NTP-CERHR Monograph on the 

Potential Human Reproductive and 

Developmental Effects 

of Butyl Benzyl Phthalate (BBP)

 March 2003 NIH Publication No. 03-4487

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Table of Contents

Preface ............................................................................................................................................. i

Introduction..................................................................................................................................... ii

NTP Brief on Butyl Benzyl Phthalate (BBP) ..................................................................................1

References........................................................................................................................................4

Appendix I. NTP-CERHR Phthalates Expert Panel 

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

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

Appendix II. Phthalates Expert Panel Report on BBP

Preface ............................................................................................................................. II-i

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

General Toxicological and Biological Parameters .........................................................II-4

Developmental Toxicity Data........................................................................................II-10

Reproductive Toxicity ...................................................................................................II-17

Data Summary & Integration........................................................................................II-21

References.....................................................................................................................II-35

Tables............................................................................................................................II-40

Appendix III. Public Comments on the Phthalates Expert Panel Reports

AdvaMed........................................................................................................................ III-1

American Chemistry Council (12-7-2000).................................................................... III-5

American Chemistry Council (12-11-2000).................................................................. III-7

American Chemistry Council (4-13-2001).................................................................. III-58

Discovery Medical, Inc ................................................................................................ III-66

Environmental Working Group (11-3-2000)................................................................ III-67

Environmental Working Group (12-8-2000)................................................................ III-69

William Faber............................................................................................................... III-71

Healthy Environments & Product Safety Branch ........................................................ III-81

Health Care Without Harm .......................................................................................... III-83

Beverly Smith............................................................................................................... III-87

Swedish Chemical Inspection Agency......................................................................... III-88
i

The National Toxicology Program (NTP) 

established the NTP Center for the Evaluation 

of Risks to Human Reproduction (CERHR) 

in 1998. The 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. The 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

The CERHR broadly solicits nominations of 

chemicals for evaluation from the public and 

private sectors. The CERHR follows a formal 

process for review and evaluation of nominated 

chemicals that includes multiple opportunities 

for public comment. 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.

• availability of scientific evidence for 

reproductive and/or developmental toxicity. 

The CERHR convenes a scientific expert 

panel that meets in a public forum to review, 

discuss, and evaluate the scientific literature 

on the selected chemical. Public comment 

is invited prior to and 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. The CERHR expert panels use explicit 

guidelines to evaluate the scientific literature 

and prepare the expert panel reports. Expert 

panel reports are made public and comments 

are solicited. 

Next, the CERHR prepares the NTP-CERHR 

monograph. The NTP-CERHR monograph 

includes the NTP brief on the chemical evaluated, the expert panel report, and all public 

comments. 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 health. The NTPCERHR monograph is made publicly available 

electronically on the CERHR web site and in 

hard copy or CD-ROM from the CERHR.

Preface

1 Information about the CERHR is available on the 

web at http://cerhr.niehs.nih.gov or by contacting 

the director:

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

Research Triangle Park, NC 27709 

919-541-3455 [phone] 

919-316-4511 [fax]

shelby@niehs.nih.gov [email] 

Information about the NTP is available on the web 

at <http://ntp-server.niehs.nih.gov> or by contacting the NTP Office of Liaison and Scientific Review at the NIEHS:

liaison@starbase.niehs.nih.gov [email]

919-541-0530 [phone]
ii 

In 1999, the CERHR Core Committee, an advisory committee composed of representatives 

from NTP member agencies, recommended 

seven phthalates for expert panel review. 

These chemicals were selected because: 

(a) there is the potential for human exposure 

from their widespread use and occurrence within the environment, 

(b) they have a high production volume, 

(c) there is substantial scientific literature 

addressing the reproductive and/or 

developmental toxicities of these chemicals, and 

(d) they are of concern to the public. 

These seven phthalates are as follows:

• di(2-ethylhexyl)phthalate (DEHP) 

• di-isononyl phthalate (DINP) 

• di-isodecyl phthalate (DIDP)

• di-n-butyl phthalate (DBP) 

• butyl benzyl phthalate (BBP) 

• di-n-octyl phthalate (DnOP)

• di-n-hexyl phthalate (DnHP)

Phthalates are a group of similar chemicals 

widely used to soften and increase the flexibility of plastic consumer products such as 

shower curtains, medical devices, upholstery, 

raincoats, and soft squeeze toys. They are not 

bound to the plastics and can leach into the surrounding environment. The scientific literature 

on the reproductive and developmental toxicities of several phthalates is extensive. In addition, there is widespread public concern about 

the safety of phthalates. 

As part of the evaluation of phthalates, the 

CERHR convened a panel of scientific experts 

(Appendix I) to review, discuss, and evaluate 

the scientific evidence on the potential reproductive and developmental toxicities of each 

phthalate. There were three public meetings 

of this panel (August 17-19 and December 15-

17, 1999 and July 12-13, 2000). The CERHR 

received numerous public comments on the 

phthalates throughout the evaluation process. 

The NTP has prepared an NTP-CERHR monograph for each phthalate. This monograph 

includes the NTP brief on BBP, a list of the 

expert panel members (Appendix I), the expert 

panel’s report on BBP (Appendix II), and all 

public comments received on the expert panel’s 

reports on phthalates (Appendix III). The NTPCERHR monograph is intended to serve as a 

single, collective source of information on the 

potential for BBP to adversely affect human 

reproduction or development. Those interested 

in reading this report may include individuals, 

members of public interest groups, and staff of 

health and regulatory agencies. 

The NTP brief included within this report 

presents the NTP’s interpretation of the potential for exposure to BBP to cause adverse 

reproductive or developmental effects in people. It is based upon information about BBP 

provided in the expert panel report, the public 

comments, and additional scientific information available since the expert panel meetings. 

The NTP brief is intended to provide clear, 

balanced, scientifically sound information on 

the potential for BBP exposures to result in 

adverse health effects on development and 

reproduction. 

Introduction
iii

While there are biological and practical reasons for considering developmental toxicity 

and reproductive toxicity as 2 separate issues, 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, post-natal 

development, sexual maturity, and, again, 

production of sperm and eggs. 

In the past, toxic effects were often studied in a “life stage specific” manner. Thus, 

concerns for developmental toxicity were 

addressed by exposing pregnant mothers 

and looking for adverse effects in fetuses. 

Developmental toxicity was detected as 

death, structural malformations, or reduced 

weights of the fetuses just prior to birth. Reproductive toxicity was studied by exposing 

sexually mature adults to the chemical of interest and effects were detected as impaired 

capacity to reproduce. Over the years, toxicologists realized that exposure during one 

part of the life cycle could lead to adverse 

effects that might only be apparent at a different part of the life cycle. For example, exposure of a sexually mature individual to an 

agent 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 induced genetic damage might lead to 

death or a genetic disorder in the offspring. 

In this example, chemical-induced damage 

is detected in the next generation. In contrast, the reproductive system begins developing well before birth and continues until 

sexual maturity is attained. Thus, exposure 

of sexually immature animals, either before 

or following birth, to agents or conditions 

that adversely affect development of the 

reproductive system can result in structural 

or functional reproductive disorders. These 

effects may only become apparent after the 

exposed individual reaches the age of puberty or sexual maturity.

Thus, in the case of genetic damage induced 

in eggs or sperm, what might be considered 

reproductive toxicity 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 

these examples it is difficult to make a clear 

distinction between developmental and reproductive toxicity. This issue is important 

in considering the phthalate evaluations 

because evidence of developmental toxicity affecting reproductive capacity in later 

stages of the life cycle is reported for at least 

3 of the phthalates -BBP, DBP, and DEHP.

Developmental Toxicity versus 

Reproductive Toxicity
1

NTP Brief

What is BBP?

BBP is a clear, slightly viscous liquid with the 

chemical formula C19H20O4 and the structure shown in Figure 1. It is one of a group 

of industrially important chemicals known as 

phthalates. Phthalates are primarily used as 

plasticizers to add flexibility to plastics. The 

largest use of BBP is in the production of 

vinyl tiles. It is also used in a variety of other 

products such as food conveyor belts, artificial 

leather, automotive trim, and traffic cones. 

There is no evidence that BBP is used in toys 

or medical devices.

BBP is produced by sequentially reacting butanol and benzyl chloride with phthalic anhydride. U.S. annual production figures for BBP 

were not available.

Are People Exposed to BBP?*

Yes. There are several ways that people may be 

exposed to BBP at home or at work. Human 

exposure can occur during the manufacture of 

BBP, during the manufacture of BBP-containing products, during the use of such products, 

or through the presence of BBP in the environment.

Environmental exposures can occur through 

air, water, or food. Most people are probably 

exposed to BBP primarily through food. BBP 

migrates into foods, particularly fatty foods, 

from BBP-containing materials that are used 

to process food. 

The expert panel estimated that the U.S. general population is exposed to approximately 

2 μg/kg bw/day (micrograms per kilogram 

body weight per day). This reflects a total daily 

exposure of approximately 140 μg per person 

per day. By comparison, a small drop of water 

weighs approximately 30,000 μg and a grain of 

table salt weighs approximately 60 μg.

A recent study not available to the expert panel 

determined the amount of BBP metabolites in 

human urine (Blount et al., 2000). Kohn et al. 

(2000) and David (2000) used the data from 

that study to estimate daily exposure levels of 

BBP. Kohn et al. estimated that 95% of people 

exposed to BBP are exposed to 4 μg/kg bw/day 

or less, very close to the expert panel’s estimate. 

In another recent study (Anderson et al., 2001), 

it was shown that people efficiently absorb, 

metabolize, and excrete BBP. Volunteers given 

an oral dose of BBP excrete approximately 

75% of the dose in urine within 24 hours. Most 

of the dose is excreted as the mono-benzyl 

phthalate metabolite, with only a minor fraction excreted as the mono-butyl phthalate.

Workers producing BBP or BBP-containing 

products can be exposed through skin contact 

or inhalation. It has been estimated that such 

exposures might be as high as 286 μg/kg bw/

day, but are generally thought to be far below 

this level. 

Can BBP Affect Human Development or 

NTP Brief on Butyl Benzyl Phthalate 

(BBP)

O

O

O

O

Figure 1. Chemical structure BBP

* Answers to this and subsequent questions may 

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

or Unknown
2

NTP Brief

Clear evidence of adverse effects

Some evidence of adverse effects

Limited evidence of adverse effects

Developmental Toxicity

Reproduction?

Probably. Although, there is no direct evidence 

that exposure of people to BBP adversely 

affects reproduction or development, studies 

reviewed by the expert panel and subsequently 

published studies with laboratory rodents 

show that exposure to BBP can adversely 

affect development, including development of 

the male reproductive tract (Fig. 2). 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. 

Scientific decisions concerning human health 

risks are generally based on what is known 

as “weight-of-the-evidence.” Recognizing the 

lack of human data and the evidence of BBP 

effects in laboratory animals, the NTP judges 

the scientific evidence sufficient to support the 

levels of concern for effects on development 

and reproduction expressed below (Fig. 3). 

Summary of Supporting Evidence 

As presented in the expert panel report, studies in rats and mice have shown that prenatal 

exposure to high levels of BBP can result in 

a range of effects that include prenatal mortality, reduced growth, and skeletal, visceral, and 

external malformations. Reproductive toxicity 

studies in male rats reported that oral exposure 

to BBP can result in reduced sperm counts, histological changes in the testes, and reduced fertility. Such effects were seen at very high doses, 

typically greater than 1000 mg/kg bw/day. In 

BBP-exposed females, reproductive effects 

may have occurred but this was not clear due to 

the design of the study. 

Following completion of the expert panel 

report, results of a two-generation reproductive toxicity study of BBP in Sprague-Dawley 

rats were reported (Nagao et al., 2000). Male 

and female rats were exposed orally to BBP 

at doses of 0, 20, 100, or 500 mg/kg bw/day. 

Reproductive performance was not affected 

at any dose level. The key findings involved 

developmental effects in the offspring. These 

effects included reduced birth weights of both 

males and females, decreased anogenital distance in males, delayed preputial separation 

in males and, in postpubertal males, reduced 

serum testosterone levels, decreased spermatocytes and other histopathological changes in 

the testes. Females were less susceptible than 

males to adverse developmental effects on the 

reproductive tract. Most, but not all, of these 

effects were observed only in the highest dose 

group. The authors conclude that no effects 

Figure 2. The weight of evidence that BBP causes adverse developmental or 

reproductive effects in laboratory animals 

Developmental Toxicity

Reproductive Toxicity (Females)

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 Toxicity

Reproductive Toxicity (Males)
3

NTP Brief

were observed at 20 mg/kg bw/day. The only 

effects at 100 mg/kg bw/day were reduced 

pup weights for both males and females and 

increased relative kidney weight and decreased 

relative heart weight in males.

In the rodent developmental toxicity studies 

available to the expert panel the highest doses 

at which no effects were observed were 182 

mg/kg bw/day in mice and 185 mg/kg bw/day 

in rats. Noteworthy in the Nagao et al. study 

was that, although reproductive tract changes 

were observed in pups of both sexes, there were 

no effects on the capacity of these animals to 

reproduce when they reached sexual maturity.

In another study (Piersma et al., 2000), two 

gavage treatment regimens (gestation days 5 

through 16 or 5 through 20) were used to study 

the developmental toxicity of BBP in rats. 

Study groups included 10 pregnant dams and 

10 dose levels that ranged from 0-2100 mg/kg 

bw/day. Data were submitted to a benchmark 

approach for calculating Critical Effect Doses 

(CED) based on the authors’ selection of Critical Effect Sizes. The calculated CEDs for the 

five fetal endpoints considered (frequency of 

resorptions, fetal weight, extra lumbar rib 13, 

testicular dislocation, and fetal relative testis 

weight) were approximately the same as, or 

higher than, the NOAELs determined by the 

expert panel. 

BBP was studied to determine if it produced 

antiandrogenic-like effects on sexual development in male rats exposed from gestation day 

14 to postnatal day 3 (Gray et al., 2000). Pregnant dams were exposed by gavage to 750 mg/

kg bw/day. Exposure induced shortened anogenital distance in male but not female pups, 

reduced testis weights, female-like areolas/

nipples in some male pups, as well as some 

malformations in male reproductive tracts. This 

study demonstrates the antiandrogenic effects 

of BBP but, the use of a single high dose limits 

its utility in evaluating the potential for BBP to 

affect human reproduction or development. 

Are Current Exposures to BBP High 

Enough to Cause Concern?

Probably not. More data are needed to better 

understand human BBP exposure levels and 

Figure 3. NTP conclusions regarding the possibilities that human development 

or reproduction might be adversely affected by exposure to BBP 

Serious concern for adverse effects

Concern for adverse effects

Some concern for adverse effects

Minimal concern for adverse effects

Negligible concern for adverse effects

Reproductive effects (adult females) Insufficient hazard and/or exposure data

Reproductive effects (adult males)2

Developmental effects1

1Based on Kohn et al. (2000) estimated exposure of women of reproductive age 

(median, 1.2; 95th percentile, 4.5; maximum, 7.8 μg /kg bw/day)

2Based on Kohn et al. (2000) estimated exposures of the U.S. general population 

(median, 0.88; 95th percentile, 4.0; maximum, 29 μg /kg bw/day).
4

NTP Brief

how these exposures vary across the population. 

Although the general U.S. population presently 

appears to be exposed to BBP at levels that are 

not of immediate concern for causing adverse 

reproductive or developmental effects, data are 

not available to permit conclusions regarding 

the possibility of effects in various age groups, 

occupations, or socioeconomic strata. Based on 

the expert panel report, and more recent data 

on rodent toxicity and human exposure, the 

NTP offers the following conclusions.

The NTP concludes that there is minimal 

concern for developmental effects in fetuses 

and children.

This is based on the observation of no effects in 

rats at 20 mg/kg bw/day and the human exposure estimates of Kohn et al. (see footnotes to 

Fig. 3)

The NTP concurs with the CERHR Phthalates Expert Panel that there is negligible 

concern for adverse reproductive effects in 

exposed men. 

The data are insufficient to reach conclusions 

for exposed women.

References:

Anderson WAC, Castle L, Scotter MJ, Massey 

RC, Springall C. A bio-marker approach to 

measuring human dietary exposure to certain 

phthalate diesters. Food Additives & Contaminants, 18:1068-107 (2001).

Blount BC, Silva MJ, Caudill SP, Needham LL, 

Pirkle JL, Sampson EJ, Lucier GW, Jackson RJ, 

Brock JW. Levels of seven urinary phthalate 

metabolites in a human reference population. 

Environmental Health Perspectives, 108:979-

982 (2000).

David RM. Exposure to phthalate esters. Environmental Health Perspectives, 108:A440 (2000).

Gray LE, Ostby J, Furr J, Price M, Rao Veeramachaneni DN and Parks L. Perinatal exposure 

to the phthalates DEHP, BBP, and DINP, but not 

DEP, DMP, or DOTP, alters sexual differentiation of the male rat. Toxicological Sciences, 58: 

350-365. (2000).

Kohn MC, Parham F, Masten SA, Portier CJ, 

Shelby MD, Brock JW, Needham LL. Human 

exposure estimates for phthalates. Environmental Health Perspectives, 108: A440-A442 

(2000).

Nagao T, Ohta R, Marumo H, Shindo T, 

Yoshimura S and Ono H. Effect of butyl benzyl 

phthalate in Sprague-Dawley rats after gavage 

administration: a two-generation reproductive 

study. Reproductive Toxicology, 14:513-532 

(2000).

Piersma, AH, Verhoef, A, te Biesebeek, JD, 

Pieters, MN, Slob, W. Developmental toxicity of butyl benzyl phthalate in the rat using a 

multipledose study design. Reproductive Toxicology, 14: 417-425 (2000).

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.
I-1

Appendix I

Appendix I. NTP-CERHR Phthalates 

Expert Panel Report on BBP

A 16-member panel of scientists covering dis-

ciplines such as toxicology, epidemiology, and 

medicine was recommended by the Core Com-

mittee and approved by the Associate Director 

of the National Toxicology Program. Over the 

course of a 16-month period, the panel criti-

cally reviewed more than 500 documents on 7 

phthalates and identified key studies and issues 

for plenary discussions. At three public meet-

ings

1, the expert panel discussed these studies, 

the adequacy of available data, and identified 

data needed to improve future assessments. At 

the final meeting, the expert panel reached con-

clusions 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 

October 10, 2000, and the deadline for public 

comments was December 11, 2000 (Federal 

Register 65:196 [10 Oct. 2000] p60206). The 

Phthalates Expert Panel Report on BBP is 

provided in Appendix II and the public com-

ments received on that report are in Appendix 

III. Input from the public and interested groups 

throughout the panel’s deliberations was in-

valuable in helping to assure completeness and 

accuracy of the reports. The Phthalates Expert 

Panel Reports are also available on the CERHR 

website <http://cerhr.niehs.nih.gov>. 1Phthalate Expert Panel meeting dates were: 

August 17-19, 1999, in Alexandria, VA; December 

15-17, 1999, in Research Triangle Park, NC; and 

July 12-13, 2000, in Arlington, VA. 
I-2

Appendix I

Robert Kavlock, Ph.D. (chair)

EPA/ORD

Research Triangle Park, NC

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

Brown University

Providence, RI

Robert Chapin, Ph.D.

NIEHS

Research Triangle Park, NC

Michael Cunningham, Ph.D.

NIEHS

Research Triangle Park, NC

Elaine Faustman, Ph.D.

University of Washington

Seattle, WA

Paul Foster, Ph.D.

Chemical Industry Institute of Toxicology

Research Triangle Park, NC

Mari Golub, Ph.D.

Cal/EPA

Davis, CA

Rogene Henderson, Ph.D.

Inhalation Toxicology Research Institute

Albuquerque, NM

Irwin Hinberg, Ph.D.

Health Canada

Ottawa, Ontario, Canada

Ruth Little, Sc.D.

NIEHS

Research Triangle Park, NC

Jennifer Seed, Ph.D.

EPA/OPPT

Washington, DC

Katherine Shea, M.D.

North Carolina State University

Raleigh, NC

Sonia Tabacova, M.D., Ph.D.

FDA

Rockville, MD

Shelley Tyl, Ph.D.

Research Triangle Institute

Research Triangle Park, NC

Paige Williams, Ph.D.

Harvard University

Cambridge, MA

Tim Zacharewski, Ph.D.

Michigan State University, 

East Lansing, MI

Appendix I. NTP-CERHR Phthalates Expert Panel 

(Name and Affiliation)
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Appendix II

NTP-CERHR EXPERT PANEL REPORT 

on

Butyl Benzyl Phthalate

October 2000 NTP-CERHR-DINP-00
TABLE OF CONTENTS

1.0 CHEMISTRY, USAGE, AND EXPOSURE..................................................................................................1

1.1 Chemistry ....................................................................................................................................1

1.2 Exposure and Usage ....................................................................................................................1

2.0 GENERAL TOXICOLOGICAL AND BIOLOGICAL PARAMETERS .....................................................4

2.1 General Toxicity ..........................................................................................................................4

2.1.1 Human Data ......................................................................................................................4

2.1.2 Experimental Animal Data................................................................................................4

2.2 Toxicokinetics .............................................................................................................................7

2.3 Genetic Toxicity ..........................................................................................................................8

3.0 DEVELOPMENTAL TOXICITY DATA....................................................................................................10

3.1 Human Data...............................................................................................................................10

3.2 Experimental Animal Toxicity...................................................................................................10

3.2.1 Prenatal Development.....................................................................................................10

3.2.2 Postnatal Development....................................................................................................13

4.0 REPRODUCTIVE TOXICITY ...................................................................................................................17

4.1 Human Data...............................................................................................................................17

4.2 Experimental Animal Toxicity...................................................................................................17

5.0 DATA SUMMARY & INTEGRATION......................................................................................................21

5.1 Summary .............................................................................................................................21

5.1.1 Human Exposure.............................................................................................................21

5.1.1.1 Utility of Data to the CERHR Evaluation...........................................................21

5.1.2 General Biological and Toxicological Data ....................................................................21

5.1.2.1 Utility of Data to the CERHR Evaluation...........................................................23

5.1.3 Developmental Toxicity ..................................................................................................25

5.1.3.1 Utility of Data to the CERHR Evaluation...........................................................26

5.1.4 Reproductive Toxicity .....................................................................................................29

5.1.4.1 Utility of Data to the CERHR Evaluation...........................................................30

5.2 Integrated Evaluation ................................................................................................................32

5.3 Expert Panel Conclusions .........................................................................................................33

5.4 Critical Data Needs ...................................................................................................................34

6.0 REFERENCES............................................................................................................................................35

7.0 TABLES ......................................................................................................................................................40

Appendix II
Appendix II

II-i

PREFACE

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

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, including development, 

caused by agents to which humans may be exposed.

The following seven phthalate esters were selected for the initial evaluation by the Center: butyl 

benzyl phthalate, di(2-ethylhexyl) phthalate, di-isodecyl phthalate, di-isononyl phthalate, di-n-butyl 

phthalate, di-n-hexyl phthalate, and di-n-octyl phthalate. Phthalate esters are used as plasticizers in 

a wide range of polyvinyl chloride-based consumer products. These chemicals were selected for the 

initial evaluation by the CERHR based on their high production volume, extent of human exposures, 

use in children’s products, published evidence of reproductive or developmental toxicity, and public 

concern.

This evaluation is the result of three public Expert Panel meetings and 15 months of deliberations 

by a 16-member panel of experts made up of government and non-government scientists. This 

report has been reviewed by the CERHR Core Committee made up of representatives of NTP-participating agencies, by CERHR staff scientists, and by members of the Phthalates Expert Panel. 

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

evidence that a given exposure or exposure circumstance may pose a hazard to reproduction and the 

health and welfare of children; (2) provide objective and scientifically thorough assessments of the 

scientific evidence that adverse reproductive/development health effects are associated with exposure to specific chemicals or classes of chemicals, including descriptions of any uncertainties that 

would diminish confidence in assessment of risks; and (3) identify knowledge gaps to help establish 

research and testing priorities.

The Expert Panel Reports on phthalates will be a central part of the subsequent NTP report that will 

also include public comments on the Panel Reports and any relevant information that has become 

available since completion of the Expert Panel Reports. The NTP report will be transmitted to the 

appropriate Federal and State Agencies, the public, and the scientific community.

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:

CERHR

Sciences International, Inc.

1800 Diagonal Road, Suite 500

Alexandria, VA 22314-2808

Telephone: 703-838-9440
II-ii

Appendix II

A Report of the CERHR Phthalates Expert Panel: 

Name Affiliation

Robert Kavlock, PhD (Chair) National Health and Environmental Effects Research Laboratory/ 

 USEPA, Research Triangle Park, NC

Kim Boekelheide, MD, PhD Brown University, Providence, RI

Robert Chapin, PhD NIEHS, Research Triangle Park, NC

Michael Cunningham, PhD NIEHS, Research Triangle Park, NC

Elaine Faustman, PhD University of Washington, Seattle, WA

Paul Foster, PhD Chemical Industry Institute of Toxicology, RTP, NC

Mari Golub, PhD California Environmental Protection Agency, Sacramento, CA

Rogene Henderson, PhD Lovelace Respiratory Research Institute, Albuquerque, NM

Irwin Hinberg, PhD Health Canada, Ottawa, Ontario, Canada

Ruth Little, ScD NIEHS, Research Triangle Park, NC

Jennifer Seed, PhD Office of Toxic Substances/USEPA, Washington, DC

Katherine Shea, MD, MPH Duke University, Durham, NC

Sonia Tabacova, MD, PhD Food and Drug Administration, Rockville, MD

Rochelle Tyl, PhD, DABT Research Triangle Institute, Research Triangle Park, NC

Paige Williams, PhD Harvard University, Boston, MA

Timothy Zacharewski, PhD Michigan State University, East Lansing, MI

With the Support of CERHR Staff:

NTP/NIEHS

Michael Shelby, PhD Director, CERHR

Christopher Portier, PhD Acting Associate Director, NTP

Gloria Jahnke, DVM Technical Consultant

Lynn Goldman, MD Technical Consultant

Sciences International, Inc.

John Moore, DVM, DABT Principal Scientist 

Annette Iannucci, MS Toxicologist 

Ann Walker, MS, ELS Information Specialist and Technical Editor
Appendix II

II-1

1.0 CHEMISTRY, USAGE, AND EXPOSURE

1.1 Chemistry

Figure 1: Chemical Structure of Butyl Benzyl Phthalate

Butyl benzyl phthalate (BBP) (CAS 85-68-7) is produced by sequentially reacting butanol and benzyl chloride with phthalic anhydride (1).

Table 1: Physicochemical Properties of BBP

Property Value

Chemical Formula C19H20O4

Molecular Weight 312.35

Vapor Pressure 6 x 10-7 mmHg at 25 oC

Melting Point -40.5 oC

Boiling Point 370 oC

Specific Gravity 1.12

Solubility in Water slight – 2.7 mg/L

Log Kow 4.59

(1)

1.2 Exposure and Usage

According to the American Chemistry Council (ACC, formerly CMA) (1), the largest use of BBP 

is in vinyl tile. BBP is also a plasticizer in PVC used to manufacture food conveyor belts, carpet 

tile, artificial leather, tarps, automotive trim, weather stripping, traffic cones, and is used to a limited extent in vinyl gloves. BBP is also used in some adhesives. BBP may be released to the environment during its production and also during incorporation into plastics or adhesives. Because 

BBP is not bound to the final product, it can be released during the use or disposal of the product. 

Phthalates that are released to the environment can be deposited on or taken up by crops that are 

intended for human or livestock consumption, and thus, can enter the food supply.

General Population Exposure

General population exposure to BBP through food has been estimated by at least two authoritative 

sources: the International Program on Chemical Safety (IPCS) (2) and the UK Ministry of AgriculO

O

O

O
II-2

Appendix II

ture, Fisheries, and Food (MAFF) (3-5).

BBP may enter food by environmental uptake during crop cultivation or by migration from processing equipment or packaging materials. IPCS (2) concluded that BBP exposure to the general 

population is based almost entirely on food intake; these food exposure estimates were based on a 

survey of 100 food items that were purchased in four Ontario, Canada supermarkets between 1985 

and 1988. BBP was only found in yogurt (0.6 μg/g), cheddar cheese (1.6 μg/g), butter (0.64 μg/g), 

and crackers (0.48 μg/g). Assumptions used to estimate exposure included a 70 kg body weight, and 

a daily consumption of 13.61 g butter, 3.81 g cheddar cheese, 1.54 g yogurt, 22.73 g pork, and 3.45 

g crackers. Adult BBP intake was estimated at 2 μg/kg bw/day and it was stated that exposure to 

infants and children could be up to three-fold higher. 

MAFF (5) estimated adult BBP exposure through dietary intake based on a 1993 survey of fatty 

foods in the United Kingdom. BBP was detected in carcass meat (0.09 μg/g), poultry (0.03 μg/g), 

eggs (0.09 μg/g), and milk (0.002 μg/g). In calculating dietary food exposures, MAFF assumed 

that these types of food likely account for 85% of dietary phthalate intake. Food intake levels were 

obtained from the Dietary and Nutritional Study of British Adults, but the values were not reported 

by MAFF. Mean and high-level BBP intakes were estimated at 8 μg/person/day and 20 μg/person/

day, respectively. Specific details describing the calculations and assumptions used were not provided. Using the IPCS-assumed adult body weight of 70 kg (2), the exposure values were converted 

to 0.11-0.29 μg/kg bw/day.

MAFF also addressed BBP exposure in infants resulting from the consumption of infant formula. 

A survey published in 1996 reported BBP levels of <0.0044-0.24 μg/g in infant formulas purchased 

in the UK, while a later survey reported BBP levels of <0.003-0.015 μg/g (3, 4). It is speculated 

that the drop in BBP concentration occurred because infant formula manufacturers were urged to 

reduce phthalate levels after the MAFF published the results of the 1996 survey (3). Based on the 

results from the 1998 survey and using an assumed body weight of 2.5-3.5 kg at birth and 7.5 kg at 

6 months of age, exposure levels were estimated for infants. Formula intake rates were determined 

from manufacturer instructions. Exposure levels for infants were estimated at 0.2 μg/kg bw/day at 

birth and 0.1 μg/kg bw/day at 6 months of age. Infants in the United States are likely exposed to 

lower levels of BBP through formula. In a survey of infant formulas conducted in 1996, BBP levels 

were below the detection limit of 0.005 μg/g (6). 

BBP was only detected in one sample (2.8 μg/L) collected in 1991 in a survey of 300 drinking 

water sites in two Canadian provinces from 1985 to 1994. IPCS (2) considered exposure to BBP 

through drinking water negligible; exposure through soil intake was also considered negligible.

Mouthing of toys and other BBP-containing objects is a potential source of oral phthalate exposure 

in children. However, BBP is stated not to be used in toys (7). In an analysis of 17 plastic toys, BBP 

was only detected in a PVC doll’s head at 0.02% by weight (8), a level that suggests contamination 

rather than planned use.

Off-gassing from building materials has been reported as a potential source of BBP exposurethrough inhalation; however, exposure has been postulated to be minimal because of BBP’s low 
Appendix II

II-3

vapor pressure. The available data, though minimal, support this view. IPCS (2) reported that 

median air levels of 0.034-0.035 ng/m3 were measured in a survey of 125 California homes. BBP 

levels in outdoor air were also measured for 65 of these homes and the median BBP level was 

below the detection limit of 0.051 ng/m3. The 90th percentile levels of BBP in outdoor air ranged 

from 5.3 to 6.7 ng/m3 for daytime to evening. IPCS (2) considered BBP exposure through inhalation to be negligible. Pfordt and Bruns-Weller (9) measured BBP levels in 3 flooring samples and 

found BBP in each sample at levels ranging from 10-250 μg/g.

Dermal contact with products containing BBP is possible, but absorption through skin is most 

likely minimal. Studies in rats have demonstrated that absorption of BBP through skin is fairly slow 

(approximately 27% in 7 days) (10). An in vitro study conducted with rat and human skin has demonstrated that permeability of human skin to other phthalates (DBP and DEHP) is much lower than 

that of rat skin (11).

Interpretation of exposure levels for the general population requires caution. The exposure estimates by IPCS and MAFF differed by approximately one order of magnitude. The basis for discrepancies in dietary exposure estimates is difficult to determine for several reasons, including: 

use of different food types in calculations (e.g., fatty foods vs. a variety of foods); use of different 

assumptions in calculations; varying BBP levels in foods from different countries; and changing 

BBP levels in food over time. Dietary intake can vary widely depending on the types of foods eaten 

and the types of materials in which the foods are packaged. It is noted that the food levels reported 

by MAFF were collected 12-15 years ago and may not reflect current exposure levels. 

Medical Exposure

BBP is not approved by the U.S. Food and Drug Administration for use in medical devices.

Occupational Exposure

Exposure in occupational settings can occur through skin contact and by inhalation of vapors and 

dusts.

Phthalates are manufactured within closed systems, but exposure to workers can occur during filtering or loading/unloading of tank cars (1). Higher exposures to phthalates can occur during the incorporation of the phthalate into the final product if the process is run at a higher temperature than 

is used in the manufacturing process. The ACC has estimated exposure to BBP in the workplace 

based upon an assumed level of 1 mg/m3 during the production of phthalates and 2 mg/m3 during 

the manufacture of flexible PVC. An exposure level was estimated by using assumptions of a 10 

m3/day inhalation rate and a 70 kg body weight. The resulting exposure estimates were 143 μg/kg 

bw/workday and 286 μg/kg bw/workday for workers employed in phthalate manufacturing and flexible PVC production operations, respectively. As stated in the General Exposure section, absorption 

of BBP through skin is expected to be minimal. 

The summary for Section 1 is located in Section 5.1.1.
II-4

Appendix II

2.0 GENERAL TOXICOLOGICAL AND BIOLOGICAL PARAMETERS

2.1 General Toxicity

2.1.1 Human Data

BBP was not observed to be a primary irritant or sensitizer in skin patch tests with volunteers (2). 

There are no human data on the general toxicity of BBP alone. Occupational exposures to phthalate 

mixtures containing BBP have been associated in single studies with respiratory/neurological 

effects and cancer (2). In a large, population-based case-control study (12), a significant increase in 

the risk of multiple myeloma has been found among workers employed for 5 or more years in PVC 

production. In the general population, a significant increase in the risk of bronchial obstruction 

during the first 2 years of life has been related to presence of PVC flooring (adjusted O.R≥1.89) 

in a case control study of 251 children and an equal number of matched controls (13). The consequences of exposure to children have not been studied.

2.1.2 Experimental Animal Data

Multiple studies in mice and rats are available describing the acute, sub-chronic, and chronic toxicity of BBP. These studies assess oral as well as inhalation routes of exposure. There is a 90-day 

dietary toxicity study in dogs that includes effects that are possibly related to decreased food consumption.

Acute Studies

Acute toxicity of BBP is low; an oral LD50 value for BBP in rats is reported as 2-20 g/kg (2). Rabbit dermal and ocular studies revealed no significant concern for BBP-induced sensitization or irritation (14).

Sub-chronic Studies

Agarwal et al. (15) published a study that explored previous NTP results indicating effects on male 

fertility and the hematopoietic system (Table 7-1). Adult male F344 rats, 10 per group, were fed 

diets containing 0, 0.625, 1.25, 2.5, or 5.0% BBP for 14 days. Using actual pre-treatment body 

weights (200 g) and reported food intake during the 14-day dosing period, equivalent doses of 0, 

447, 890, and 1,338 mg/kg bw/day were calculated for the 3 lower dose groups. Since the high-dose 

group actually lost weight during the study, average weight during the study was used to calculate 

a dose of 1,542 mg/kg bw/day. All treated rats showed a dose-related increase in relative liver and 

kidney weights. No histopathology or hematology changes were observed at the 447 or 890 mg/kg 

bw/day dose levels. However, at doses of 1,338 and 1,542 mg/kg bw/day, relative decreases in testes, seminal vesicle, and thymus weight were noted; relative epididymis weight was reduced at the 

high dose. Dose-related histopathological changes in seminal vesicles, testes, and prostate were 

observed, as was a decrease in bone marrow cellularity at the two highest doses. Mild multifocal 

hepatitis and cortical lymphocytolysis in the thymus were also observed at the high dose. Increases 

in luteinizing hormone (LH) were observed at the lowest dose and two highest doses tested. An increase in follicle stimulating hormone (FSH) was observed in the two highest doses, and a decrease 

in testosterone was observed at the high dose. The decreased body weight seen at the two highest 

doses may be due to unpalatability of food; decreased food intake was documented. The severity of 
Appendix II

II-5

the reduced food intake and attendant weight loss precludes associating effects with BBP, or BBP 

and inanition, at the high dose. The systemic LOAEL determined from these studies is 447 mg/kg 

bw/day based on increases in organ weight (liver, kidney) and increased LH levels.

Three-month feeding studies were conducted in 4-6 week-old Wistar and Sprague-Dawley (SD) 

rats fed diets with 2,500-12,000 or 2,500-20,000 ppm BBP, respectively (14) (Table 7-2). Male 

Wistar rats (27-45 rats/sex/group) received doses of 151, 381, or 960 mg/kg bw/day; female doses 

were 171, 422, or 1,069 mg/kg bw/day. At the low dose, an increase in liver to body weight ratio 

was seen in both sexes. No histopathology or hematology changes were noted. At the mid-dose, a 

decrease in body weight was noted in both sexes and increases in liver and kidney to body weight 

ratios were seen. Pancreatic tissues showed islet cell enlargement, vacuolization, congestion, inflammation, and minor fibrosis. Less frequently, additional pancreatic changes were observed, such 

as acinar cell atrophy, inflammation, and pyknotic nuclei. A decrease was observed in urinary pH in 

male rats only. At the highest doses tested, 960 (M) and 1,069 (F) mg/kg bw/day, hepatic necrosis 

and anemia were observed in addition to the effects seen at lower doses. Cecal enlargement, a finding of uncertain toxicological importance, was reported in this study. The LOAEL for this study 

was 151–171 mg/kg bw/day based on weight change in the liver. 

In this same study, Sprague-Dawley (SD) rats (10/sex/group) were tested at doses of 0, 188, 375, 

750, 1,125, or 1,500 mg/kg bw/day. Sprague-Dawley rats were less sensitive to BBP than were Wistar rats, as no pancreatic, hepatic, or testicular lesions, or cecal enlargement were observed. There 

were no changes in urinary pH or hematological parameters. The NOAEL was set at 375 mg/kg 

bw/day and the LOAEL at 750 mg/kg bw/day based on increases in organ weight ratios for kidney 

(male) and liver (female) (14).

A 13-week inhalation study was also conducted in groups of 6-8 week-old SD rats (25/sex/group) 

(14) (Table 7-2). The rats were exposed to BBP mists (>90% of aerosol particles <10 μm) at concentrations of 51, 218, or 789 mg/m3 for 6 hours/day, 5 days/week. Using EPA (16) assumptions 

for rat body weights and daily inhalation rates, estimated exposure doses were 9.2, 39.4, and 143 

mg/kg bw/day for males and 9.8, 42.0, and 152 mg/kg bw/day for females. NOAELs of 39.4 (M) 

and 42.0 (F) mg/kg bw/day were identified in this study. A LOAEL was determined at the highest 

doses tested, 143 (M) and 152 (F) mg/kg bw/day; this LOAEL was based on increases in liver and 

kidney organ to body weight changes. Serum glucose levels were also reduced at this dose in male 

rats only. No body weight changes or histopathological changes were observed.

The NTP (17) reported results of a 26-week dietary exposure study in 6-week-old F344/N male 

rats (Table 7-3). Groups of 15 male rats were fed BBP in the diet at concentrations of 0, 300, 900, 

2,800, 8,300, or 25,000 ppm for 26 weeks. The authors calculated doses of 30, 60, 180, and 550 

mg/kg bw/day for the 4 lowest exposure levels. A dose was not calculated in the highest exposure 

group because food intake could not be measured due to an excess scattering of feed. However, a 

dose of 1,650 mg/kg bw/day was estimated by CERHR based on intake levels observed in the lower 

dose groups. In the high-dose group, decreases in total body weight (due to decreased food intake) 

were observed, as were increases in relative liver and kidney weights. An increased incidence of 

macrocytic anemia was observed on days 30-180. The testis was determined the primary target 

organ based on weight, sperm concentration, and histopathological findings at the high dose. De-
II-6

Appendix II

creases in relative testis, absolute epididymis, and absolute seminal vesicle weight were observed, as 

were atrophy of seminiferous tubules and degenerative changes in testis and epididymis. No histologic changes in other body tissues were seen at this dose. The testis from animals in the lower dose 

groups were examined histologically and no effects were observed; lowered sperm counts were not 

seen at the 60, 180, or 550 mg/kg bw/day doses. Absolute and relative liver weight was increased at 

550 mg/kg bw/day. A NOAEL was established at 180 mg/kg bw/day1. The LOAEL of 550 mg/kg 

bw/day reflects increases in mean cell hemoglobin after 60-180 days of treatment that may be associated with the macrocytic anemia observed at the next higher dose.

In a 3-month feeding study, 3 adult male and female beagle dogs/group were fed diets with 

10,000–50,000 ppm BBP (males: 400, 1,000, or 1,852 mg/kg bw/day; females: 700, 1,270, or 1,973 

mg/kg bw/day, as calculated by study authors) (14). Food palatability complicated interpretation 

of reduced body weights in low-and high-dose males and mid-and high-dose females. No other 

changes were observed for hematological or urinalysis measurements. In high-dose animals there 

were no histopathological effects in liver, testes, or pancreas. 

Chronic Exposure Studies

Two sets of chronic feeding studies have been performed by the NTP (17, 18).

Potential BBP carcinogenicity was examined in both B6C3F1 mice and F344/N rats (18). Four-tofive week-old B6C3F1 mice (50/sex/group) were dosed through feed at concentrations of 0, 6,000, 

or 12,000 ppm for 106 weeks. Using EPA assumptions for B6C3F1 mouse body weight and food 

intake (body weight: 0.03733 kg [M], 0.0353 kg [F]; food intake: 0.0064 kg/day [M], 0.0061 kg/

day [F]), dose levels of 0, 1,029, and 2,058 mg/kg bw/day and 0, 1037, and 2,074 mg/kg bw/day 

were calculated for males and females, respectively. No treatment-related changes in survival or 

neoplastic developments were seen. Dose-related decreases in body weight were seen in both male 

and female mice. There were no lesions observed in male or female reproductive organs.

F344/N rats (50/sex/dose) were fed diets containing 0, 6,000, or 12,000 ppm BBP (18) for 106 

weeks. Using EPA assumptions for F344 rat body weight and food intake, respectively (M:0.380 

kg, 0.030 kg/day; F:0.229 kg, 0.021 kg/day), dose levels of 0, 474, and 948 mg/kg bw/day and 0, 

550, and 1,100 mg/kg bw/day were estimated for males and females, respectively. Male rats were 

sacrificed 29-30 weeks into the study because of increases in premature death. Internal hemorrhaging was suspected as the cause of these deaths. Body weight gain and food intake were decreased 

in both males and females. The female rats were allowed to continue through the 106 weeks of 

exposure; at necropsy the females exhibited an increased incidence of mononuclear cell leukemia 

(MNCL). Spleens were examined in the high-dose group and were found to be congested and infiltrated with mononuclear cells. MNCL has been associated with splenomegaly and sometimes hepatomegaly. No evidence of hepatomegaly was reported in these studies.

In another 2-year NTP bioassay (17) groups of 60 male Fischer 344/N rats (6 weeks old) were fed 

1The NTP (17) report stated that epididymal sperm concentration was determined for the lowest and two highest of the treated groups. CMA reports that an audit revealed the original laboratory report, that is the data 

source for the NTP Report, states that epididymal sperm counts were determined from the three highest dose 

groups. The data from the original laboratory report are used in this evaluation.
Appendix II

II-7

BBP in the diet at concentrations of 0, 3,000, 6,000, or 12,000 ppm (0, 120, 240, or 500 mg/kg bw/

day) and 60 females (6 weeks old) per group were fed concentrations of 0, 6,000, 12,000, or 24,000 

ppm (0, 300, 600, or 1,200 mg/kg bw/day) (Table 7-4) (17). After 2 years of exposure to BBP, 

increases in relative kidney weights were observed in male rats at 120 mg/kg bw/day and represented the lowest observable changes in this study (17). Additional dose-related increases included 

relative epididymis weights at the 240 mg/kg bw/day dose and relative liver weight at the 500 mg/

kg bw/day dose in male rats, with total body weight changes in rats occurring only at the highest 

dose tested, 500 mg/kg bw/day. At the highest dose level, histopathological changes included renal 

tubule pigmentation, hepatic granulomas, and focal pancreatic hyperplasia with “some evidence” 

of pancreatic carcinogenicity based on increased incidence of acinar cell adenoma and adenoma or 

carcinoma (combined). No testicular changes were observed; however, decreases in red blood cells 

(RBC) and increases in hemoglobin were observed 6 months into the study. 

Female F344/N rats exposed to BBP for 2 years showed nephropathy at the 2 lowest doses tested 

(300 and 600 mg/kg bw/day). At 1,200 mg/kg bw/day, the animals exhibited decreases in body 

weight and increases in liver and kidney organ to body weight ratios. They also exhibited renal 

tubule pigmentation (15-24 months), nephropathy, microcytic anemia (15 months), decreases in 

triiodothyronine, and “equivocal evidence of carcinogenicity” based on pancreatic acinar cell adenoma and urinary bladder transitional cell epithelial papilloma. Pancreatic effects may have been 

due to chronic stimulation of pancreatic lipase secretion. 

In a parallel study at the same laboratory, BBP’s ability to induce hepatic peroxisomes was evaluated in female F344/N rats (17). Two enzyme markers for peroxisome proliferation, palmitoyl CoA 

oxidase and carnitine acetyl transferase, were significantly elevated after 1 month and 1 year of 

exposure in animals exposed to 6,000 ppm BBP and higher (~300 mg/kg bw/day), although the 

level of induction was lower than that observed after a 3-week exposure to DEHP. The discussion in 

the NTP report highlights the fact that BBP is a mild peroxisome proliferator compared to DEHP or 

to hypolipidemic drugs such as clofibrate.

From these 2-year studies, LOAELs for non-cancer, general toxicity effects were determined at 120 

(M) and 300 (F) mg/kg bw/day based on kidney organ weight changes in the male and nephropathy in the females. At 500 (M) and 1,200 mg/kg bw/day (F), the highest doses tested, respectively, 

“some to equivocal evidence” of pancreatic (male and female) and urinary bladder carcinogenicity 

(female) was observed in rats. No testicular changes were observed at any of the doses tested; however, increases in epididymal weight were seen at the 2 highest doses (240 and 500 mg/kg bw/day). 

This change in epididymal weight was observed in the absence of total body weight change at the 

240 mg/kg bw/day exposure dose.

2.2 Toxicokinetics

Phthalate Moiety Toxicokinetics

Absorption

Dermal: In a study of dermal absorption of a series of phthalate diesters (10), 14C-BBP (157 μmol/

kg) was applied to the skin (clipped back) of male F344 rats and the area covered with a perforated 
II-8

Appendix II

cap. Absorption was estimated by the radioactivity eliminated in urine and feces over 7 days, which 

equaled 27% for BBP. Most of the remainder of the radioactivity was found at the site of application.

Oral: Oral administration of 5 g of BBP/kg to dogs resulted in 10% absorption (19). Administration of single oral doses of 2, 20, 200, or 2,000 mg/kg to male Fischer 344 rats showed a dose-dependent increase in the fraction of dose eliminated via the feces (20% at doses from 2-200 mg/kg; 

72% at 2,000 mg/kg) and a dose-dependent decrease in the fraction eliminated via the urine (75% 

at a dose of 2-200 mg/kg and 22% at 2,000 mg/kg), suggesting that absorption through the gut was 

limited at the high dose (20).

Inhalation: There are no reports of the absorption of BBP administered by inhalation. By analogy 

with other phthalates, di(2-ethylhexyl)phthalate and diisodecylphthalate, BBP would be expected to 

be absorbed from the lung as the parent compound (21, 22).

Biotransformation

Oral studies in rats indicate that BBP is rapidly metabolized by gut enterases to its monoester metabolites (monobutyl and monobenzyl phthalates), which are absorbed and are either excreted in 

urine as the ester or conjugated with glucuronic acid and then excreted via the urine as the glucuronate (19, 20, 23). Urinary metabolites in rats following oral administration of 3.6 mmol BBP/kg/

day (1,125 mg/kg bw/day) for 3 days indicated that 70% of the metabolites were monoesters while 

the remainder were monoester conjugates. The monobutyl ester is generally present in the highest 

amount; in one study, the ratio of monobutyl to monobenzyl phthalate was 5:3 (23). The glucuronidation pathway appears to be saturated at high doses, as noted by the decrease in the glucuronide 

metabolite relative to the monoester metabolites at high doses (2,000 mg/kg in rats) versus low 

doses (20 mg/kg in rats). 

BBP and dibutyl phthalate (DBP) share a common metabolite, monobutyl phthalate (MBuP); information from DBP germane to the monoester, and therefore also to BBP, will be presented throughout this evaluation. In addition to the monoesters, the esterase cleavage products, phenol (from the 

benzyl moiety) and butanol (from the butyl moiety), will be included. 

Distribution

Tissue distribution was non-specific for the small amount of dermally absorbed BBP (10).

Excretion

Excretion of absorbed BBP and its metabolites is rapid, with approximately 90% eliminated in 

24 hours, approximately 80% in urine and 20% in feces, at low doses (2-200 mg/kg). The half-life 

of BBP in blood is 10 minutes. The blood half-life of the monoester metabolites of BBP is approximately 6 hours (20). Following intravenous (IV) administration of 20 mg/kg of 14C-BBP, 55% of 

the dose was excreted into bile while 34% was excreted in the urine (20).

Side Chain-associated Toxicokinetics

Phenol and butanol are products of hydrolysis of the monoesters. Phenol metabolism to polyphenols is well known: butanol is a primary alcohol that is easily oxidized to butyric acid (n-butanoic 

acid) by alcohol dehydrogenase and aldehyde dehydrogenase. Further metabolism (by α-oxidation 
Appendix II

II-9

pathways) converts butyric acid into acetyl-CoA conjugates in intermediary metabolism pathways 

with no toxicological importance (24).

2.3 Genetic Toxicity

The NTP (17) reviewed the genetic toxicity of BBP. An increase in mutations was not observed following treatment of Salmonella and L5178Y mouse lymphoma cells with BBP in the presence or 

absence of S9 activation. BBP treatment with and without S9 activation did not result in sister chromatid exchanges or chromosomal aberrations in Chinese hamster ovary cells. However, induction 

of sister chromatid exchanges and increased chromosomal aberrations in bone marrow cells were 

observed following a single intraperitoneal (IP) injection of mice with 1,250–5,000 mg/kg bw BBP. 

There were no increases in sex-linked recessive lethal mutations in the germ cells of Drosophila fed 

or injected with BBP. 

Subsequent to the NTP review, BBP tested negative in the L5178Y mouse lymphoma mutation assay with and without activation, and in the Balb/3t3 cell transformation assay (25). Ashby et al. (26) 

reported negative results in a micronucleus assay in rats. The IPCS (2) review included the publication of Ashby et al. and concluded: “Although the weight of evidence of genotoxicity is clearly negative, available data are inadequate to unequivocally conclude that BBP is not clastogenic. However, 

in the available studies, the activity has been weak and is often consistent with secondary effects of 

the chemical on DNA.” 

The summary for Section 2, including general toxicity, toxicokinetics, and genetic toxicity, is 

located in Section 5.1.2.
II-10

Appendix II

3.0 DEVELOPMENTAL TOXICITY DATA

3.1 Human Data

There were no human data located for Expert Panel review.

3.2 Experimental Animal Toxicity

Eleven complete studies and two abstracts were evaluated. Two studies performed through the NTP, 

were standard prenatal assessment (segment II) studies of BBP administered in the diet of rats and 

mice. A third was an oral gavage Segment II study in rabbits. There were five studies by Ema et al. 

in Wistar rats where BBP was administered in the diet or by gavage. Three studies of BBP evaluated 

drinking water exposure to Wistar rats during gestation and lactation with assessment of adult F1 

males. One abstract evaluated BBP exposure by subcutaneous injection to two strains of male mice 

(B6C3F1 and CD-1) with subsequent mating to unexposed females (dominant lethal assessment).

3.2.1 Prenatal Development

A dietary study in CD (Sprague-Dawley) rats (27) involved exposure of 30 pregnant rats per group 

to 0, 0.5, 1.25, and 2.0% BBP (0, 420, 1,100, and 1,640 mg/kg bw/day) on gestation day (gd) 6-15. 

The dams were killed on gd 20, necropsied, and pups examined and evaluated (Table 7-5). Maternal 

toxicity was expressed in reduced body weights and decreased weight gain, decreased absolute feed 

consumption (but increased relative feed consumption in g/kg/day), increased relative liver weight 

(with no histopathological changes), and increased relative water intake at the 1,100 and 1,640 mg/

kg bw/day doses. Relative kidney weights were increased at the 1,640 mg/kg bw/day dose. However, the kidneys were not examined histologically. Clinical signs of maternal toxicity, including 

ataxia and abnormal gait, were also observed at this dose.

At 1,640 mg/kg bw/day, there were increased resorptions and concomitant reduced numbers of live 

fetuses per litter, reduced fetal body weight, and increased fetal malformations. Urogenital malformations, analyzed separately, were increased; they included distended ureters and distended or 

absent kidneys. Other fetal malformations at the high dose were anophthalmia (missing eyes), fused 

or malaligned vertebrae, and fused ribs. There were increased incidences of fetal variations per litter at both the 1,100 and 1,640 mg/kg bw/day doses.

Significant developmental toxicity occurred at the 1,100 and 1,640 mg/kg bw/day doses; teratogenicity was observed at 1,640 mg/kg bw/day. Maternal toxicity was observed at doses that caused developmental toxicity. The maternal and developmental NOAELs were identified at 420 mg/kg bw/day.

Ema et al. (28) exposed pregnant Wistar rats, 15-18/group, to BBP in the diet at 0, 0.25, 0.5, 1.0, 

and 2.0% (intakes of 0, 185, 375, 654, and 974 mg/kg bw/day, respectively) on gd 0-20. Dams were 

killed on gd 20 and evaluated in a Segment II study design (Table 7-6). There were also pair-fed 

controls matched with the animals in the highest dose group. No dams died in any group. Adjusted 

maternal body weight gains (not including gravid uterus weight) and feed consumption were reduced at doses of 654 and 974 mg/kg bw/day. All dams at 974 mg/kg bw/day had fully resorbed 

litters. There was no treatment-related pre-implantation loss or teratogenicity. The authors concluded that the maternal NOEL was 375 mg/kg bw/day and the developmental toxicity NOEL was 

654 mg/kg bw/day. The Expert Panel did not agree with the author’s identification of developmental 
Appendix II

II-11

effect levels given that live litter size was reduced at 375 mg/kg bw/day (11.3 vs. control value of 

13.9) and 654 mg/kg bw/day (12.3 vs. control value of 13.9); fetal body weights (by sex per litter) 

were significantly reduced at 654 mg/kg bw/day. The data did support a developmental NOAEL of 

185 mg/kg bw/day. 

In a second Segment II study, Ema et al. (29) treated 10 Wistar rats/group with BBP by gavage 

with 0, 500, 750, or 1,000 mg/kg bw/day on gd 7–15 (Table 7-7). Dams and fetuses were evaluated 

following sacrifice on gd 20. Maternal body weight gains were reduced at doses of 750 and 1,000 

mg/kg bw/day, but the corrected weight gain (maternal body weight excluding the gravid uterus) 

was decreased only at the high dose. Food intake was reduced at all dose levels. Four dams in the 

high-dose group died and entire litters were resorbed in the six surviving dams. Complete litter 

resorptions were observed in 3/10 dams in the 750 mg/kg bw/day group. Other effects at that dose 

included increased fetal death due to postimplantation loss, reduced fetal weight, and increased external, skeletal, and internal malformations. The malformations consisted primarily of cleft palate, 

fused sternebrae, and dilated renal pelves. The maternal and fetal NOAEL was identified as 500 

mg/kg bw/day. 

The Segment II dietary study in CD-1 mice (30) involved exposure of 30 pregnant mice per 

group to 0, 0.1, 0.5, and 1.25% BBP (0, 182, 910, and 2,330 mg/kg bw/day), on gd 6-15 (Table 

7-8). Maternal toxicity was expressed as reduced weight gain at the two highest doses (910 and 

2,330 mg/kg bw/day), and increased relative liver and kidney weights and increased relative water 

intake at the high dose. No histopathological changes were observed in the liver or kidneys.

Embryofetal effects included increased incidences of resorptions and late fetal deaths, with concomitant reductions in live fetuses per litter, and increased malformations (external and skeletal) 

at 910 and 2,330 mg/kg bw/day. Malformations included exencephaly, short tail, cardiovascular 

defects, fused ribs, and abnormal or fused sternebrae and vertebrae. Fetal body weight per litter 

was decreased and fetal variations were increased at the 2,330 mg/kg bw/day dose. As with rats, 

maternal and developmental toxicity was present at the two highest doses. The maternal and developmental NOAEL was 182 mg/kg bw/day.

A Segment II developmental toxicity study (31) was also performed in New Zealand white rabbits. The does, 17/group, were administered BBP (Santicizer 160) orally by gelatin capsule on gd 

6-18 at 0, 3.0, or 10 mg/kg bw/day. Does were terminated on gd 29. There was no demonstrable 

maternal toxicity. There was no demonstrable developmental toxicity, such as effects on fetal body 

weight, 24-hour survival, or treatment-related external or visceral malformations. Skeletal findings 

in toto were considered equivalent across groups.

Mechanistic Studies

Ema et al. has published a series of articles that focus on three issues: 1) direct vs. indirect toxicity 

of BBP; 2) the dose and time dependency of the prenatal effects of BBP exposure; and 3) study of 

the toxic properties of the two monoester metabolites of BBP. 

Direct vs. indirect toxicity: 

Ema (32, 33) exposed Wistar rats to BBP at 2.0% in diet (974 mg/kg bw/day) on gd 0-20, gd 0-11 
II-12

Appendix II

or gd 11-20. Pair-fed controls received the same amount of diet as treated rats. All dams exposed on 

gd 0-20 had fully resorbed litters. The pair-fed controls exhibited maternal weight gains comparable 

to the BBP group, but no treatment-related fetal malformations or resorptions were observed. Dams 

fed BBP on gd 0-11 also had fully resorbed litters. No increase in postimplantation loss was found 

in rats exposed on gd 11-20, but the fetuses in this group exhibited malformations, predominantly 

cleft palate and fused sternebrae. Thus, resorption does not appear to be related to decreased food 

consumption, but is an effect of the chemical, per se.

Time-and dose-dependency: 

In another dietary study using 2.0 % BBP on gd 0–7, gd 7–16, and gd 16–20 (34), postimplantation 

loss was increased after exposure on gd 0-7 or 7-16; teratogenicity was observed (predominantly 

cleft palate and fused sternebrae) after exposure on gd 7–16 (34). Ema et al. (29) also dosed Wistar 

rats by gavage with BBP in olive oil at 0, 500, 750, or 1,000 mg/kg bw/day on gd 7-15. No live fetuses were present at 1,000 mg/kg bw/day and malformations (cleft palate, fused sternebrae, dilated 

renal pelves) occurred at 750 mg/kg bw/day accompanied by increased in utero death, decreased 

fetal body weight, and maternal toxicity (reduced weight gain and feed consumption). At 500 mg/

kg bw/day, maternal feed consumption during the exposure period was reduced, but no embryofetal 

effects were observed.

To investigate further the observed embryolethality and teratogenicity, Ema et al. (35) exposed Wistar rats to BBP in the diet at 2.0% (954 mg/kg bw/day) during gd 0-7g, gd 0-9, or gd 0-11. Pre-implantation loss was equivalent across all groups. Postimplantation loss was highest for groups treated on gd 0-11. Uterine and ovarian weights were reduced, as was plasma progesterone in all groups 

(except that ovarian weight was unaffected on gd 7). The authors suggest that the post-implantation 

loss in early pregnancy was mediated by reduced plasma progesterone levels from impairment in 

luteal function. 

It appears that postimplantation death or the development of malformations is dependent upon both 

the dose and time during gestation when the exposure occurs.

Studies on monoesters:

Ema et al. evaluated the developmental toxicity of the two metabolites of BBP: MBuP (36-38) and 

mono-n-benzyl phthalate (MBeP) (39) when administered by gavage to Wistar rats. 

Ema et al. (38) gavaged Wistar rats with MBuP at 0, 250, 500, and 625 mg/kg bw/day on gd 7–15. 

Maternal toxicity was present at the two highest doses, expressed as reduced body weight gains and 

reduced feed consumption. At these doses there were also significant increases in postimplantation 

loss/litter, and decreases in live fetuses/litter and fetal body weight per litter. Fetal malformations 

were also increased at these doses, with cleft palate, deformed vertebral column, and dilated renal 

pelves the predominant findings.

Ema et al. (36) followed-up with evaluation of stage specificity studies. Wistar rats were dosed with 

MBuP at 0, 500, 625, or 750 mg/kg bw/day on gd 7–9, gd 10–12, or gd 13–15. Embryolethality 

was increased at all doses for all dosing intervals. No teratogenicity was observed from the gd 10–

12 dosing interval. Increased incidences of fetal external malformations were present in the groups 
Appendix II

II-13

treated with 500 and 750 mg/kg bw/day on gd 7–9 and 13–15. Increased skeletal malformations 

were observed in groups treated with 500, 625, and 750 mg/kg bw/day on gd 7–9, and with 625 and 

750 mg/kg bw/day on gd 13–15. Deformed cervical vertebrae were predominant in groups treated 

on gd 7–9. Cleft palate and fused sternebrae were observed in groups treated on gd 13–15. These 

results are consistent with the findings for DBP and BBP, and imply that MBuP (and/or subsequent 

metabolites) may account for the developmental toxicity (embryolethality and malformations) for 

both DBP and BBP.

Ema et al. (39) also administered MBeP by gavage at 0, 250, 313, 375, 438, and 500 mg/kg bw/day 

to pregnant Wistar rats on gd 7–15. Decreased maternal weight gain during dosing was present 

at doses from 313 to 500 mg/kg bw/day, and reduced feed consumption was present from 250 to 

500 mg/kg bw/day. Increased postimplantation loss was present at 438 and 500 mg/kg bw/day. Increased incidences of fetal external malformations were present at 438 and 500 mg/kg bw/day, skeletal malformations were present at 313–500 mg/kg bw/day, and visceral (“internal”) malformations 

at 375–500 mg/kg bw/day. The most common fetal findings were effects on cervical and thoracic 

vertebrae, ribs, and kidney (dilated renal pelves at 375 and 438 mg/kg bw/day, and hypoplasia of 

the kidney at 500 mg/kg bw/day).

These studies establish a maternal and developmental NOAEL for MBuP of 250 mg/kg bw/day. For 

MBeP, no maternal NOAEL was identified (effects were observed at 250 mg/kg bw/day); the developmental NOAEL was 250 mg/kg bw/day under the conditions of the study. The finding of fetal 

kidney effects at 375–500 mg/kg bw/day for MBeP is of concern since the CD rat study (27) also 

found fetal kidney malformations at the high dietary dose (1,640 mg/kg bw/day) and the kidney is 

a known target organ in adult rats. Cervical ribs are also of concern due to their rarity and proposed 

mechanism of disruption in gene expression.

An additional study by Ema et al. (37) compared effects of BBP and DBP administered by gavage 

to pregnant Wistar rats at 0, 750, 1,000 or 1,250 mg/kg bw/day on gd 7–9, gd 10–12, or gd 13–15. 

Increased postimplantation loss was observed for both compounds at all doses from all exposure 

periods. Malformations were observed in groups treated with both phthalate esters at ≥750 mg/kg 

bw/day on gd 7–9 (vertebral column and ribs) and on gd 13–15 (cleft palate and fused sternebrae). 

No malformations were observed with either compound at any dose when they were administered 

on gd 10–12. The authors concluded that “the similarity in dependence of gestational days of treatment on the manifestations of developmental toxicity and on the spectrum of fetal malformations 

caused by BBP and DBP suggests that they may act by the same mechanism, possibly via a common metabolite of these two parent compounds.”

3.2.2 Postnatal Development

Imajima et al. (40) gavaged pregnant Wistar-King A (WKA) rats with MBuP in sesame oil at 0 or 

300 mg/day on gd 15–18 (equivalent to approximately 1,000 mg/kg bw/day) (Table 7-16). Male 

offspring were evaluated on gd 20 and on postnatal days (pnd) 30–40 to determine the position of 

the testes. In control males, all testes were located in the lower abdomen on gd 20 (19 pups, 3 litters) and had descended into the scrotum on pnd 30–40 (15 pups, 3 litters). In stark contrast, in 

males exposed in utero to MBuP, on gd 20 all testes were located high in the abdominal cavity 

(15 pups, 3 litters) with significantly higher testes ascent. On pnd 30–40, MBuP exposed males ex-
II-14

Appendix II

hibited cryptorchidism (22/26 pups, 5 litters with uni-or bi-lateral undescended testes); 87% of the 

undescended testes were in the abdominal cavity, the remaining 13% were located at the external 

inguinal ring. Testis descent is under androgenic control; the authors suggest that phthalate esters 

may interfere with FSH stimulation of cAMP accumulation in Sertoli cells, resulting in the reduced 

secretion of Mullerian inhibiting substance, a putative mediator in transabdominal migration of the 

testis. 

The Panel is aware of data indicating that DEHP, BBP, and diisononylphthalate (DINP), but not 

diethylphthalate (DEP), or dimethylphthalate (DMP), produced reproductive tract malformations 

in male offspring of rats gavaged with 750 mg/kg bw/day in corn oil on gd 14 to pnd 3 (41). DEHP 

and BBP are approximately equipotent, resulting in 91 and 84% of the male offspring with multiple 

malformations, respectively; DINP resulted in 7.7% of the offspring males affected (p<0.04) versus 

0% in controls. DBP is also active as an anti-androgen with comparable potency to DEHP and BBP. 

Since BBP and DBP share a common metabolite, MBuP, the study by Mylchreest et al. (42), in 

which pregnant rats were orally dosed on gd 12–21 with DBP at 0, 0.5, 5, 50, 100, or 500 mg/kg 

bw/day, is germane. The male offspring were evaluated until puberty. The maternal NOAEL was 

500 mg/kg bw/day. The developmental NOAEL was 50 mg/kg bw/day, based on the presence of 

retained nipples and areolae in pre-weanling males at 100 mg/kg bw/day and malformations of the 

male reproductive tract, testicular lesions (Leydig cell hyperplasia and one Leydig cell adenoma), 

increased incidence of undescended testes, reduced anogenital distance, and retained nipples and 

areolae in males at 500 mg/kg bw/day. 

3.2.3 Postnatal Function

This section discusses a series of studies in which pregnant rats were exposed to low doses in drinking water. Two primary issues emerged: effects on male reproductive organs and perinatal mortality. 

Sharpe et al. (43) reported on adult male offspring from Wistar rat dams exposed 2 weeks prior to 

mating, and during gestation and lactation, to BBP (in ethanol) in drinking water at 1 mg/L (Table 

7-9). This study combined data from the same dams bred twice, with exposure continuing, to assess the effects of BBP. At weaning, male offspring were reared to adulthood, with no further BBP 

exposure and assessed for reproductive effects. Maternal BBP intake was calculated by weighing 

water bottles for three 48-hour intervals. On pnd 1-2, pnd 10-12, and pnd 20-21, BBP intake was 

estimated at 0.126, 0.274, and 0.336 mg/kg bw/day (the latter two measurements were confounded 

by pups drinking the treated water). At 90-95 days of age, male offspring had significantly smaller 

testes, but exhibited no effects on body, kidney, or ventral prostate weights. Testicular morphology 

and seminiferous epithelial tubule cross-sections were unaffected, but the authors reported reduced 

daily sperm production when compared to controls. This laboratory subsequently reported unexplained fluctuation in testicular weight of control rats (44).

Ashby et al. (26) attempted to replicate the Sharpe et al. (43) findings with larger group sizes and 

better control and characterization of the dosing material. They exposed 18 AP (Wistar) rats during gestation and lactation to 1 mg/L BBP in drinking water and assessed the F1 male offspring 

as adults (Table 7-10). They found no effects of BBP exposure on any endpoints assessed, including testis weights, daily sperm production, caudal epididymal sperm count, accessory sex organ 
Appendix II

II-15

weights, or relative incidence of gonadotrophs (FSH-positive cells) in the pituitary for male or female offspring. This study employed only one dose level. Additional details about study results are 

included in Table 7-10. 

Another replication of the Sharpe et al. (43) study was attempted by TNO (45) (Table 7-11). They 

exposed Wistar outbred (Crl:(WI)WU BR) rats, 28 females/group, to BBP in the drinking water at 

0.1, 1, and 3 mg/L during premating, gestation, and lactation periods. Doses to dams were estimated at 0, 0.012, 0.14, and 0.385 mg/kg bw/day. No effects were observed on mating index, female fecundity or fertility, or on prenatal postimplantation loss in the parental generation. The study failed 

to reproduce any effects on F1 male reproductive organ weights or daily sperm production rates 

when the F1 offspring reached adulthood. A decreased number of normal epididymal sperm was 

found in the low-dose group, and was not considered treatment related. Epididymal sperm motility 

was normal. Preputial separation in males and estrous cyclicity in females were also unaffected by 

BBP treatment. Following an evaluation of the Sharpe et al, Ashby et al., and TNO studies, the Expert Panel recommended that the reproductive effects in F1 males reported by Sharpe et al. (43) not 

be used in assessing the reproductive toxicity of BBP. The bases for the recommendation are: 

1) lack of dose-response data (e.g., a single-dose study); 

2) no analytical information of BBP levels in drinking water; 

3) the original laboratory could not replicate their original findings; and 

4) two other respected laboratories have been unable to replicate the effects.

Although no reproductive effects were observed in the TNO (45) study, an increase in postnatal 

pup mortality was noted. There was a significant decrease in postnatal pup survival (by total pups/

group) in the 1 and 3 mg/L BBP groups and the DES-positive control (Table 2). According to the 

authors, the values for BBP were not statistically significant on a per litter basis. The same lab immediately repeated the study according to the same protocol, except that only controls and the 1 and 

3 mg/L doses were tested. Pup losses in the second study were significantly decreased compared 

to control at the 1 mg/L dose and again significantly increased compared to control at the 3 mg/L 

level (Table 2). Again, statistical significance was reported by the authors as not being achieved 

when analyzed on a per litter basis. Interestingly, significant effects on decreased pup survival (by 

total pups/group) were reproduced at the 3 mg/L level. The Panel is aware that the concurrent control values for pnd 0–4 pup loss in these two studies exceeded the historical control values for this 

laboratory, and that other studies performed at this laboratory during this general time period also 

experienced high pup losses on pnd 0–4, even in the vehicle control groups.

As reported in an abstract, Parks et al. (47) dosed Sprague-Dawley rats by gavage with 750 mg/

kg bw/day of BBP, DEHP, or corn oil (vehicle) from gd 14 through pnd 3. On pnd 2, anogenital 

distance (AGD), and testes weight were measured. Testes weights and AGD were significantly 

decreased, and the incidence of retained areolae on pnd 13 was increased for both DEHP-and BBPexposed male pups. 

Developmental effects were also reported in a reproductive screening study by Piersma (48) and are 

addressed in Section 4. 
II-16

Appendix II

Table 2: Combined Postnatal Mortality in Two TNO Studies with Wistar Rats

Maternal BBP doses in 

Drinking Water: 

mg/L (mg/kg bw/day)

0 0.1 (0.012)a 1.0 (0.14) 3.0 (0.385)

Study 1 Pnd 0-4 pup loss/

total pups at birthb 17/252 (25) 2/233 (23) 30c

/212 (23) 36c/248 (24)

Study 2 Pnd 0–4 pup loss/

total pups at birthb 42/299 (26) Not determined 19d/248 (23) 70c

/277 (26)

Combined pnd 0-4 Pup loss 

/total pups at birthb 59/551 (51) 2/233 (23) 49/460 (46) 106/525 (50)

% Pnd 0-4 pup loss 10.7% 0.86% 10.65% 20.19%

a

 This dose only tested in one study; all other doses tested in two studies.

b Number in parentheses equals total number of litters.

c

 Significant increase when analyzed by group not significant when analyzed by litter.

d Significant decrease when analyzed by group not significant when analyzed by litter.

Table 3: Pre and Postnatal Mortality in Bayer Study

Drinking Water (ppm) Diet (ppm)

0 1.0 3.0 0 1.0 3.0

BBP intake

 (mg/kg bw/day) 0 0.17 0.54 0 0.11 0.34

Postimplantation loss 

per group: number of 

resorptions (number of 

implantations) 

24(269) 30(281) 40(300) 18(282) 33(316) 25(300)

% Postimplantation loss 

per group (not statistically 

significant)

8.92 10.68 13.33 6.38 10.44 8.22

Postnatal viability index 

% (pnd 0–4) 97.1 100.0 99.6 98.5 99.6 99.3

The summary for Section 3 is located in Section 5.1.3.
Appendix II

II-17

4.0 REPRODUCTIVE TOXICITY

4.1 Human Data

There were no human data available on the reproductive toxicity of BBP alone. Occupational exposure to phthalate mixtures containing BBP in PVC production has been associated with increased 

incidence of menstrual disorders and spontaneous abortions among female workers (49). 

4.2 Experimental Animal Toxicity

Six studies were reviewed in the evaluation of the reproductive toxicity of BBP. No study was 

definitive and no multigeneration-reproduction study has been published for BBP. Three studies 

measured reproductive performance. One other reported claims of low-level effects of BBP on 

reproductive development (discussed in Section 3), but these effects have not been reproduced by 

separate laboratories.

An assessment of the reproductive toxicity of BBP was reported by Piersma (48) (Table 7-13). This 

standard general and reproductive toxicity screen, conducted according to the OECD 421 protocol, provides useful indications as to major toxic effects. Male and female WU rats (10/sex/group), 

10–11 weeks old at the start of exposure, were gavaged for 14 days with BBP in corn oil at dose 

levels of 0, 250, 500, or 1,000 mg/kg bw/day, and then paired (1:1) and allowed to mate for a maximum of 14 days while dosing continued. Once evidence of mating was observed, the animals were 

separated. Males continued to be dosed daily, and were then killed and necropsied after a total dosage period of 29 days. Reproductive organs were removed and placed in Bouins fixative. Dosing of 

females continued until pnd 6, after which the females were killed and necropsied and ovaries and 

uteri examined. Pups were counted, sexed, weighed, and examined for external malformations on 

pnd 1 and 6 and then killed.

Body weight gain for the F0 males was reduced at the high dose (by 21%), whereas the body 

weight gain of the F0 females was increased in the second week of dosing (12 g/week compared to 

4 g/week for the controls). During pregnancy, the body weight gain of the dams was significantly 

reduced at the high dose (by 40%). The numbers of animals achieving a pregnancy were 9, 8, 7, and 

4 (of 10) in the 0, 250, 500, and 1,000 mg/kg bw/day groups, respectively. Postnatal pup mortality 

did not differ across dose groups, but average litter sizes at birth were 9.4, 11.4, 8.4, and 1.5 in the 

0, 250, 500, and 1,000 mg/kg bw/day groups, respectively, with statistical significance achieved at 

the highest dose. Absolute pup weight was significantly reduced at birth in the high-(29%) and middose (7%) groups. Testicular degeneration accompanied by interstitial cell hyperplasia was significantly increased in the high-dose F0 males. Ovary structure was not affected by treatment. 

In Piersma et al. (48), the high-dose group had lower fertility (decreased numbers of litters and 

decreased numbers of pups per litter) in the F0 generation with marked histopathology in the testes, 

but not in the ovaries. F1 pup weight was reduced at birth in the mid-and high-dose groups and a 

developmental NOAEL of 250 mg/kg bw/day was identified. The reproductive NOAEL was identified as 500 mg/kg bw/day. The Expert Panel’s confidence in the quality of the study is moderate to 

high; however, because of the design limitations, such as a lack of measures in the F1 generation, 

there is uncertainty that these doses correctly represent the reproductive NOAEL. 
II-18

Appendix II

A one-generation reproduction study following OECD guideline 415 was performed in Wistar rats 

that were mated twice and produced two litters (50) (Table 7-14). BBP was administered in the diet 

at levels of 0, 0.2, 0.4, and 0.8% to 12 male and 24 female rats per group for 10 and 2 weeks prior 

to the first mating, respectively. Seven to thirteen days after the first litter was weaned (at pnd 21), 

the study was repeated with the same rats. Average doses to males during the premating period were 

estimated by authors at 0, 108, 206, or 418 mg/kg bw/day. Average female doses during the premating, gestation, and lactation periods were estimated at 0, 106, 217, or 446 mg/kg bw/day; 0, 116, 

235, or 458 mg/kg bw/day; and 0, 252, 580, or 1,078 mg/kg bw/day, respectively. There were no 

treatment-related clinical signs or mortality. There were periods of reduced body weight or weight 

change in females in the high-dose group during gestation and lactation in each of the two matings. A decrease in food consumption during the gd 0–14 period in both matings was considered 

a substance-related effect. A slight decrease in the number of treated females with litters observed 

in the first mating was not observed in the second mating. Mean pup weight was slightly decreased 

in the high-dose group during lactation; this decrease reached statistical significance at pnd 21 in 

the second litter. The authors attributed the pnd 21 finding to direct consumption of BBP in diet 

by the pups after pnd 14. All standard reproductive indices (fertility, implantation, and fecundity) were within normal ranges. At necropsy, tissues from male and female reproductive organs 

were collected and fixed in 4% buffered formalin. Microscopic examination of hematoxylin-and 

eosin-stained slides from these tissues was performed for control and high-dose rats. Relative liver 

weights were increased in high-dose females, but examination revealed that the liver and reproductive tissues were normal. The authors concluded that the NOAEL for reproductive performance was 

418 mg/kg bw/day in males and 446 mg/kg bw/day in females, with the parental NOAEL being 

206 mg/kg bw/day in males and 217 mg/kg bw/day in females.

The NTP (17) (Table 7-15) described a 10-week modified mating study. Male F344 rats, 6 weeks 

old at the commencement of the study, were exposed to BBP (15/group) in the diet at levels of 

0, 300, 2,800, or 25,000 ppm for 10 weeks (which delivered approximate doses at 0, 20, 200, 

2,200 mg/kg bw/day) and then allowed to recover for 2 days. The rats were then housed individually with two untreated females during a 7–day mating period and females were removed on the 

first day of a vaginal plug or sperm detection. Females were necropsied on gd 13. After the mating 

period, 10 and 11 days after receiving the last dose in feed, the males were necropsied and a full histological examination made at 0 and 25,000 ppm only. However, the testis and epididymis, seminal 

vesicle, and prostate were examined in all groups. The fixative used to preserve the testis was not 

indicated. Epididymal sperm analysis was also performed on the males; sperm samples were collected for evaluation at the end of the study.

Mean body weights of the high-dose males were 71% of control values at the end of the study, representing a significant reduction. Food consumption differences between the control and high-dose 

groups at the end of the study were only modestly decreased with treatment when proportionality to 

body weight is considered. Liver and thymus to body weight ratios were increased in the 2,200 mg/

kg bw/day group, whereas absolute and relative testis and prostate weights were reduced. There was 

marked degeneration in the testis and epididymis at this dose. One animal in the low-dose group 

had marked testicular atrophy and others had fewer sperm in the epididymis. Epididymal sperm 

concentration was: 87, 70, and 0.1 % of control at the 20, 200, and 2,200 mg/kg bw/day groups, 

respectively. Other sperm parameters (motility, morphology) were not measured in the high-dose 
Appendix II

II-19

group due to the absence of sperm; sperm motility and morphology were not different from controls in the other treatment groups. Although 10/30 females mated to high-dose males were spermpositive during the mating trial, none were pregnant at necropsy. The pregnancy measures of the 

two lower dose groups were similar to control values.

In the NTP study, the high-dose group (2,200 mg/kg bw/day) had a high rate of infertility 

(decreased numbers of pregnancies) with marked histopathology in the testes and epididymides 

and a lowered sperm count. Effects in the 200 mg/kg bw/day group were restricted to a significant 

reduction in sperm count. However, it was subsequently noted that sperm counts might have been 

affected by a shorter recovery period from the time between mating to necropsy in the 200 mg/kg 

bw/day group compared to the other dose groups (51). Judd et al. provide the most recent example 

of a significant body of literature indicating that sperm levels in the cauda epididymis are significantly reduced by ejaculation; in some cases counts are reduced to <50% of control values (52, 

53). Because epididymal sperm counts in rats have been found to require at least 4–7 days to return 

to normal after mating (54), and 13/15 rats in the 200 mg/kg bw/day group were killed less than 

4 days after the detection of a vaginal plug in their mates, while only 7 control males were killed 

in this same period, the reduction in sperm count in the 200 mg/kg bw/day group in this 10-week 

study must be considered questionable. Additionally, an expert panel reviewing methods of sperm 

analysis stated that at least a week should transpire between mating and necropsy in order to avoid 

ejaculation-induced confounding of sperm count data (55). The effects at 2,200 mg/kg bw/day 

are considered both treatment-and dose-related. A NOAEL of 200 mg/kg bw/day was selected by 

the Expert Panel. This may not correctly represent the NOAEL because of the lack of measures to 

assess effects in females and the lack of assessment of reproductive systems in the F1 generation. 

Parallel to the 10-week modified mating study (17), a 26-week sub-chronic study was performed where male F344 rats received BBP in the diet at doses of 0, 300, 900, 2,800, 8,300, and 

25,000 ppm (0, 30, 60, 188, 550, and 1,650 mg/kg bw/day). The results of this study are presented 

in the section on General Toxicity and in Table 7-3. While a mating sequence was not part of the 

26-week-study design, all other protocol parameters associated with male effects (organ weights, 

tissues for microscopic evaluation, and epididymal spermatozoal parameters) were identical to the 

NTP 10-week study. A comparison of results shows similarity in effects on body weight gain, organ 

weights, histopathological findings, and sperm motility. Interestingly, while sperm concentration 

in the 200 mg/kg bw/day group was reduced by 30% in the 10-week study, the values for the 550 

mg/kg bw/day group in the 26-week study were not reduced. All other measures at this dose were 

similar to controls. Results of the other two doses, compared to their contemporary controls, were 

similar.

Agarwal et al. (15) (Table 7-1) examined the effect of BBP on the male reproductive system of 

adult rats. Fischer F344 rats (10 males per group) aged 12–13 weeks were administered BBP at 

levels of 0, 0.625, 1.25, 2.5, and 5% (0, 447, 890, 1,338, and 1,542 mg/kg bw/day) in the diet for 

14 days and killed on day 15. Details of the study and effects on systemic endpoints are provided in 

Chapter 2. Reproductive effects at the two highest doses included significant weight and histological changes to the testis and accessory sex glands accompanied by changes in circulating FSH and 

LH levels. An oral NOAEL for reproductive toxicity in this 14-day study in adult male F344 rats 

was 1.25 % (890 mg/kg bw/day). Expert Panel confidence in the quality of the study is moderate; 

 
II-20

Appendix II

within design limitations, the study is well conducted and reported. Panel confidence is low that 

these dose levels correctly represent the NOAEL due to the short exposure time and because guidelines for this type of study do not require assessment of younger animals or the F1 generation. 

Studies on postnatal male fertility, with animals exposed indirectly through maternal consumption, 

as reported by Sharpe et al. (43), and subsequent publications by Ashby et al. (26) and TNO (45), 

that failed to reproduce the original findings are presented and discussed in Section 3.2. 

According to an abstract, a dominant lethal study was performed (56) on B6C3F1 and CD-1 male 

mice administered BBP by subcutaneous injections on days 1, 5, and 10 of the study at doses 

equivalent to 400–600, 1,280-1,840, and 3,200–4,560 mg/kg bw/day (triethylene melamine was the 

positive control). The males were then paired with untreated females every 4 days through day 49; 

female uterine contents were evaluated on gd 17. BBP did not affect prenatal deaths or fertility in 

either strain at any dose.

Mode of Action

Several studies have examined the ability of selected phthalate esters to compete with labeled 

estradiol (E2) for binding to the estrogen receptor (ER). Sources of ER protein included rat uterine 

cytosol (57), rainbow trout hepatic cytosol (58), recombinant human ERs (rhER) overexpressed in 

SF9 insect cells using the baculovirus system (59, 60), and rainbow trout ERs expressed in yeast 

(61). Tritiated 17ß-estradiol (E2) was used in the tissue cytosol binding assays while a high affinity fluorescent E2 derivative was used in the rhER binding assays. BBP has been shown to bind to 

the estrogen receptor (ER) of rat (57) or trout (58). The relative binding affinity is approximately 

10,000–100,000 times less than (E2). 

Selected phthalate esters have been examined in a number of in vitro gene expression assays systems. The assays have used stably transfected cells (57), transiently transfected cells (57, 58), yeast 

based assays (57, 61-63), and vitellogenin induction in rainbow trout hepatocyte cultures (61). 

BBP induces weak activity in in vitro estrogen-mediated gene expression assays in mammalian cell 

transfection experiments at 10 μM, the highest concentration examined (57). In a yeast assay of 

estrogen-mediated gene expression, the potency of BBP was 1x106–5x107 less than that of E2, but 

its metabolites MBuP and MBeP demonstrated no estrogenic activity (63). However, no effects on 

uterine wet weight and vaginal epithelial cell cornification were observed in 10 Sprague-Dawley 

rats/group gavaged with 20, 200, and 2,000 mg/kg bw/day for 4 days (57). Moore (64) reviewed the 

data on the estrogenic potential of phthalates and concluded that the estrogenic ability of phthalates 

identified in the in vitro studies is “not relevant to humans or to the environment.”

The summary for Section 4 is located in Section 5.1.4.
Appendix II

II-21

5.0 DATA SUMMARY & INTEGRATION

5.1 Summary

5.1.1 Human Exposure

BBP is used in PVC construction materials, automotive materials, and food conveyor belts (1). 

There appears to be no significant use of BBP in toys or medical equipment. It is believed that only 

negligible amounts of BBP are present in air due to its low volatility; a limited number of air monitoring studies support this view. In a survey of California homes, the median air level of BBP was 

measured at 0.034–0.035 ng/m3, and median outdoor air levels of BBP were below the detection 

limit of 0.051 ng/m3. However, inhalation exposure to BBP in flexible PVC manufacturing facilities has been estimated at 286 μg/kg bw/workday (1). Exposure through contact of BBP-containing 

materials with skin is negligible due to the relatively slow absorption through skin (2, 10, 11). 

The IPCS (2) concluded that consumption of food containing trace levels of BBP is the only significant source of exposure to the general population. Based on a survey of Canadian foods, IPCS 

estimated that exposure of adults to BBP is 2 μg/kg bw/day and that exposure levels in children 

could be up to three-fold higher. Exposures in children may be higher due to dietary differences and 

intake of BBP through mouthing of BBP-containing objects. MAFF estimated the BBP exposure of 

adults through diet at 0.11–0.29 μg/kg bw/day and exposure of infants through formula at 0.1–0.2 

μg/kg bw/day. In all exposure estimates, it was evident that exposure to the general population, 

including children, is well below 10 μg/kg bw/day. Discrepancies in food exposure estimates may 

be due to the inherent variability of food eaten by individuals based on age, sex, ethnicity, time of 

sampling, and geographical locations. 

5.1.1.1 Utility of Data to the CERHR Evaluation

BBP exposures resulting from food intake were estimated by two authoritative sources. There are 

limitations in these estimates. One agency used 12-15 year-old data which may not reflect current 

exposure, and the food data were collected in Europe and Canada and estimates may not accurately reflect US dietary patterns.

5.1.2 General Biological and Toxicological Data

Toxicity. 

There are no human data on exposure to BBP alone. Exposures to BBP-containing phthalate mixtures have been associated with elevated respiratory/neurological morbidity and increased risk of 

cancer in occupationally exposed population groups (2); a single controlled epidemiological study 

has found an increased risk of bronchial obstruction in young children related to indoor exposures 

from PVC floor covering (13); BBP is a common component of PVC. 

In animals BBP is not acutely toxic by the oral or dermal route as evidenced by the LD50 value 

exceeding 2 g/kg bw (2). Several subchronic and chronic dietary studies in rats reported consistent 

adverse effects on body weight and in kidney, liver, and testes (14, 15, 17). The earliest response 

was an increase in kidney or liver to body weight ratio(s) observed at doses of 120-151 mg/kg 

bw/day and higher. Histological changes in the liver were observed in some studies at doses of 
II-22

Appendix II

960 mg/kg bw/day and higher and changes in kidneys were observed in the chronic study at doses 

of 500 (M) – 1,200 (F) mg/kg bw/day. Anemia was observed at doses of 500 mg/kg bw/day and 

higher. The pancreas may also be a target organ in rats, as pancreatic lesions were reported in a subchronic study at 381 mg/kg bw/day. Lesions in testes, seminal vesicles, epididymis, and/or prostate 

were noted after exposure of rats to 1,338 mg/kg bw/day or higher. In an inhalation study in rats, 

increases in liver and kidney weights were reported at the maximum dose of 789 mg/m3 (∼150 mg/

kg bw/day) (14). BBP is considered a weak inducer of peroxisome proliferation in rats.

In repeat-dose studies, mice were less sensitive to toxic effects than were rats. Dietary studies of 

up to 2-years’ duration in B6C3F1 mice showed dose-related reductions in body weight at doses of 

1,029 mg/kg bw/day and higher (18). There was no clinical or histological evidence of toxicity in 

tissues, including male and female reproductive organs. Male dogs also appear to be less sensitive 

than rats because oral doses up to 1,852 mg/kg bw/day for 90 days resulted in reduced body weight 

but produced no histopathological effects in testes or liver (14).

A 2-year dietary study found no evidence of carcinogenicity in B6C3F1 mice and only a marginal 

increase in mononuclear cell leukemia in F344 female rats (18). In a second study in F344 rats, 

there was some evidence of pancreatic carcinogenicity in males exposed to 500 mg/kg bw/day and 

equivocal evidence of pancreatic and urinary bladder carcinogenicity in females exposed to 1,200 

mg/kg bw/day (17). 

Toxicokinetics. 

There are no data from studies in humans. There are no inhalation studies in any species. BBP is 

rapidly absorbed (at least 75% at doses of 2-200 mg/kg) in orally-dosed rats; this dropped to 22% 

at 2,000 mg/kg, suggesting saturation at high doses (20). BBP is absorbed slowly through the skin 

(27% in 7 days) of rats (10). BBP is rapidly metabolized to monobutyl and monobenzyl esters; 

by analogy to other phthalate esters, this probably occurs by pancreatic lipase and esterases in the 

small intestine. The monobutyl ester is usually present in higher amounts, 5:3, than is the monobenzyl ester (2). These monoesters are typically conjugated with glucuronic acid and then excreted in 

the urine (19, 20, 23). The glucuronidation pathway appears to be saturated at high doses, as noted 

by the decrease in the glucuronide metabolite relative to the monoester metabolites at high doses 

(2,000 mg/kg in rats) versus low doses (20 mg/kg in rats). There is no evidence of accumulation in 

tissues. Excretion of the absorbed BBP and its metabolites is rapid, with approximately 90% elimination in 24 hours. The half-life of BBP in blood is 10 minutes; the blood half-life of the monoester 

metabolites of BBP is approximately 6 hours.

Genetic toxicity. 

A recent review by the IPCS (2) stated: “Although the weight of evidence of genotoxicity is clearly 

negative, available data are inadequate to unequivocally conclude that BBP is not clastogenic. 

However, in the available studies, the activity has been weak and is often consistent with secondary 

effects of the chemical on DNA.” 
Appendix II

II-23

5.1.2.1 Utility of Data the CERHR Evaluation

The oral subchronic studies in rats and mice are adequate for the evaluation of general toxicity 

induced by BBP. The database is adequate to determine that the liver is a target organ of toxicity. 

Some studies were conducted according to GLP standards and relevant exposure routes were utilized. The examination of hepatic effects was adequate and included a limited evaluation of peroxisomal proliferation in rats. There is an inhalation study in rats.

There are acceptable toxicokinetic data for BBP, consisting of absorption, distribution, metabolism, 

and excretion data following oral and dermal exposure in the rat.
II-24

Appendix II

Table 4: Summaries of NOAELs and LOAELs and Major Effects in Oral General Toxicity Studies

Protocol and BBP Doses 

(mg/kg bw/day) 

NOAEL

(mg/kg bw/day)

LOAEL (mg/kg bw/day)

and Effects Major Effects at Higher Doses

14-day repeat dose dietary study 

in adult male Fischer 344 rats. 

10 rats/group. 

Doses 0, 447, 890, 1,338, or 1,542 

(15)

None 447

↑LH

↑Liver and kidney weight.

↓Testes, seminal vesicle, 

epididymis weight

Histopathological effects in testes, 

seminal vesicles, and prostate

↑LH and FSH 

↓Testosterone

↑Liver, kidney, and thymus weight

Histological changes in liver and 

thymus

↓Bone marrow cellularity.

3-month repeat dose dietary study 

in Wistar rats

4–6 weeks old at start of study

27–45 rats/sex/group

Doses – M: 0, 151, 381, 960 

F: 0, 171, 422, 1,069 

(14)

None M: 151; F: 171

↑Liver (4%) weight

↑Liver and kidney weight

Liver lesions

Pancreatic lesions

Anemia

↓Urine pH (M)

No testicular lesions. 

90-day repeat dose dietary study 

in adult Beagles. 

3/sex/group

Doses – M: 0, 400, 1,000, 1,852 

F: 0, 700, 1,270, 1,973 

(14)

M: None

F: 700

M: 400

F: 1,270

Decreased body weight.

No histological effects in liver or 

testes.

26-week dietary study in adult 

male Fischer 344/N rats

6-weeks-old at start of study

15 rats/group

Doses – 0, 30, 60, 180, 550, 1,650

(17)

180 550

↑Liver weight

↑Hemoglobin

↓Testis, seminal vesicle, & 

epididymis weight

Lesions in testis and epididymis. 

↓ Sperm counts

↑ Liver and kidney weight

Anemia

2-year dietary study in Fischer 

344/N rats. 

6-weeks-old at start of study

60 rats/sex/group

Doses – M: 0, 120, 240, 500 ;

F: 0, 300, 600, or 1,200 

(17)

None M: 120; F: 300

↑Kidney weight (M)

Nephropathy (F). 

↑ Liver weight

↑ Kidney weight

Nephropathy (F)

Anemia

↓ Thyroid hormone (F). 

Some evidence of pancreatic 

cancer (M)

Equivocal evidence of urinary 

bladder and pancreatic cancer (F)

No testicular lesions.

2-year dietary study in B6C3F1

mice.

4–5 weeks old at start of study. 

50 mice/sex/group.

Doses – M: 0, 1,029, 2,058 ;

F: 0, 1,037, 2,074 

(18)

None M: 1,029; F: 1,037

↓Weight gain.

↓ Weight gain.

No changes in survival or 

neoplasm development.

No lesions in male or female 

reproductive organs.
Appendix II

II-25

5.1.3 Developmental Toxicity

Studies of prenatal development consistently show BBP to be embryolethal and teratogenic following exposure to high oral doses in rats and mice on gd 6–15 or 7–15. The incidence of these effects 

is dependent on dose and developmental age. A maternal and developmental NOAEL in CD-1 

mice was 182 mg/kg bw/day (30). The Expert Panel noted that there was wide spacing between 

the NOAEL and the LOAEL of 910 mg/kg bw/day in this study. Effects at the LOAEL and higher 

doses included increased resorptions and late fetal deaths, reduced number of live fetuses per litter, 

and increased external and skeletal malformations. The developmental NOAELs in Sprague Dawley 

and Wistar rats ranged from 420 to 500 mg/kg bw/day, respectively (27, 29). Effects at doses of 750 

mg/kg bw/day and higher included increased prenatal mortality, reduced fetal growth, and increased 

fetal variations and skeletal, visceral, and external malformations. Extending the exposure period to 

gd 0–20 in Wistar rats resulted in a developmental NOAEL of 185 mg/kg bw/day. An oral prenatal 

study in rabbits revealed no maternal or developmental toxicity at doses up to 10 mg/kg bw/day; 

however, utility of the results is limited since no maximum tolerated dose was established (31).

Using a prenatal study design similar to that used with BBP (29), the monoesters MBuP and MBeP 

were investigated (38, 39). The developmental toxicity observed with the monoesters was qualitatively similar to that produced by BBP. These data suggest that both monoesters can contribute to 

the developmental toxicity associated with BBP. Differences in the doses selected for study do not 

permit a close quantitative comparison of the dose-response relationship between the two monoesters or with BBP. A rat study, using an MBuP dose of 1,000 mg/kg bw/day, reported a subsequent 

interference with testicular migration and descent (40). 

Studies in rats indicate that prenatal effects are directly related to the chemical and are not due to 

decreased food consumption (32). The mechanism of action for resorption has been proposed as 

reduced circulating progesterone due to impaired luteal function (35).

The effect of low-dose exposure during mating, gestation, and lactation in Wistar rats has been 

studied. An increase in postnatal pup mortality was reported (45) for rats treated with BBP through 

drinking water at 1 and 3 mg/L (0.14 and 0.385 mg/kg bw/day). The study was immediately 

repeated at the same laboratory and only the result at the highest dose (3 mg/L) was replicated. 

In both studies, statistical significance was not achieved with the litter as the unit of analysis. The 

Panel noted that concurrent control values for pnd 0–4 pup loss in these two studies exceeded the 

historical control values for this laboratory. Further, other studies performed at this laboratory 

during this time period also experienced high pup losses on pnd 0–4, even in the vehicle control 

groups. Increased pup mortality was not observed in similar studies by Sharpe et al. (43) and Ashby 

et al. (26) who dosed Wistar rats with 1 mg/L BBP in drinking water. In addition, a subsequent study 

by Bayer (46) did not result in increased postnatal pup loss at BBP doses of 1 or 3 ppm in drinking 

water or feed. For the Bayer study, maternal BBP intakes during the first week of lactation (the time 

of pup losses in the TNO studies) were 0.11 μg/kg bw/day and 0.34 μg/kg bw/day through diet and 

0.170 μg/kg bw/day and 0.540 μg/kg bw/day through drinking water. 

The Panel is therefore presented with a developmental LOAEL from the two TNO (45) studies of 

approximately 0.385 mg/kg bw/day (3 ppm) and a NOAEL of 0.140 mg/kg bw/day (1 ppm) based 
II-26

Appendix II

on F1 pup losses on pnd 0–4. There is very low confidence in these values due to the discrepancy 

between the results of the data when analyzed by group (statistically significant) versus by litter (not 

statistically significant) and due to the lack of effects in the Bayer (46) study. The NOAEL/LOAEL 

values from the TNO study are approximately 3 orders of magnitude lower (~0.3–0.4 mg/kg bw/

day) than the NOAEL values for reproductive/developmental NOAELs in other studies (~185 mg/

kg bw/day for developmental toxicity and 500 mg/kg bw/day for reproductive toxicity).

5.1.3.1 Utility of Data to the CERHR Evaluation

The data in rats and mice are adequate for a prenatal assessment of fetal growth, lethality, and 

teratogenicity. One study examined prenatal effects following exposure during late pregnancy. None 

of the studies included a postnatal evaluation of androgen-regulated effects (e.g., nipple retention, 

testicular descent, or preputial separation) that were the most sensitive indicators of developmental toxicity with DBP. BBP and DBP share a common monoester metabolite. Prenatal studies with 

BBP monoesters (MBuP and MBeP) were sufficient to determine that both metabolites contribute to 

developmental toxicity. Because of differences in doses administered to the mice and rats, it is not 

possible to compare sensitivity between the two species.
Appendix II

II-27

Table 5: Summaries of NOAELs and LOAELs and Major Effects in Developmental Toxicity Studies

Protocol and Study NOAEL

(mg/kg bw/day)

LOAEL (mg/kg bw/day) Developmental Effects 

Observed at Higher 

Maternal Developmental Dose Levels 

Prenatal dietary study in 

Sprague-Dawley rats.

30 per group received 0, 

420, 1,100, or 1,640 mg/

kg bw/day on gd 6–15.

Dams and pups examined late in gestation.

(27) 

Maternal: 420

Developmental:420

1,100

↓ Body weight gain.

↑ Liver weight.

1,100

↑ Variations.

↑ Prenatal mortality.

↓ Fetal weight.

↑ Visceral, skeletal, and 

external malformations.

↑ Variations.

Prenatal dietary study in 

Wistar rats. 

15–18/group received 

0, 185, 375, 654, or 974 

mg/kg bw/day on gd 

0–20. 

Dams and pups examined late in gestation.

(28)

Maternal: 375

Developmental: 185

654

↓ Weight gain.

375

↑ Prenatal mortality.

↑ Prenatal mortality.

↓ Decreased fetal 

weight.

Prenatal gavage studies 

conducted in Wistar rats. 

10/group received BBP 

0, 500, 750, or 1,000 mg/

kg bw/day (0, 1.60, 2.40, 

3.20 mmol/kg bw/day) 

on gd 7–15. Dams and 

pups examined late in 

gestation.

(29)

Maternal: 500

Developmental: 500

(1.60 mmol/kg 

bw/day)

750 (2.40 mmol/kg 

bw/day)

↓ Weight gain.

750 (2.40 mmol/kg 

bw/day)

↑ Prenatal mortality.

↓ Fetal weight.

↑ External and skeletal malformations.

Complete prenatal mortality.

The same study was 

conducted with MBuP 

at doses of 0, 250, 500, 

or 625 mg/kg bw/day (0, 

1.13, 2.25, 2.81 mmol/kg 

bw/day).

(38)

Maternal: 250

Developmental: 250

(1.13 mmol/kg 

bw/day)

500 (2.25 mmol/kg 

bw/day)

↓ Weight gain.

500 (2.25 mmol/kg 

bw/day)

↑ Prenatal mortality.

↓ Fetal weight.

↑ External and skeletal malformations.

↑ Visceral variations.

↑ Prenatal mortality.

↓ Fetal weight.

↑ External and skeletal 

malformations.

↑ Visceral variations.

The same study was 

conducted with MBeP 

at doses of 0, 250, 313, 

375, 438, or 500 mg/kg 

bw/day (0, 0.976, 1.22, 

1.46 mmol/kg bw/day).

(39)

Maternal: None

Developmental: 250

(0.976 mmol/kg 

bw/day)

250

(0.976 mmol/kg 

bw/day)

↓ Food intake. 

313

(1.22mmol/kg bw/

day)

↑ Skeletal malformations.

↑ Prenatal mortality.

↑ Internal, external, 

and skeletal malformations.
II-28

Appendix II

Table 5 (continued)

Protocol and Study NOAEL

(mg/kg bw/day)

LOAEL (mg/kg bw/day) Developmental Effects 

Observed at Higher Maternal Dose Levels Developmental

Prenatal dietary study 

in CD-1 mice.

30 per group received 0, 

182, 910, or 2,330 mg/

kg bw/day on gd 6–15.

Dams and pups examined late in gestation.

(30)

Maternal: 182

Developmental: 182

910

↓ Weight gain.

910

↑ Prenatal mortality.

↑ Visceral, skeletal, 

and external malformations.

↓ Fetal weight.

↑ Prenatal mortality.

↑ Visceral, skeletal, 

and external malformations.

↑ Variations.

Postnatal drinking water 

study in Wistar rats. 

28/group received 0, 

0.012, 0.140, or 0.385 

mg/kg bw/day from 2 

weeks prior to mating 

throughout gestation 

and lactation. Pups were 

examined postnatally

Maternal: 0.385

Developmental: 0.14

No adverse maternal 

systemic or reproductive effects.

0.385

↑ Pup death on pnd 

1–4 (12% in treated versus 0.8% in 

control).

No higher doses.

The experiment was 

repeated with the mid 

and high dose to verify 

increased postnatal 

mortality.

(45)

↑ Pup death on pnd 

1–4 (17% in treated versus 10% in 

control) in repeat 

experiment.

Postnatal drinking water 

and dietary study. 21-

25/group were treated 

from 2 weeks prior 

to mating throughout 

gestation and lactation. 

Lactational doses were 

0, 0.11–0.16, and 0.34–

0.49 through diet and 

0, 0.17–0.24, and 0.54–

0.80 through drinking 

water. Pups were examined postnatally.

(46)

Maternal: 0.34–

0.49 (diet), 0.54–

0.80 (drinking 

water)

Developmental: 

0.34–0.49 (diet), 

0.54–0.80 (drinking water)

No adverse maternal 

systemic or reproductive effects.

No significant effects 

on development 

including pup mortality.

 No higher doses.
Appendix II

II-29

5.1.4 Reproductive Toxicity

There are no conclusive data in humans that assess the reproductive effects from exposure to BBP 

alone. All experimental animal studies that assess reproduction have been performed in the rat. 

Male reproductive toxicity

Male reproductive performance was evaluated in three rat studies by the oral route of exposure (17, 

48, 50). There were no effects in reproductive performance in 10 WU rats exposed to up to 500 

mg/kg bw/day by gavage for 2 weeks prior to mating. Decreased fertility and testicular histopathology were seen at 1,000 mg/kg bw/day (48). No adverse effects were noted in Wistar rats exposed 

through diet with up to 418 mg/kg bw/day from 10 weeks prior to mating until the birth of a second 

litter (50). Reduced sperm counts were noted in F344 rats exposed to 200 mg/kg bw/day through 

diet for 10 weeks, but reproductive performance was not affected (17). The sperm count effects in 

the 200 mg/kg bw/day group were considered questionable and not used to determine a NOAEL 

because: 1) that group had a shorter recovery time from mating to necropsy and the required time to 

restore cauda epididymal sperm counts following ejaculation was not reached for most animals (51, 

54); and 2) no effects on sperm count were reported following exposure to 550 mg/kg bw/day in a 

26-week study by the same laboratory (17). 

Histopathology of male reproductive organs has also been examined in subchronic and chronic 

F344 rat studies by the oral route; the lowest dose that produced testicular lesions was 1,338 mg/kg 

bw/day in rats exposed through diet (15). The reproductive organs of male B6C3F1 mice were unaffected at dietary doses up to 2,058 mg/kg bw/day and testes of beagle dogs were not affected at 

dietary doses up to 1,852 mg/kg bw/day. The Expert Panel selected a reproductive NOAEL of 500 

mg/kg bw/day for adult male rats. There is uncertainty as to the dose that is without effect on the 

developing male reproductive tract. The Expert Panel noted that a primary BBP metabolite, MBuP, 

is likely the active toxicant in DBP studies where exposure during in utero development or during 

the neonatal period of life led to reproductive effects (42). Given that MBuP is a metabolite of BBP, 

the DBP data are relevant to BBP. Similar studies with MBeP, the other metabolite of BBP have not 

been performed. The existing studies with BBP did not critically examine pups during the sensitive 

postnatal phases of life. It is probable that such studies would likely result in a lower NOAEL. 

Female reproductive toxicity 

In a reproductive toxicity screening study in WU rats, decreases in the number of females conceiving and in the number of live pups per litter were observed at an oral gavage dose of 1,000 mg/kg. 

Clear testicular effects in males suggest that the effect may be due in part to toxicity in the male 

(48). Five hundred mg/kg bw/day was a NOAEL for female fertility in this study, but these data 

were from a screening study. No effects on implantation, reproductive organ morphology, fertility, 

or fecundity were seen in a one-generation reproductive toxicity study in Wistar rats that received 

the highest dietary dose (0.8%), comparable to a BBP intake value 446 mg/kg bw/day in diet during 

the mating phase of the study (50).

Mode of Action

BBP has been shown to bind to the estrogen receptor (ER) of rat and trout (57, 58). The relative 

binding affinity is approximately 10,000-100,000 times lower than 17-estradiol (E2). BBP also 

induces weak activity in in vitro estrogen-mediated gene expression assays at 10 μM, the highest 
II-30

Appendix II

concentration examined (57). In a yeast assay of estrogen-mediated gene expression, the potency of 

BBP was 1x106–5x107 less than that of E2, but its metabolites MBuP and MBeP demonstrated no 

estrogenic activity (63). However, no effects on uterine wet weight and vaginal epithelial cell cornification were observed in Sprague-Dawley rats gavaged with 20, 200, and 2,000 mg/kg bw/day for 

4 days. Moore (64) reviewed the data on the estrogenic potential of phthalates and concluded that 

the estrogenic ability of phthalates identified in the in vitro studies is “not relevant to humans or the 

environment.”

5.1.4.1 Utility of Data to the CERHR Evaluation

The data in rats are adequate for an assessment of reproductive toxicity in adults. Studies are available that evaluate both structure and reproductive function. In studies with DBP, a phthalate that is 

also metabolized to MBuP, male rats exposed while in utero and during lactation were most sensitive to DBP-induced effects on reproductive structure and function (65). Therefore, the most sensitive age for reproductive toxicity was not addressed for BBP. The data was sufficient to demonstrate 

the testes as a target organ.
Appendix II

II-31

Table 6: Summaries of NOAELs, LOAELs, and Major Effects in Reproductive Toxicity Studies

Protocol & Study NOAEL 

(mg/kg bw/day)

Reproductive 

LOAEL 

(mg/kg bw/day) 

and effects

Systemic LOAEL 

(mg/kg bw/day) 

and Effects

Reproductive 

Effects at 

Higher Doses

One-generation reproductive 

screening assay in WU rats. 

10 pairs/group received 0, 250, 

500, or 1,000 mg/kg bw/day 

by gavage from 2 weeks prior 

to mating for a total of 29 days 

(males) or until pnd 6 (females).

(48)

Reproductive:

500

Systemic: 

500

1,000

↓Fertility

Testicular 

lesions

↓Litter size

1,000

↓Weight gain

No higher 

doses in study

One-generation dietary reproductive toxicity assay in Wistar rats 

with 12 males and 24 females/

group. Males were treated 10 

weeks prior to mating with 0, 

108, 206, or 418 mg/kg bw/day. 

Females were treated from 2 

weeks prior to mating (0, 106, 

217, or 446 mg/kg bw/day), 

through gestation (0, 116, 235, 

or 458 mg/kg bw/day) and lactation (0, 252, 580, or 1,078 mg/kg 

bw/day). 

(50)

Reproductive:

418 (M);

446 (F)

Systemic: 

206 (M);

217 (F)

No structural 

or functional 

effects at any 

dose 

418 (M);

446 (F)

↓Weight gain (F)

↑Liver weight

No higher 

doses in study

One-generation modified mating 

study in male F344 rats.

15 males/group treated with 

BBP through diet at 0, 20, 200, 

or 2,200 mg/kg bw/day for 10 

weeks and then mated with untreated females.

(17)

Reproductive: 

200 

Systemic: 

200

 2,200

↓Sperm counts

↓Fertility

Testicular and 

epididymal 

lesions

↓Testis and 

prostate 

weight

2,200

↓Weight gain

↑Liver weight

Anemia

No higher 

doses in study
II-32

Appendix II

5.2 Integrated Evaluation

BBP is primarily used in PVC utilized in the manufacture of construction materials, automotive 

materials, and food conveyor belts. Exposure of the general population through inhalation is negligible due to the low volatility of BBP. Inhalation exposure to BBP in flexible PVC manufacturing 

facilities has been estimated at 286 μg/kg bw/workday. Exposure through contact with skin is negligible due to the relatively slow absorption. The IPCS has concluded that consumption of food containing trace levels of BBP is a significant source of exposure to the general population. Estimates 

based on BBP levels in Canadian and UK foods indicate that exposure to the general population, 

including children, is below 10 μg/kg bw/day. 

There are no human toxicokinetic or toxicity studies for BBP. Studies in rats demonstrate that 

orally-administered BBP is rapidly converted to the monoester metabolites, MBuP and MBeP, and 

their respective alcohols within the gut. At low doses, 2–200 mg/kg, approximately 80% of the 

administered dose is metabolized and the metabolites are absorbed into systemic circulation. The 

remainder of the dose is excreted in feces unchanged. Absorbed metabolites are glucuronidated and 

rapidly excreted in urine with no evidence of accumulation. The Expert Panel assumes the toxicokinetic studies in rats to be relevant to human exposure of BBP through food. There are no inhalation 

toxicokinetic studies.

Prenatal exposure studies in rats and mice have indicated that oral exposure on gd 6–15 or 7–15 to 

high doses of BBP (> 500 mg/kg bw/day) results in reduced fetal growth, prenatal mortality, and 

visceral, skeletal, and external malformations. NOAELs of 182 mg/kg bw/day and 500 mg/kg bw/

day were identified for mice and rats exposed on gd 6- or 7-15, respectively; however, a comparison 

of sensitivity between species is not possible due to variations in doses administered. Exposure of 

Wistar rats during the entire gestation period resulted in a developmental NOAEL of 185 mg/kg 

bw/day. Oral prenatal studies with the BBP metabolites MBuP and MBeP have demonstrated qualitatively similar results to BBP and suggest that the metabolites are associated with the observed 

developmental toxicity. None of the studies examined the postnatal effects on the male reproductive 

system. This is of concern because standard prenatal studies do not detect effects such as altered 

anogenital distance, retained nipples, delays in acquisition of puberty (preputial separation), and 

malformation of the post-pubertal male reproductive system. Such effects have been observed with 

DBP, the monoester metabolite of which is the same as one of the metabolites of BBP. Therefore, 

the Expert Panel is not confident in the NOAELs obtained from the existing BBP developmental 

studies. In studies using DBP, a NOAEL of 50 mg/kg bw/day was identified with male reproductive 

tract anomalies observed at higher doses. 

The data indicate that BBP is a reproductive toxicant in adult male rats as evidenced by testicular 

lesions, reduced sperm counts, and increased infertility following exposure to oral doses exceeding the NOAEL of 500 mg/kg bw/day. Effects on the reproductive system of adult female rats are 

less certain. There were no reproductive effects in female rats exposed orally to 446–1,078 mg/kg 

bw/day from 2 weeks prior to mating through lactation. However, in a second study, the number 

of females conceiving litters was reduced following exposure to 1,000 mg/kg bw/day by gavage. 

The data do not permit clear delineation as to whether this was male-or female-related, although 

clear evidence of testicular toxicity was seen. The Expert Panel notes that the database does not 

allow for a complete evaluation of reproductive effects due to the lack of a multigeneration study 
Appendix II

II-33

that exposes animals during the development of the reproductive system. Lower NOAELs may be 

observed in studies with late gestational exposure and complete postnatal examination of the male 

reproductive system.

The Expert Panel believes the database is sufficient to judge that oral exposure to BBP can cause 

reproductive toxicity in adult rats and developmental toxicity in rats and mice. These data are 

assumed to be relevant to humans. The Panel is not confident that the lowest dose at which developmental toxicity, specifically effects on the developing male reproductive tract, has been established. 

Lastly, the Panel is aware of studies performed at CDC using urine from human subjects. Results 

of these studies were given in an oral presentation in Copenhagen, Denmark, in May, 2000. MBuP 

values in the urine of women of child-bearing age were among the higher values. Such data, when 

published, should serve to improve our ability to assess phthalate exposure in the general population.

5.3 Expert Panel Conclusions

BBP is used in the manufacture of vinyl tile and PVC to make food conveyor belts, carpet tile, 

tarps, weather stripping, and, to a limited extent, vinyl gloves and adhesives. BBP can be released 

into the environment during production, incorporation into products, use, and disposal.

The best estimate of exposure to the general public is 2 μg/kg bw/day from food in adults, with 

exposures to infants and children possibly up to three-fold higher, with negligible exposures from 

infant formula, dermal absorption, drinking water, or soil intake. Occupational exposure is estimated at 286 μg/kg bw/workday. Median indoor air levels (from 1 study of 125 southern California 

homes) were 0.034–0.035 ng/m3, outdoor ambient air levels from 65 of these homes were 5.3–6.7 

ng/m3 for the 90th percentile, and below the estimated detection limit of 0.051 ng/ m3 for the 

median BBP level. The Expert Panel has low-to-moderate confidence in the completeness of the 

exposure database from which these estimates were made, based on the range of values provided by 

different sources for the same route of exposure and on the age of the data available for exposures 

for food and food packaging. 

With regard to developmental and reproductive toxicity, the database is sufficient to judge that 

oral exposure to BBP can cause developmental toxicity in rats and mice, and reproductive toxicity in rats. The current database is insufficient to fully characterize the potential hazard. The lowest 

NOAELs identified by the Panel for developmental toxicity were 182 mg/kg bw/day in CD-1 mice 

and 185 mg/kg/day in Wistar rats. Given the low exposures to adults and the high dose designated 

as the NOAEL, the Panel agrees that there is an adequate database to provide negligible concern 

for male reproductive effects from adult exposure. There is not an adequate database to determine 

NOAELs/LOAELs for male or female reproductive effects from perinatal exposure. BBP and DBP 

have a common metabolite, MBuP, and the Panel noted that orally-administered DBP causes male 

reproductive tract malformations at 100 mg/kg bw/day (LOAEL). Data gaps did not permit the 

Panel to ascribe a level of concern for postnatal consequences from perinatal exposure to BBP. 

Multigeneration studies now in progress include endocrine-sensitive endpoints and should provide 

a robust dataset from which to determine the LOAEL/NOAEL and allow subsequent assignment of 

the level of confidence in these values, and of the level of concern.
II-34

Appendix II

5.4 Critical Data Needs

Critical data needs are discussed under two categories: experimental studies and human exposure.

Experimental Studies

1) Multigeneration study. There is a priority need for a multigenerational study that evaluates 

effects on reproductive development, fertility, and reproductive system structures, including 

endocrine sensitive parameters, with continuous exposure across multiple generations. Female 

reproductive effects need to be evaluated explicitly.

The Expert Panel is aware that a two-generation study under current testing guidelines and with 

evaluation of endocrine-sensitive endpoints in rats was recently completed in Japan and that a similar study is underway in the United States. It is likely that data needs cited in 1) above would be fulfilled by the results from these studies. 

Human Exposure

1) No studies of humans were found. Occupationally-exposed cohorts might be located, but would 

be of limited utility if the major exposure source is food. Priority should be given to studies on 

occupational exposures and general population indoor exposures from BBP-releasing materials. 

2) Better exposure data. The Panel is aware of emerging data for human exposure (by analysis of 

urinary phthalate metabolites from a human reference population) that may alter existing exposure estimates, particularly for women of child-bearing age. 
Appendix II

II-35

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Appendix II

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29. Ema M, Itami T, Kawasaki H. Teratogenic evaluation of butyl benzyl phthalate in rats by 

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32. Ema M, Itami T, Kawasaki H. Evaluation of the embryolethality of butyl benzyl phthalate 

by conventional and pair-feeding studies in rats. J Appl Toxicol 11:39-42(1991).

33. Ema M, Itami T, Kawasaki H. Embryolethality and teratogenicity of butyl benzyl phthalate 

in rats. J Appl Toxicol 12:179-183(1992).

34. Ema M, Itami T, Kawasaki H. Effect of period of exposure on the developmental toxicity of 

butyl benzyl phthalate in rats. J Appl Toxicol 12:57-61(1992).

35. Ema M, Kurosaka R, Amano H, Ogawa Y. Embryolethality of butyl benzyl phthalate during 

early pregnancy in rats. Reprod Toxicol 8:231-236(1994).

36. Ema M, Kurosaka R, Harazono A, Amano H, Ogawa Y. Phase specificity of developmental 

toxicity after oral administration of mono-n-butyl phthalate in rats. Arch Environ Contam 

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37. Ema M, Kurosaka R, Amano H, Ogawa Y. Comparative developmental toxicity of n-butyl 

benzyl phthalate and di-n-butyl phthalate in rats. Arch Environ Contam Toxicol 28:223-

228(1995).

38. Ema M, Kurosaka R, Amano H, Ogawa Y. Developmental toxicity evaluation of mono-nbutyl phthalate in rats. Toxicol Lett 78:101-106(1995).

39. Ema M, Harazono A, Miyawaki E, Ogawa Y. Developmental toxicity of mono-n-benzyl 

phthalate, one of the major metabolites of the plasticizer n-butyl benzyl phthalate, in rats. 

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40. Imajima T, Shono T, Zakaria O, Suita S. Prenatal phthalate causes cryptorchidism postnatally by inducing transabdominal ascent of the testis in fetal rats. J Pediatr Surg 32:18-

21(1997).

41. Gray LE, Ostby J, Furr J, Price M, Veeramachaneni DNR, Parks L. Perinatal exposure to 

the phthalates in DEHP, BBP and DINP but not DEP, DMP or DOTP alters sexual differentiation of the male rat. Toxicol. Sci. (in press)(2000).

42. Mylchreest E, Wallace DG, Cattley RC, Foster P. Dose-dependent alterations in androgenregulated male reproductive development in rats exposed to di(n-butyl)phthalate during late 
II-38

Appendix II

gestation. Toxicol Sci(2000).

43. Sharpe RM, Fisher JS, Millar MM, Jobling S, Sumpter JP. Gestational and lactational expsoure of rats to xenoestrogens results in reduced testicualr size and sperm production. Environ Health Perspect 103:1136-1143(1995).

44. Sharpe RM, Turner KJ. Endocrine disruptors and testis development. Environ Health Perspect 106:A221(1998).

45. TNO NaFRI. Oral developmental reproduction study with butyl benzyl phthalate in Wistar 

rats. Volume 1 of 3: European Council for Plasticizers and Intermediates, 1998.

46. Bayer AG. Butyl benzyl phthalate (BBP) -Developmental reproduction study in Wistar rats 

with application in the diet or drinking water 28215: Bayer AG, Institute of Toxicology, 

Carcinogenicity and Genotoxicity, 1998.

47. Parks LG, Ostby JS, Lambright CR, Abbott BD, Gray LE, Jr. Perinatal butyl benzyl 

phthalate (BBP) and bis(2-ethylhexyl) phthalate (DEHP) exposures induce antiandrogenic 

effects in Sprague-Dawley (SD) rats. Biol Reprod 60:153(1999).

48. Piersma AH, Verhoef A, Dortant PM. Evaluation of the OECD 421 reproductive toxicity 

screening test protocol using butyl benzyl phthalate. Toxicology 99:191-197(1995).

49. Aldyreva MV, Klimova TS, Izumova AS, Timofievskaya LA. The influence of phthalate 

plasticizers on the generative function (translation). Gig Tr Prof Zabol 19:25(1975).

50. TNO NaFRI. Dietary one-generation reproduction study with butyl benzyl phthalate in rats: 

Monsanto, 1993.

51. Barter RA. Correspondence to R. Chapin concerning Solutia’s review of NTP Report 458, 

1997, 1999.

52. Judd JE, Berndtson WE, Castro ACS. Extragonadal sperm reserves, sperm depletion rates, 

numbers of sperm per mating, and fertility with successive matings by intact or unilaterally 

vasectomized rats. J Androl 18:698-707(1997).

53. Ratnasooriya WD, Wadsworth RM. Effect of mating on sperm distribution in the reproductive tract of the male rat. Gamete Research 17:261-266(1987).

54. Ruangsomboon D, Visutakul P. Effect of mating on sperm numbers and weight of the epididymis. Contraception 32:217-221(1985).

55. Seed J, Chapin RE, Clegg ED, Dostal LA, Foote RH, Hurtt ME, Klinefelter GR, Makris SL, 

Perreault SD, Schrader SM, Seyler D, Sprando R, Treinen KA, Veeramachaneni DNR, Wise 

LD. Methods for assessing sperm motility, morphology, and counts in the rat, rabbit, and 

dog: A consensus report. Reprod Toxicol 10:237-244(1996).

56. Bishop JB, Teaf CM, Bhoosan B. Assessment of fetal death rate among in utero progeny of 
Appendix II

II-39

B6C3F1 and CD-1 mice after subcutaneous injections of males with butyl benzyl phthalate 

(BBP). Environ Mutagen 9:15(1987).

57. Zacharewski TR, Meek MD, Clemons JH, Wu ZF, Fielden MR, Matthews JB. Examination 

of the in vitro and in vivo estrogenic activities of eight commercial phthalate esters. Toxicol 

Sci 46:282-293(1998).

58. Jobling S, Reynolds T, White R, Parker MG, Sumpter JP. A variety of environmentally 

persistent chemicals, including some phthalate plasticizers, are weakly estrogenic. Environ 

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59. Bolger R, Wiese TE, Ervin K, Nestich S, Checovich W. Rapid screening of environmental 

chemicals for estrogen receptor binding capacity. Environ Health Perspect 106:551-7(1998).

60. Nakai M, Tabira Y, Asa D, Yakabe Y, Shimyozu T, Noguchi M, Takatsuki M, Shimohigashi 

Y. Binding characteristics of dialkyl phthalates for the estrogen receptor. Biochemical and 

Biophysical Research Communications 254:311-314(1999).

61. Petit F, Le Goff P, Cravedi J-P, Valotaire Y, Pakdel F. Two complementary bioassays for 

screening the estrogenic potency of xenobiotics: Recombinant yeast for trout estrogen 

receptor and troup hepatocyte cultures. Journal of Molecular Endocrinology 19:321-

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62. Coldham NG, Dave M, Sivapathasundaram S, McDonnell DP, Connor C, Sauer MJ. Evaluation of a recombinant yeast cell estrogen screening assay. Environ Health Perspect 105:

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vitro. Environ Health Perspect 1997 105:802-811(1997).

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183-92(2000).

65. Wine R, Li L-H, Barnes LH, Gulati DK, Chapin RE. Reproductive toxicity of di-n-butyl 

phthalate in a continuous breeding protocol in Sprague-Dawley rats. Environ Health Perspect 105:102-107(1997).
II-40

Appendix II

7.0 TABLES

7.1 BBP General Toxicity, Male Rats...........................................................................................41

7.2 BBP General Toxicity, Rats....................................................................................................42

7.3 BBP General Toxicity, Male Rats...........................................................................................43

7.4 BBP General Toxicity, Rats....................................................................................................44

7.5 BBP Developmental Toxicity, Rats........................................................................................45

7.6 BBP Developmental Toxicity, Rats........................................................................................46

7.7 BBP Developmental Toxicity, Rats........................................................................................47

7.8 BBP Developmental Toxicity, Mice .......................................................................................48

7.9 BBP Developmental Toxicity, Rats........................................................................................49

7.10 BBP Developmental Toxicity, Rats........................................................................................50

7.11 BBP Developmental Toxicity, Rats........................................................................................51

7.12 BBP Developmental Toxicity, Rats........................................................................................52

7.13 BBP Reproductive Toxicity Screening Study, Rats................................................................53

7.14 BBP Reproductive Toxicity, Rats...........................................................................................54

7.15 BBP Reproductive Toxicity, Male Rats..................................................................................55

7.16 MBuP Developmental Toxicity, Rats .....................................................................................56

7.17 References..............................................................................................................................57
Appendix II

II-41

Table 7-1: BBP General Toxicity, Male Rats

Species, 

Strain, and 

Source

Experimental 

Regimen

Animal

Number Dose* Body Weight Organ Weight Histopathology Hematopoietic System Chemistry Other

Fischer 344 RatAgarwal 1985(1)

Adult male rats were fed diets with BBP at 0, 0.625, 1.25, 2.5, or 5.0% for 14 days, then were sacrificed and necropsied.

10 010 447a NE ↑Li and Ki NE NE ↑LH LOAEL10 890a NE ↑Li and Ki NE NE NE NE10 1,338a ↓ ↓Te and SV

↑Li and Ki↓Th

Dose-related increase in severity of morphological changes in seminal vesicles, testes and prostate

↓Bone marrow 

cellularity

↑FSH↑LH

↓Food 

consumption

10 1,542b ↓ ↓Te, SV, Ep

↑Li, Ki↓Th

Mild multifocal chronic hepatitis in liver Cortical lymphocytolysis in thymus (atrophy)

↓Bone marrow 

cellularity

↓Test↑FSH↑LH

↓Food 

consumption

*Dose in mg/kg bw/day. aDoses calculated using pre-treatment body weights (200 g) and average food consumed per group during 14-day study. bDose calculated from average body weight during study (since there was a weight loss) and food consumed during the 14-day study.

NE = No Effects↑= Statistically significant increase ↓=Statistically significant decrease 

Li = Liver Te = TestesKi = Kidney Ep = EpididymisTh = Thymus SV = Seminal Vesicle

FSH=Follicle Stimulating HormoneLH = Luteinizing HormoneTest=Testosterone
II-42

Appendix II

Table 7-2: BBP General Toxicity, Rats

Species, 

Strain, and 

Source

Experimental 

Regimen

Animal

Number/ 

Sex

Dose** Body Weight Organ Weight Histopathology Hematology Chemistry Other

Sprague Dawley Rat

4−6 week-old rats were fed diets with BBP at 2,500−20,000 ppm for 3 months, then were sacrificed and necropsied.

10 010 188 NE NE NE NE NA10 375 NE NE NE NE NA NOAEL10 750 NE ↑Ki(M), Li(F) NE NE NA LOAEL10 1,125 ↓(M)* ↑Ki(M), Li NE NE NA10 1,500 ↓* ↑Ki(M), Li NE in liver, testes, 

or pancreas

NE NA

Wistar Rat 4−6 week-old rats 

were fed diets with BBP at 2,500−12,000 ppm for 3 months and sacrificed and necropsied.

27−45 027−45 151(M)−171(F) ↓(M)* ↑Li and Ce(F) NE NE NE LOAEL27−45 381(M)−422(F) ↓* ↑Li and 

Ce(F), Ki

Pancreatic lesions NE NE ↓Urinary 

pH (M)

27−45 960(M)−1,069(F) ↓* ↑Ce(F), Li, Ki Hepatic necrosis 

and pancreatic lesions

Anemia(M) NE ↓Urinary 

pH (M)

SpragueDawley RatHammond 1987 (2)

6−8 week-old rats inhaled BBP mists at 50, 218, or 789mg/m3 for 6 hours/day, 5 days/week for 13 weeks, then were sacrificed and necropsied.

25 025 9.2(M)/9.8(F) NE NE NE NE NE25 39.4(M)/42(F) NE NE NE NE NE NOAEL25 143(M)/152(F) NE ↑Li, Ki NE NE ↓Serum 

glucose 

(M, 13wk)

LOAEL

↑= Statistically significant increase ↓=Statistically significant decrease *Statistical significance is unknown**Dose in mg/kg bw/day

F=FemaleM=MaleNA=Not AnalyzedNE=No Effect

aOrgan to body weight ratioCe=CecumKi=KidneyLi=Liver

wk=Week
Appendix II

II-43

Table 7-3: BBP General Toxicity, Male Rats

Species, 

Strain, and 

Source

Experimental Regimen Animal Number Dose* Body Weight Organ Weight Histopathology Epididymal Sperm Count Hematology Other

Fischer 344/N RatNTP 1997(3)

Sub-chronic study (26 wk) 6-week-old male rats fed diets with BBP at 0, 300, 900, 2,800, 8,300, and 25,000 ppm. Hematological measurements taken every 30 days. Rats were killed and necropsied at the end of the study, epididymal sperm counts were taken.

13 014 30 NE NE NA NA NE14 60 NE NE NA NE NE14 180 NE NE NA NE NE NOAEL15 550 NE ↑Lib NE NE ↑Hb day 60–180 LOAEL11 1,650a ↓ ↑Li, Kib

↓Teb↓SV, Epc

Testicular and epididymal degeneration and seminiferous tubule atrophy.

↓ Sperm 

counts

↑Macrocytic 

anemia days 30−180

*Dose in mg/kg bw/day.aThe dose for the highest exposure level could not be calculated but was estimated from lower doses, assuming equal body weight and food intake.bOrgan to body weight ratio.cAbsolute organ weight.↑= Statistically significant increase ↓=Statistically significant decrease NA=Not analyzed NE=No effects Ep=Epididymis Ki=Kidney

Li=Liver

Hb=HemoglobinSV=Seminal VesicleTe=Testes
II-44

Appendix II

Table 7-4: BBP General Toxicity, Rats

Species, 

Strain, and 

Source

Experimental Regimen Animal Number Dose** Body Weight Organ Weight* Histopathology Hematology Chemistry Other

Fischer 344/N RatNTP 1997(3)

6-week-old rats were fed diets with BBP at 0, 3,000, 6,000, and 12,000ppm (M); 0, 6,000, 12,000, and 24,000 ppm (F) for 2 years. Hematological analysis was conducted at 6, 8, and 15 months and hormone levels were measured at 6, 15, and 24 months. Organ weights were measured at 15 months and histopathology was evaluated at 15 and 24 months. 

0

60 Male:

120 

NE ↑Ki NE NE NE

60 Male:

240

NE ↑Ki

↑Ep

NE NE NE

60 Male:

500

↓ ↑Ki, Li

↑Ep

Renal tubule pigmentation

(15−24 mo)

Hepatic granuloma 

(24 mo)

No testicular effectsFocal pancreatic 

hyperplasia and some evidence of pancreatic carcinogenicity

↓RBC (6 mo)↑Hb (6 mo)

NE ↑Skin 

lesions

60 Female:

300

NE NE Nephropathy (24 mo) NE NE

60 Female:

600

NE NE Nephropathy (24 mo) NE NE

60 Female:

1,200

↓ Renal tubule pigmentation 

(15–24 mo)

Nephropathy (24 mo)Equivocal evidence of 

pancreatic and urinary bladder carcinogenicity 

↑Microcytic 

anemia (15 mo)

↓Triiodothyronine 

(6−15 mo)

 *Organ to body weight ratio∗∗Dose in mg/kg bw/day↑= Statistically significant increase ↓=Statistically significant decrease 

NA=Not analyzedNE=No effectsmo=Month

Ep=EpididymisKi=KidneyLi=Liver

Hb=HemoglobinRBC=Red Blood Cell
Appendix II

II-45

Table 7-5: BBP Developmental Toxicity, Rats

Species, 

Strain, and 

Source

Experimental Regimen Num- bersa Dose* Maternal effects Fetal effects

CD RatField 1989(4)

Prenatal developmental toxicity study. BBP administered in feed on gd 6−15, at 0, 0.5, 1.25, 2.0%. Sacrificed on gd 20. Dams weighed on gd 0, 3, 6, 9, 12, 15, 18, and 20. Maternal liver, kidney, and intact uterus were weighed, corpora lutea were counted and implantation sites examined. All fetuses were weighed and examined for gross external, visceral, and skeletal malformations. 

28 027 420 NOAEL NOAEL30 1.100 ↓ Weight gain (37%)

↑ Liver to body weight ratio↑ Food and water intake

Fetuses with variations/litter 

(41 vs 19%)

29 1,640 ↓ Weight gain (93%)

↓ Corrected weight gain 

(17%)

↑ Liver to body weight ratio 

with no pathological effects

↑Kidney to body weight ratio ↑ Food and water intakeClinical signs of toxicity

↓ Fetal Weight (20%)↓ Live fetuses/litter (n=10 vs 15)↑ Resorptions/litter (40 vs 4%) 

and litters with resorptions (86 vs 32%)

↑ Fetuses with variations/litter

(71 vs 19%)

↑ Fetuses with malformations

(53 vs 2%); 

↑ Litters with malformations 

(visceral, external, and skeletal, especially of the urinary tract, eyes, and spine)(96 vs 25%) 

*Dose in mg/kg bw/day.aNumber of dams pregnant at sacrifice.↑=Statistically significant increase ↓=Statistically significant decrease gd=gestation day
II-46

Appendix II

Table 7-6: BBP Developmental Toxicity, Rats

Species, 

Strain, and 

Source

Experimental Regimen Num- bersa Dose * Maternal effects Fetal effects

Wistar RatsEma 1990(5)

Prenatal developmental toxicity study.Rats were fed diets with DBP at 0, 0.25, 0.5, 1.0, 2.0% from gd 0−20. Body weights and food intake were measured daily. Dams were sacrificed on gd 20. Implantation sites were examined. Pups were sexed, weighed, and evaluated for external malformations. Two-thirds of fetuses were examined for skeletal malformations and 1/3 for visceral malformations.

15 (15) 017 (17) 185 NE NOAEL15 (15) 375 NOAEL ↓Live fetuses/litter 

(n=113 vs 139)

13 (13) 654 ↓Weight gain (35%)

↓ Adjusted weight gain (96%)↓Food Intake

↓Fetal weight (7%)↓Live fetuses/litter 

(n=123 vs 139)c

13 (0) 974 Weight loss (15 g)

Adjusted weight loss (21 g)b↓Food Intake

Complete postimplantation loss 

in all litters

Treatment-related increases in 

malformations, variations, or retardations were not seen at any dose

*Dose in mg/kg bw/day.aNumber of pregnant rats (Number of litters evaluated).bBody weight not including gravid uterus weight. cNot statistically significant

NE=No Effectn=Numbergd=gestation day

↓=Statistically significant decrease ↑=Statistically significant increase 
Appendix II

II-47

Table 7-7: BBP Developmental Toxicity, Rats

Species, 

Strain, and 

Source

Experimental Regimen Num- bersa Dose * Maternal effects Fetal effects

Wistar RatsEma 1992(6)

Prenatal developmental toxicity study.Rats were gavaged with BBP on gd 7−15. Body weights and food intake were measured daily. Dams were sacrificed on gd 20. Implantation sites were examined. Pups were sexed, weighed, and evaluated for external malformations. Two-thirds of fetuses were examined for skeletal malformations and 1/3 for visceral malformations.

10 (10) 010 (10) 500 NOAEL NOAEL

10 (7) 750 ↓ Body weight gain

↓ Food intake

Complete resorption in 3/10 litters↑ Fetal death/litter (n=11 vs 1)↑ Postimplantation loss/litter (82 vs 8%)↓ Fetal weight (18%)↑ Malformations:

• external (12 fetuses/7 litters vs 0), • skeletal (5 fetuses/4 litters vs 1)• internal (3 fetuses/3 litters vs 0) 

10 (0) 1,000 ↑ Death (4 dams)

↓ Corrected body weight gainb↓ Food intake

Complete resorption in 6/6 litters

*Dose in mg/kg bw/day.aNumber of pregnant rats (Number of litters evaluated).bBody weight not including gravid uterus weight. 

n=Numbergd=gestation day

↓=Statistically significant decrease ↑=Statistically significant increase 
II-48

Appendix II

Table 7-8: BBP Developmental Toxicity, Mice

Species, 

Strain, and 

Source

Experimental Regimen Num- bersa Dose* Maternal effects Fetal effects

CD-1 MicePrice 1990(7)

Prenatal developmental toxicity study. BBP administered in feed at 0, 0.1, 0.5, 1.25% on gd 6−15. Sacrificed on gd 17. Dams weighed on gd 0, 3, 6, 9, 12, 15, and 17. Maternal liver, kidney, and intact uterus were weighed, corpora lutea were counted and implantation sites examined. All fetuses were weighed and examined for gross external, visceral, and skeletal malformations.

2928 182 Maternal NOAEL Developmental NOAEL30 910 ↓ Weight gain (15%) ↑ Late fetal deaths/litter (29 vs 07%)

↑ Non-live implants/litter (15 vs 8%)b↓ Live fetuses/litter (n=12 vs 13)↑ Fetuses/litter with malformations 

(14 vs 4%)

↑ Litters with malformations 

(60 vs 31%)

27 2,330 ↓ Weight gain (71%)

↓ Corrected weight gain 

(25%)

↑ Water intake↑ Liver and kidney to body 

weight ratio with no pathological effects

↑ Resorptions/litter (91 vs 7%); ↑ Litters with resorptions (100 vs 55%)↑ % Non-live implants/litter (93 vs 8%)↑ Litters with non-live implants 

(100 vs 59%)b

↓ Live fetuses/litter (n=3 vs 13)↓ Fetal weight (17%)↑ Fetuses/litter with malformations 

(89 vs 4%)

↑ Litters with malformations, especially 

external and skeletal defects of the tail, ribs, sternebrae and vertebrae(100 vs 31%)

↑ Fetuses with variations/litter (98 vs 29%)

*Dose in mg/kg bw/dayaNumber of pregnant dams evaluated at sacrificebNon-live implants include resorptions and late fetal deaths

n=Numbergd=gestation day

↓=Statistically significant decrease ↑=Statistically significant increase 
Appendix II

II-49

Table 7-9: BBP Developmental Toxicity, Rats

Species, 

Strain, and 

Source

Experimental Regimen Num- bersa Dose* Maternal Effects Fetal effects

Wistar RatSharpe 1995(8)

Pre- and post-natal developmental toxicity study. Female rats were exposed to BBP through drinking water at 0 or 1 mg/L for 2 weeks prior to mating, and during mating, gestation and lactation. Rats were mated to untreated males. Dams were allowed to litter. Litter sizes were evaluated at birth. At 90−95 days of age, male offspring were sacrificed and organ weights were determined. 

5 05 0.126−0.336 NA ↑Body weight on pnd 22 (11%). 

↓Absolute testes weight (10%) and testes

to body weight ratio (8%).

6 0.0011 DESb ↓Body weight on pnd 22. 

↓Absolute testes weight and testes 

to body weight ratio

After the first litters were weaned, the experiment was repeated in the same dams. Additional parameters monitored included testicular morphology in 2 pups/group and sperm counts in 7−12 pups/group.

6 05 0.126−0.336 NA ↑Body weight on pnd 22 (14%).

↓Absolute testes weight (7%) and testes 

to body weight ratio (7%).

↓Daily sperm production (∼10−21%).

5 0.0011 DESb ↑Body weight on pd 22.

↓Absolute testes weight and testes 

to body weight ratio.

↓Daily sperm production.

*Dose in mg/kg bw/dayaTotal litters evaluated. The number of treated dams was not stated.bPositive DES control, dose estimated by CERHR.

NA=Not Analyzed↓=Statistically significant decrease ↑=Statistically significant increase 
II-50

Appendix II

Table 7-10: BBP Developmental Toxicity, Rats

Species, 

Strain, and 

Source

Experimental Regimen Num- bersa Dose* Maternal effects Offspring effects

Wistar AP RatAshby 1997(10)

Pre- and post-natal developmental toxicity study. Rats were exposed to BBP through drinking water at 1 mg/L during gestation and lactation (gd 1−pnd 20). Body weights were measured on gd 1, 4, and 22 and pd 3, 7, 14, and 20. Water intake was measured daily. Dams were allowed to litter and following weaning of pups, were killed and necropsied. Liver enzyme activity, hematology, and micronucleated erythrocytes were assessed. Pups were sexed, weighed, and evaluated for sexual maturation. Uterotrophic effects were examined in groups of 10 female rats on pnd 21 and 24. The majority of pups were sacrificed and necropsied on pnd 90 and 10 males/group were sacrificed on pnd 137. Sperm analysis was conducted at necropsy. FSH-positive pituitary cells were counted in 9 rats/sex.

1918 0.183 NE ↑ Male pup weight on pnd 2 (13%).

↑ Anogenital distance in males on pnd 2 (4%)b.↓ Age of vaginal opening (34 vs 35.1 days)b.↑ Liver to body weight ratios in males (4%).No effects on sperm counts, testes weight, or 

uterotrophic response.

5 0.0086 

DESa

↓Body weight ↓Body weight.

↑Uterine weight and uterotrophic response.↑Absolute ovarian weight.↓Anogenital distance in males and females.↓Age of vaginal opening.↑Age of preputial separation.↓Decrease testis, epididymis, seminal vesicle, 

and prostate weight.

↓Decreased sperm count.

*Dose in mg/kg bw/dayaPositive DES controlbAuthors considered effects to be related to increased pup weight.

gd=gestation daypnd=postnatal dayNE=No Effect

↓=Statistically significant decrease ↑=Statistically significant increase 
Appendix II

II-51

Table 7-11: BBP Developmental Toxicity, Rats

Species, 

Strain, and 

Source

Experimental Regimen Num- bersa Dose* Maternal effects Fetal effects

Wistar RatTNO 1998(9)

Pre- and post-natal developmental toxicity study.Female rats were exposed to BBP through drinking water at 0.1, 1, or 3 mg/L for 2 weeks prior to mating, and during mating, gestation and lactation. Rats were mated for 1 week to untreated males, that were only exposed to BBP while breeding. Body weights and food intake were measured weekly and water intake was measured daily. Dams were allowed to litter and following weaning of pups, were killed, necropsied, and implantation sites were examined.Pups were weighed, examined for abnormalities, evaluated for sexual maturation and function, and necropsied at 89–101 days of age.

25 023 0.012 NE NE22 0.140 NE ↑Pup death on pnd 1−4 (14 vs 08%)

(Pup death/litter not significant)

↑Large pups (pnd 4)

24 0.385 NE ↑Pup death on pnd 1−4 (12 vs 08%) 

(Pup death/litter not significant)

↑Cold pups (pnd 1)↑Large pups (pnd 4)↑Hair lossNo effects on sperm morphology, number, 

or motility; estrous cycles; or sexual maturation at any dose level

21 0.0011−0.0055

DESa

↓Gestational weight 

gain

↑Duration of pregnancy

↑Pup death (pnd 1−4)↓Live pups/litter↓Decreased weight gain↑Age of preputial separation ↓ Normal sperm↓Sperm count (significance not known)↓Testes weight

The study was repeated with BBP to verify postnatal pup deaths

26 022 0.140 ↓Pup death on pnd 1−4 (46 vs 10%)24 0.385 ↑ Pup death on pnd 1−4 (17 vs 10%)

↑Stillborn pups (n=28 vs 13)

(Both effects/litter were insignificant)

*Dose in mg/kg bw/dayaPositive DES control, dose estimated by CERHR

NE=No Effectn=Number

↓=Statistically significant decrease ↑=Statistically significant increase 
II-52

Appendix II

Table 7-12: BBP Developmental Toxicity, Rats

Species, 

Strain, and 

Source

Experimental Regimen Numbers* Dose** Maternal effects Fetal effects

Wistar RatBayer 1998(11)

Pre- and post-natal developmental toxicity study. Female rats were exposed to BBP through drinking water or diet at 0, 1, or 3 ppm for 2 weeks prior to mating, and during mating, gestation and lactation. Rats were mated for up to 3 weeks to untreated males, that were only exposed to BBP while breeding. Body weights and food and water intake were measured every 3−7 days. Dams were allowed to litter and following weaning of pups, were killed, necropsied, and examined for implantation sites. At birth, pups were counted, weighed, and examined for abnormalities. Pups were evaluated for survival and weight gain until pnd 21, when they were sacrificed and necropsied.

21−22 022−25 0.08−0.09/0.06−0.07/0.11−0.06a

0.10−0.12/0.11−0.11/0.17−0.24b

No significant effects on fertility, body weight gain or food and water intake.

Non-significant increase in resorptions in both dose groups. No significant effects on litter size, pup viability from birth to pnd 4, and pup weight.

24 0.27−0.28/0.19−0.25/0.34−0.49c

0.34−0.35/0.35−0.35/0.54−0.80d

*Number of females that gave birth to a live litter/exposure media.**Dose in mg/kg bw/day.pnd=postnatal day

aExposure through 1 ppm diet during prebreed/gestation/lactation.bExposure through 1 ppm drinking water during prebreed/gestation/lactation.cExposure through 3 ppm diet during prebreed/gestation/lactation.dExposure through 3 ppm drinking water during prebreed/gestation/lactation.
Appendix II

II-53

Table 7-13: BBP Reproductive Toxicity Screening Study, Rats

Species, 

Strain, and 

Source

Experimental Regimen Dose* Paternal Effects Maternal Litters

WU RatPiersma 1995(12)

Reproduction screening study. BBP administered by gavage to male and females rats 10−11 weeks old for 2 weeks prior to mating. Males were dosed for a total of 29 days and females were dosed until pnd 6. Rats were housed together 1:1 for a maximum of 2 weeks. Body weight and food intake were measured weekly. Dams delivered and nursed pups. F0 were evaluated for fertility and reproductive function, and were killed and necropsied at end of dosing period. Implantation sites were examined and histopathology was conducted. Litters were examined for external malformations, counted, sexed, weighed, and sacrificed and discarded on pnd 6.

0 9/10 females conceived.

250 NE 8/10 females conceived.500 NE 7/10 females conceived. ↓Pup weight on pnd 1 (7%).

1,000 ↓Weight gain (21%).

↓ Testis and epididy–

mis weight in F0males (14%).

↑Leydig cell hyperplasia and testicular 

degeneration.

4/10 females conceived.↓Gestational weight gain 

(42%).

↓Live pups/litter at birth 

(n=2 vs 9) and pnd 6 (n=1 vs 9).

↓Pup weight on pnd 1 and 6 

(29% and 43%).

*Dose in mg/kg bw/day↓=Statistically significant decrease ↑=Statistically significant increase 

NE=No Effectn=Numberpnd=postnatal day
II-54

Appendix II

Table 7-14: BBP Reproductive Toxicity, Rats

Species, 

Strain, and 

Source

Experimental Regimen Animal Number Dose b Effects

Wistar RatTNO 1993(13)

One generation reproductive toxicity study. BBP administered in feed at 0, 0.2, 0.4, 0.8% for 10 weeks and 2 weeks before mating in males and females, respectively, and throughout rest of study. Body weight and food intake measured weekly. One male and two females housed together for 3 weeks. Dams nursed pups through pnd 21. Dams were rebred after first litter was weaned. Study was repeated in the same rats. Litters examined counted, sexed, and weighed. After weaning, F1 examined for external abnormalities and sacrificed. F0 rats were killed and necropsied. Histopathology examined in liver and reproductive tissue of control and high-dose group.

12(M)/

21−20(F)a

0

12(M)/

17−22(F)

108/106116/252

NE

12(M)/

20−21(F)

206/217235/580

NE

12(M)/

17−22(F)

418/446

458/1,078

↑Liver to body weight ratios in F0 females.↓Weight gain of F0 females during gestation and 

lactation.

↓F1b pup weight on pnd 21 (12%).No effects on implantations, reproductive organ 

morphology, or fertility, fecundity, and gestation indices.

aNumber of males and females delivering first and second litter, respectively.bDoses (in mg/kg bw/day) for males during premating / females during premating / females during gestation / females during lactation. NE=No Effectpnd=postnatal day↑=Statistically significant increase ↓=Statistically significant decrease 
Appendix II

II-55

Table 7-15: BBP Reproductive Toxicity, Male Rats

Species, 

Strain, and 

Source

Experimental Regimen Animal Number Dose* Effects

F344/N RatNTP 1997(3)

Sub-chronic reproductive toxicity study (10 wks), in 6-week-old males. BBP was administered in feed at 0, 300, 2,800, and 25,000 ppm for 10 weeks prior to mating. Body weight and food intake were measured weekly. Each male was mated to 2 untreated females for 7 days. Reproductive parameters included fertility and fetal mortality. Males were then killed and examined for hematological, sperm, and histopathological effects. Females were killed and examined for corpora lutea and implantation sites on gd 13 or 13 days after mating.

15 015 20 NE15 200 NOAEL15 2,200 ↓ Sperm concentration (>99%).

Evidence of mating in 10/13 females; no pregnancies.↓Prostate and testes to body weight ratio.↓Epididymis and seminal vesicle weight.Testicular and epididymal degeneration.↓Body weight gain (29%).↑Liver and thymus to body weight ratio.Mild macrocytic anemia response.

*Dose in mg/kg bw/daygd=gestation day↓=Statistically significant decrease ↑=Statistically significant increase 
II-56

Appendix II

Table 7-16: MBuP Developmental Toxicity, Rats

Species, 

Strain, and 

Source

Experimental Regimen Numbersa Dose* Maternal effects Developmental effects

Wistar-King A rats.Imajima et al. (1997)(14)

Pre-and post-natal developmental toxicity study with prenatal exposure.Rats were gavaged with 0 or 300 mg/day MBuP in sesame oil from gd 15–18.

Testicular descent was evaluated in male offspring on gd 20 or pnd 30–40.

19/15 015/26 1,000 Not reported. Testicular ascent on gd 20.

↑ Chryptorchidism in 22/26 male pups on 

pnd 30–40 with 87% of the undescended testes in abdominal cavity and 13% in the inguinal ring.

*Dose in mg/kg bw/day aNumber of male fetuses evaluated on gd 20 / pnd 30–40gd=gestation daypnd=postnatal day↑=Statistically significant increase
Appendix II

II-57

References

1. Agarwal DK, Maronpot RR, Lamb J, IV, Kluwe WM. Adverse effects of butyl benzyl phthalate 

on the reproductive and hematopoietic systems of male rats. Toxicology 35:189-206(1985).

2. Hammond BG, Levinskas GJ, Robinson EC, Johannsen FR. A review of the subchronic toxicity 

of butyl benzyl phthalate. Toxicol Ind Health 3:79-97(1987).

3. NTP. Toxicology and carcinogenesis studies of butyl benzyl phthalate (CAS no. 85-68-7). in 

F344/N rats (feed studies). Rep nr. NTP TR 458, NIH Publication No. 97-3374: U.S. Department of Health and Human Services, Public Health Service, National Institute of Health, 1997.

4. Field EA, Price CJ, Marr MC, Myers CB. Developmental toxicity evaluation of butyl benzyl 

phthalate (CAS No. 85-68-7) administered in feed to CD rats on gestational days 6 to 15 NTP89-246. Research Triangle Park: National Toxicology Program, 1989.

5. Ema M, Murai T, Itami R, Kawasaki H. Evaluation of the teratogenic potential of the plasticizer 

butyl benzyl phthalate in rats. J Appl Toxicol 10:339-43(1990).

6. Ema M, Itami T, Kawasaki H. Teratogenic evaluation of butyl benzyl phthalate in rats by gastric 

entubation. Toxicol Lett 61:1-7(1992).

7. Price CJ, Field EA, Marr MC, Myers CB. Final report on the developmental toxicity of butyl 

benzyl phthalate (CAS No. 85-68-7) in CD-1-Swiss mice. NTP-90-114. Research Triangle 

Park: National Toxicology Program, National Institute of Environmental Health Sciences, 1990.

8. Sharpe RM, Fisher JS, Millar MM, Jobling S, Sumpter JP. Gestational and lactational expsoure 

of rats to xenoestrogens results in reduced testicualr size and sperm production. Environ Health 

Perspect 103:1136-1143(1995).

9. TNO NaFRI. Oral developmental reproduction study with butyl benzyl phthalate in Wistar rats. 

Volume 1 of 3: European Council for Plasticizers and Intermediates, 1998.

10. Ashby J, Tinwell H, Lefevre PA, Odum J, Paton D, Millward SW, Tittensor S, Brooks AN. Normal sexual development of rats exposed to butylbenzyl phthalate from conception to weaning. 

Regul Toxicol Pharmacol 56:102-118(1997).

11. Bayer AG. Butyl benzyl phthalate (BBP) - Developmental reproduction study in Wistar rats 

with application in the diet or drinking water 28215: Bayer AG, Institute of Toxicology, Carcinogenicity and Genotoxicity, 1998.

12. Piersma AH, Verhoef A, Dortant PM. Evaluation of the OECD 421 reproductive toxicity screening test protocol using butyl benzyl phthalate. Toxicology 99:191-197(1995).

13. TNO NaFRI. Dietary one-generation reproduction study with butyl benzyl phthalate in rats: 

Monsanto, 1993.

14. Imajima T, Shono T, Zakaria O, Suita S. Prenatal phthalate causes cryptorchidism postnatally by 

inducing transabdominal ascent of the testis in fetal rats. J Pediatr Surg 32:18-21(1997).
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