects at doses of 2500 mg/kg/day, then the level of 

vitamin C in the average human diet would be protective of similar exposures to DEHP (test 

exposures were more than 100,000-fold higher than CDC data demonstrate actually occur in 

humans). In other words, the vitamin C levels in the marmoset diet in the Tomonari et al. study 

 

6

 CAL. CODE REGS. tit. 22, § 12306(i)(5). 

6

Appendix III
were similar to normal levels in the human diet and, consequently, whether vitamin C had a 

protective effect is not directly relevant to a risk assessment. 

Marmosets, like all primates, require that their diet be supplemented with vitamin C 

(ascorbic acid) (NRC, 2003). Flurer et al. (1987) reported that marmosets need more vitamin C 

than do humans, suggesting that a minimum of 20 mg/kg/day (the same amount cited by NRC, 

2003) should be provided in the diet. Flurer et al. also stated that they consider the optimal 

vitamin C content in the diet of the marmoset to be 2,000 ppm. The diet used in Tomonari et al. 

provided 1g vitamin C per 1,000 g feed (0.1%, or 1,000 ppm, or about 80 mg/day), an amount 

recommended in the published literature (Layne and Power, 2003), and only one-half that 

recommended by Flurer et al. Thus, the amount of vitamin C used in the Tomonari et al. study 

was not excessive relative to the marmoset’s dietary requirements and any potential protection 

conferred by the vitamin C would not be out of line with the degree of protection afforded the 

marmoset by its natural diet. 

Moreover, it is not clear whether a vitamin C-supplemented diet even impacts DEHPinduced testicular effects. Ishihara et al. (2000) demonstrated that rats given vitamins C and E in 

drinking water (about 450-500 mg/kg/day vitamin C) exhibited reduced testicular effects, 

relative to animals not receiving vitamins, from exposure to 20,000 ppm (1,000 – 1,500 

mg/kg/day) DEHP in the diet. The absolute testes weights of DEHP/vitamin treated animals 

were significantly lower than controls (although testes-to-body weight ratios were comparable to 

controls), but significantly higher than DEHP-exposed rats that did not receive vitamins C and E. 

In addition, testicular pathology of DEHP/vitamin rats was improved relative to DEHP rats, 

though not entirely normal (spermatogenesis was present, but not at control levels; severe 

aspermatogenesis was not observed in DEHP/vitamin animals). Thus, the combination of 

vitamins C and E afforded some protection to the rats against the reproductive toxicity of high 

doses of DEHP to rats. 

Similarly, in Ablake et al. (2004), CD-1 male mice were fed a diet containing 2% DEHP 

for 15 days and then fed a DEHP-free diet with or without supplementation of 3.0 mg/mL 

vitamin C and 1.5 mg/ML vitamin E in drinking water for another 50 days. The results showed 

that the DEHP-treatment induced aspermatogenesis, but that the damaged seminiferous 

epithelium spontaneously recovered whether the vitamins were provided or not, indicating that 

the DEHP-induced aspermatogenesis was reversible. In addition, the supplementation of 

vitamins C and E in the diet significantly accelerated regeneration of the injured seminiferous 

epithelium, suggesting that the vitamins have a therapeutic effect on DEHP-induced 

aspermatogenesis. 

However, the potential protective effect of vitamin C in Ishitara et al. and Ablake et al. 

cannot be distinguished from that of vitamin E because, in both studies, the two vitamins were 

provided together. Verma and Nair (2001) showed that mice pretreated with vitamin E showed 

little or no signs of testicular toxicity following treatment with aflatoxin. On the other hand, 

Cave and Foster (1990) reported that very high levels of vitamin C (2 mM) were required for any 

protective effect against m-dinitrobenzene or m-nitrosonitrobenzene toxicity on Sertoli cells in 

vitro. Hence, it is possible that vitamin C had little impact on testicular toxicity, and that vitamin 

E played the larger role in the protective effect observed by Ishihara et al. in rats and Ablake et 

al. in mice. 

7

Appendix III
Even if vitamin C does protect rats and mice against the effects of DEHP exposure, 

because rodents produce their own vitamin C the protective effect of dietary vitamin C in 

primates would have to be much greater than in rodents to account for the results of Tomonari et 

al. For example, since rats produce about 150 mg/kg/day of their own vitamin C (Chatterjee, 

1973), the rats in the Ishihara et al. study were effectively exposed to a total vitamin C dose of 

about 600 – 650 mg/kg/day. Comparing the results of Ishihara et al. to Tomonari et al., rats 

given about 600 mg/kg vitamin C (plus 225 mg/kg vitamin E) exhibited smaller testes and 

reduced spermatogenesis after exposure to 1,000 mg/kg/day DEHP whereas marmosets given 

only about 360 mg/kg/day vitamin C had normal-sized testes and comparable spermatogenesis to 

controls (based on sperm counts) when ingesting 2,500 mg/kg/day DEHP. Thus, if the 

hypothesis is that dietary vitamin C accounted for the lack of effects seen in Tomonari et al., as 

opposed to a difference in the marmosets’ sensitivity to DEHP, then a much smaller dose of 

vitamin C (50 – 66% of the amount given to the rats) would have to have protected the 

marmosets against 2 – 2.5 times the amount of DEHP given to rats. Put another way, vitamin C 

would have to be about 3 – 5 times more protective in primates than rodents to account for the 

results of Tomonari et al. 

Indeed, if such a small amount of vitamin C in the diet had a complete protective effect 

against the high doses of DEHP given the marmosets, one might question the possible impact of 

DEHP exposure on human health. The RDA for vitamin C is 75 mg/person/day for women and 

90 mg/person/day for men (NRC, 2003), although the mean daily intake is about 100 mg/day 

based on NHANES III and CSF II surveys (NRC, 2003). If 80 mg/day was as protective to 

primates as suggested, then the risk to humans would appear quite low since human exposures to 

DEHP are at least 100,000 times lower than the amount received by the marmosets (McKee et 

al., 2004), and the human diet contains higher levels of vitamin C. Even if one were to calculate 

the protective potential of that much vitamin C on a mg/kg body weight basis, the 360 mg/kg/day 

dose of vitamin C (hypothetically) protected the marmosets from testicular effects at 2,500 

mg/kg DEHP (roughly a 7-fold protection factor). Applying this protection factor to an average 

human intake of 1.3 – 1.4 mg/kg/day vitamin C (90 – 100 mg/day for a 70 kg person), humans 

would be at no risk of testicular effects from DEHP exposures up to 6 mg/kg/day or roughly 

10,000 times the mean exposures as determined by the CDC (Blount et al., 2000; CDC, 2001; 

CDC, 2003). 

Thus, it seems unlikely that the amount of vitamin C provided the marmosets in 

Tomonari et al. invalidates the study’s findings of no effect. Further, even if vitamin C had a 

protective effect, it is unlikely that any human other than one severely deficient in vitamin C 

would be at risk of adverse effects from exposure to the amounts of DEHP found in the 

environment. 

In addition, contrary to OEHHA’s statement that “[s]erum levels of vitamin C in common 

marmosets are markedly higher (2.56 mg/100 ml in average; Flurer and Zucker, 1987; 1989) 

than most other mammals (0.63 mg/100ml in average in humans; Hampl et al., 2004),” human 

and marmoset serum levels of vitamin C are not that different. Hampl et al. (2004) indicate that 

mean vitamin C levels in human plasma range from about 0.64 mg/dL (36.3 μM) to 0.97 mg/dL 

(55 μM), with an average of about 0.8 mg/dL (44 μM). The serum vitamin C level of 2.6 mg/dL 

cited as “average” by OEHHA is derived from marmosets that were given 2,000 ppm dietary 

vitamin C (Flurer et al., 1987). This level is four times the minimum requirement cited by Flurer 

8

Appendix III
et al., and twice the recommended level of Layne and Power (2003). The marmosets in the 

Tomonari DEHP study were fed only 1,000 ppm vitamin C, with no reported vitamin C-related 

ill effects. Visual inspection of the Figure 2 in the Flurer et al. study (1987) indicates that, for 

marmosets, a 1,000 ppm diet results in a plasma vitamin C level of about 1.9 mg/dL. Therefore, 

it is equally, if not more, appropriate to conclude that average marmoset serum vitamin C levels 

are about 1.9 mg/DL, which is only a 2-fold difference from humans, not 4-fold as indicated by 

OEHHA. Even this small difference may not be statistically significant as the HPLC 

methodology used by the two different groups of investigators incorporated different detection 

systems, electrochemical detection (Hampl et al., 2005) and spectrophotometry (Flurer et al., 

1987). 

In any event, intracellular levels, not plasma levels, are probably responsible for any 

protective effect that vitamin C may afford. Intracellular ascorbate levels are about 100-fold 

greater than those found in plasma (Tsukaguchi et al., 1999). Intracellularly, vitamin C serves to 

maintain prosthetic ions in their reduced forms (e.g., Fe++), scavenges free radicals to protect 

tissues from oxidative damage, and functions as a cofactor in a number of enzyme systems 

involved in the synthesis of collagen, microsomal drug metabolism, and the processing of certain 

neurotransmitters and peptide hormones (Marcus and Coulston, 1990; Tsukaguchi et al., 1999). 

As summarized by the National Research Council (1989), vitamin C is absorbed in the intestine 

by a sodium-dependent transport system and distributed to body tissues via blood as an unbound 

anion. From the blood, vitamin C is taken up by cells via a saturable, high affinity, sodiumdependent, transport system that results in intracellular vitamin C levels in the mM range. This 

transport system has been identified in a variety of cell types including leukocytes (Moser, 

1987), endothelial cells (May and Qu, 2005), lung cells (Castranova et al., 1993), and Leydig 

cells (Moger, 1987). 

Because intracellular levels are what matter, it is the kinetic parameters (e.g., Km, Vmax) 

of the vitamin C transport systems in marmosets and humans, not the absolute plasma levels, that 

will determine whether plasma vitamin C levels provide any protection from DEHP-induced 

testicular toxicity. While these kinetic differences are not known, evolutionary pressures 

typically result in enzyme systems that operate most efficiently under typical biological 

conditions, which may vary significantly among species. Thus, the average plasma vitamin C 

levels in marmosets (1.9 mg/dL) and humans (0.8 mg/dL) noted at required dietary levels for 

each species (NRC, 1989; Layne and Power, 2003) probably afford each a comparable degree of 

protection, if any. In other words, directly comparing plasma vitamin C levels across species is 

probably not a reliable indicator of the relative degree of protection those plasma levels might 

afford each species. 

In summary, the need for supplemental vitamin C in primate and human diets reinforces 

the similarity between the two primate species. Since the amount of vitamin C administered in 

Tomonari et al. was in line with dietary recommendations, and since there is no reliable way to 

compare serum vitamin C levels across species, there is no reason to question the results of the 

study, and no reason to consider the results not relevant to assessing potential health effects in 

humans. The administration of medically appropriate amounts of vitamin C to the marmosets 

certainly would not appear to provide any scientific reason to prefer rodent data over the primate 

data for human hazard and risk assessment. Further, one might question whether it would have 

been scientifically appropriate, or even ethical, to withhold vitamin C from the marmosets. 

9

Appendix III
Indeed, had vitamin C been withheld or administered in artificially low doses, interpretation of 

any adverse findings would be difficult at best. 

D. Based on Pharmacokinetic Differences, Marmosets Are Less Susceptible 

Than Rodents to Developmental Toxicity from Phthalate Exposure 

OEHHA Comment: “In general, findings from [several studies cited by the Panel] 

clearly indicate that pharmacokinetic features of DEHP are qualitatively similar between 

marmosets and rats.” 

OEHHA concludes that data from several studies indicate that there are no DEHP 

pharmacokinetic differences between marmosets and rats, and that these studies do not support 

the Panel’s statement in its earlier comments that “primates are less susceptible than rodents for 

developmental toxicity based on metabolism, absorption and elimination.” On the contrary, 

several studies do support the conclusion that, based on pharmacokinetic differences, marmosets 

are less susceptible than rodents to developmental toxicity from phthalate exposure. 

Rhodes et al. (1986) reported that marmosets dosed with dietary DEHP at 2,500 

mg/kg/day achieved a maximum absorbed dose that was 10 to 25-fold lower than that of equally 

dosed rats. Similar results were obtained in studies in cynomologus monkeys (Astill, 1989). 

Both findings are supported by results of a recent study (Kurata et al., 2005) in which juvenile 

rats and marmosets were gavaged with 100 mg/kg DEHP. Plasma radioactivity measurements 

taken up to 24 hr post-dosing indicated that rats absorbed 20 to 100-fold more DEHP than 

marmosets. While this radiolabel study could not differentiate between DEHP and its 

metabolites, the results of Kessler et al. (2004) bear on this issue. In Kessler et al., pregnant and 

nonpregnant rats and marmosets were given oral doses of 30 or 500 mg/kg/day DEHP. In both 

species, MEHP was present in the blood at much higher levels than DEHP. In rats, the 

normalized areas under the concentration-time curves (AUCs) for MEHP were 100-fold higher 

than the normalized AUCs for DEHP; in marmosets, however, this difference was only about 10-

fold. There was also a significant interspecies difference in plasma MEHP levels. Peak blood 

levels of MEHP in rats were 2 to 4-fold higher than those in marmosets, while AUC 

measurements indicated that MEHP levels in rats were 4 to 12-fold higher than those of 

marmosets. Thus, current evidence indicates that, when exposed to similar levels of DEHP, rats 

experience much higher levels of the toxicologically relevant metabolite, MEHP, than do 

marmosets. This indicates that marmosets, and other primates, are less susceptible than rodents 

to developmental toxicity from phthalate exposure based on pharmacokinetic differences. 

II. THE MARMOSET DATA COMPRISE SCIENTIFICALLY VALID DATA, NOT 

CONSIDERED BY NTP-CERHR, WHICH ESTABLISH THAT THE 

PHTHALATES DO NOT MEET THE CRITERIA FOR LISTING UNDER 

PROPOSITION 65 

As discussed above, OEHHA stated in its response to the Panel’s earlier comments that 

the marmoset data in Tomonari et al. (2004) were “relevant for DBP, BBP, DnHP, and DIDP” 

and thus considered by OEHHA to be “‘new data’ that were not considered by the authoritative 

body.” After acknowledging this fact, OEHHA rejected the marmoset data, stating that several 

of the marmoset’s reproductive features make marmosets an unacceptable model for 

10

Appendix III
investigating developmental toxicity in humans. However, as the preceding section makes clear, 

OEHHA’s rejection of the marmoset study is unfounded; the marmoset is a suitable model for 

investigating the potential developmental toxicity of phthalates to humans. Because the 

marmoset study provides new data that were not considered by the authoritative body, and those 

data clearly establish that the association between adverse reproductive effects in humans and the 

phthalates is not “biologically plausible,” OEHHA’s decision to list these phthalates fails to meet 

the listing requirements of Proposition 65. 

Under Proposition 65 Section 12306(h), to list the phthalates OEHHA must first 

determine that an authoritative body has “formally identified” the phthalates as causing 

reproductive toxicity.7

 OEHHA must further determine that the studies considered by NTPCERHR satisfy the Section 12306(g) criteria for “as causing reproductive toxicity.”8

 According 

to regulations, a chemical is identified “as causing reproductive toxicity” when: 

(1) Studies in humans indicate that there is a causal relationship between the 

chemical and reproductive toxicity; or 

(2) Studies in experimental animals indicate that there are sufficient data, 

taking into account the adequacy of the experimental design and other 

parameters such as, but not limited to, route of administration, frequency 

and duration of exposure, numbers of test animals, choice of species, 

choice of dose levels, and consideration of maternal toxicity, indicating 

that an association between adverse reproductive effects in humans and 

the toxic agent in question is biologically plausible.9

NTP-CERHR has not concluded that studies in humans indicate a causal relationship 

between these phthalates and reproductive toxicity (the first criterion). Rather, as the OEHHA 

listing package recognizes, NTP-CERHR relied upon studies in rodents in reaching its ostensible 

conclusions. Therefore, in this case only Section 12306(g)(2) is relevant. As such, the 

phthalates should be listed only if the data from experimental animals indicate that an association 

between adverse reproductive effects and the phthalates is “biologically plausible.” 

Proposition 65 also contains a provision, Section 12306(h), which states: 

The lead agency [OEHHA] shall find that a chemical does not 

satisfy the definition of “as causing reproductive toxicity” if 

scientifically valid data which were not considered by the 

authoritative body clearly establish that the chemical does not meet 

the criteria of subsection (g), paragraph (1) or subsection (g), 

paragraph (2). 10

 

7

 See CAL. CODE REGS. tit. 22, § 12306(d). 

8

 See id. at § 12306(g)(1)-(2). 

9

 Id.

10 Id. at 12306(h). 

11

Appendix III
Thus, if scientifically valid data from experimental animals which were not considered by NTPCERHR clearly establish that an association between adverse reproductive effects and the 

phthalates is not “biologically plausible,” OEHHA must find that the phthalates do not satisfy the 

definition of “as causing reproductive toxicity.” The marmoset data in Tomonari et al., which 

were acknowledged by OEHHA to be new data that were not considered by NTP-CERHR, 

clearly establish that an association between adverse reproductive effects and the phthalates is 

not “biologically plausible.” 

Tomonari et al. (2004; and MCSI, 2003) conducted a repeated oral dose study of the 

effects of DEHP treatment on the development of the male reproductive tract in common 

marmoset monkeys (Callithrix jacchus). The animals were administered 0, 100, 500 or 2500 

mg/kg/day by gavage on a daily basis for 65 weeks, from weaning (about three months) until 

about 18 months of age. This exposure period covered the entire sexual maturation phase as 

marmosets reach sexual maturity at about 400 to 450 days (57-65 weeks). During the treatment 

period, the testosterone levels in all treated groups were similar to those of control groups. At 

the end of the treatment period, the animals were examined for gross and histologic evaluation of 

principal organs. The testes and accessory organs were subjected to light and electron 

microscopic examination, and measurements of hormone levels and sperm counts were carried 

out. 

No treatment-related abnormalities were observed in microscopic and functional 

examinations of the marmosets’ testes, and there were no treatment-related effects on sperm 

count. In addition, histochemical examination after 3β hydroxysteroid dehydrogenase staining 

did not reveal any alteration in steroid synthesis. The only significant effect observed, a dosedependent increase in P450 content, was considered to be an adaptive change and not an adverse 

affect. Thus, this study demonstrated that daily administration of high doses of DEHP (up to 

2,500 mg/kg/day) spanning the entire period of sexual maturation had no effect on male 

reproductive tract development in the marmoset. 

Therefore, the empirical data from marmosets, which were shown in the preceding 

section to be valid for assessing human reproductive toxicity, indicate that primates are at least 

much less sensitive to the effects of phthalates than are rodents and may in fact be refractory, as 

there was no evidence of effects at the highest levels tested. A similar lack of effect was noted 

by Kurata et al. (1998) in adult marmosets treated with 2,500 mg/kg DEHP for 13 weeks, and by 

Pugh et al. (2000) in adolescent cynomolgus monkeys treated with 500 mg/kg DEHP for 14 

days. 

Humans are primates, and therefore data from primate studies are likely much more 

indicative of what effect can be anticipated in humans than data from rats. The recent marmoset 

data, along with the data of Kurata et al. and Pugh et al., demonstrate that an association between 

phthalates and adverse reproductive effects in humans is not biologically plausible. Thus, 

scientifically valid data from experimental animals which were not considered by NTP-CERHR 

clearly establish that of the criteria for “as causing reproductive toxicity” are not met. Therefore, 

the phthalates fail to meet the Proposition 65 listing criteria. 

12

Appendix III
III. NTP-CERHR FOUND MINIMAL TO NEGLIGIBLE RISK OF HUMAN 

REPRODUCTIVE TOXICITY FOR THESE PHTHALATES; THEREFORE THE 

PROPOSITION 65 LISTING CRITERIA ARE NOT MET 

OEHHA’s stated basis for listing these phthalates is the monographs published in 2003 

by NTP-CERHR. (NTP-CERHR, 2003a,b,c,d). However, in representing NTP-CERHR’s 

conclusions in these monographs, OEHHA fails to acknowledge qualifying language, which 

results in an overstatement of NTP-CERHR’s findings of phthalate toxicity. For example, for 

DBP, BBP and DIDP, respectively, NTP-CERHR stated: 

In this case, recognizing the lack of human data and the clear 

evidence of effects in laboratory animals . . ., the NTP judges the 

scientific evidence sufficient to conclude that DBP may adversely 

affect human reproduction or development if exposures are 

sufficiently high. 

(NTP-CERHR, 2003b, emphasis added); and 

The NTP believes it is reasonable and prudent to conclude that the 

results reported in laboratory animals indicate a potential for 

similar or other adverse effects in human populations if exposures 

are sufficiently high. 

(NTP-CERHR, 2003a, emphasis added); and 

In this case, recognizing the lack of human data and the evidence 

of effects in laboratory animals, the NTP judges the scientific 

evidence sufficient to conclude that DIDP is a developmental 

toxicant and could adversely affect human development if the 

levels of exposure were sufficiently high.

(NTP-CERHR, 2003d, emphasis added). 

Thus, for each case in which NTP-CERHR made a determination of concern about a 

phthalate’s potential reproductive or developmental toxicity (it made no concern determination 

for DnHP), it qualified its determination by indicating the potential for toxicity only at 

“sufficiently high” exposure levels. Because of these qualifications, NTP-CERHR’s findings of 

potential toxicity are inextricably tied to exposure levels, which, as explained at length by the 

Panel in its earlier comments (Section III, Table 1), are not “sufficiently high” to indicate a 

potential for human effects. OEHHA fails to acknowledge the significance of NTP-CERHR’s 

use of this qualifying language. 

In addition to failing to acknowledge language linking risk to exposure, OEHHA ignores 

the fact that NTP-CERHR found minimal or negligible concern for human developmental or 

reproductive toxicity for DBP, BBP and DIDP. About DBP, NTP-CERHR stated: 

The NTP concurs with the CERHR Phthalates Expert Panel that 

there is minimal concern for developmental effects when pregnant 

13

Appendix III
women are exposed to DBP levels estimated by the Panel (2-10 

μg/kg bw/day);11

and 

 “The NTP concurs with the CERHR Phthalates Expert Panel that 

there is negligible concern for reproductive toxicity in exposed 

adults. 

(NTP-CERHR, 2003b, emphasis added). 

About BBP, NTP-CERHR stated: 

The NTP concludes that there is minimal concern for 

developmental effects in fetuses and children; 

and 

The NTP concurs with the CERHR Phthalates Expert Panel that 

there is negligible concern for adverse reproductive effects in 

exposed men. 

(NTP-CERHR, 2003a, emphasis added). 

About DIDP, NTP-CERHR stated: 

 “The NTP concurs with the CERHR Phthalates Expert Panel that 

there is minimal concern for developmental effects in fetuses and 

children ; 

and

“The NTP concurs with the CERHR Expert Panel that there is 

negligible concern for reproductive toxicity in exposed adults 

(emphasis added). 

(NTP-CERHR, 2003d, emphasis added).12

 

11 Based upon estimated DBP exposures among some women of reproductive age, the NTP did 

have “some concern” for DBP causing adverse effects to development to fetus of women so 

exposed. However, the exposure estimates causing “some concern” were based on preliminary 

urinary metabolite data from the CDC for a small, nonrepresentative sample of women (Blount et 

al., 2000; Kohn et al., 2000). CDC scientists subsequently analyzed data for women of 

childbearing age in a much larger and statistically representative sample (Manori et al., 2004). 

Those results showed that women of reproductive age had DBP exposure levels the same as or 

lower than other age groups of women. 

14

Appendix III
Moreover, regarding the toxicity of DnHP, NTP-CERHR stated: 

The NTP judges the scientific evidence insufficient to reach a 

conclusion regarding the potential for DnHP to adversely affect 

human development or reproduction. 

(NTP-CERHR, 2003c). Because NTP-CERHR reached no conclusion with regard to the 

potential of DnHP to adversely affect human reproduction or development, OEHHA’s justified 

its listing decision by relying on “the generally accepted assumption that ‘an agent that produces 

an adverse developmental effect in experimental animal studies will potentially pose a hazard to 

humans following sufficient exposure during development . . . .’” (citing EPA, 1991, emphasis 

added). In so doing, OEHHA both ignores the authoritative body’s explicit failure to conclude 

that DnHP adversely affects human development or reproduction and, again, fails to 

acknowledge qualifying language necessarily linking adverse affects to “sufficient exposure.” 

Therefore, even more so than for DBP, BBP and DIDP, OEHHA relies on an overstatement of 

the authoritative body’s assessment of DnHP’s toxicity to justify its listing. 

These statements by NTP-CERHR do not satisfy the prong of the first regulatory 

criterion that the authoritative body’s report must “conclude[] that the chemical causes 

reproductive toxicity” because for purposes of Proposition 65, the reproductive toxicity must 

plausibly be in humans.13 As discussed in Section II of these comments, the Proposition 65 

regulations define a conclusion of “as causing reproductive toxicity” as one that satisfies the 

requirement that studies in experimental animals indicate that there are sufficient data to show 

that an association between adverse reproductive effects in humans and exposure to the chemical 

in question is “biologically plausible.”14 NTP-CERHR does not “conclude” that the phthalates 

cause reproductive toxicity in humans under this definition. Instead, their statements conclude 

only that such effects occur in rodents. There is no finding that an association between the 

phthalates and adverse reproductive effects is biologically plausible. As such, the suggestion to 

treat the phthalates as potentially reproductively toxic in humans is merely a default assumption, 

not a conclusion of biological plausibility. 

As a result, OEHHA’s decision to list these four phthalates under Proposition 65 is based 

on its incomplete, and therefore overstated, representation of NTP-CERHR’s conclusions as to 

the phthalates’ potential toxicity. NTP-CERHR found only that these phthalates have the 

“potential” to adversely affect humans if concentrations are “sufficiently high,” and stated that it 

had minimal or negligible concern for developmental or reproductive effects in humans. By 

 

12 The NTP-CERHR stated that “[t]hese conclusions are based on the assumption that the general 

US population is exposed to DIDP at less than 30 μg/kg bw/day.” In its August 2004 comments, 

the Panel explained that, based on analogy to DINP exposures, urinary metabolite data indicated 

that the best estimate for ambient exposure to DIDP is ≤ 1 μg/kg/day (McKee et al., 2004). This 

exceedingly low exposure level is supported by another study which found that urinary levels of 

DIDP following exposure from the use of personal hygiene products were below detectable 

limits. (Stock et al., 2001; Stock personal communication). Thus, the NTP-CERHR’s overall 

conclusions of minimal to negligible concern from DIDP exposures are well supported. 

13 See CAL. CODE REGS. tit. 22, § 12306(d)(1). 

14 Id. at § 12306(g)(2). 

15

Appendix III
leaving out this language, OEHHA changes fundamentally the nature of NTP-CERHR’s 

conclusions. Because NTP-CERHR did not conclude that the phthalates cause reproductive 

toxicity, the Proposition 65 listing criteria are not met for these four phthalates. 

IV. OEHHA HAS NO STATUTORY MANDATE TO LIST CHEMICALS SUCH AS 

THESE PHTHALATES, WHICH POSE NO SIGNIFICANT RISK TO HUMAN 

HEALTH; THEREFORE, LISTING OF THESE PHTHALATES MAKES 

LITTLE SENSE FROM A PUBLIC POLICY PERSPECTIVE 

As stated in its August 2004 comments, the Panel believes it makes little sense to list 

these phthalates under Proposition 65, as it would be poor public policy to list chemicals for 

which the data clearly demonstrate no significant risk to public health. The Panel’s earlier 

comments demonstrated that low risk by showing that exposures to phthalates from all sources 

are well below what are likely to be Maximum Allowable Dose Levels (MADLs) for DBP, BBP, 

DnHP and DIDP. OEHHA’s response to those comments states that OEHHA has not calculated 

MADLs for these chemicals and so has no basis to agree or disagree with the Panel’s comment. 

This is a somewhat disingenuous response. The procedure for calculating a MADL is 

straightforward – select the most sensitive relevant study of sufficient quality, divide its no 

observed effect level by 1000, and multiply by either 70 or 58 kilograms, depending on whether 

the applicable reproductive effect is upon the male or upon the female or conceptus.15 This is 

precisely what the Panel did to generate likely MADL values. The Panel knows of no study 

OEHHA could select that would give a substantially lower MADL value. However, even if 

OEHHA were to calculate MADLs an order of magnitude below those calculated by the Panel, 

average exposures to the phthalates – from all sources – would still be well below the MADL. 

The Panel’s primary point, therefore, remains valid: the risks from phthalates are so low that it is 

highly unlikely that a Proposition 65 warning would be necessary for any product containing 

these phthalates. 

OEHHA also states that the question of whether exposures are below the MADL has no 

bearing on an authoritative bodies listing – that it is a question for consideration when and if the 

phthalates are listed. The Panel disagrees. There is no statutory mandate that OEHHA list each 

and every substance which an authoritative body has concluded to cause reproductive toxicity in 

animals. The statute states:

On or before March 1, 1987, the Governor shall cause to be published a list of 

those chemicals known to the state to cause cancer or reproductive toxicity within 

the meaning of this chapter, and he shall cause such list to be revised and 

republished in light of additional knowledge at least once per year thereafter. 

(CAL. HEALTH & SAFETY CODE § 25249.8, emphasis added) 

Part of the additional knowledge OEHHA could apply is knowledge that a given chemical is 

unlikely ever to pose a risk to human reproduction due the large gap between effect levels in test 

animals and human exposures. In addition, as discussed in Section III of these comments, the 

very authoritative body on which OEHHA relies for its proposed listings of DBP, BBP, DnHP 

and DIDP has found minimal to negligible concern that these phthalates will cause reproductive 

 

15 See CAL. CODE REGS. tit. 22, § 12803. 

16

Appendix III
effects in humans. In such a case, the Panel believes that good policy judgment dictates that the 

chemicals not be listed. 

OEHHA, in its response to the Panel’s earlier comments, describes Proposition 65 as a 

“public right-to-know statute” and an “informational resource” for the public about chemicals 

“known to cause reproductive toxicity.” Yet, as discussed at length in the Panel’s earlier 

comments, and expanded upon here, the toxicity and exposure data indicate it is highly unlikely 

that human exposures to these phthalates will in fact cause such effects. Thus, rather than the 

Proposition 65 list serving as a reliable “informational resource” about risks from phthalates, it 

would be misleading with respect to these substances. 

Moreover, inclusion of chemicals on the Proposition 65 list inevitably leads to public and 

regulatory concern about the chemicals thus listed. As a result, even though a company’s 

product may result in phthalate exposures below the maximum allowable dose level, exempting 

the product from warning requirements, 16 the stigma associated with using a chemical listed as 

“known to the State” to cause reproductive/developmental toxicity often forces companies to 

eliminate use of the listed chemical. Yet, where a chemical is well studied, such that its risks are 

well characterized, as for phthalates, use of an unlisted substitute chemical will not necessarily 

result in a public health benefit. The substitute may be unlisted because it is not as well-studied, 

so that its own hazards have not yet been discovered. It makes little sense to drive companies to 

make such a substitution where the data show that risks from the chemical are extremely low, as 

is the case for the phthalates. 

The use of a given chemical in products results from a balancing of safety, performance 

and cost. Reformulation away from the chemical is likely to cause degradation in at least one of 

those factors. The Panel strongly reiterates that sound public policy would avoid promoting such 

consequences through Proposition 65 listing, where human exposures to substances – such as 

DBP, BBP DnHP and DIDP – have been shown to pose a very low risk of reproductive or 

developmental toxicity. 

CONCLUSION

The data presented in the Panel’s earlier comments, and expanded upon here, support a 

conclusion that DBP, BBP, DnHP and DIDP do not pose a significant risk of reproductive or 

developmental toxicity in humans. The recent marmoset data and NTP-CERHR’s statements 

demonstrate that the Proposition 65 listing criteria are not met for these four phthalates. Further, 

it would not be good public policy to list such low-risk chemicals. The Panel therefore believes 

that OEHHA should not list these phthalates under Proposition 65. 

 

16 See CAL. HEALTH & SAFETY CODE § 25249.10(c); 22 CCR. § 12801. 

17

Appendix III
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20

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Comparative pharmacokinetics and subacute toxicity of di(2-ethylhexyl) phthalate (DEHP) in 

rats and marmosets : extrapolation of effects in rodents to man. Environ Health Perspect 65:299-

308. 

Rune, GM, Pretzer D, De Souza P, Bollmann U and Merker HJ (1992). Ultrastructure of adult 

and juvenile marmoset (Callithrix jacchus) Sertoli cells in vivo and in vitro. J Androl 13:560-

570. 

Sharpe RM, Walker M, Millar MR, Atanassova N, Morris K, McKinnell C, Saunders PTK and 

Fraser HM (2000). Effect of neonatal gonadotropin-releasing hormone antagonist administration 

on Sertoli cell number and testicular development in the marmoset: comparison with the rat. Biol 

Reprod, 62:1685-1693. 

Stock A, Silva M, Hodge C, Malek N, Reidy, J and Brock, J (2001). A pilot study assessing 

phthalate exposure after personal hygiene usage. The Toxicologist 60:20. 

Tomonari Y, Kurata Y, Kawasuso T, David RM, Gans G, Katoh M. 2004. Testicular toxicity 

study of di(2-ethylhexyl) phthalate (DEHP) in juvenile common marmoset. The Toxicologist 

72:385. 

Tsukaguchi H, Tokui T, Mackenzie B, Berger UV, Chen XZ, Wang Y, Brubaker RF and 

Hediger MA (1999). A family of mammalian Na+-dependent L-ascorbic acid transporters. 

Nature 399: 70-75. 

Verma RJ and Nair A (2001). Vitamin E ameliorates aflatoxin-induced biochemical changes in 

the testis of mice. Asian J Androl 3:305-9. 

21

Appendix III
Weinbauer GF, Aslam H, Krishnamurthy H, Brinkworth MH, Einspanier A, Hodges JK (2001). 

Quantitative analysis of spermatogenesis and apoptosis in the common marmoset (Callithrix 

jacchus) reveals high rates of spermatogonial turnover and high spermatogenic efficiency. Biol 

Reprod 64:120-6. 

Wistuba J, Mundry M, Luetjens M and Schlatt S (2004). Cografting of hamster and marmoset 

testicular tissue into nude mice does not overcome blockade of early spermatogenic 

differentiation in primate grafts. Biol Reprod 71:2087-2091. 

Zuhkle U and Weinbauer G (2003). The common marmoset as a model in toxicology. Toxicol 

Pathol 31(suppl): 123-127. 

22

Appendix III
January 19, 2006 

To: Marian Stanley, ACC 

From: Suzette Tardif, Ph.D. 

Subject: Findings regarding female reproductive physiology from the Mitsubishi 

Study #B000496, “Sixty-five week repeated oral dose toxicity study of 

DEHP in juvenile common marmosets” 

I have reviewed the findings from the above-referenced study relative to female 

reproductive physiology – specifically the findings relative to ovarian weight at the end 

of the study and to circulating estradiol concentrations throughout the study. I do not 

believe that these findings can be used to support or refute the conclusion that DEHP 

treatment resulted in precocious puberty in female marmosets, primarily due to the 

extremely low body weights of the majority of the subjects. My reasoning is as follows: 

1. The weights of female subjects at the end of the study (week 66 of study, when the 

animals would be approximately 78 weeks, or 17 months, old) is extremely low, 

particularly for the control group and for females receiving the lowest DEHP dose (100) 

– see Figure 1. The average weights for females in the four groups are all well below 

the average for a healthy marmoset at 17 months of age (350-370 grams). I believe this 

reflects the fact that the study procedures, involving daily gavage for many weeks in a 

row, resulted in many basically unhealthy animals with impaired growth. 

1=0; 2=100, 3=500; 4=2500

Treatment

0.0 1.0 2.0 3.0 4.0 5.0

Body weight

360

340

320

300

280

260

240

220

FIGURE 1. 

Appendix III
2. While the difference in average weights across the treatment groups is not significant 

at p<0.05 (F= 2.849, p= 0.065), there is a trend for females in the two highest DEHP 

doses to have higher weights (see Figure 1). In our colony, we would consider any 17 

month old animal below 275 grams as unsuitable for research use. If we use this 

criterion on the Mitsubishi study population, then only 3 out of 6 subjects in the control 

and low dose groups would be suitable, while 5/6 and 5/5 of the animals in the higher 

dose groups would be suitable. 

3. Estradiol concentrations appear to be bimodally distributed, with most under 50pg/ml 

and a few from 130-1300 pg/ml. I believe this bimodal distribution reflects the difference 

between pre-pubertal and post-pubertal state. The range for earliest age at which 

subjects displayed estradiol concentrations reflective of likely post-pubertal state is from 

around 51 weeks (or 12 months) of age to 65 weeks (or 17 months) of age. The lower 

limit on this range (12 months of age) is well within the norm for age of puberty for 

female marmosets. Animals who appear to remain prepubescent up to 17 months of 

age (n=5) and who appear to be pre-pubescent throughout the length of the study 

(n=11) are abnormal, displaying what I would call delayed puberty. This condition 

appears to be related to body weight – see Figure 2 

At least one E2 value > 130

Body weight

220 240 260 280 300 320 360340

2.4

2.2

2.0

1.8

1.6

1.4

1.2

1.0

.8

Observed

Logistic

Figure 2. Logistics regression of puberty occurrence (as assessed by at least one 

estradiol concentration over 130 pg/ml; 1=no; 2=yes) versus body weight. Body weight 

was a significant determinant of puberty occurrence (F = 10.64, df= 21, p=0.004). 

Appendix III
4. Ovarian weight is largely reflective of body weight. Relative ovarian weight (ovarian 

weight/body weight) is also still largely reflective of body weight – see Figure 3. 

Body weight

320300280260240220 340 360

Ovarian wt relative to body weight

1.2

1.0

.8

.6

.4

.2

dose

2500

500

100

0

Figure 3. 

In a general linear model analysis of relative ovarian weight x treatment, with body 

weight as a covariate, there is a marginal relationship between treatment and relative 

ovarian weight (F=2.787, df=3, p=0.070). If those subjects below 275 grams are 

removed (see #2), then there is no indication of a relation between treatment and 

relative ovarian weight (F=1.097, df=3, p=0.391), however the sample size is so small 

for the control and low dose group that the validity of the comparison becomes 

questionable. 

The higher ovarian weights were generally associated with higher body weights and 

occurrence of ovulation/corpus luteum formation – i.e., they are normal in a sexually 

mature female. The fact that these higher ovarian weights were seen in the group with 

the highest dose exposure is, I believe, simply due to the fact that this group contained 

more normal weight animals who, therefore, were more likely to have ovulated. Normal 

ovarian function in marmosets includes the development and maintenance of a large, 

steroidogenic interstitial gland. The persistent presence of this gland, along with the 

cyclical presence of corpus lutea, means that sexually mature females will have higher 

ovarian weights. I disagree with the study conclusion that such features are “usually 

observed in more mature females.” At the age at which these females were sacrificed, I 

would have expected most, if not all, of them to display evidence of sexual maturity. Appendix III
February 3, 2006 

Dr. Michael D. Shelby, CERHR Director 

NIEHS 

P.O. Box 12233 

MD EC–32 Research Triangle Park, 

North Carolina 27709 

Submitted electronically to shelby@niehs.nih.gov 

Dear Dr. Shelby, 

The following comments are submitted on behalf of the more than one million members 

and supporters of People for the Ethical Treatment of Animals (PETA), in response to the 

NTP-CERHR expert panel update on the reproductive and developmental toxicity of di(2-

ethylhexyl) phthalate (DEHP) which was prepared in November, 2005. PETA is the 

world’s largest animal rights organization and is committed to using the best available 

science to protect animals from suffering and to promote the acceptance of alternatives to 

animal testing. 

Summary of comments 

Recommendations for several of the data needs listed in the update’s summaries and 

conclusions section call for additional studies on animals, including primates. However, as 

noted in the American Chemistry Council (ACC) comments to the draft update (2005), 

these data needs have been sufficiently met by existing studies, including a number of 

recent studies that do not appear to have been fully considered by the expert panel. Existing 

data clearly demonstrate that primates, including humans, are much less sensitive than rats 

to the developmental and reproductive effects of DEHP. Considering that estimates of 

human exposures calculated by the ACC (2005) from recent CDC biomonitoring data are 

1,000 to 10,000-fold lower than NOELs determined in rats, the existing weight of evidence 

is clearly sufficient to establish conservative NOELs for all relevant human populations 

and exposures to safeguard the public health without subjecting additional animals to 

suffering and death. 

Low level dose-response data exist for DEHP and MEHP 

Under the heading “Significance of Perinatal Exposure”, section 3.1, the need for 

additional dose-response data correlating mono-(2-ethylhexyl) phthalate (MEHP) levels and 

developmental reproductive effects in rats is identified. However, the dose-responses of 

rats to DEHP and its toxicologically active metabolite MEHP at low levels are wellcharacterized and sufficient to establish a developmental NOEL of 46 mg/kg. Li et al. 

(2000) investigated the effects of low doses of DEHP and MEHP on the testicular 

development of rat pups. DEHP doses of 20, 100, 300 and 500 mg/kg were administered to 

3-day-old rat pups as a single dose by oral gavage. The investigators found that the lowest 

dose of DEHP that produced changes in neonatal testicular cells was 100 mg/kg. 

Appendix III
2

The results of a recent NTP continuous breeding study (2005) are consistent with these 

findings. Although the purpose of this study was to assess potential reproductive effects, 

developmental effects were also measured. DEHP doses of 10, 30, 100, 300, 1,000, 7,500, 

and 10,000 ppm were administered in feed to groups of 17 male and 17 female rats. 

Reproductive effects were noted in the 7500 ppm and 10,000 ppm groups. Developmental 

effects, such as decreases in the weights of sex organs and histological abnormalities, were 

also observed at these concentrations. While mention is made of small sex organs in several 

rat pups in the 300 and 1000 ppm groups, the authors viewed the toxicological significance 

of these findings as questionable since organ weights were normal, no other reproductive 

effects were noted, and the incidence of these findings was low. As a result, the 

developmental NOEL, as suggested by the expert panel, is most likely 1000 ppm, 

calculated to be no more than 46 mg/kg/day based on feed consumption. 

The effect of intravenous exposure to DEHP on the development of reproductive organs in 

male rat pups was investigated by Cammack et al. (2003). No effects of any kind were 

observed in animals treated with 60 mg/kg/day, while testicular changes were noted in the 

300 and 600 mg/kg/day dose groups. Other groups were dosed daily by oral gavage at 300 

and 600 mg/kg/day. The investigators noted that testes changes were generally more severe 

among animals dosed orally than intravenously. These consistent findings across studies 

support a conservative developmental reproductive NOEL of 46 mg/kg/day for oral 

exposure to DEHP and 60 mg/kg/day for intravenous exposure. 

ADME data in primates explains reduced sensitivity 

Under the heading “Extension of PBPK Model”, section 4, the need to extend ADME data 

across species into primates is identified and under the “Additional Data Needs” heading in 

vivo metabolic data on lipase across species is listed. There is ample data to conclude that 

primates are much less sensitive to the developmental and reproductive effects of DEHP 

than are rodents. In addition, the mechanisms responsible for this lower sensitivity can be 

understood on the basis of existing ADME data. A recent study by Tomonari et al. (2004) 

found that exposure to very high levels of DEHP – 2500 mg/kg/day – resulted in no 

observed effects on testicular development in marmosets. In addition, a recent human study 

found no developmental effects of DEHP in adolescents who had been exposed to high 

medical treatment related levels as neonates (Rais-Bahrami et al., 2004). 

Studies by Rhodes et al. (1986), Astill (1989) and Kurata et al. (2005) demonstrated that 

DEHP is absorbed 10 to 100-fold less efficiently in marmosets and cynomolgous monkeys 

than in rats. Furthermore, at higher doses absorption efficiency decreased with the peak 

blood level for MEHP in marmosets leveling off at 20 mg/L. Lipase is the enzyme that 

catalyzes the hydrolysis of DEHP to its toxicologically active metabolite MEHP. Ito et al. 

(2005) found that lipase activity in rats was more than 10-fold higher than in marmosets, 

and the Vmax/Km ratio was nearly 200-fold greater. In addition, MEHP and its metabolites 

remained in their more active, free forms in rodents, while in primates they were 

conjugated with glucuronide (Silva et al., 2003; Kato et al., 2004; Kurata et al., 2005). 

Glucuronide conjugation decreases their toxicological activity and increases their water Appendix III
3

solubility resulting in faster excretion in the urine. This conclusion is supported by the 

results of Kessler et al. (2004) who demonstrated that while the area under the plasma 

concentration versus time curves (AUCs) for MEHP was 3 to 10-fold higher in rats than in 

marmosets, the peak blood level was only 1 to 3-fold higher, a result of faster excretion of 

MEHP in marmosets. The observed lower sensitivity of primates to the developmental and 

reproductive effects of DEHP can therefore be explained by less efficient absorption, lower 

activity of lipase, and increased glucuronidation of MEHP and its metabolites resulting in 

faster excretion. This explanation is consistent with the observation that the AUC for 

MEHP was 100-fold higher than the AUC for DEHP in rats but only 10-fold higher in 

marmosets (Kessler, et al., 2004). Also, a much higher proportion of oral DEHP doses was 

excreted as DEHP in the feces in marmosets than in rats. 

Estimated human exposures are 1,000 to 10,000-fold lower than the experimental 

NOEL 

In its comments to the draft update, the ACC calculated human exposure to DEHP based 

on recent CDC urinary metabolite biomonitoring data. The range of exposures for all U.S. 

populations was conservatively estimated to be 3-30 μg/kg/day. This range is 1,000 to 

10,000-fold lower than the experimentally determined rat oral NOEL of 46mg/kg/day. 

The need for additional dose-response data correlating MEHP levels and developmental 

reproductive effects in rats has been met by studies of Li et al. (2000), Cammack et al. 

(2003) and NTP (2005). The results of each of these studies are consistent with establishing 

a conservative developmental reproductive NOEL in rats of 46 mg/kg/day for oral 

exposure to DEHP and 60 mg/kg/day for intravenous exposure. Developmental 

reproductive effects of DEHP exposure were not observed in humans (Rais-Bahrami et al., 

2004) or other primates (Tomonari et al., 2004) even at much higher exposure levels, such 

as those that have been found in medical treatment-related exposures. 

This lower sensitivity is adequately explained by species differences in the absorption, 

metabolism and excretion of DEHP and its active metabolites (Rhodes et al., 1986; Astill, 

1989; Silva et al., 2003; Kato et al., 2004; Kurata et al., 2005). Considering that estimates 

of human exposures calculated from recent CDC biomonitoring data are 1,000 to 10,000-

fold lower than NOELs determined in rats, the NTP can reasonably conclude that more 

animal data will not increase public safety and that the developmental NOELs based on 

existing data are sufficient. 

Please feel free to contact me at 610-586-3975 or via e-mail at JosephM@peta.org if you 

have any questions. 

Sincerely, 

Joseph Manuppello 

Research Associate, Research & Investigations 

People for the Ethical Treatment of Animals 

Appendix III
4

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Rais-Bahrami K, Nunez S, Revenis ME, Luban NL, Short BL. 2004. Follow-up study of 

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membrane oxygenation (ECMO) support. Environ Health Perspect 112:1339-1340. 

Rhodes C, Orton TC, Pratt IS, Batten PL, Bratt H, Jackson SJ, Elcombe CR. 1986. 

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Toxicologist 72:385. 

Appendix III