NTP-CERHR Monograph on the

Potential Human Reproductive

and Developmental Effects 

of 

Bisphenol A

September 2008 NIH Publication No. 08–5994

Center For The Evaluation of Risks

To Human Reproduction

National Toxicology Program

U.S. Department of Health and Human Services

Table of Contents

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

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

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

NTP Brief on Bisphenol A ...............................................................................................................1

What is Bisphenol A? ...........................................................................................................1

Are People Exposed to Bisphenol A?...................................................................................1

Can Bisphenol A Affect Human Development or Reproduction?........................................6

Are Current Exposures Bisphenol A High Enough to Cause Concern?.............................34

NTP Conclusions................................................................................................................38

Appendix A: Interpretation of Blood Monitoring Studies..................................................40

References...........................................................................................................................45

Appendix I. NTP-CERHR Bisphenol A Expert Panel...................................................................I-1

Appendix II. Expert Panel Report on Bisphenol A ......................................................................II-1

Appendix III: Public Comments and Peer Review Report on Bisphenol A................................ III-1

iii
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v

Preface

The National Toxicology Program (NTP) 1

established the NTP Center for the Evaluation

of Risks to Human Reproduction (CERHR) in

June 1998. The purpose of the CERHR is to

provide timely, unbiased, scientifically sound

evaluations of the potential for adverse effects

on reproduction or development resulting from

human exposures to substances in the environment. The NTP-CERHR is headquartered at

the National Institute of Environmental Health

Sciences (NIEHS) and Dr. Michael Shelby is

the director.2

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

sectors. Chemicals are selected for evaluation

based on several factors including the following:

potential for human exposure from use

and occurrence in the environment

extent of public concern

production volume

extent of database on reproductive and

developmental toxicity studies

CERHR follows a formal processfor review and

evaluation of nominated chemicalsthat includes

multiple opportunities for public comment.

Briefly, CERHR convenes a scientific expert

panel that meets in a public forum to review,

discuss, and evaluate the scientific literature on

the selected chemical. Public comment isinvited

priorto and during themeeting.The expert panel

produces a report on the chemical’sreproductive

•

•

•

•

and developmental toxicities and provides its

opinion of the degree to which exposure to the

chemical is hazardous to humans. The panel

also identifies areas of uncertainty and where

additional data are needed. Expert panel reports

are made public and comments are solicited.

Next, CERHR preparesthe NTP Brief.The goal

of the NTP Brief isto provide the public, as well

as government health, regulatory, and research

agencies, with the NTP’s conclusions regarding

the potential for the chemical to adversely

affect human reproductive health or children’s

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

Brief and the Expert Panel Report. The NTPCERHR Monograph is made publicly available

on the CERHR website and in hardcopy or CD

from CERHR.

12

1

NTP is an interagency program headquartered

in Research Triangle Park, NC at the National

Institute of Environmental Health Sciences, a

component of the National Institutes of Health.

2

Information about the CERHR is available on the

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

Michael Shelby, Ph.D.

Director, CERHR

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

Research Triangle Park, NC 27709

919-541-3455 [phone]

919-316-4511 [fax]

shelby@niehs.nih.gov [email]
vi

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vii

The NationalToxicology Program (NTP) Center

for the Evaluation of Risks to Human Reproduction (CERHR) conducted an evaluation of the

potential for bisphenol A to cause adverse effects

on reproduction and development in humans. The

CERHR Expert Panel on Bisphenol A completed

its evaluation in August 2007.

CERHR selected bisphenol A for evaluation

because of the:

Widespread human exposure

Public concern for possible health ef­fects

from human exposures

High production volume

Evidence of reproductive and developmental toxicity in laboratory animal

studies

BisphenolA (CAS RN: 80–05–7) is a high production volume chemical used primarily in the

production of polycarbonate plastics and epoxy

resins. Polycarbonate plastics are used in some

food and drink containers; the resins are used

as lacquers to coat metal products such as food

cans, bottle tops, and water supply pipes. To

a lesser extent bisphenol A is used in the production of polyester resins, polysulfone resins,

polyacrylate resins, and flame retardants. In addition, bisphenol A is used in the processing of

polyvinyl chloride plastic and in the recycling

of thermal paper. Some polymers used in dental

sealants and tooth coatings contain bisphenol

A. The primary source of exposure to bisphenol

A for most people is assumed to occur through

the diet. While air, dust, and water (including

skin contact during bathing and swimming) are

other possible sources of exposure, bisphenol A

in food and beverages accounts for the majority

of daily human exposure. The highest estimated

daily intakes of bisphenol A in the general population occur in infants and children.

•

•

•

•

The results of this bisphenol A evaluation are

published in an NTP-CERHR Monograph that

includes the (1) NTP Brief and (2) Expert Panel

Report on the Reproductive and Developmental

Toxicity of BisphenolA.Additional information

related to the evaluation process, including the

peer review report for the NTP Brief and public

comments received on the draft NTP Brief and

the final expert panel report, are available on the

CERHR website (http://cerhr.niehs.nih.gov/).

See bisphenol A under “CERHR Chemicals” on

the homepage or go directly to http://cerhr.niehs. 

nih.gov/chemicals/bisphenol/bisphenol.html).

The NTP reached the following conclusions on

the possible effects of exposure to bisphenol A

on human development and reproduction. Note

that the possible levels of concern, from lowest to

highest, are negligible concern,minimal concern,

some concern, concern, and serious concern.

The NTP has some concern for effects on the 

brain, behavior, and prostate gland in fetuses, 

infants, and children at current human exposures to bisphenol A. 

The NTP has minimal concern for effects on the 

mammary gland and an earlier age for puberty 

for females in fetuses, infants, and children at 

current human exposures to bis­phenol A.

The NTP has negligible concern that exposure of 

pregnant women to bisphenol A will result in fetal 

or neonatal mortality, birth defects, or reduced 

birth weight and growth in their offspring. 

The NTP has negligible concern that expo­sure 

to bisphenol A will cause reproductive effects in 

non-occupationally exposed adults and minimal

concern for workers exposed to higher levels 

in occupational settings.

Abstract

NTP-CERHR MONOGRAPH ON THE POTENTIAL HUMAN REPRODUCTIVE 

AND DEVELOPMENTAL EFFECTS OF BISPHENOL A
viii

NTPwilltransmittheNTP-CERHRMonograph

on Bisphenol A to federal and state agencies,

interested parties, and the public and make

it available in electronic PDF format on the

CERHR web site (http://cerhr.niehs.nih.gov) 

and in printed text or CD from CERHR:

Dr. Michael D. Shelby

Director, CERHR

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

Research Triangle Park, NC 27709

919- 541- 3455 [phone]

919- 316- 4511 [fax]

shelby@niehs.nih.gov [email]
ix

Introduction

BisphenolA (CAS RN: 80–05–7) is a high production volume chemical used primarily in the

production of polycarbonate plastics and epoxy

resins. Polycarbonate plastics are used in food

and drink packaging; the resins are used as lacquers to coat metal products such as food cans,

bottle tops, and water supply pipes. To a lesser

extent bisphenol A is used in the production of

polyester resins, polysulfone resins, polyacrylate

resins, and flame retardants. In addition, bisphenol A is used in the processing of polyvinyl

chloride plastic and in the recycling of thermal

paper. Some polymers used in dental sealants

and tooth coatings contain bisphenol A.

In 2007, the CERHR Expert Panel on Bisphenol

A evaluated bisphenol A for reproductive and

developmental toxicity. Because most people in

the United States are exposed to bisphenol A

and a number of studies have reported effects

on reproduction and development in laboratory

animals, there is considerable interest in its possible health effects on people. For these reasons,

the CERHR convened an expert panel to conduct an evaluation of the potential reproductive

and developmental toxicities of bisphenol A.

This monograph includes the NTP Brief on Bisphenol A, a list of the expert panel members

(Appendix I), and the Expert Panel Report on

bisphenol A (Appendix II). The monograph is

intended to serve as a single, collective source

of information on the potential for bisphenol

A to adversely affect human reproduction or

development.

The NTP Brief on Bisphenol A presents the

NTP’s opinion on the potential for exposure to

bisphenol A to cause adverse reproductive or

developmental effects in people. The NTP Brief

is intended to provide clear, balanced, scientifically sound information. It is based on information about bisphenol A provided in the expert

panel report, public comments, comments from

peer reviewers3 and additional scientific information available since the expert panel meeting.

3

Peer review of this brief was conducted by the NTP

Board of ScientificCounselors(sup­plemented with

eight non-voting ad hoc review­ers) on June 11,

2008. The peer report is available at http://cerhr.

niehs.nih.gov/chemicals/bis­phenol/bisphenol.

html.
NTP Brief on Bisphenol A

[CAS NO. 80 –05–07]

Center For The Evaluation of Risks

To Human Reproduction

National Toxicology Program

U.S. Department of Health and Human Services

iii

Table of Contents

What is Bisphenol A?........................................................................................................................1

Are People Exposed to Bisphenol A?................................................................................................1

Can Bisphenol A Affect Human Development or Reproduction? ...................................................6

Supporting Evidence.............................................................................................................7

How Was This Conclusion Reached?..............................................................................9

Human Studies..............................................................................................................15

Laboratory Animal Studies...........................................................................................16

Are Current Exposures to Bisphenol A High Enough to Cause Concern?.....................................34

Supporting Evidence...........................................................................................................34

Daily Intake Exposure Estimates..................................................................................34

Exposure Comparisons Based on Daily Intake.............................................................36

Exposure Comparisons Based on Blood Concentrations of Free Bisphenol A.............37

NTP Conclusions............................................................................................................................38

List of Figures

Figure 1: Chemical structure of bisphenol A....................................................................1

Figure 2a: The weight of evidence that bisphenol A causes adverse

developmental or reproductive effects in humans.............................................7

Figure 2b: The weight of evidence that bisphenol A causes adverse

developmental or reproductive effects in laboratory animals...........................8

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

or reproduction might be effected by exposure to bisphenol A........................8

List of Tables

Table 1: Summary of ranges of estimated daily intakes in people

based on sources of exposure.................................................................................2

Table 2: Urinary concentrations and corresponding “back calculated”

daily intakes of bisphenol A in people ..................................................................5

Table 3: Blood and breast milk biomonitoring of bisphenol A in people............................6

Appendix A: Interpretation of Blood Biomonitoring Studies........................................................40

References.......................................................................................................................................45
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NTP Brief on Bisphenol A

What is Bisphenol A?

Bisphenol A (BPA) is a chemical produced in

large quantities for use primarily in the production of polycarbonate plastics and epoxy resins

(Figure 1).

HO

CH3

CH3

OH

Figure 1.

Chemical structure of Bisphenol A

(C15H16O2; molecular weight 228.29)

It exists at room temperature as a white solid

and has a mild “phenolic” or hospital odor.

Polycarbonate plastics have many applications

including use in certain food and drink packaging, e.g., water and infant bottles, compact

discs, impact-resistant safety equipment, and

medical devices. Polycarbonate plastics are typically clear and hard and marked with the recycle

symbol “7” or may contain the letters “PC” near

the recycle symbol. Polycarbonate plastic can

also be blended with other materials to create

molded parts for use in mobile phone housings,

household items, and automobiles. Epoxy resins

are used as lacquers to coat metal products such

as food cans, bottle tops, and water supply pipes.

Some polymers used in dental sealants or composites contain bisphenol A-derived materials.

In 2004, the estimated production of bisphenol

A in the United States was approximately 2.3

billion pounds, most of which was used in polycarbonate plastics and resins.

CERHR selected bisphenol A for evaluation

because it has received considerable attention in

recent years due to widespread human exposures

and concern for reproductive and developmental

effects reported in laboratory animal studies.

Bisphenol A is most commonly described as

being “weakly” estrogenic; however, an emerging body of molecular and cellular studies indicate the potential for a number of additional

biological activities. These range from interactions with cellular receptors that have unknown

biological function to demonstrated effects on

receptor signaling systems known to be involved

in development.

The NTP Brief on Bisphenol A is intended to be

an environmental health resource for the public

and regulatory and health agencies. It is not a

quantitative risk assessment nor is it intended to

supersede risk assessments conducted by regulatory agencies. The NTP Brief on Bisphenol A

does not present a comprehensive review of the

health-related literature or controversies related

to this chemical. Only key issues and study findings considered most relevant for developing

the NTP conclusions on concerns for potential

reproductive and developmental human health

effects of bisphenol A are discussed. Literature

cited includesthe most relevantstudiesreviewed

in the CERHR Expert Panel Report on Bisphenol A and relevant research articles published

in the peer-reviewed literature subsequent to the

deliberations of the expert panel.

Are People Exposed to

Bisphenol A?

Yes. Based on the available data the primary

source of exposure to bisphenol A for most

people is through the diet. While air, dust, and

water (including skin contact during bathing

and swimming) are other possible sources of

exposure, bisphenol A in food and beverages

accounts for the majority of daily human exposure [(1); reviewed in (2, 3)]. Bisphenol A can

migrate into food from food and beverage con4

Answers to this and subsequent questions may

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

Unknown
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tainers with internal epoxy resin coatings and

from consumer products made of polycarbonate

plastic such as baby bottles, tableware, food containers, and water bottles. The degree to which

bisphenol A migrates from polycarbonate containers into liquid appears to depend more on

the temperature of the liquid than the age of the

container, i.e., more migration with higher temperatures (4). Bisphenol A can also be found in

breast milk (5). Short–term exposure can occur

following application of certain dental sealants

or composites made with bisphenol A-derived

material such as bisphenol A dimethacrylate

(bis-DMA). In addition, bisphenol A is used

in the processing of polyvinyl chloride plastic

and in the recycling of thermal paper, the type

of paper used in some purchase receipts, selfadhesive labels, and fax paper (6, 7). Bisphenol A can also be found as a residue in paper

and cardboard food packaging materials (7).

Workers may be exposed by inhalation or skin

contact during the manufacture of bisphenol A

and bisphenolA-containing products, e.g., polycarbonate and polyvinyl plastics, thermal paper,

epoxy or epoxy-based paints and lacquers and

tetrabrominated flame retardants (6).

Estimating human exposure to bisphenol A is

generally done in one of two ways. Concentrations of bisphenol A can be measured directly

in human blood, urine, breast milk, and other

fluids or tissues (“biomonitoring”). Researchers

can use biomonitoring information, such as the

concentration of bisphenolA in urine, to estimate

(“back calculate”) a total intake that reflects all

sources of exposure, both known and unknown.

Scientists can also add, or aggregate, the amounts

of bisphenol A detected in various sources, i.e.,

food and beverage, air, water, dust.The approach

of aggregating exposure to estimate daily intake

requires sources of exposure to be known and

measured. In general, estimates based on biomonitoring are preferred for calculating total

intake because all sources of exposure are integrated into the fluid or tissue measurement and

do not have to be identified in advance. Estimates

based on sources of exposure are useful to help

discern the relative contributions of various

exposure pathways to total intake.

The highest estimated daily intake of bisphenol

A in the general population occur in infants and

children (Table 1).

Table 1. 

Summary of Ranges of Estimated Daily Intakes in People Based on Sources of Exposure

Population Bisphenol A

μg/kg bw/day Assumptions References

Infant 0–6 months

Formula-fed

1–11* 1 assumes body weight of 4.5 kg and formula intake of

700 ml/day with 6.6 μg/L [maximum concentration detected in U.S. canned formula (23, 24)] (2)

(2, 25 –27)

11 assumes body weight of 6.1 kg and formula intake of

1060 ml/day with (1) 50 μg/L bisphenol A/day migrating

into formula from polycarbonate bottles (8.7 μg/kg bw/

day); and (2) 14.3 μg bisphenol A/day ingested from powdered infant formula packed in food cans with epoxy linings(2.3 μg/kg bw/day) [0.143 kg powder/day (the amount

of powder required to reconstitute a volume of formula of

1060 ml/day) containing 14.3 μg bisphenol A (100 μg bisphenol A/kg powder)]. 8.7+2.3=11 μg/kg bw/day (25)

(continued on next page)
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Population Bisphenol A

μg/kg bw/day Assumptions References

Infant Breast-fed 0.2–1* 0.2 assumes body weight of 6.1 kg and breast milk intake

of 1060 ml/day with 0.97 μg/L bisphenol A [maximum

concentration of bisphenol A detected in Japanese breast

milk samples (28)](25)

(2, 25) 

1 assumes body weight of 4.5 kg and breast milk intake

of 700 ml/day with 6.3 μg/L free bisphenol A [maximum

concentration of free bisphenol A detected in U.S. breast

milk samples (5)](2)

6–12 months 1.65–13* 1.65 assumes body weight of 8.8 kg with (1) 7 μg/L bisphenol A/day from formula intake of 700 ml/day with

10 μg/L (0.8 μg/kg bw/day); and (2) 7.6 μg/kg bisphenol

A/day from ingestion of 0.38 kg canned food/day with 20

μg/kg (~0.85 μg/kg bw/day). 0.8+0.85=1.65 (26)

(24 – 27)

13 assumes body weight of 7.8 kg, formula intake of 920

ml/day, and food consumption of 0.407 kg/day with (1) 50

μg/L bisphenol A migrating into formula from polycarbonate bottles (5.9 μg/kg bw/day); (2) 12.4 μg bisphenol

A/day ingested from powdered infant formula packed in

food cans with epoxy linings (1.6 μg/kg bw/day) [0.124

kg powder/day (the amount of powder required to reconstitute a volume of formula of 920 ml/day) containing 12.4

μg bisphenol (100 μg bisphenol A/kg powder)]; (3) 40.7

μg bisphenol A/day ingested from canned food (5.2 μg/kg

bw/day) [0.407 kg food/day containing 40.7 μg bisphenol

A (100 μg bisphenol A/kg food)]; and (4) 2.04 μg bisphenol A/day migration from polycarbonate tableware (0.26,

or ~0.3 μg/kg bw/day )[0.407 kg food/day containing 2.04

μg bisphenol A (5 μg bisphenol A/kg food)] 5.9+1.6+5.2

+0.3=13.0 μg/kg bw/day (25)

Child 1.5–6 years 0.043–14.7 0.043isthemean(range:0.018–0.071μg/kgbw/day)based

on individual body weight and measured concentrations of

bisphenol in indoor and outdoor air, dust, soil, and liquid

and solid food from day care and home and the assumption

of 100% absorption (29)

(1, 25 – 27, 

29, 30) 

14.7 assumes body weight of 14.5 kg and consumption

of 2 kg canned food/day with (1) 200 μg bisphenol A/day

ingested from canned food (~14 μg/kg bw/day) [2 kg food/

day containing 200 μg bisphenol A (100 μg bisphenol A/

kg food)]; and (2) 10 μg bisphenol A/day migration from

polycarbonate tableware (~ 0.7 μg/kg bw/day) [2 kg food/

day containing 10 μg bisphenol A (5 μg bisphenol A/kg

food)] 14+0.7=14.7(27)

(continued on next page)
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Infants and children have higher intakes of

many widely detected environmental chemicals

because they eat, drink, and breathe more than

adults on a pound for pound basis. In addition,

infants and children spend more time on the floor

than adults and may engage in certain behaviors,

such as dirt ingestion or mouthing of plastic items

that can increase the potential for exposure.

Biomonitoring studies show that human exposure to bisphenol A is widespread (Table 2).

The National Health and Nutrition Examination

Survey (NHANES) 2003–2004 conducted by

the Centers for Disease Control and Prevention

(CDC) found detectable levels of bisphenol A

in 93% of 2517 urine samples from people 6

years and older (8). This study did not include

children younger than 6 years of age. The CDC

measured the “total” amount of bisphenol A in

urine, a value that includes both bisphenol A

and its metabolites. The CDC NHANES data

are considered representative of exposures in

Population Bisphenol A

μg/kg bw/day Assumptions References

Adult General

Population

0.008–1.5** 0.008 assumes body weight of 74.8 kg and is based on

measured concentrations of bisphenol A in 80 canned and

bottled food items and a 24 – hour dietary recall in ~4400

New Zealanders (31)

(24–27, 

30, 31)

1.5 assumes body weight of 60 kg and (1) 70 μg bisphenol

A/day from canned food (1.2 μg/kg bw/day) [3 kg/day total

consumption (1 kg solid food with 50 μg bisphenol A/kg

and 2 L beverage with 10 μg bisphenol A /L)]; and 15 μg

bisphenol A/day migration from polycarbonate tableware

(0.25, or ~0.3 μg/kg bw/day ) [3 kg food/day containing 15

μg bisphenol A (5 μg bisphenol A/kg food)] 1.2+0.3=1.5

μg/kg bw/day (25)

Occupational 0.043–100 0.043 is based on back calculating from a median urinary

bisphenol A concentration of 1.06 μmol/mol creatinine

(2.14 μg/g creatinine) from Hanaoka et al. (32). A daily

intake of 0.043 μg/kg bw/day is based on the assumption of

1200 mg/day creatinine excretion (2.57 μg/day bisphenol

excreted) and a body weight of 60 kg (2).

(2, 27, 33)

100 is the maximal estimated exposures in U.S. powder

paint workers based on time weighted averages of 0.001–

1.063 mg/m3, an inhalation factor of 0.29 m3/kg day (33),

100% absorption from the respiratory system, and 8 hours

worked per day (2).

**A study by Miyamoto et al. (30) reported much lower estimated intakes for infants (0.028 to 0.18 μg/kg

bw/day); however, these estimates were excluded from the summary table because (1) insufficient detail was

presented in the study to understand the assumptions used to derive these values, and (2) the authors assumed

no bisphenol A in breast milk, an assumption not supported by data from the CDC (5) and Sun et al. (28).

**In 2003, the European Union (27) calculated an extreme worst – case scenario of ~9 μg/kg bw/day based on

1.4 μg/kg bw/day from food plus ~7 μg/kg bw/day from wine. The high estimated intake from wine (0.75 L

wine/day with 650 μg bisphenol A /L=325 μg bisphenol A/day, or ~7 μg/kg bw/day, from wine) was based

on an extraction study conducted with an epoxy resin that is sometimes used to line wine vats. A study

published subsequent to the evaluation by the European Union identified a maximum concentration of 2.1 μg

bis­phenol A/L in wine (34).
5

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the United States because of the large number

of people included in the survey and the process

used to select participants. In addition, the analytical techniques used by the CDC to measure

bisphenol A are considered very accurate by the

scientific community. There is some indication

that exposure to bisphenol A may be increasing.

The median levels of bisphenolA in human urine

doubled (from 1.3 μg/L to 2.7 μg/L) and the 95th

percentile values tripled (from 5.2 μg/L to 15.9

μg/L) between NHANES III (1988 –1994) and

NHANES 2003–2004. Many smaller studies

also report detection of bisphenol A in urine,

blood, and other body fluids and tissues from

people in the United States, Europe, and Asia

[(9–12); studies published prior to mid-2007

are reviewed in (2, 3, 13)]. Because bisphenol

A does not persist for long periods of time in

the body, its widespread detection in people

indicates that exposures occur frequently.

Bisphenol A can be detected in the blood of

pregnant women, amniotic fluid, placental tissue, and umbilical cord blood indicating some

degree of fetal exposure (12, 14–17). Concentrations of bisphenol A measured in breast milk

and the blood of pregnant women in the United

States are presented in Table 3.

Table 2. Urinary Concentrations and Corresponding “Back Calculated” 

Daily Intakes of bisphenol A in People (United States)

Population Urinary Concentration of 

Total bisphenol A [μg/L]* (8)

Estimated Intake of bisphenol A 

[μg/kg bw/day]**( 35)

All 2.7 (1.3 – 15.9/149) 0.0505 (0.0235 – 0.2742/3.47)

6–11 years 3.7 (1.7–16.0/46.1) 0.0674 (0.0310 – 0.3105/0.55)

12–19 years 4.2 (1.9 – 16.5/149) 0.0773 (0.0378 – 0.3476/3.47)

20–39 years 3.1 (1.5–15.4/61.4) 0.0563 (0.0272 – 0.289./0.84)

40–59 years 2.4 (1.1–15.5/75.2) 0.0415 (0.0179 – 0.2335/0.88)

60+ years 1.9 (0.8–13.3/52.4) 0.0334 (0.0163 – 0.2331/0.88)

Female 2.4 (1.2–15.7/80.1) 0.0443 (0.0190 – 0.2705/1.40)

Male 3.2 (1.4 – 16.0/149) 0.0572 (0.0269 – 0.2778/3.47)

Data is shown as median (25th – 95th percentile range/maximum)

**The CDC data for ages 20 – 39 and 40–59 years were not presented in the study by Calafat et al. (8). Lakind

et al. (35) obtained these values from data files available on the CDC website (http://www.cdc.gov/nchs/

about/major/nhanes/nhanes2003 –2004/lab03_04.htm ). Lakind et al. (35) conducted a separate analysis

of the CDC data and calculated mean and percentile values within 0.2 μg/L of those presented by Calafat et 

al. (8). The NTP obtained maximum urine concentrations for each category from the CDC data files. The

highest urinary concentrations and estimated intakes in Table 2 represent data from the same individual.

** Lakind et al. (35) assumed that daily intake of bisphenol A was equivalent to daily excretion. Daily excretion

was calculated by multiplying the urine concentration of bisphenol A (μg/L) by 24–hour urinary output

volume. Daily urinary volume was assumed to be 600 ml for children aged 6 –11 years, 1200 for males

and females aged 12 – 19, 1200 for adult females, and 1600 for adult males. Body weight data from the

2003–2004 NHANES database was used to calculate daily intake adjusted for body weight. The NTP

calculated the maximum estimated daily intakes by multiplying the maximum detected urine concentration

for each category by the corresponding default urine output volume used by Lakind et al. and then dividing

this number by the individual’s body weight provided in the CDC data files.
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It is helpful in interpreting the biomonitoring

data for bisphenolA to understand how the body

processes and excretes it once exposure occurs.

Following ingestion, the majority of bisphenol A is quickly bound to glucuronic acid to

produce bisphenol A-glucuronide, a metabolic

process called glucuronidation that is carried

out by enzymes primarily in the liver [reviewed

in (2)]. Glucuronidation makes bisphenol A

more soluble in water and, therefore, easier to

eliminate in the urine and also minimizes its

ability to interact with biological processes in

the body.To a lesser extent, unconjugated parent

(commonly referred to as “free”) bisphenol

A is converted to other metabolites, primarily

bisphenol A sulfate. Understanding the degree

to which bisphenol A is metabolized is very

important in determining whether bisphenol A

poses a potential risk to human reproduction

and development. While free bisphenol A and

its major metabolites (bisphenol A-glucuronide

and bisphenol A-sulfate) can all be measured in

humans, only free bisphenol A is considered to

be biologically active. Bisphenol A is metabolized more quickly following oral exposure compared to non-oral exposures such as inhalation

because of “first pass effects” (see below).

5.Unmetabolized bisphenolA is commonly referred

to as “free”; however, the majority of “free” bisphenol A circulating in human blood is bound to

plasma proteins.

There is evidence in laboratory rodents that very

young animals metabolize bisphenol A less efficiently than adult animals (18 –20). Neonatal

rats have higher circulating concentrations of

free bisphenol A in their blood compared to

older animals given an equal exposure, presumably due to an underdeveloped ability to glucuronidate early in life (18). However, neonatal

rats do have the capacity to metabolize and

eliminate bisphenol A. The specific enzymes

that glucuronidate bisphenol A have not been

identified in people, but there is evidence of

postnatal maturation for a number of glucuronidation enzymes in humans. For this reason, a

reduced ability or efficiency to glucuronidate

is generally predicted for human fetuses and

infants [reviewed in (2)]. However, a number of

the enzymes involved in metabolizing bisphenol

A to bisphenol A sulfate in humans are known

and have been shown to be active in fetal and

neonatal life (21, 22), suggesting that this metabolic pathway may be more important than glucuronidate early in life relative to adulthood.

Can Bisphenol A Affect Human 

Development or Reproduction? 

Possibly. Although there is no direct evidence

that exposure of people to bisphenolA adversely

affects reproduction or development, studies

with laboratory rodents show that exposure to

high dose levels of bisphenolA during pregnancy

and/orlactation can reduce survival, birth weight,

Table 3. Blood and Breast Milk Biomonitoring of bisphenol A in People (United States)

Biological 

Medium

Population

(sample size)

Free bisphenol A (μg/L)

Mean or Median 

[range]

Total bisphenol A (μg/L)

Mean or Median 

[range]

Reference

Blood Pregnant women

(40)

Mean: 5.9

[0.5 – 22.4] (12)

Breast milk Lactating women

(20)

Mean: 1.3;

Median: 0.4

[<0.3 (LOD) – 6.3]

Mean: 1.3;

Median: 1.1

[<0.3 (LOD) –7.3]

(5)

LOD=limit of detection
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and growth of offspring early in life, and delay

the onset of puberty in males and females.These

effects were seen at the same dose levelsthat also

produced some weight loss in pregnant animals

(“dams”).These “high” dose effects of bisphenol

A are not considered scientifically controversial

and provide clear evidence of adverse effects on

development in laboratory animals. However, the

administered dose levels associated with delayed

puberty (≥50 mg/kg bw/day), growth reductions

(≥300 mg/kg bw/day), or survival (≥500 mg/kg

bw/day) are far in excess of the highest estimated

daily intake of bisphenolA in children (<0.0147

mg/kg bw/day), adults(<0.0015 mg/kg bw/day),

or workers (0.100 mg/kg bw/day) (Table 1).

In addition to effects on survival and growth

seen at high dose levels of bisphenolA, a variety

of effects related to neural and behavior alterations, potentially precancerous lesions in the

prostate and mammary glands, altered prostate

gland and urinary tract development, and early

onset of puberty in females have been reported in

laboratory rodents exposed during development

to much lower doses of bisphenol A (≥0.0024

mg/kg bw/day) that are more similar to human

exposures. In contrast to the “high” dose developmental effects of bisphenol A, there is scientific controversy over the interpretation of the

“low” dose findings. When considered together,

the results of “low” dose studies of bisphenol A

provide limited evidence for adverse effects on

development in laboratory animals (see Figures

2a & 2b).

Recognizing the lack of data on the effects of

bisphenol A in humans and despite the limitations in the evidence for “low” dose effects in

laboratory animals discussed in more detail

below, the possibility that bisphenol A may alter

human development cannot be dismissed (see

Figure 3).

Supporting Evidence

The NTP finds that there is clear evidence of

adverse developmental effects at “high” doses of

bisphenolA in the form of fetal death, decreased

litter size, or decreased number of live pups per

litter in rats (≥500 mg/kg bw/day) (36, 37) and

mice (≥875 mg/kg bw/day) (38–40), reduced

growth in rats (≥300 mg/kg bw/day) (36, 37)

and mice (≥600 mg/kg bw/day) (38, 39, 41), and

delayed puberty in male mice (600 mg/kg bw/

day) (41), male rats(≥50 mg/kg bw/day) (37, 42)

and female rats (≥50 mg/kg bw/day) (37, 43).

In addition to these “high” dose effects on survival and growth, the NTP recognizes that there

are studies that provide evidence for a variety of

effects at much lower dose levels of bisphenol

Figure 2a. The weight of evidence that bisphenol A causes adverse 

developmental or reproductive effects in humans

Clear evidence of adverse effects

Some evidence of adverse effects

Limited evidence of adverse effects

Developmental and reproductive toxicity Insufficient evidence for a conclusion

Limited evidence of no adverse effects

Some evidence of no adverse effects

Clear evidence of no adverse effects
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Figure 3. NTP conclusions regarding the possibilities that human development 

or reproduction might be effected by exposure to bisphenol A

1

Based on reduced survival in fetuses or newborns (≥500 mg/kg bw/day) (36 –40), reduced fetal or birth

weight or growth of offspring early in life (≥300 mg/kg bw/day) (36, 37, 41), and delayed puberty in female

rats (≥50 mg/kg bw/day) and male rats and mice (≥50 mg/kg bw/day) (37, 41–43).

2Based on possible decreased fertility in mice (≥875 mg/kg bw/day) (40); altered estrous cycling in female

rats (≥600 mg/kg bw/day) (110), and cellular effects on the testis of male rats (235 mg/kg bw/day) (111).

3

Based a variety of effects related to neural and behavior alterations (≥10 μg/kg bw/day) (44 –50), lesions

in the prostate (10 μg/kg bw/day) (51) and mammary glands (0.0025 –1 mg/kg bw/day) (52, 53); altered

prostate gland and urinary tract development (10 μg/kg bw/day) (54), and early onset of puberty (2.4 and

200 μg/kg bw/day) (48, 55).

Figure 2b. The weight of evidence that bisphenol A causes adverse 

developmental or reproductive effects in laboratory animals

“High” dose developmental toxicity1 Clear evidence of adverse effects

Reproductive toxicity2 Some evidence of adverse effects

“Low” dose developmental toxicity3 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

Serious concern for adverse effects

Concern for adverse effects

Developmental toxicity for fetuses, infants & children

(effects on the brain, behavior and prostate gland) Some concern for adverse effects

Developmental toxicity for fetuses, infants & children

(effects on mammary gland & early puberty in females)

Reproductive toxicity in workers

Minimal concern for adverse effects

Reproductive toxicity in adult men and women

Fetal or neonatal mortality, birth defects, 

or reduced birth weight and growth

Negligible concern for adverse effects

Insufficient hazard and/or exposure data
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A related to neural and behavioral alterations in

rats and mice (≥0.010 mg/kg bw/day) (44 –50),

preneoplastic lesions in the prostate and mammary gland in rats (0.010 mg/kg bw/day and

0.0025 mg/kg bw/day, respectively) (51 –53),

altered prostate and urinary tract development

in mice (0.010 mg/kg bw/day) (54), and early

onset of puberty in female mice (0.0024 and

0.200 mg/kg bw/day) (48, 55).

These “low” dose findings in laboratory animals

have proven to be controversial for a variety of

reasons including concern for insufficient replication by independent investigators, questions on

thesuitabilityofvariousexperimentalapproaches,

relevance of the specific animal model used for

evaluating potential human risks, and incomplete

understanding or agreement on the potential

adverse nature of reported effects. These issues

have been extensively addressed elsewhere (2, 

56–60) and were considered by the NTP when

evaluating the bisphenol A literature.

How Was This Conclusion

Reached?

Scientific decisions concerning health risks

are generally based on what is known as the

“weight-of-evidence.” In the case of bisphenol

A, evidence from the limited number of studies in humans exposed to bisphenol A is not

sufficient to reach conclusions regarding possible developmental or reproductive hazard. In

contrast, there is a large literature of laboratory

animal studies. These include studies of traditional designs carried out to assess the toxicity

of bisphenolA, as well as a wide variety of studies examining the possibility that exposure to

“low” doses of bisphenol A, defined in the NTP

Brief on Bisphenol A as ≤5 mg/kg bw/day (61),

during critical periods of development might

result in adverse health outcomes later in life

due to its estrogenic or other biological properties. Many of these latter studies were designed

not as toxicology studies but rather to probe

very specific experimental questions, and their

results are not always easily interpreted with

regard to how they contribute to the weight-ofevidence for human health risks.

Many of the laboratory animal studies of bisphenol A have technical or design short­comings or

their reports do not provide sufficient experimental details to permit an assessment of technical adequacy (2). As discussed in more detail

below, the NTP did not establish strict criteria

for determining which studies from the bisphenol A literature to consider for the evaluation.

Rather, in an effort to glean information that

might contribute to understanding the numerous

reported effects of bisphenol A, NTP evaluated

many individual study reports. Attention was

paid to issues of sample size, control for litter

effects, and various other aspects of experimental design; however, experimental findings were

initially evaluated in relation to their biological plausibility and consistency across studies

by multiple investigators. Studies were then

evaluated as to their adequacy of experimental

design and the likelihood that any inconsistent

outcomesresulted from differences orshortcomings in experimental design. The NTP considered several overarching issues when evaluating

the bisphenol A literature:

Are the in vivo effects biologically plausible?

Historically, bisphenolA has been characterized

as being weakly estrogenic. For this reason the

most common type of positive control compounds used in bisphenol A studies are potent

estrogens. There is wide variability in in vitro 

estrogenic potency estimates for bisphenol A,

although the mean estimate is ~1,000 to 10,000

times less potent than positive control compounds (2). However, a number of the “low”

dose studies suggest that bisphenol A has a

higher in vivo potency than would be predicted

based on binding to estrogen receptor alpha.

The lack of concordance in potency estimates

based on estrogen receptor binding and in vivo

biological activity has been a point of debate
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in considering the biological plausibility of a

number of the reported low dose effects. The

NTP does not necessarily consider it appropriate to consider the reported biological effects of

bisphenol A exclusively within the context of

estrogen receptor α or β binding. An increasing

number of molecular or cell-based (“in vitro”)

studies suggest that attributing the effects

of bisphenol A solely to a classic estrogenic

mechanism of action, or even as a selective

estrogen receptor modulator (SERM)6, is overly

simplistic. In addition to binding to the nuclear

estrogen receptors ERα and ERβ, bisphenol

A has been reported to interact with a variety

of other cellular targets [reviewed in (2, 62)]

including binding to a non-classical membranebound form of the estrogen receptor (ncmER)

(63 –65), a recently identified orphan nuclear

receptor called estrogen-related receptor gamma

ERR-γ (66 –70), a seven-transmembrane estrogen receptor called GPR30 (71), and the aryl

hydrocarbon receptor (AhR) (72, 73).

Several in vitro studies show that bisphenol

A can act as an androgen receptor antagonist

(72, 74 – 80) and is reportedly mitogenic in a

human prostate carcinoma cell line through

inter­actions with a mutant tumor-derived form

of the androgen receptor (81). Bisphenol A also

interacts with thyroid hormone receptors (TRs)

and, based on in vitro studies, is reported to

either inhibit TR-mediated transcription (82),

inhibit the actions of triiodothyronine (T3) or

its binding to TRs (83, 84), or stimulate cell

proliferation in a thyroid hormone responsive

cell line (85). One in vivo study suggests that

bisphenol A acts as a selective TRβ antagonist

(86). Bisphenol A may also inhibit activity of

aromatase, the enzyme that converts testosterone to estradiol (72, 87).

6

A selective estrogen receptor modulator (SERM)

is a compound that binds nuclear estrogen receptors and acts as an estrogen agonist in some

tissues and as an estrogen antagonist in other

tissues.

The toxicological consequences of the nonnuclear estrogen receptor interactions identified so far are unclear. In some instances, the

physiologic role of the receptor is unknown or

not well characterized, i.e., ERR-γ, GPR30,

which makes interpreting the consistency of the

data impossible with respect to the implicated

mechanism based on the cellular or molecular

studies and the observed in vivo toxicology. In

other instances, the binding affinity of bisphenol

A for the receptor is sufficiently low that no or

minimal influences on biological processes in 

vivo would be expected. However, even when

the physio­logical effects are generally understood, e.g., AR binding, aromatase function,

scientists can only speculate as to the possible

in vivo impacts when multiple receptor or other

cellular interactions are considered together.

Nevertheless, the identification of a growing

number of cellular targets for bisphenol A may

help explain toxicological effects that are not

considered estrogenic or predicted simply based

on the lower potency of bisphenolA compared to

estradiol. Effects mediated through the ncmER

are of interest because of its role in regulating

pancreatic hormone release and because bisphenol A has been shown to activate this receptor in 

vitro at a concentration of 1 nM, which is similar

to the active concentration of the potent estrogen

diethylstilbestrol (63, 65).

Are the in vivo effects reproducible?

Two issues become evident when considering the

topic ofreproducibility of effectsin the bisphenol

A literature. In some cases, the reproducibility

of certain effects has been questioned because

attempts at replication by other researchers

using similar experimental designs did not necessarily produce consistent findings. This leads

to reduced confidence in the utility of the effect

for identifying a hazard. Numerous reasons

have been suggested to explain the inconsistent

findings including differences in sensitivity of

the rodent model, i.e., species, strain, breeding

stock, the author’s funding source, the degree
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of lab­oratory expertise, and variations in diet,7

husbandry and route of admin­istration. However, it is not known if these factors account for

the inconsistencies. In other cases, particularly

for findings based on studies with very specific

experimental questions, variations in experimental design are large enough to conclude that

the reproducibility of the finding is essentially

unknown. A number of these effects have not

been addressed in traditional toxicity studies

carried out to assess the toxicity of bisphenol

A. Typically, the safety studies do not probe for

potential organ effects with the same degree of

specificity or detail as those studies with specific

experimental questions. The NTP evaluated the

biological plausibility of findings with unknown

reproducibility in light of supporting data at the

mechanistic, cellular, or tissue level.

Another issue is that the “low” dose studies

generally have not tested higher dose levels of

bisphenol A, i.e., >1 mg/kg.Testing over a wide

range of dose levels is necessary to adequately

characterize the dose-response relationship.

Typically, effects are easier to interpret when

the dose-response curve is monotonic and the

incidence, severity, or magnitude of response

increases as the dose level increases. Effects that

have biphasic, or non-monotonic dose response

curves, have been documented in toxicology,

endocrinology and other scientific disciplines

(90, 91), but can be more difficult to interpret,

which often limits their impact in risk assessments or other health evaluations.Testing higher

dose levels may also identify additional effects

7Understanding the impact of variations in dietary

phytoestrogen content in laboratory animal studies of estrogenic compounds, including bis­phenol

A, is an active area of inquiry (88). Recent research suggests that bisphenol A may alter DNA

methyl­ation (an epigenetic mechanism to alter

phenotype) following exposure during development and that this effect may be offset by dietary

exposure to methyl donors or the phyto­estrogen

genistein (89).

that aid in interpreting the “low” dose finding

with respect to potential health risk.

Do the in vivo effects represent adverse 

health findings in laboratory animals 

and/or humans?

A general limitation in the “low” dose literature for bisphenol A is that many studies have

addressed very specific experimental questions

and not necessarily established a clear linkage

between the “low” dose finding and a subsequent adverse health impact. For example,

when an effect is observed in fetal, neonatal, or

pubertal animals, investigations may not have

been conducted to determine if the effect persists or manifests as a clear health effect later in

life. Establishing a linkage to an adverse health

impact is important because many of the “low”

dose findings can be described as subtle, which

can make them difficult to utilize for risk assessment purposes.An additional factor in considering the adversity of a finding is determining if

the experimental model is adequate for predicting potential human health outcomes.

How should studies that use a non-oral 

route of administration be interpreted? 

Because the majority of exposure to bisphenol

A occurs through the diet (1), laboratory animal

studies that use the oral route of administration

are considered the most useful to assess potential effects in humans. However, a large number

of the laboratory animal studies of bisphenol A

have used a subcutaneous route of administration to deliver the chemical, either by injection

or mini-pumpsthat are implanted under the skin.

The consideration of these studiesin health evaluations of bisphenol A has proven controversial

(2, 92). There is scientific consensus that doses

of bisphenol A administered orally and subcutaneously cannot be directly compared in adult

laboratory animals because the rate of metabolism of bisphenol A differs following oral and

non-oral administration.There is also consensus

that fetal and neonatal rats do not metabolize
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bisphenol A as efficiently as adult rats at a given dose because the enzyme systems that are

responsible for the metabolism of bisphenol A

are not fully mature during fetal or neonatal life.

However, there is scientific debate on whether

the reduced metabolic capability of neonatal rats

is sufficient to adequately metabolize low doses

of bisphenol A.

In adult rats and monkeys, bisphenol A is

metabolized to its biologically inactive form,

or glucuronidated, more quickly when administered orally than by a non-oral route, e.g., subcutaneously, intraperitoneally, or intravenously

(93– 95). This is because bisphenol A administered orally first passes from the intestine to

the liver where it undergoes extensive conjugation primarily with glucuronic acid before

reaching the systemic circulation (“first pass

metabolism”). Because non-oral administration bypasses the liver, and therefore first pass

metabolism, these routes of dosing in adult rats

and monkeys result in higher circulating concentrations of biologically active, free bisphenol

A compared to oral administration. Although

not tested directly in adult laboratory mice, the

impact of first pass metabolism is predicted to be

similar. Thus, a subcutaneous dose is expected

to have a greater biological effect than the same

dose delivered by mouth in adult laboratory animals, including in the offspring of dams treated

with bisphenol A during pregnancy.

Studies that administer bisphenol A through

non-oral routes are most useful for human health

evaluations when information on the fate, e.g.,

half-life, and concentration of free bisphenol A

in the blood or other tissue is also available.

For example, if the peak and average daily concentrations of free bisphenol A in blood were

measured following non-oral administration,

these values could then be compared to levels

of free bis­phenol measured in rodent studies

where bisphenol A is administered orally or to

levels measured in humans. However, none of the

reproductive and developmental toxicity studies

that treated animals by non-oral routes of administration determined the circulating levels of free

bis­phenol A or its metabolites. As a result, studies that treat laboratory animals using non-oral

routes of administration have often been considered of no or of limited relevance for estimating

potential risk to humans (2, 27, 56).

As discussed previously (see “Are People

Exposed to Bisphenol A?”), fetal and neonatal

rats do not metabolize bisphenol A as efficiently

as the adult and, as a result, have higher circulating concentrations of free bisphenol A for some

period of time compared to adults receiving the

same dose (18 – 20).The peak concentrations of

free bisphenol A in the blood of 4-day old male

and female rat pups orally dosed with 10 mg/kg

are 2013 and 162-times higher than the peak

blood levels measured in male and female adult

rats treated with the same mg/kg dose (18). A

measure of how long it takes the body to eliminate free bisphenol A, referred to as “half-life,”

was also slower at this dose in neonatal rats:

>6.7 hours in male or female pups compared to

well under an 1 hour in adult animals (18).Thus,

for a given administered dose, blood levels of

bisphenol A are higher in neonatal rats than in

adults, and remain so longer following exposure. However, neonatal rats do have the ability to metabolize bisphenol A as indicated by

the presence of bisphenol A glucuronide in the

blood and the inability to detect the free form

within the measurement sensitivity of the assay

by 12 to 24-hours after treatment in females and

males respectively (18).

Neonatalrats appearto be able tomore efficiently

metabolize bisphenol A when given at lower

dose levels than at higher dose levels. Although

Domoradzki et al. (18) also treated neonatal

and adult animals with a lower dose level of bisphenolA, 1 mg/kg, making a direct comparisons

based on age at exposure was not possible at

that dose because free bisphenol A was too low
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to be quantified in the blood of adults. However,

in 4-day old male and female rats treated with 1

mg/kg of bisphenolA, 98–100% of administered

bisphenolA was detected as bisphenolA-glucuronide8 compared to 71–82% at 10 mg/kg, i.e.,

a smaller proportion of administered bisphenol

A is glucuronidated at 10 mg/kg compared to 1

mg/kg. This would be expected when the limited capacity of young animals to metabolize

bisphenol A is overwhelmed by higher dose levels of the compound. These data suggest more

efficient metabolism by neonatal rats at 1 mg/kg

compared to 10 mg/kg and imply that the age

at exposure differences described above may

be less profound in the “low” dose range (≤5

mg/kg bw/day).

Taken together these data indicate that, compared to adults at a given dose, neonatal rats(and

presumably mice) metabolize bisphenol A more

slowly and suggest that differences in circulating levels of free bisphenol A arising from oral

and subcutaneous routes of administration as a

result of “first-pass metabolism” are reduced in

fetal or infant animals compared to adults. This

prediction is supported by a recent study that did

not detect differences in the blood concentration of free bisphenol A as a function of route

of administration (oral versus subcutaneous

injection) in 3-day old female mice following

treatment with either 0.035 or 0.395 mg/kg of

bisphenol A (92).

Additional research is needed to understand the

metabolism of bisphenol A in both laboratory

animals and humans. For example, a complete

assessment of the UDP-glucuronosyltransferase

(UGT) and sulfotransferase (SULT) isoforms

involved in the glucuronidation and sulfation

of bisphenol A is needed for both rodents and

humans. UGT2B1 has been identified asthe prin8

Based on percentage of plasma area under the

curve (AUC) for radioactivity that was bisphenol

A glucuronide.

ciple UGT isoform that metabolizes bis­phenolA

to bisphenol A glucuronide in the rat (20). This

isoform shows low expression and activity during development. However, it isimportant to note

that the Matsumoto et al. study only characterized UGT2B1 activity during development and

did not include other members of the UGT2B

family. Thus, the understanding of bisphenol

A metabolism during development in the rat

is still incomplete. In addition, it is difficult to

translate the rat findings to humans because the

UGT isoform(s) that metabolize bisphenol A in

humans have not been identified. Humans have 7

members ofthe UGT2 family that have functional

activity, 1 UGT2A and 6 UGT2B isoforms.

In contrast, there is information on the SULT

isoformsthat metabolize bisphenolA in humans.

In humans, SULT1A1 has been identified as the

SULT with the highest catalytic activity towards

bisphenol A, although SULT1E1, SULT2A1

and a SULT1C isoforms are also capable of

catalyzing bisphenol A-sulfate formation (21).

In humans, SULT1A1 activity is comparable

in fetal and postnatal liver although there are

differences in localization (hematopoietic stem

cells during fetal life and hepatocytes after

birth). Characterizing the ontogeny of individual

UGT and SULT enzymes is complex as specific

isoforms show unique patterns of expression

during development and also vary with respect

to preferred substrates and associated catalytic

activity. As a result, it is unknown if the various metabolic pathways provide for “sufficient”

metabolism of low doses of bisphenol A in

humans exposed during fetal life and infancy.

Although infants can metabolize bisphenol A, it

is likely that significant variation in the developmental profile, e.g., rate and extent of metabolic

capacity, would be observed at the population

level. The issue of sulfation is also important

given the role of sulfation pathways in regulating endogenous compounds that are involved in

controlling the growth and function of some of

the reproductive tissues identified as targets of
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bisphenol A. For example, this raises the possibility that bisphenol A-sulfate conjugates may

interfere with estriol biosynthesis during fetal

development (96).

While more research in this area is warranted,

data from studies where bisphenol A was given

by subcutaneous injection were considered as

useful in the NTP evaluation as oral administration when treatment occurred during infancy

when the capacity to metabolize bisphenol A is

low. Studies in adult animals, including pregnant dams, that administered bisphenol A by

subcutaneous injection or by a subcutaneous

mini-pump were considered informative for

identifying biological effects of bisphenol A but

not for quantitatively comparing exposures in

laboratory animals and humans.

What is the impact of limitations in 

experimental design and how should 

studies with these limit­ations be 

interpreted? 

The impact on study interpretation due to

limitations in experimental design has been a

significant point of discussion for bisphenol A,

especially for the issues of (1) small sample size,

(2) a lack of experimental or statistical control

for litter effects, and (3) failure to use a positive

control (2, 97).

In general, studies with larger sample sizes

will have more power to detect an effect due to

bisphenol A exposure than studies with small

sample sizes. For this reason, “negative” results

from small sample size studies are viewed with

caution. On the other hand, “negative” results

from studies with larger sample sizes are usually

considered more credible (98). However, there

is no single sample size that can be identified

as appropriate for all endpoints. The ability to

detect an effect is affected by the background

incidence, e.g., tumor or malformation rates in

control animals, variability of a particular endpoint, and the magnitude of the effect. A sample

size of at least six may be reasonable for many

endpoints with low or moderate degrees of

variability, such as body weight, but could be

insufficient to detect statistically significant

differences in endpoints with a higher degree

of variability such as hormone level or sperm

count, or that occur infrequently such as malformations or tumor formation. These factors

can make consistent detection of relatively small

changes especially difficult on endpoints that

have a high degree of inherent variability.

Lack of statistical or experimental control

for litter effects was perhaps the single most

common technical shortcoming noted in the

developmental toxicity studies evaluated by the

CERHR Expert Panel for Bisphenol A (2). Adequate control for litter effects when litter­mates

are used in an experiment is considered essential

in developmental toxicology. In 2000, the NTP

co-sponsored a workshop with the U.S. Environmental Protection Agency referred to as the

“Low Dose Endocrine Disruptors Peer Review.”

As part of the peer review, a group of statisticians reanalyzed a number of “low” dose studies (98). Based on studies that used littermates,

they determined that litter or dam effects were

generally present such that pups within a litter

were found to respond more similarly than pups

from different litters. The overall conclusion on

this issue was that “[f]ailure to adjust for litter

effects (e.g., to regard littermates as independent observations and thus the individual pup

as the experimental unit) can greatly exaggerate

the statistical significance of experimental findings.” Studies that did not adequately control

for litter effects were given less weight in the

NTP evaluation and were generally only used

as supportive material.

The NTP concurs with the opinion of several

scientific panelsthat positive control groups can

be very useful to evaluate the sensitivity and

performance of a given experimental model (2, 

60, 98). However, the NTP does not consider
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use of a positive control to be a required study

design component particularly in animal model

systemsthat are well characterized regarding the

background incidence of “effects” and their variability. For bisphenolA studies, potent estrogens,

such as diethylstilbestrol, ethinyl estradiol, 17βestradiol, and estradiol benzoate, are the most

commonly used positive control chemicals given

bisphenol A’s historical classification as a weak

estrogen. Failure to obtain predicted responses

with these chemicalsis generally interpreted as a

“failed” experiment, perhapsreflecting the selection of a relatively insensitive animal or experimental model or insufficient chemical challenge.

Studies where no responses are observed in the

positive control group have generally contributed less weight to evaluations of bisphenol A

(2, 60). The significance of a “failed” positive

control for bisphenol A varies from endpoint

to endpoint and reflected more negatively on a

study in the NTP evaluation when the predicted

effect on reproductive tissue or function was not

observed at dose levelsthatshould be sufficiently

high to produce an effect. In addition, although

potent estrogens are used as positive controlsfor

bisphenol A, as discussed earlier an increasing

number of molecular or cell-based studies suggest that interpreting the toxicological effects of

bisphenol A solely within the context of their

consistency with a classic estrogenic mechanism

of action is overly simplistic.

Human Studies

Only a very small number ofstudies have looked

at associations between bisphenol A exposure

and disorders of reproduction or developmental

effects in humans [(12, 99, 100), studies prior to

mid-2007 reviewed in (2, 3)].The human studies

have looked at the relationship between urine or

blood concentrations of total or free bisphenol A

and a variety of health measures including levels

of certain hormones that help regulate reproduction (32, 101), markers of DNA damage

(102), miscarriage (103), chromosomal defects

in fetuses (104), fertility and obesity in women

(16, 99, 105), effects on the tissue that lines the

uterus (“endometrium”) (99, 106), polycystic

ovary syndrome (101, 105), and birth outcomes

and length of gestation (12, 100).

In these studies, there are reports of associations

between higher urine or blood concentrations of

bisphenol A and lower levels of follicle – stimulating hormone in occupationally exposed

men (32), higher levels of testosterone in men

and women (101, 105), polycystic ovary syndrome (101, 105), recurrent miscarriage (103),

and chromosomal defects in fetuses (104). In

addition, one study reported that patients with

endometrial cancer and complex endometrial

hyperplasia had lower blood levels of bisphenol

A than healthy women and women with simple

endometrial hyperplasia (106). Bisphenol A

was not associated with decreased birth weight

or several other measures of birth outcome

in two recent studies (12, 100). Drawing firm

conclusions about potential reproductive or

developmental effects of bisphenolA in humans

from these studies is difficult because of factors

such assmallsample size, cross-sectional design,

lack of large variations in exposure, or lack of

adjustment for potential confounders. However,

the CERHR Expert Panel on Bisphenol A (2) 

concluded that several studies collectively suggest hormonal effects of bisphenol A exposure

(32, 101, 105) including one in occupationally

exposed male workers likely exposed through

multiple routes including inhalation (32).

The NTP concurs with findings of the recent

evaluations (2, 3) that while these studies may

suggest directions for future research, there is

currently insufficient evidence to determine if

bisphenol A causes or does not cause reproductive toxicity in exposed adults. There is also

insufficient evidence from studies in humans to

determine if bisphenol A does or does not cause

developmental toxicity when exposure occurs

prenatally or during infancy and childhood.
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Laboratory Animal Studies

In contrast to the limited literature evaluating

possible effects of bisphenolA in humans, the scientific literature on the toxic effects of bisphenol

A in laboratory animalsis extensive and expanding. For example, between February 2007 (the

cut-off date for literature included in the CERHR

Expert Panel Report on Bis­phenol A) and April

11, 2008, more than 400 new articles related to

bisphenol A were identified by PubMed search.

All new studiesrelated to the potential reproductive and developmental effects of bisphenol A

were considered during preparation of the NTP

Brief on BisphenolA. However, only those studies that were considered the most informative

for developing NTP conclusions are cited in

the Brief. In addition to the new literature cited,

many key studies reviewed in the expert panel

report are cited herein.

Reproductive Toxicity Studies

The reproductive toxicity studies of bisphenol

A include assessment of fertility, sperm counts,

estrous cycling, and growth or cellular damage

in reproductive tissues. Reproductive toxicity

can be studied in animals exposed during adulthood, during development, or both. Conclusions

on reproductive toxicity presented in this section

of the NTP Brief on Bisphenol A are limited to

the assessment of fertility in laboratory animals,

regardless of when exposure occurred, and other

indicators of reproductive effects in animals

exposed only during adulthood. Assessments of

aspects of the reproductive system other than

fertility in animals exposed during development

are discussed under the headings of “High”

Dose and “Low” Dose Developmental Toxicity

Studies below.

Studies show that bisphenol A does not reduce

fertility in laboratory animals exposed in adulthood and/or during development at dose levels

up to 500 mg/kg bw/day in rats (37, 107). Fertility may be negatively impacted at higher dietary

doses (≥875 mg/kg bw/day) in mice exposed

as adults as indicated by a decreased number

of litters per breeding pair (40), although two

multigenerational reproductive toxicity studies

did not report effects on fertility in mice at doses

up to 1669– 1988 mg/kg bw/day (39, 41).There

are occasional reports of decreased fertility in

smaller sample size studies of rodents exposed

to much lower dose levels of bisphenol A during adulthood, such as oral treatment with 0.025

and 0.100 mg/kg bw/day in male mice (108). In

the Al-Hiyasat et al. study, decreased pregnancy

rates and increased incidence of resorptions in

untreated female mice were attributed to effects

in treated adult males, i.e., reductions in the

number of testicular or epididymal sperm and

hypothesized impaired sperm quality. However,

the magnitude of the impact on weight– corrected testicular or epididymal sperm number,

~16 to 37%, is not generally considered severe

enough to account for the observed pregnancy

rate decrease of ~33 to 40%.9

At high oral dose levels, adult exposure to

bisphenol A caused reproductive toxicity in

the form of altered estrous cycling in female

rats (≥600 mg/kg bw/day)10 (110) and cellular

effects on the testis of male rats (235 mg/kg

bw/day) (111). In addition, more subtle effects

on maternal behavior, i.e., decreased duration

of licking and grooming of pups, are reported

at a lower oral dose in treated adult female rats

(0.04 mg/kg bw/day) (112).

“High” Dose Developmental Toxicity 

Studies (>5 mg/kg bw/day)

Results from developmental toxicity studies

in mice and rats show adverse effects on pup



Sperm counts in laboratory rodents and rabbits

generally have to be severely impacted to cause

infertility. Rats may still be fertile with a 90%

reduction in sperm count (109).

10Animals were treated with 1000 mg/kg bw/day

for 1-week and then the dose was reduced to 600

mg/kg for 22– 25 additional days.
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survival and growth following maternal exposure to dose levels of bisphenol A defined by

the NTP as “high” (>5 mg/kg bw/day). In rats,

a ~20–36% decrease in the number of pups

per litter is reported following maternal dosing

with ≥500 mg/kg bw/day (36, 37). Increases in

fetal death and post-implantation loss are seen

in rats treated with 1000 mg/kg bw/day during

pregnancy (36). Reductions in fetal weight or

growth during postnatal life occur at oral dose

levels of ≥300 mg/kg bw/day in rats (36, 37).

In mice, developmental toxicity is generally

reported at higher oral doses in the form of fetal

death, decreased number of live pups, reduced

fetal or pup body weight at ≥875 mg/kg bw/day

(38–40), and reductions in body weight during postnatal life in the F1 generation (but not

the F2 generation) at 600 mg/kg bw/day (41).

Fetal death in mice has also been observed in

a recent study that reported embryo lethality

following subcutaneous dosing with 10 mg/kg

bw/day bisphenol A to pregnant mice (113).

Occasionally, decreases in pup survival have

been reported at much lower oral dose levels,

such as 0.0024 mg/kg bw/day in mice (114).

However, this effect is not typically reported at

oral doses in this range even in studies from the

same laboratory using a similar dosing regimen

and the same source of mice (115).

Delayed onset of puberty (assessed by day of

vaginal opening) has been reported in the female

offspring of rats orally treated with bisphenol A

at 50 mg/kg bw/day during gestation (43) or 500

mg/kg bw/day during gestation and lactation

(37). In the study by Tyl et al. (37), this effect

has been attributed to a decrease in body weight

also observed at that dose and has not necessarily been considered a direct developmental effect

(27). However, decreased body weight was not

observed in females at the dose where delayed

vaginal opening was reported by Tinwell et al. 

(43). This high dose effect of delayed vaginal

opening is not the predicted effect of exposure

to an estrogenic compound. It is worth noting

that Tinwell et al. (43) did not detect any difference in onset of puberty in female rats when

age at first estrus assessed by vaginal smear was

used as the marker of puberty. Other “high” dose

studies report no effect on onset of puberty in

female rats exposed during gestation and lactation at maternal oral doses ranging from 3.2 to

~1000 mg/kg bw/day (116 –119). One “high”

dose study reported an accelerated onset of

puberty in female rats following subcutaneous

injection of bisphenol A during early postnatal

life at 105 and 427 mg/kg bw/day (120). Delayed

puberty in male rats treated during development

has also been reported at oral doses of ≥50 mg/

kg bw/day (37, 42). This effect was associated

with decreased body weight in the study by Tyl

et al. (37), but not in the study by Tan et al. (42).

A delay in puberty of 1.8 days has also been

reported in male mice at 600 mg/kg bw/day in a

2-generation reproductive toxicity study (41).

With the exception of a possible morphological alteration of the urethra (discussed below)

(54), bisphenol A has not been shown to cause

malformations, such as skeletal birth defects or

abnormally shaped or absent organs, in rats or

mice at oral doses up to 1000 and 1250 mg/kg

bw/day, respectively (36, 38). An indication of a

possible developmental delay, apparent delayed

bone formation (“ossification”), was reported at

an oral dose level of 1000 mg/kg bw/day (36). A

more subtle effect, cellular changes in the liver,

in developmentally exposed animals has been

reported at ≥50 mg/kg bw/day (41).

“Low” Dose Developmental Effects 

(≤5 mg/kg bw/day)

Neural and Behavioral Alterations

The NTP concurs with the CERHR Expert Panel

on BisphenolA that there is a sufficiently consistent body of literature to suggest that perinatal or

pubertal exposure to “low” doses of bisphenolA

causes neural and behavioral alterations in rats

and mice, especially related to the development
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of normal sex-based differences between males

and females(“sexual dimorphisms” or “sexually

dimorphic”).

Research on the effects of bisphenol A on the

brain and behavior does not have as long a history as the assessment of reproductive tissues,

but is now an active area of study that has been

growing quickly in the past few years. Currently, the literature is composed of a collection

of findings based on behavioral assessments,

morphometric and cell-based measurements of

the brain of laboratory animals, and in vitro 

studies to identify molecular and cellular targets

and mechanisms of action. From these studies,

themes are emerging that suggest exposures to

bisphenol A can produce a loss or reduction of

sexual dimorphisms in non-reproductive behaviors and in certain regions of the brain as well

as effects on the dopaminergic system. Neural

effects are also implicated from mechanistic

studies that show bisphenol A can interfere with

thyroid hormone signaling.

Sexual dimorphisms include differences in the

size, cellular composition, or molecular expression patterns of specific regions or structures

in the brain. The studies detecting bisphenol

A-induced changes in sexually dimorphic brain

structures generally report a reduction or loss of

sexual dimorphisms, for example, in the locus

ceruleus (LC; a brain region involved in mediating responses to stress) (121, 122), and the

bed nucleus of the stria terminalis (involved in

regulating emotional behavior) (123). Similar

effects are reported in some, but not all, studies

(124 –126) of the anteroventral periventricular

nucleus (AVPV), a brain region that provides

input to gonadotropin-releasing hormone neuronsinvolved in regulating ovulation.The lowest

administered doses delivered to either pregnant

dams or neonatal animals associated with these

effects range from ~0.03 mg/kg bw/day (oral)

(122), 0.000025 mg/kg bw/day (subcutaneous mini-pump) (125) to ~100 mg/kg bw/day

(subcutaneous injection) (124). Changes are not

reported for all sexually dimorphic structures.

One well-known sexually dimorphic structure

reportedly not affected even at doses up to 320

mg/kg bw/day in rats is the sexually dimorphic

nucleus in the preoptic area (SDN-POA), a brain

region that has a homologue in humans and is

known to be modified by gonadal hormones

during perinatal life (116, 118, 121, 122, 126, 

127). Interpreting the potential human health or

behavioral significance of effects on sexually

dimorphic brain regions can be difficult. For

example, the bed nucleus of the stria terminalis

is described as being responsive to reproductive

hormones and generally involved in regulating

emotional behavior (128), but the specific functions of this brain region in rats, and therefore

the impact of loss of sexual dimorphism, remain

unclear.

Effects on behavior have been assessed by a

wide variety of experimental tests. Reported

behavioral changes in rats or mice relate to play

(129), maternal behavior (44, 112), aggression

(130, 131), cognitive function (132), motor activity (133, 134), exploration (46), novelty-seeking

(45, 46,135), impulsivity (135), reward response

(45, 135– 137), pain response (138), anxiety and

fear (46, 48, 50, 139), and social interactions

(140). Many of these behaviors, including activity, anxiety, exploration, and novelty seeking are

sexually dimorphic to some degree. The lowest

oral dose associated with behavioral changes is

0.01 mg/kg bw/day (via treatment to the pregnant dam) (44 –46) and a number of behavioral

changes have been reported following developmental exposure to oral doses between 0.01 and

1 mg/kg bw/day (48, 50, 112, 129–132, 135, 

138, 140–142).

With the exception of a study that showed a

slight increase in receptive behavior in females

and an impairment of sexual performance

in males (130), the loss of behavioral sexual

dimorphisms does not relate to reproductive
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behavior(116, 122, 143). For instance,responses

to novelty and exploratory behavior are sexually

dimorphic behaviors where female mice tend to

display more of these behaviors than males (46, 

135). Bisphenol A seems to dampen this sexdifference by reducing the expression of these

behaviors in female mice (“defeminization” or

“masculinization”)exposedduringdevelopment,

either through gestation via the dam with oral

doses of 0.01 mg/kg bw/day or through gestation

until weaning at 0.04 mg/kg bw/day (46, 135).

While a loss of sexual dimorphism seems to

be one general trend observed in the behavior

literature, findings for other effects can be more

difficult to interpret. A number of studies have

looked at the relationship between developmental exposure to bisphenol A and increased

activity.The studiesthat most directly support an

effect of increased activity administered bisphenolA directly into the brain (133, 134, 144, 145).

This route of administration limits the ability to

interpret these studiesin relation to human exposure levels as well as to compare the findings to

results from other studies that use more typical

routes of administration. Other studies using

similar behavior assessments have not reported

differences in spontaneous motor activity in the

offspring of dams orally treated with a range of

doses from 0.1–400 mg/kg bw/day (50, 146).

Indications of increased activity based on other

types of behavioral tests are also mixed. Some

studiesreport no impact of bisphenolA treatment

on activity (107, 142, 147), increased morphineinduced locomotion in animals treated during

development with bisphenol A (136, 148), no

difference between control and bisphenol A

treated animals in response to methylphenidate,

a drug used to treat attention deficit hyperactivity disorder (ADHD) (147), and decreased

amphetamine-induced activity in bisphenol Atreated male rats (46). The literature provides

more consistent support for a loss of sexual

dimorphism in locomotor activity. Bisphenol

A exposure during development eliminated statistically significant sex differences observed in

control animals where females are more active

than males (122, 125), or caused significant

differences in activity consistent with a loss of

sexual dimorphism, i.e., increased activity in

male, but not female rats (149).

Certain behavioral effects such as alterations in

locomotor activity, reward behavior, response

to novelty, motivation, cognition, and attention

can display some degree of sexual dimorphism

but also implicate involvement of the dopaminergic system, a monoaminergic neurotransmitter. Inter­actions with the dopaminergic system

are supported by findings that bisphenol A can

alter the gene expression of D1, D3, and D4

dopamine receptors (137, 145, 150) and dopamine transporters (145, 151, 152). In addition,

several studies report that perinatal exposure to

bisphenol A can alter (usually decrease) expression of the rate–limiting enzyme for dopamine

synthesis, tyrosine hydroxylase (TH), that catalyzes the conversion of tyrosine to a pre-cursor

of dopamine, dihydroxyphenylalanine (DOPA),

in several regions of the brain including the substantia nigra (145, 153), the anteroventral periventricular nucleus of the hypothalamus(AVPV)

(124), midbrain (151), limbic area (152), and

rostral periventricular preoptic area (125).

Additional support for the brain as a target of

bisphenol A is provided by a number of studies that report neural alterations at the cellular

level including interactions with or changes in

measures of expression of a number of receptors involved in brain function, such as estrogen

receptors ERα and ERβ (47, 154–156), gammaaminobutyric acid type A (GABAA) (157, 158),

progesterone (159, 160), aryl hydrocarbon receptor (AhR), retinoic acid receptor (RAR) alpha,

retinoid X receptor (RXR) alpha (161–163),

and thyroid receptors (82–86). Other studies

report effects on neuronal migration or organization (164, 165), synaptogenesis (166, 167),

GABA-induced currents (158), neuronal cell
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death (168), synaptic plasticity (169); thyroid

receptor-mediated differentiation of oligo­dendro­cytes (170), and reduced proliferation of

neural progenitor cells (171).

The NTP concurs with the CERHR Expert Panel

on Bisphenol A that the results of neurological

and behavioral studies of exposures of laboratory animals to bisphenolA during development

raise questions about possible risks to human

development.Although the technical merit of the

scientific literature on brain and behavior varies,

a number of the “low” dose studies have been

considered by various evaluation groups to be

experimentally well-conducted.11 For example,

the CERHR Expert Panel on Bisphenol A classified the studies by Kwon et al. (116), Negishi

et al. (50), Della Seta et al. (49), Palanza et al. 

(44), and Ryan and Vandenbergh (48) as having

“high utility” in its evaluation.

The NTP also concurs with the CERHR Expert

Panel on Bisphenol A that additional research

is needed to more fully assess the functional,

long-term impacts of exposures to bisphenol A

on the developing brain and behavior. Overall,

the current literature cannot yet be fully interpreted for biological or experimental consistency or for relevance to human health.12 Part

of the difficulty for evaluating consistency lies

in reconciling findings of different studies that

use different experimental designs and different

specific behavioral tests to measure the same

dimension of behavior.

11The studies by Negishi 2004 et al. (50), Carr et 

al. (132), Ryan and Vandenberg (48), and Adriani et al. (135) were described as sufficiently

reliable for regulatory use in a minority opinion

expressed by Denmark, Sweden, and Norway in

the latest European Union risk assessment (6).

Dr. Michael Baum, an ad hoc reviewer in the

NTP Board of Scientific Counselors peer review,

considered the studies by Ryan and Vandenberg

(48), Gioiosa et al. (46), and Rubin et al. (125)

to be exceptionally well-conducted.

For some of the reported effects, there is some

degree of consistency in the rodent studies (96).

For example, Rubin et al. (125) reported that

perinatal exposure to low doses of bisphenol

A significantly reduced the number of tyrosine

hydroxylase (dopaminergic) neurons in the

AVPV of female mice to the values observed in

control and bisphenol-treated males. Because

the AVPV is involved in regulating ovulation in

rodents this finding is consistent with an earlier

study by this same group that reported disrupted

estrous cyclicity in adult female mice following

perinatal exposure to bisphenol A (173). However, the manifestation or translation of these

effects to primates or humans is unclear because

there is no homologous hypothalamic structure

to the AVPV in humans.12

Another issue that complicates translation of

the rodent findings to primates and humans is

species differences in the role of estradiol in

regulating sexual differentiation of the brain

12The following “low” dose studies were cited in

the “Characterization of Risk to Human Health”

section of the Health Canada Draft Screening

Assessment for Bisphenol A (172): Palanza et 

al. 2002 (44), Laviola et al. 2005 (45), Gioiosa

et al. 2007 (46), Farabollini et al. 2002 (130),

Della Seta et al. 2005 (112), Adriani et al. 2003

(135), Negishi et al. 2004 (50), and Carr et al.

2003 (132). From these studies, Health Canada

concluded that “While collectively these studies

provide evidence that exposure to bisphenol A

during gestation and early postnatal life may be

affecting neural development and some aspects

of behaviour in rodents, the overall weight of evidence was considered limited from the perspective of rigour (e.g., study design limitations such

as conduct of behavioural assessments at a single

time point); power (e.g., limited number of animals per test group), corroboration/consistency

(limited consistency of studies) and biological

plausibility (e.g., certain studies involve use of a

single dose, lack of dose response relationship).

These limitations make it difficult to determine

actual significance of findings to human health

risk assessment.”
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(96). In brief, estradiol has a clearer role in

regulating male-typical brain and behavioral

sexual differentiation in rodents compared to

primates and humans. The sexual dimorphism

of a number of the neural and behavioral endpoints affected by bisphenol A exposure, i.e.,

AVPV and LC volume/cell number, locomotor

activity, exploration in the plus maze, have been

shown to depend on estradiol formed perinatally in the male rodent brain via aromatization of testosterone secreted from the fetal and

neonatal testes. To the extent that these effects

of bisphenol A are due to its interactions with

the estrogen receptor the translation of these

findings to humans is not clear because there

is currently no evidence that estrogen receptor

signaling plays an essential role in male-typical

brain and behavioral sexual differentiation in

primates including humans (96).

However, as discussed previously a number of

studies suggest that bisphenol A may also exert

biological effects by mechanisms that do not

involve estrogen receptor binding. Forthisreason,

future neural and behavioralstudies of bisphenol

A should not focus exclusively on estrogen receptor-mediated endpoints. For example, a number of

male-typical brain and behavioral sexual dimorphisms in rodents depend on androgen receptor

signaling, i.e., spinal nucleus of the bulbocavernosus, the posterodorsal medial amygdalar

nucleus, the ventromedial hypothalamic nucleus,

the suprachiasmatic nucleus, forebrain response

to pheromones, play fighting and the preference

of males to seek out female vs same-sex (male)

urinary odors.These androgen receptor-mediated

behaviors are considered to have more direct

relevance to humans and should be more thoroughly assessed following bisphenolA exposure

given suggestionsthat bisphenolA can modulate

androgen receptor activity or expression levels.

In addition,severalstudiessuggest that bi­s­phenol

A may interfere with the dopaminergic system.

The CERHR Expert Panel on Bis­phenol A did

not consider most of these studies useful in

the evaluation by due to experimental design

limitations and/or use of a non-oral route of

administration.Additional research that includes

assessment of dopaminergic-related endpointsto

address the limitations in the current literature

would be helpful.

Future studies should also take precautions to

distinguish between “organizational” and “activational” effects of hormones. Organizational

effects are permanent and induced by hormones

during perinatal life whereas activational effects

are acute, generally reversible, and occur

throughout life (174). Many sexual and other

behaviors reflect both organizational and activational influences of hormones (175). Observed

behavioral effects of perinatal bisphenol A

could reflect organizational changes in the brain

accomplished during the normal perinatal period

of brain sexual differentiation or, alternatively,

they could simply reflect group differences in

plasma levels of circulating sex hormones at the

time of adult testing. Only a small number of the

studies in the bisphenol A literature controlled

for the influence of differences in plasma levels

of circulating sex hormones at the time of testing,

for example by testing ovarectomized females,

using females at the same stage of the estrouscycle, or conducting assessments on pre-pubertal

animals (46, 48, 125).

Mammary Gland

There is evidence from rodentstudiessuggesting

that perinatal exposure to bisphenol A via subcutaneous mini-pump at administered doses of

0.0025 to 1 mg/kg bw/day causes tissue changes

(“lesions”) in the mammary gland that may signal

an increased susceptibility to develop mammary

gland tumorslater in life (52, 53).Although these

lesions have been described as preneoplastic, currently no data are available that assess whetherthe

reported lesions progress to invasive carcinoma.

For this reason, the evidence is not sufficient to

conclude that bisphenol A is a rodent mammary
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gland carcinogen or that bisphenol A presents a

breast cancer hazard to humans.

While bisphenol A has not been shown to cause

cellular changes or cancer of the mammary gland

in female rats and mice exposed as adults (176),

two recent studies suggest that exposure of rats

to bisphenol A during gestation may lead to the

development of “preneoplastic” lesions in adulthood, ductal hyperplasia and carcinoma in situ,

that may potentially progress to tumors (52, 53).

[Technical comment: During preparation and

peer review of the draft NTP Brief on Bisphenol A a number of pathologists questioned the

classification of the lesions with cribriform-like

structures described as carcinoma in situ in the

studies by Murray et al. and Durando et al. In

addition, the degree of hyperplasia reported

in these studies was described during the peer

review as being of a relatively mild form and

not necessarily of the type that presents the

most concern for development of invasive breast

cancer in women, i.e., focal areas of atypical

hyperplasia (see the Peer Review Report for Bisphenol A (96) for additional discussion)].

In the study by Murray et al. (53), rats were

treated with 0.0025–1 mg/kg bw/day bisphenol

A during pregnancy by subcutaneous mini-pump.

Significant increases in the incidence of hyperplastic ducts were reported in all dose groups of

female offspring on postnatal day 50 and only in

the lowest dose group of 0.0025 mg/kg bw/day

on postnatal day 95 (sample sizes range from

4–6). Lesions described as carcinoma in situ

were reported in female offspring in the 0.25

and 1 mg/kg bw/day groups on postnatal day 50

(25% incidence for both treatment groups) and

postnatal day 95 (33% incidence for both treatment groups). These findings are supported by a

study by Durando et al. (52)13 where pregnant

rats were treated with 0.025 mg/kg bw/day, again

using a subcutaneous mini-pump. In this study,

the percent of hyperplastic ducts wassignificantly

increased in the female offspring at both postnatal

days 110 and 180 (~2–5-fold).A non-significant

increase in the incidence of ductal carcinoma in 

situ was noted following adult treatment with

a subcarcinogenic dose of N-nitroso-N-methylurea, a chemical used in cancer research to assess

susceptibility to carcinogens (2/15 compared to

0/10 in control animals).13

These findings are generally consistent with

other reports of changes in mammary gland

growth and development following perinatal

exposure to bisphenol A that are related to an

altered rate of maturation, e.g., advanced fat pad

maturation, delayed lumen formation, enhanced

duct growth, adoption of a pregnancy-like state,

enhanced responsivenessto secondary estrogenic

exposures, and potentially increased susceptibility to carcinogenesis, e.g., increased number or

density of terminal end buds and ducts (52, 53, 

177–183).These findings have been interpreted

by some authors as indicating that developmental exposure to bisphenol A causes effects on

breast tissue maturation that may lead to a predisposition to disease onset later in life (52, 53, 

181–183, 191).

13The study by Durando et al. (52) implied that

99.9% DMSO was used in the mini-pump

[“Pumps are designed to deliver 25 bisphenol

A (Sigma-Aldrich de Argentina S.A., Buenos

Aires, Argentina) or only DMSO (99.9% molecular biology grade, Sigma-Aldrich de Argentina S.A.)”]. The manufacturer of the mini-pump

does not recommend use of DMSO concentrations greater than 50% because it can degrade

the pump reservoir material and potentially result

in tissue inflammation and edema. For this reason, the CERHR Expert Panel on Bis­phenol A

considered this study critically flawed (2). The

NTP concurs that use of a high concentration

of DMSO is a technical short-coming, but is

not convinced that this factor could account for

the observed results. The NTP also considered

the possibility that potential pump degradation

could result in variations in administered dose,

but concluded that the study was still useful to

consider in the context of other findings.
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With the exception of an oral dosing study conducted by Moral et al. (183) that reported an

increased number of mammary gland terminal

ducts in the female offspring of rats treated

during gestation with 0.250 mg/kg/day, the

cellular and tissue-level effects on the mammary gland occurred in studies where bis­phenol

A was administered by subcutaneous treatment

via mini-pump at doses of 0.000025 to 10 mg/

kg/day (52, 53, 177, 179 – 182).

Certain aspects of mammary gland cancer differ

between rats and humans, e.g., metastases are

uncommon in rodents, but ductal hyperplasia

and carcinoma in situ, are generally recognized

asintermediary stepsin chemical-induced mammary gland cancer in the rat and as preneoplastic

lesions in the human (184 – 187). The appearance of ductal hyperplasia and carcinoma in situ

are similar enough between rats and humans that

these findings in the rat are considered relevant

to humans (185). In humans, a greater than

mild degree of ductal hyper­plasia and ductal

carcinoma in situ are associated with increased

relative risk of developing invasive breast carcinoma. It is important to note that the development of these lesions does not guarantee the

formation of tumors or cancer in rats or humans

and they are most appropriately interpreted as

risk factors. If similar changes occur in women,

the increased relative risks for developing invasive breast cancer range from 1.5 to 5-fold for

moderate and atypical ductal hyperplasia and

8.0 to 10.0-fold for ductal carcinoma in situ

(188).The relative risk is based on a comparison

to women of the same age in the general population. For example, a 50-year old woman has

a 1 in 39 chance of developing invasive breast

cancer in the next 10 years. If a 50-year woman

has atypical ductal hyperplasia, a form of ductal

hyperplasia associated with a moderate level of

increased relative risk (4 to 5-fold), then her

chance of developing invasive breast cancer in

the next 10 years increases to approximately 1

in 10 to 1 in 8.

The current literature is notsufficient to establish

the reproducibility of the ductal lesion findings

by multiple independent investigators. Bisphenol A was not shown to induce neoplastic or

non-neoplastic lesions in the mammary gland of

female rats (~74 and 135 mg/kg bw/day) or mice

(650 and 1300mg/kg bw/day)in two-year dietary

cancer bioassays where exposure was initiated

in young adult animals (5-weeks of age) (176).

However, these studies did not include perinatal

exposure and the NTP recognizes that adult-only

exposure may not be sufficient to detect chemical carcinogensin hormonally-responsive tissues

such as the mammary gland (187). Most of the

toxicology studies of bisphenol A that included

assessment of females following developmental

exposure either (1) did not report examination

of the mammary gland (37, 43, 120, 189, 190),

or (2) collected mammary gland tissue but did

not prepare the tissue in a manner that would

readily reveal these changes, i.e., whole mounts

(41, 107). The limited assessment of the mammary gland in these studies is critical because it

is not clear that, if present, intraductal epithelial

proliferations would have been detected during

the routine histopathologic examinations. More

severe mammary lesions were not reported in

these studies.Although severe lesions or tumors

could be detected during routine necropsy, the

studies by Ema et al. (107) and Tyl et al. (41)

were primarily designed to detect effects on

reproduction and development and not tumor

incidence. Animals were not followedup for a

sufficiently long period of time to necessarily

expect to observe tumors in control animals or

differences in tumor incidence between treatment groups. In both of these studies, mammary

gland tissues in the parental (F0) and F1 generations of females were only examined after

weaning of their pups and the animals would

have been well under one year of age at the time

of tissue collection.

TheNTPconcurswith recentreviews(2, 191)that

additional data are needed to more completely
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understand the possible long-term consequences

of disrupting mammary gland development in

animals by bisphenol A exposure and its significance for human health. Namely, long-term follow-up studies with sufficient statistical power

should be conducted to evaluate if the ductal

hyperplasia and carcinoma in situ progress to

mammary gland tumors, preferably without the

use of a secondary chemical challenge in adulthood. In addition, conducting the appropriate

pharmacokinetic studiesto better understand the

distribution of bisphenol A to target tissues with

the subcutaneous mini-pump would aid in interpreting the results. While researchers predict that

circulating levels of total and free bisphenol A

in the subcutaneous mini-pump studies would

be quite low based on the administered dose (≤1

mg/kg bw/day), the lack of supporting pharmacokinetic information limits the ability to make

comparisons to human exposures.

Prostate Gland and Urinary Tract 

There issome evidence that perinatal exposure to

bisphenol A in rodents may alter prostate gland

and urinary tract development and predispose the

prostate to develop hormonally-induced pre-neoplastic lesions later in life. The evidence is not

sufficient to conclude that bisphenolA is a rodent

prostate gland carcinogen or that bisphenol A

presents a prostate cancer hazard to humans.

In mice, exposure of pregnant dams to bisphenol A at an oral dose of 0.010 mg/kg bw/day

has been shown in one study to alter prostate

development in offspring by increasing the

number of prostatic ducts, ductal volume, and

the proliferation of a cell population implicated

in the development of prostate cancer (basal

epithelial cells) in one or more regions of the

prostate (54, 192). This study also reported a

urinary tract deformation where the urethra

narrows near the neck of the bladder, an effect

that, if permanent, could contribute to urine flow

disorders. These effects were observed in fetal

mice and it is unclear if they persist into adulthood or relate to a clear adverse health outcome.

It is important to note that other studies have not

reported severe consequences of urinary tract

constriction in adult animals exposed during

development that might be predicted based on

the finding by Timms et al. including bladder

stones, hydronephrosis, hydroureter, or other

indications of kidney toxicity.

In Sprague-Dawley rats, subcutaneous injection of neonates with 0.010 mg/kg bisphenol

A followed by adult hormone treatment14

was reported to cause 100% of the animals to

develop “low” grade (3/10 animals) or “high”

grade (7/10 animals) prostate intraepithelial

neoplasia (51).15,16 The incidence of prostate

intraepithelial neoplastic (PIN) lesions in animals that did not receive the adult hormone

14Animals were given Silastic capsule implants

packed with estradiol and testosterone that result

in serum concentrations of ~75 pg/ml estradiol

and 3 ng/ml testosterone. This hormone treatment is intended to mimic the ratio of estradiol

to testosterone in the aging male.

15The classification scheme of “low” and “high”

grade PIN lesions used by Ho et al. (2006) appears to be their own (96).

16One other study assessed bisphenol A’s ability to

predispose the prostate to develop prostate intraepithelial neoplasia lesions and tumors (193). In

this study, female F344 rats were orally dosed

with 0.05, 7.5, 30, or 120 mg/kg bw/day of bisphenol A during pregnancy and lactation. In order to induce prostate lesions and tumors, male

offspring were treated with a chemical carcinogen, 3,2’-dimethyl-4-aminobiphenyl (DMAB).

No statistically significant changes in prostate

intraepithelial neoplasia lesions or carcinomas

were observed. Differences between this study

and the report of Ho et al. may be related to age

at exposure (fetal versus neonatal and fetal), rat

strain (F344 versus Sprague–Dawley), carcinogenic insult (DMAB versus estradiol+testosterone), route of administration (subcutaneous

versus oral to dams), or other factor such as

animal husbandry and housing.
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treatment was not significantly different from

controls (2/6 versus 1/9 in control animals). Proposed biological mechanisms to account for the

effects of bisphenol A on the prostate include an

epi­genetic mode of action exemplified as altered

DNA methylation patterns in genes that help

regulate prostate development and growth as an

epi­genetic mode of action (51, 194). PIN lesions

in the male rodent have similar histopathology

to PIN lesions in men, and evidence of high

grade PIN lesions in men is considered a risk

factor for developing prostate cancer (96). The

use of adult hormone treatment to promote the

development of prostate intraepithelial neoplasia lesions complicates the interpretation of this

study when considering its relevance to human

bisphenol A exposure. However, as discussed in

more detail below, rodents are normally resistent to developing prostate cancer and the use of

hormone treatment, chemical treatment, or other

alternative animal model to obtain a more sensitive rodent model is considered an acceptable

and recommended strategy in prostate cancer

research (187).

The findings of Ho et al. (51) are consistent

with a recent report of increased expression of

cytokeratin 10 (CK10), a cell-marker associated

with squamous differentiation, in adult male

offspring of pregnant mice orally treated with

0.020 mg/kg bw/day bisphenol A during gestation (195). Chronic exposure to high doses of

potent estrogens,such as diethylstilbestrol, leads

to squamous metaplasia of the prostate, a tissue

change characterized by a multilayering of prostatic basal epithelial cells. Squamous metaplasia

is associated with benign prostatic hyperplasia

or long-term estrogen treatment in patients

with benign or malignant prostatic disease. The

induction of CK10 expression in basal epithelial

cells is an early indicator of changes leading to

estrogen-induced squamous metaplasia. While

the long-term health consequences of such an

alteration are unclear, prostatic basal epithelial

cells are important for normal growth and development and are implicated in the initiation and

early progression of prostate cancer due to their

function in maintaining ductal integrity and regulating the differentiation of luminal epithelial

cell differentiation (192). It is important to note

that prostates in the Ogural et al. study appeared

morphologically the same as control animals

based on the staining technique normally used in

pathology (hematoxylin and eosin, or H&E). A

stain specific for squamous keratin was required

to detect the change. Thus, it is unclear whether

similar changesin basal epithelial cell phenotype

were present in other studies that evaluated the

prostate using only an H&E stain.

The NTP concurs with the CERHR Expert Panel

on Bisphenol A (2) and another recent evaluation (191) that additional studies are needed to

understand the effects of bisphenol A on the

development of the prostate gland and urinary

tract. Studies should attempt to confirm these

findings and include longer periods of follow-up

to understand the significance of the structural

and cellular effects observed in fetuses and to

clarify the relevance of prostate intraepithelial

neoplastic lesions resulting from bisphenol A

exposure to the development of prostate cancer in these animals. Future research to clarify

the role of bisphenol A in the development of

prostate cancer presents a scientific challenge.

Unlike humans where prostate cancer is common, it is the most common non-skin cancer

in American men (187), rodents rarely develop

prostate cancer. Of the almost 4,550 rats and

mice used as controls in NTP 2-year inhalation

or feed studies conducted during the last decade,

only 1 cancerous tumor and 17 benign tumors

(“adenoma”) of the prostate gland were detected

(187). No substances, including bisphenol A

(176), have been identified as causing prostate

tumors in NTP studies (187).The NTP has long

recognized the limits of the traditional rodent

cancer bioassay for detecting chemical-inducedprostate tumors and organized a workshop in

May 2006 to address this issue (187). Suggested
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strategies to improve the sensitivity of rodent

models for detecting prostate cancer included

using alternative models, e.g., genetically modified, and/or initiating exposure in perinatal life.

In addition, NTP workshop participants suggested a more detailed histopathologic evaluation of the prostate because the assessment

of human carcinogenic potential may be better

determined based on chemical-induced preneoplastic changes rather than tumor incidence.

During its evaluation of bisphenol A exposure

and prostate development, the NTP also considered a number of studies in rats or mice that have

detected increased prostate weight at low doses

(115, 196) or failed to detect this effect (37, 

41, 43, 107, 116, 122, 193, 197 – 201). Prostate

weight effects have taken on a special significance in the controversy surrounding bisphenol

A because elevated prostate weight was the first

“low” dose finding reported in laboratory animals (115) and prompted numerous follow-up

studies. Attempts to understand the basis for

discordant findings has generated considerable scientific discussion and debate including

their review at the NTP Low-Dose Peer Review

workshop mentioned earlier (97). In brief, the

NTP believes that the overall conclusions of the

Bisphenol A Subpanel of the NTP Low-Dose

Peer Review remain valid with respect to “low”

dose effects on prostate weight, i.e., increased

prostate weight cannot be considered a general

or reproducible finding.

More importantly, it is not clear that prostate

weight should continue to be considered a critical endpoint in risk evaluations of bisphenol A

given the relative crudeness of this measure.

Changes in organ weight may be useful to identify potential target tissues, but become less

important when additional data relating to structural, cellular, or functional integrity are available. Prostate enlargement does not correlate

with the development of prostate histo­pathology

or cancer in rodents, and the evaluation of prostate weight without corresponding assessment of

histopathologic changes is not considered useful

for determining carcinogenic potential (202).

In addition, changes in prostate weight are not

necessarily observed in the same bisphenol

A studies that report prostatic cellular or tissue-level changes. For example, no effects on

prostatic lobe weight were observed in studies

that reported (1) increased incidence and susceptibility to develop prostate intra­epithelial

neoplastic lesions (51), (2) changes in the prostatic periductal stroma and decreases in androgen-receptor positive stromal cells and epithelial cells positive for prostatic acid phosphatase

(PAS), an enzyme produced by the prostate that

can be found in higher amounts in men with

prostate cancer (203), and (3) increased expression of CK10 in adult mice exposed as fetuses

to 0.020 mg/kg bw/day via treatment of the dam

or during adulthood to high doses of bisphenol

A (2 – 200 mg pellets implanted under the skin

for 3-weeks) (195).

Puberty and Sexual Maturation

NTP concurs with the CERHR Expert Panel on

BisphenolA that limited data are available at low

dosesto suggest an effect of accelerating the onset

of puberty in female mice. In humans, early onset

of puberty in girlsis associated with elevated risk

of developing breast cancer, early bone age maturation, and psychosocial impacts that include

influencing age at first sexual intercourse and

increasing risk for certain adolescent risk behaviors (204–206). Depending on the magnitude of

the effect, early onset of puberty in laboratory

animals can be considered an “adverse” effect

in reproductive toxicology (204).

The consistency of the literature on “low” dose

effects of bisphenol A related to puberty in

female rodents is different in rats and mice. Of

the eight studies in rats evaluated by the NTP,

seven were interpreted as being “negative” (37, 

43, 53, 107, 122, 173, 207) and one study was
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considered “positive” (52). Overall, the NTP

considered the rat data to indicate that “low”

doses of bisphenol A do not affect the onset

of puberty in female rats. A total of six “low”

dose mouse studies were identified and evaluated by the NTP. Of these, two reported effects

consistent with accelerated puberty and one

showed an alteration in events related to sexual

maturation in females and were interpreted as

“positive” and the other three were considered

“negative.”

The NTP strategy for evaluating the entire “low”

dose literature on puberty in female rodents was

to first conduct a detailed assessment of the

mouse studies to determine whether any aspect

of study design could account for the apparently

contradictory results in this species. The most

consistent difference between the “positive” and

“negative” studies in mice is the approach used

to measure a puberty-related event. Age at first

estrusisthe most accurate indicator of puberty in

rodents. This occurs at the same time as vaginal

opening in rats. However, in mice, vaginal opening does not correlate well with puberty and the

first day of detecting cornified cells in a vaginal

smear, a sign of first estrus, isthe preferred measure used to indicate the onset of puberty (208).

Although accelerated vaginal opening is an

expected response to estrogens in mice, the lack

of simultaneous occurrence of vaginal opening

and first estrussuggeststhat these events may be

differentially regulated even though they are both

estrogen responsive. Thus, the NTP considers

vaginal opening to be a marker of sexual maturation, but not a surrogate measure of puberty,

i.e., first estrus. The three mouse studies that

reported effects consistent with an acceleration

of a puberty-related event used first estrus as the

marker of puberty (48, 55, 189). In contrast, the

“negative” studies used vaginal opening (41, 178, 

197). Each study also has its own limitation that

complicates a straight-forward interpretation of

the results, e.g., small sample size, positive control response, or use of subcutaneous injection

to pregnant dams. Based on the analysis outlined

below, the NTP concluded that the “positive”

mouse studies provided limited evidence of an

acceleration of puberty.

In contrast, the NTP concluded that the rat studies do not indicate an effect on puberty at “low”

doses. The differences in outcomes cannot be

attributed to the use of an insensitive strain

or stock because a variety of rat models were

used in the “negative” studies: Sprague-Dawley,

Wistar, Wistar-Furth rats, Wistar-derived Alderley Park, CD, and Donryu. Moreover, three of

the “negative” rat puberty studies reported other

“low” dose effects (53, 122, 173). The effects of

bisphenol A on puberty in rats at “high” doses

are more inconsistent than the “low” dose studies. Only one study has reported an effect on

puberty in the predicted direction, i.e., acceleration following subcutaneous treatment on postnatal days 0 to 9 (120). Other studies reported

no effect (116–119) or a delay in puberty at ≥50

mg/kg bw/day (37, 43). Four of these studies

used a positive control group (43, 116, 118, 120).

In these studies, responses to potent estrogens

based on age at vaginal opening ranged from no

effect (116), to a statistically significantsmall or

moderate acceleration [1.7 days (43); 2.4 days

(120); 3.6 days (118, 119)].

Mouse Studies

“Positive” Mouse Studies 

The largest magnitude of effect on puberty

in female mice was reported by Ryan et al. 

(48). In this study, age at first estrus was accelerated by 4.5 daysinC57BL/6mice whose

dams were orally dosed with 0.2 mg/kg/day

bisphenol A during gestation and lactation

(GD3–PND21) (48). Acceleration in puberty of approximately 6 days was reported in

the ethinyl estradiol positive control group.

The major limitation of this study was the

relatively small sample sizes used for this

endpoint (4–5 dams per treatment group).

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Howdeshell et al. (55) reported that female

CF-1 offspring of dams orally-treated with

0.0024 mg/kg/day of bisphenol A during

gestation (GD11 –17) had a 2.5 day shorter interval between age at vaginal opening

and first estrus. This study also evaluated

whether fetal response to prenatal bisphenol

A treatment differed based on intrauterine

position (IUP). In some rodent species, females surrounded by 2 females in utero (0

male or 0M) have higher serum concentrations of estradiol and lower serum concentrations of testosterone compared to females

surrounded by 2 males (2M). Intrauterine

position effects have been reported for some

behaviors or physiological characteristics,

mostly in mice but also other rodents and

swine (209). The shorter interval of ~2.5

days between vaginal opening and first estrus in control and bisphenol A-treated animals was pri­marily attributed to a 5-day

shortening of this interval in the 0M females

leading the authors to hypothesize that animals with higher background exposures to

estradiol may be more sensitive to bisphenol

A exposure. The most significant limitation

of this study is the interpretation of the interval between vaginal opening and first estrus. No effect was observed on age at first

estrus in bisphenol A-treated mice17 or age

at vaginal opening. A shortening of the interval between these two events should not

be interpreted as an acceleration in the onset

of puberty and is more appropriately characterized as a alteration in the timing of events

related to sexual maturation.

The findings of Ryan et al. (48) and Howdeshell et al. (55) are supported by a subcutaneous injection study that noted a statistically significant 1-day earlier onset of first

17Age at first estrus was significantly accelerated

for 0M females versus controls in post hoc analyses (personal communication Frederick vom

Saal, August 13, 2008)

•

•

estrus and vaginal opening in female ICR/Jcl

mice whose mothers were treated with 0.02

mg/kg bw/day bisphenol A during gestation

(GD11–17) (189). Although the reported

magnitude of the effect in the bis­phenol Atreated animals was small, ~1-day, the authors also reported that females in both the

0.002 and 0.02 treatment groups had significantly longer estrous cycles (a “classic”

estro­genic response) compared to control animals. Accelerations in vaginal opening and

puberty and lengthened estrous cycles were

also observed in the diethylstilbestrol positive control groups. The interpretation of this

study is limited by the small magnitude of

an effect on age at first estrus and use of subcutaneous injection as the route of administration to pregnant dams.

 “Negative” Mouse Studies

Ashby et al. (197) did not detect an effect

of bisphenol A on vaginal opening in the

female offspring of CF-1 mice dosed orally

with 0.002 or 0.02 mg/kg bw/day. The interpretation of this study is complicated by

the response in the positive control group

where diethylstilbestrol (0.0002 mg/kg bw/

day) caused a significant 3.6-day delay in

the age of vaginal opening compared to the

vehicle control group. A delay in puberty is

inconsistent with the predicted estrogenic

effect of accelerated puberty for the diethylstilbestrol group.

Markey et al. (178) did not report an effect

of bisphenol A on age at vaginal opening in

female CD-1 mice whose dams were dosed

with 0.000025 or 0.00025 mg/kg bw/day via

subcutaneous mini-pump. The authors noted that a portion of the mice showed partial

vaginal opening approximately 4 days earlier than control animals. In addition, both bisphenol A groups had longer estrous cycles

than control animals.

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Tyl et al. (41) reported no effect of bisphenol

A on age at vaginal opening in CD-1 mice

using a multigenerational study design at

dietary doses that ranged from 0.003 to 600

mg/kg bw/day. The positive control group

used in the bisphenol A study, 17β-estradiol

(0.08 mg/kg bw/day) caused the expected effect of accelerated vaginal opening. However,

the experimental model used in thisstudy did

not appear to be sensitive in detecting estrogenic effects at low doses. In a separate multigenerational study designed to characterize

the response in CD-1 mice to 17β-estradiol

(0.0002–0.1 mg/kg bw/day), the authors did

not report any effect on vaginal opening at

doses below ~0.03 mg/kg/day (210). In addition, estrous cycle length was unaffected

at all doses. Thus, detecting an estrogenic effect of a weaker estrogen such as bisphenol A

at very low doses (≤ ~0.03 mg/kg bw/day)

would not be expected in this animal model.

Species Differences

There are other indications of species differences between rats and mice that may contribute to the inconsistent literature for bisphenol

A. Research on the effects of pheromones in

regulating puberty suggests that puberty may

be more easily perturbed in mice compared to

rats. Puberty in female mice can be accelerated

when the mice are exposed to urine from a male.

This effect has been reported more often and

more consistently for mice than for rats (211, 

212). In addition, the IUP effect, which was an

important factor in the Howdeshell et al. study,

is better documented in mice compared to rats

suggesting that this effect may be more robust

in mice (209). IUP was not considered in other

studies in the bisphenol A literature, but it is

worth noting that the studies by Markey et al. 

(178) and Ryan et al. (48) also show indications of a subpopulation of mice that may be

more responsive to bisphenol A. As discussed

earlier, Markey et al. commented that a portion

of mice exhibited partial vaginal opening ~4

• days earlier than control animals. In the Ryan

et al. study, two of the five animals in the 0.200

mg/kg bw/day group that had a ~4.5 day statistically significant acceleration in puberty showed

a much greater acceleration than the other animals in that group, i.e., ~10 days early onset of

first estrus compared to the mean of the control

group (public comment on the draft NTP Brief

from Dr. Earl Gray, received May 23, 2008).

Other Effects Considered 

A variety of other effects in laboratory animals

have been linked to “low” dose bisphenolAexposure during development, including decreased

sperm quantity or quality, obesity, disruption

of meiosis, changes in reproductive hormone

levels, or cellular effects in reproductive tissues.

These effects had less impact in shaping NTP’s

conclusions on potential risks to humans from

bisphenol A exposure than the developmental

effects observed at “high” doses on survival and

growth and the “low” dose effects on brain and

behavior, mammary gland, prostate gland, and

onset of puberty in females described above.

In some cases, the relationship between a

specific cellular- or tissue-level finding and a

potential health effect in the whole organism is

unclear. This is because there is often uncertainty about the functional impact of a cellular or

mechanistic finding, such as the altered level of

a receptor protein or change in enzyme activity.

For example, the potential health impact that

may result from uterine changes characterized

by altered ERα and ERβ expression and from

an increase in the number and appearance of

uterine epithelial cells is unclear (213).

In other cases, the literature is not sufficiently

developed. Newbold et al. (214) recently

described a number of morphological changes

in the ovaries and uteri of 18-month old mice

that had received subcutaneous injections of

bisphenol A at doses of 10, 100, or 1000 μg/kg

on days 1–5 of life. Increases in cystic ovaries
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and cystic endometrial hyperplasia were statistically significant in the 100 μg/kg dose group but

not at 1000 μg/kg. Non-statistically significant

increases in the incidence of a variety of other

ovarian and uterine proliferative lesions and

cysts were also reported. Replication of these

findings and further study of the linkage of early

and late occurring events will be important in

establishing a better understanding of any longterm consequences of exposures of the developing organism to bisphenol A.

As mentioned earlier, NTP Briefs are not meant

to serve as comprehensive reviews of the scientific literature. Only key study findings and

issues that relate to NTP conclusions on concerns for potential reproductive and developmental health effects in humans are typically

presented. However, three reported “low” dose

health effects (obesity, decreased sperm count

or quality, and abnormalities of meiosis) that

ultimately had less impact in determining the

NTP’s conclusions are briefly discussed below

in order to illustrate the interpretive challenges

associated with this literature. Two examples

of such effects, obesity and impacts on sperm,

demonstrate findings that are not reported consistently enough to be considered reproducible.

The third example relates to abnormalities of

meiosis and is presented to demonstrate that

effects predicted from in vitro studies are not

necessarily observed in the in vivo studies.

Obesity

There is currently insufficient evidence to conclude that bisphenolA exposure during development predisposes laboratory animals to develop

obesity or metabolic diseases such as diabetes,

later in life. Obesity and metabolic disruption

have become a research focus for bisphenol A

based on several reports of increased postnatal

growth following “low” dose exposure during

development and several in vitro and in vivo

studies that report effects related to altered carbohydrate and lipid regulation.

The NTP concurs with the CERHR Expert Panel

on Bisphenol A that the effects of bisphenol A

on body weight at “low” doses are inconsistent

(2). A number of studies in rats and mice report

increases in postnatal growth following developmental exposure to bisphenol A at oral doses

of 0.0024–1.2 mg/kg bw/day (55, 155, 173, 

215) or a subcutaneous dose of 0.5 mg/kg bw/

day (179). Other “low” dose (≤5 mg/kg bw/day)

studies in rats and mice have either not detected

any significant effect on body weight (43, 48, 

50, 52, 107, 116, 122, 200, 214, 216) or reported

growth reductions (37, 115, 146, 189, 217).

Differences in study outcomes cannot easily be

attributed to the use of a potentially insensitive

rodent model or experimental protocol because

several studies that did not detect any significant

difference in body weight reported other effects

at “low” dose levels (48, 50, 52, 122, 217). The

bases for the inconsistent findings are unclear,

but may relate to factors such as diet and differences in experimental design or analysis.

The data are currently too limited to conclude

that developmental exposure to bisphenol A

causes diabetes or other metabolic disorders

later in life. Two studies in laboratory animals

have assessed endpoints related to carbohydrate

or lipid regulation. In adult male mice, a single

subcutaneous dose of 0.010 or 0.100 mg/kg bw/

day bisphenolA caused decreased blood glucose

and increased plasma insulin (218). Additionally, increased pancreatic insulin content and

insulin resistance were reported at 0.100 mg/kg

bw/day (administered orally or by subcutaneous

injection) after a slightly longer period of dosing

(4-days) (218). A recent study by Miyawaki et 

al. (215) assessed a variety of endpoints related

to carbohydrate and lipid regulation in 1-month

old mice that were exposed through maternal

treatment during gestation and lactation with

0.001 or 0.010 μg/ml bisphenol A in drinking

water (~0.26 and 2.42 mg/kg bw/during gestation). Endpoints included body weight, adipose

tissue weight, and blood concentrations of leptin,
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total cholesterol, triglycerides, non-esterified

fatty acid and glucose. Body weight and total

cholesterol were significantly increased in

female offspring in both dose groups although

adipose tissue weight and leptin levels were only

significantly increased in the 1 μg/ml treatment

group. Male offspring in the high dose group

of 10 μg/ml were significantly heavier and had

increased adipose tissue weight. Leptin levels

were not associated with either of these effects

in males. Significantly increased triglycerides

and non-esterified fatty acid and decreased

glucose were observed in male offspring in the

low dose group of 1 μg/ml. Although this study

addresses the hypothesis that developmental

exposure to bisphenolA can affect carbohydrate

and lipid metabolism in postnatal life, the inconsistent pattern of effects on serum lipid levels,

leptin, and glucose and lack of control for litter

effects18 makes the study on its own insufficient

to draw any conclusion.

More research in this area is warranted. Several in vitro studies report effects of bisphenol

A related to carbohydrate and lipid regulation

including effects on pancreatic cells that govern

the release of insulin (β-cells) and glucagon (αcells), altered differentiation of fibroblast cells

into adipocytes, and altered glucose transport

in adipocytes (219 –223). Some of the effects

on pancreatic cells are very rapid, e.g., altered

frequency of glucose-induced calcium oscillations in α- and β-cells, activation of cAMP

response element binding protein, and appear

to be mediated by ncmER (63, 65, 224). Effects

mediated through the ncmER are of interest

because bisphenol A has been shown to activate

this receptor in vitro at a concentration of 1 nM,

which is similar to the active concentration of

diethylstilbestrol (63, 65).

1816 –25 males or females were reported for each

treatment group however these animals were

derived from only 3 litters per treatment group

(215).

Decreased Sperm Count and Sperm Quality

There is currently insufficient evidence to conclude that bisphenol A exposure during development or adulthood causes decreased sperm

count or sperm quality. A large number of studies have addressed this issue but the literature is

inconsistent and not easily reconciled.

Exposure during development

There are some indications that treatment with

“high” oral doses of bisphenol A during development or young adulthood can impact sperm

quantity in laboratory rats (37, 42, 43).Tan et al. 

(42) reported that 33% of rats did not show any

evidence of having a spermatogenic cycle after

treatment in young adulthood with 100 mg/kg

bw/day of bisphenolA. Other reported decreases

in measures of testicular or epididymal sperm

count and sperm production were more modest

and ranged from 10 to 19% at doses of 50 and

500 mg/kg bw/day (37, 43). In addition, in the

three-generation rat study conducted by Tyl et 

al. (37), significant decreases in sperm parameters were only observed in certain generations

of similarly exposed males in the high dose

group of 500 mg/kg bw/day: ~18% decrease in

epididymal sperm concentration in F1 males,

~19% decrease in testicular daily sperm production in F3 males and no significant effects in the

F0 or F2 generations. Testicular or epididymal

histopathology was not detected in any treatment

group (37). Significantly decreased sperm motility and an increased percentage of abnormal

sperm was also reported following “high” dose

subcutaneous injection, ~25 mg/kg bw/day,19 to

neonatal mice in a study conducted byAikawa et 

al. (225). Again, these effects were not associated with testicular histological alterations.

Effects on sperm parameters have been reported

at lower doses administered orally or by sub19

Administered dose was 0.050 mg/pup. This is

approximately equal to 25 mg/kg/day assuming

that a neonatal mouse weighs 0.002 kg
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cutaneous injection.20 vom Saal et al. (226)

reported a ~19% decrease in testicular daily

sperm production in adult male mice exposed

to bisphenol A as fetuses via maternal dosing

with 0.02 mg/kg bw/day (higher dose levels

were not tested). Toyama et al. (227) observed

increased incidences of several measures of

abnormal sperm morphology (40–80% compared to <0.3% in controls) in mice treated with

>0.17 mg/kg or ratstreated with >0.33 mg/kg by

subcutaneous injection21 of bisphenol A every

other day during postnatal days 2 to 12.

However, a number of larger studies have not

reported effects on sperm parameters following

exposure during development at “high” or “low”

dose levels (0.0002– 600 mg/kg bw/day) (41, 

107, 199–201, 228).

Exposure during adulthood only

Several studies have reported effects on sperm

parameters in mice or rats exposed to “low”

doses of bisphenol A only during adulthood. In

rats, these effects are reported following oral

dosing of 0.02– 200 mg/kg bw/day for six days

(~24–32% decreased daily sperm production

per gram tissue) (229), 0.0002–0.02 mg/kg

bw/day for 45 days (~23–41% decrease in epididymal sperm motility; ~18 –27% decrease in

epidiymal sperm count at 0.002–0.02 mg/kg

20Talsness et al. (217) reported effects on sperm

quantity in rats exposed during gestation to 0.1

and 50 mg/kg bw/day but this study is not included in the discussion because (1) reported effects

included an increase in sperm number which was

opposite the effect observed in the positive control

group, and (2) effects on daily sperm production

appeared inconsistent over time and across dose.

21Administered doses were ≥0.001 mg/pup in the

mouse and ≥0.01 mg/pup in the rat. These doses

are approximately equal to 0.17 to 0.5 mg/kg

in the mouse and 0.33 – 1.33 mg/kg in the rat

assuming that body weight between postnatal

days 2 to 12 ranges from 0.002 to 0.006 kg in

the mouse and 0.0075 and 0.03 kg in the rat.

bw/day) (230), and 0.0002–0.02 mg/kg bw/day

for 60 days (~30–45% decrease in epididymal

sperm motility; ~12–40% decrease in epidiymal sperm count at 0.002–0.02 mg/kg bw/

day) (231). In adult mice, “low” dose effects on

sperm are observed at oral doses of 0.025–0.1

mg/kg bw/day for 30 days (~16 – 37% decrease

in weight corrected testicular or epididymal

sperm count) (108) and subcutaneous dosing

with 0.02 and 0.2 mg/kg bw/day for 6 days

(abnormal sperm morphology) (232).

Other larger studies have not reported effects

in adult animals at these doses. The 2-generation mouse study conducted by Tyl et al. (41)

reported a 15% decrease in epididymal sperm

concentration in F0 generation animals at the

highest dose tested of 600 mg/kg bw/day but

not at lower doses of 0.003 to 50 mg/kg bw/day.

Ema et al. (107) also did not detect an effect

on sperm measures in the F0 generation in a rat

multigeneration study at oral doses of 0.0002 to

0.2 mg/kg bw/day. The finding by Sakaue et al. 

(229) of a ~24–32% decrease in sperm production in adult Sprague-Dawley rats(obtained from

CLEA Japan, Inc.) was not reproduced in a study

using largersample sizes of Sprague-Dawley rats

obtained from Charles River UK (233).

The basis for the inconsistent findings is not

clear. One proposed explanation is that rodent

species, strains, and breeding stocks differ in

their responsiveness to estrogens (59). Species

and strain differences in response to estrogen

have been documented, but animal model

sensitivity varies depending upon the specific

trait being assessed [discussed in (2, 59, 200)].

Studies that include sperm assessment in the

bisphenol A literature are too varied in terms of

periods of dosing, use of positive control, e.g.,

none used, ethinyl estradiol, or 17β-estradiol,

and other aspects of experimental conduct to

determine if differences in sensitivity of the

animal model can account for the inconsistent

findings on sperm quantity and quality.
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Chromosome and Meiosis Abnormalities

Disruption of the processes that distribute chromosomes during meiosis or mitosis can result in

aneuploid cells, i.e., germ cells that have more

or fewer chromosomes than the normal haploid

number or somatic cells that have more or fewer

chromosomes than the normal diploid number.

When this happens in eggs or sperm of humans,

it can lead to such conditions as Down Syndrome in which the fetus ends up with 3 copies

of chromosome 21, rather than two copies, or a

range of syndromes associated with abnormal

numbers of sex chromosomes (normal is XX

for females, XY for males) such as Klinefelter

Syndrome (XXY males) or Turner Syndrome

(XO females). If a chemical exposure is capable

of inducing aneuploid eggs or sperm, affected

individuals would be expected to exhibit problems in achieving or maintaining pregnancy, or

to produce aneuploid offspring. While the body

of evidence from both in vitro and in vivo studies

provides evidence that bisphenol A can disrupt

certain aspects of cell division involving both

mitotic and meiotic processes, breeding studies

in laboratory animals exposed to bisphenolA do

not present results consistent with such effects.

Thus, the significance of the reported effects on

meiosis and mitosis for mammalian reproduction is not yet clear.

Two in vivo studies (234, 235) reported that

short-term oral exposure to low doses of bisphenolA (≥0.020 mg/kg bw/day) in peri­­pubertal

or pregnant mice can interfere with meiotic

divisions in development of female germ cells

(“egg” or “oocyte”). An increase in hyper­ploid

(aneu­ploid) metaphase II oocytes was ob­served

following treatment with 0.020 mg/kg bw/day.

There was not a significant increase in aneuploid

embryos. Two subsequent in vivo studies (236, 

237) attempted to replicate these findings. Consistent with the previous findings, they detected

no significant effects of bisphenolA exposure on

the frequency of aneuploidy in “zygotes” (fertilized oocytes) produced from female mice treated

before puberty or as adults with a similar range

of doses. In addition, Eichenlaub-­Ritter et al. 

(236) found no effects of bisphenol A exposure

on aneuploid oocytes and Pacchierotti et al. 

(237) found no increase in aneuploid or diploid sperm following exposure of male mice

to bisphenol A.

A number of in vitro studies using cultured

mammalian somatic cells have also looked at the

potential for bisphenol A to cause aneuploidy.

Earlier studies (238–240) consistently reported

the induction of aneuploidy in various cell lines

including SHE, V79, and MCL-5 at concentrations of bisphenol A between 50 and 200 μM

(14.4 and 57.6 μg/ml). Recent in vitro studies

reported effects of bisphenol A on maturation,

but not induction of aneuploidy, in mouse oocytes

(236, 241) or cultured mammalian somatic cells

(242, 243), increased frequency of mitotic cells

with aberrant spindles (243), and various effects

on cellular and nuclear division in fertilized sea

urchin eggs (244). Although these new studies

provide further evidence of bisphenol A’s effects

on meiotic and mitotic cell division using a variety of in vitro systems and treatment concentrations, no impact of such effects on reproduction

isreported in animal breeding studies and the significance of these findings with regard to human

health hazards is not clear. If aneuploid eggs or

sperm were induced by bisphenol A, it would be

expected to result in reduced littersizesfollowing

exposure of one or both parents to bisphenol A.

Such an effect is notseen in reproductive toxicity

studies of bisphenol A in rats or mice except at

very high exposure levels (500 mg/kg bw/day or

higher) where other types of toxicities are manifest (37, 40, 41), including in the F2 generation

(37, 41). Findings ofsignificantly decreased litter

size or pregnancy loss are reported occasionally

at lower doses of bisphenol A (114, 245), but in

general, most “low” dose studies do not report

this outcome including a number of those that

report other effects of bisphenolA exposure (44, 

48, 52, 53, 115, 125, 189).
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Are Current Exposures to 

bisphenol A High Enough to 

Cause Concern?

Possibly.The “high” dose effects of bisphenol A

in laboratory animalsthat provide clear evidence

for adverse effects on development, i.e., reduced

survival, birth weight, and growth of offspring

early in life, and delayed puberty in female rats

and male rats and mice, are observed at levels

of exposure that far exceed those encountered

by humans. However, estimated exposures in

pregnant women and fetuses, infants, and children are similar to levels of bisphenol A associated with several “low” dose laboratory animal

findings of effects on the brain and behavior,

prostate and mammary gland development, and

early onset of puberty in females. When considered together, these laboratory animal findings

provide limited evidence that bisphenol A has

adverse effects on development (Figure 2b).

Exposuresin humans and laboratory animals can

be compared using approaches based on either

estimated daily intake (based on aggregating

sources of exposure or back calculating from

biomonitoring data) or measured blood concentrations of free bisphenol A. Each approach has

a unique set of assumptions and limitations.The

conclusion of similarities between exposures of

certain human populations and laboratory animals treated with “low” doses of bisphenol A

is supported by multiple approaches. For this

reason, the possibility that human development

may be altered by bisphenol A at current exposure levels cannot be dismissed.

Supporting Evidence

A considerable amount of research has been

directed towards understanding the levels of

human exposure to bisphenol A, either by estimating daily intake or by measuring bisphenolA

concentrationsin human blood, urine, breastmilk,

or other tissue. An overarching issue relevant to

the bisphenol A biomonitoring studies in both

humans and laboratory animals is the accuracy

of the laboratory methods used to measure the

compound (see Appendix A). There is concern

that measurements of bisphenol A, especially

free bisphenol A, may be too high due to problemsrelated to sample preparation orstorage and

the analytical technique employed [reviewed in

(2, 13)].The NTP recognizes the possibility that

the published values of free bisphenol A may, in

some cases, not accurately represent the “true”

concentrations of free bisphenol A in the blood

or body fluids of humans or laboratory animals.

However, because of the similarity among values

reported with different analytical methods, with

the exception of studies that use an enzymelinked immunosorbent assay (ELISA), the NTP

accepts the published values as sufficiently reliable for use in this evaluation.

Daily Intake Exposure Estimates

The vast majority of bisphenol A exposure is

through the diet, estimated at ~99% (1); therefore, estimates of daily intake in humans can be

compared to oral doses used in laboratory animal

studies where effects considered relevant to human

health were observed. Estimates of daily intake are

derived using two general approaches.Researchers

can use information on the amount of bisphenolA

detected in varioussources of exposure (i.e., food,

food packaging, air, water, dust, etc.) and sum, or

aggregate, the measurements to estimate a total

daily intake (“aggregating sources of exposure”

method). Alternatively, biomonitoring information, such as the concentration of bisphenol A in

urine, can be used to estimate, or “back calculate”,

a total intake that reflects all sources of exposure,

both known and unknown. Both approaches for

estimating daily intake rely on various assumptions and default values such as average body

weight, amount of food or beverage consumed,

daily volume of urine output, or ability of a single

measurement to characterize exposure.

Infants & children less than 6 years of age 

For infants and children less than 6 years of age,

estimates of daily intake were based on aggregat-
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ing sources of exposure (Table 1). No biomonitoring data, i.e., blood or urine concentration of

bisphenol A, are available for these life stages

[reviewed in (2)].An estimated daily intake of ~1

μg/kg bw/day for both breast-fed and formulafed infants was calculated by the CERHR Expert

Panel for Bisphenol A (2). Higher “worst case”

daily intake estimates of 11–13 μg/kg bw/day

during the first year of life have been calculated

for infants(25). In children 1.5 to 6 years of age,

the range of estimated daily intakes based on

aggregating sources of exposure is 0.043–14.7

μg/kg bw/day, with 14.7 μg/kg bw/day representing a worst case scenario (27, 29).

Although biomonitoring data are not available

for infants and children less than 6 years of age,

blood and urine levels of free bisphenol A are

predicted to be higher in these age groups compared to pregnant women or other adult populations. This is based on information re­lated to

age-specific differences in daily intake of bisphenol A and in the ability to metabolize the

chemical. More specifically, it is based on observations of (1) higher urinary measurements

of total bisphenol A in children (6 –11 years of

age) compared to adolescents and adults (8), (2)

higher estimated daily intakes of bisphenol A

for infants and children (2, 25, 27) compared

to estimated daily intakes for adults (2, 25, 35),

and (3) predicted higher blood concentrations of

free bisphenol A in infants compared to adults

at a given daily intake level based on less efficient metabolism of bisphenol A in rat fetuses

and neonates (18 –20), and very low or absent

activities in human fetuses and premature or

full-term infants of the isozymes that govern

glucuronidation (246 –248).

Adults and children aged 

6 years and above 

Daily intake estimates for adults and children

aged 6 years and older are based on (1) back calculations from the most recent CDC NHANES

data on urinary concentrations of total bisphenol

and (2) aggregating sources of exposure (Table

1 and Table 3). Of these estimates, the NTP has

more confidence in the estimates based on back

calculating from urinary biomonitoring data

because all sources of exposure are integrated

into the fluid measurement and thus do not have

to be identified in advance. However, it is worth

noting that the estimates for non-occupationally

exposed adults based on aggregating sources

of exposure encompass the range estimated

from back calculating from urine [aggregating

sources of exposure: 0.008–1.5 μg/kg bw/day

(Table 1); and back calculating based on urine:

0.233–0.289 μg/kg bw/day for various categories of adults ages 20+ at the 95th percentile

(35)]. Fewer studies have estimated daily intakes

for children older than 6 years of age and adolescents. In Japanese children and adolescents

between the ages of 7 and 19 years, the range

of estimated daily intakes based on aggregating

sources of exposure is 0.36 to 0.55 μg/kg bw/day

(30), which is only slightly higher than the estimated range of daily intakes for American children and adolescents based on back calculating

from urinary concentration of total bisphenol A

[0.311–0.348 μg/kg bw/day for children ages

6–11 and 12–19 at the 95th percentile (35)].

Estimated daily intake based

on blood biomonitoring

The NTP also considered the appropriateness of

estimating daily intake based on back calculations from free bisphenol A measured in human

blood and concluded that the scientific uncertainties are currently too large to support this

exercise (see Appendix A). In brief, estimated

daily intakes in adults based on this approach

are much greater (~500 μg/kg– 1.54 mg/kg bw/

day for a 65 kg human) (3, 249) than estimates

of daily intake based on aggregating routes of

exposure (0.008 –1.5 μg/kg bw/day) (25, 31) or

from back calculating from urinary data (adults

aged 20– 60+: medians 0.0563–0.0334 μg/kg
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bw/day; 95th percentiles 0.289– 0.233) (35). In

addition, data from an intentional dosing study

conducted by Tsukioka et al. (250)22 provides

further support for daily intakes in humans of

<1 μg/kg. Several explanations have been proposed to account for the discrepancy between

estimated intake based on blood and urine but

they are not sufficient to fully explain it.

Exposure Comparisons

Based on Daily Intake

The “high” dose effects of bisphenol A that

represent clear evidence for adverse effects on

development, i.e., reduced survival (≥500 mg/

kg bw/day) (36 – 40), reduced birth weight and

growth of offspring early in life (≥300 mg/kg

bw/day) (36 –39, 41), and delayed puberty in

female rats and male rats and mice (≥50 mg/

kg bw/day) (37, 41– 43), are observed at dose

levels that are more than 3,500-times higher

than “worst case” daily intakes of bisphenol A

in infants and children less than 6 years of age

(≥50 mg/kg bw/day versus 0.008–0.0147 mg/

kg bw/day). The differences in exposures are

much greater, more than 160,000-times different, when the high oral dose level is compared to

estimated daily intakes for children ages 6–11

and adult women (as an indicator of exposure for

pregnant women) at the 95th percentile of 0.311

and 0.271 μg/kg bw/day, respectively (35).

However, a number of “low” dose developmental effects have been reported in mice treated

orally with bisphenol A including effects on

behavior (≥10 μg/kg bw/day) (44–50), prostate

22Tsukioka et al. (250) used GC/MS with trimethyl­silyation (TMS) derivatization (LOQ 0.1

mg/L). Brock et al. (251) report that use of TMS

may produce interfering peaks in the chromatogram. Sample workup included glucuronidase

treatment, solvent extraction, and solid phase

clean-up. Few details were presented in the

Tsukioka et al. (250) study on sample preparation process, such as storage temperature.

gland and urinary tract development (10 μg/kg

bw/day) (54), and early onset of puberty (2.4

and 200 μg/kg bw/day) (48, 55). In addition,

subcutaneousinjection with 10 μg/kg bw/day of

bisphenol A during neonatal life in rats results

in development of hormonally induced preneoplastic lesions in the prostate later in life (51).23

This non-oral study is considered relevant for

comparing exposures because, as discussed previously, the differences in the rate of bisphenol

A metabolism seen in adult rats based on route

of administration (oral versus non-oral) appear

to be greatly reduced in neonatal rats and mice

(18, 92). As stated earlier, these findings, when

considered together, provide limited evidence

for adverse effects of bisphenol A exposure on

development in laboratory animals (Figure 2b).

In infants, the doses of 2.4 and 10 μg/kg bw/

day are 2.4 – 10 times higher than the estimated

daily intake of ~1 μg/kg bw/day calculated by

the CERHR Expert Panel for Bisphenol A (2).

Higher “worst case” daily intakes have been calculated for infants by the European Food Safety

Authority of 11–13 μg/kg during the first year

of life (25). To the extent these estimates are

accurate, then dose levels of 2.4 and 10 μg/kg

bw/day slightly exceed (1.1 to 5.4-times) worst

case estimates. The doses of 2.4 and 10 μg/kg

bw/day are approximately 7.7–32 and 8.9– 37

times higher than the estimated daily intakes of

0.311 μg/kg bw/day for children (ages 6–11

years) and 0.271 μg/kg bw/day for adult women

at the 95th percentile (35).

23Preneoplastic lesions in the mammary gland, i.e.,

ductal hyperplasia and carcinoma in situ, have

been reported in rats treated as fetuses with 2.5

μg/kg bw/day via a subcutaneous pump implanted in the dam (52, 53); however, as discussed

previously, studies that administer bisphenol A

via subcutaneous pump are considered informative for identifying potential biological effects of

bisphenolA, but not for quantitatively comparing

exposures in laboratory animals and humans.
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Exposure Comparisons Based 

on Blood Concentrations of 

Free Bisphenol A

No studies in laboratory animals have measured

circulating levels of free bisphenol A in the

blood following a dosing schedule that mimics

human exposures, i.e., long-term dietary lowdose exposure occurring numerous times during the day. However, a number of studies have

detected quantifiable levels of free bisphenol A

in the blood of adult rodents following a single

oral administration of bisphenol A, typically at

doses considered high when compared to estimated human daily intakes (500–1,000,000

μg/kg for rodents versus <14.7 μg/kg bw/day

for humans) (3, 27, 35, 249).These studies were

used by Vandenberg et al. (3) to estimate circulating blood levels of free bisphenolA in rodents

at a lower oral dose of 50 μg/kg based on the

assumption of linear proportionality between

administered dose and circulating concentration

of free bisphenol A. The estimated peak blood

levels of free bisphenol A in the first 30 minutes after dosing at 50 μg/kg ranged from 0.01

to 1.14 μg /L (median 0.11 μg /L) (3). Based

on this estimate, peak concentrations of free

bisphenol A in mice or rats treated with 2.4 or

10 μg/kg bw/day of bisphenol A are projected

to be lower than the free blood concentrations

measured in humans, including pregnant women

(12, 15). See Appendix A for further details on

these calculations.
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NTP Conclusions

The NTP reached the following conclusions on

the possible effects of exposure to bisphenol A

on human development and reproduction. Note

that the possible levels of concern, from lowest to

highest, are negligible concern,minimal concern,

some concern, concern, and serious concern.

The NTP has some concern for effects on the 

brain, behavior, and prostate gland in fetuses, 

infants, and children at current human exposures to bisphenol A. 

The NTP concurs with the conclusion of the

CERHR Expert Panel on Bisphenol A that the

scientific evidence supports a conclusion of

some concern for exposures in fetuses, infants,

and children based on a number of laboratory

animal studies reporting that “low” level exposure to bisphenol A during development can

cause changes in the brain and behavior. In addition, the NTP has some concern for exposures

to these populations based on effects on the

prostate gland observed in laboratory animals.

This level of concern for effects on the prostate

gland is higher than that expressed by the Expert

Panel and is based primarily on new supportive

data related to (1) the interpretation of studies

that use a non-oral route of administration in

neonatal rodents, and (2) an additional publication reporting subtle cellular changes in the

prostate gland.These reports were not published

when the Expert Panel completed its deliberations. These studies in laboratory animals provide only limited evidence for adverse effects

on development and more research is needed to

better understand their implications for human

health. However, because these effectsin animals

occur at bisphenol A exposure levels similar to

those experienced by humans, the possibility

that bisphenol A may alter human development

cannot be dismissed.

The NTP has minimal concern for effects on the 

mammary gland and an earlier age for puberty 

for females in fetuses, infants, and children at 

current human exposures to bisphenol A.

The NTP concurs with the conclusion of the

CERHR Expert Panel on Bisphenol A that

the scientific evidence supports a conclusion

of minimal concern for exposures in fetuses,

infants, and children based on a number of

laboratory animal studies reporting that “low”

level exposure to bisphenol A during development can alter the timing of events related to

sexual maturation in females. In addition, the

NTP has minimal concern for exposures to these

populations based on effects on the mammary

gland observed in laboratory animals. This

level of concern for effects on the mammary

gland is higher than that expressed by the Expert

Panel and is based primarily on (1) information

received through public comments and (2) a

new supportive study reporting subtle changes

in the undifferentiated structures of the mammary gland.These studies in laboratory animals

provide only limited evidence for adverse effects

on development and more research is needed to

better understand their implications for human

health. However, because these effectsin animals

occur at bisphenol A exposure levels similar to

those experienced by humans, the possibility

that bisphenol A may alter human development

cannot be dismissed.

The NTP has negligible concern that exposure of 

pregnant women to bisphenol A will result in fetal 

or neonatal mortality, birth defects, or reduced 

birth weight and growth in their offspring. 

The NTP concurs with the conclusion of the

CERHR Expert Panel on Bisphenol A that there

is negligible concern that exposure of pregnant
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women to bisphenol A will result in fetal or

neonatal mortality, birth defects or reduced

birth weight and growth in their offspring. In

laboratory animals, exposure to very high levels of bisphenol A during pregnancy can cause

fetal death and reduced birth weight and growth

during infancy. These studies provide clear evidence for adverse effects on development, but

occur at exposure levels far in excess of those

experienced by humans.Two recent human studies have not associated bisphenol A exposure in

pregnant women with decreased birth weight or

several other measures of birth outcome. Results

from several animal studies provide evidence

that bisphenol A does not cause birth defects

such as cleft palette, skeletal malformations, or

grossly abnormal organs.

The NTP has negligible concern that exposure 

to bisphenol A will cause reproductive effects in 

non-occupationally exposed adults and minimal 

concern for workers exposed to higher levels in 

occupational settings.

The NTP concurs with the conclusion of the

CERHR Expert Panel on Bisphenol A that there

is negligible concern that exposure to bisphenol

A causes reproductive effects in non-occupationally exposed adults and minimal concern 

for workers exposed to higher levels in occupational settings Data from studies in humans

are not sufficient to determine if bisphenol A

adversely affects reproduction when exposure

occurs during adulthood. A number of studies,

when considered together, suggest a possible

effect on reproductive hormones, especially in

men exposed to higher levels of bisphenol A in

the workplace. Laboratory studies in adult animals show adverse effects on fertility, estrous

cycling, and the testes at exposure levels far in

excess of those experienced by humans. A number of other effects, such as decreased sperm

counts, are reported for the reproductive system

at lower doses in animals exposed only during

adulthood, but these effects have not been shown

to be reproducible. Laboratory animal studies

consistently report that bisphenol A does not

affect fertility.

These conclusions are based on 

information available at the time this 

brief was prepared. As new information 

on toxicity and exposure accumulates, 

it may form the basis for either lowering 

or raising the levels of concern 

expressed in the conclusions.
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Free bisphenol A has been measured in the

blood of pregnant women at concentrations

up to 22.4 μg/L (12). How to account for the

detection of free bisphenol A in human blood is

an area of scientific debate. In a controlled and

intentional dosing study in humans, free bisphenol A was not detected in the blood or urine of

a small number of adult subjects (n=9) orally

dosed with 5 mg/person bisphenol A, ~54–90

μg/kg (252). This dose range is approximately

200 to 400-fold higher than the estimates of

daily intake based on urinary biomonitoring

data for adults (95th percentile of 0.233–0.289

μg/kg bw/day) (35). The findings by Völkel et 

al. (252) lead to the prediction that the capacity

for conjugation reactions is so large in humans

that free bisphenol A should not be present in

detectable concentrations in the blood of nonoccupationally exposed adults. However, biomonitoring studies of the general population

report detecting free bisphenol A in the blood,

including from pregnant women (12, 15), urine

(253), and breast milk (5). Despite the relatively

high limit of detection of the analysis method

for free bisphenol A of 2.28 μg/L (10 nM) for

blood in the 2002 study by Völkel et al. (252),

it is a source of scientific uncertainty why free

bisphenol A was not detected in this study in

light of reports of mean blood concentrations

of free bisphenol A up to 4.4 μg/L (15) and 5.9

μg/L (12) in pregnant women in the general

population.

This discrepancy has contributed to the concern

expressed by some scientists that the reported

detections of free bisphenol A are artifacts

of problems related to sample preparation or

storage and the analytical technique employed

(2, 13). Ideally, methods should measure only

bis­phenol A and not other compounds (“specificity”). There is scientific consensus that measurements of bisphenol A based on the enzymelinked immunosorbent assay (ELISA) are the

least reliable and non-specific due to potential

cross-reactivity with structurally-similar compounds (2, 3, 13).

241 Analytical methods should

also be able to detect bisphenol A at low concentrations (“sensitivity”). In addition, measurements of free bisphenol A should be based on

analytical methods that accurately distinguish

between the concentrations of free bisphenol A

and its conjugated metabolites.

There is concern that current measurements of

free bisphenol A may be too high (2, 13). This

could occur, for example, if the method used

misidentified other chemicals as bisphenol A

or if there was background contamination from

laboratory ware. Alternatively, the procedures

used to process the samples could introduce bias

in measurement even if the analytical method

employed is high quality. Measurements of free

bisphenol A could be overestimated if the samples were processed in a manner that allowed the

conjugated metabolites to revert back to the free

form of bisphenol A. For example, conjugated

bisphenol A in urine only appears to be stable

when stored at room temperature for ~24 hours.

After 2–4 days at this temperature conjugated

bisphenol A begins to degrade and the percent

detected in samples decreases ~8 to 30%, i.e.,

higher concentrations of free bisphenolA would

be detected over time (254).

However, free bisphenol A has been detected in

10% of human urine samples [range=<limit of

detection (0.3)–0.6 μg/L; n=30] (253) and in

24Analytical techniques used to measure bisphenol A include gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass

spec­tro­metry (LC/MS), high performance liquid

chrom­atography (HPLC) with fluorescence or

electrochemical detection, and enzyme-linked

immunosorbent assay (ELISA).

Appendix A: 

Interpretation of Blood Biomonitoring Studies
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60% of breast milk samples [mean=1.3 μg/L;

median=0.4 μg/L; range=<limit of detection

(0.3)–6.3 μg/L; n=20] (5) by researchers at

the CDC who use analytical methods considered by many scientists to be very accurate. A

recent analytical methods report by the CDC

provides further support that the ratio of free to

total bisphenol A may be higher in breast milk

compared to other biological media. In thissmall

study which used only 4 human milk samples,

free bisphenolA was detected in allsamples and

ranged from 49 to 99% of the total concentration

(255).A proposed explanation to account for the

detection of free bisphenol A in breast milk is

that the free form of bisphenol A is more lipophilic than the conjugated forms and therefore

more likely to sequester in breast milk (5, 255).

In addition, Tsukioka et al. (250) were able to

detect free bisphenolA in the urine of all human

subjectstreatedwith~0.83μg/kg,whereasVölkel

et al. (252)wasunable todetect anyfreebisphenol

A in subjects treated with doses 65–108-times

higher, ~54–90 μg/kg. It cannot be definitively

determined if the detection of free bisphenol A

in urine in the study byTsukioka et al. (250) was

due to the analytical method employed or partial

cleavage of glucuronide during sample storage,

preparation or analysis. However, Tsukioka et 

al. (250) also detected total and free bisphenol

A in the urine ofsubjectsthat were not intentionally treated [total bisphenol A: 0.82 μg/L (range

0.14–5.47; n=91); free bisphenol A: 0.08 μg/L

(range 0.01–0.27 ng/m; n=11)], and these values are lower than CDC measurements of total

[2.6 μg/L for all subjects in the NHANES study

(8)] and free bisphenol A [10 of 30 subjects at

<LOD(0.3)–0.6 μg/L (253)].

CDC researchers recently published an analytical methods study that reported detecting

bisphenol A in only one of the 15 commercial

samples tested (the concentrations of total and

free in the one sample were similar, 1.5 ng/ml)

(256). However, caution should be exercised in

interpreting this information for exposure analysis because information on sample collection,

handling and storage protocols is not available

for these commercial samples.The NTP considers it noteworthy that the studies reporting the

highest blood concentrations of bisphenol A in

humans using non-ELISA analytical methods

both relied on samples collected from pregnant

women in the process of delivery (12, 15). The

use of bisphenol A in polyvinyl chloride plastic

has been documented (6) and, if still occurring,

could result in exposuresin medicalsettingssuch

as in women during delivery who are often connected to an IV. Ifsuch a scenario were occurring

then the reported concentrations of bisphenol

A in pregnant women may be accurate but not

necessarily reflective of exposures throughout

gestation or to the general population.

In summary, the NTP recognizes the possibility that the published values of free bisphenol

A may, in some cases, not accurately represent

the “true” concentrations of free bisphenol A in

the blood or body fluids of humans or laboratory animals. However, because of the similarity

among values reported with different analytical

methods, with the exception of ELISA-based

studies, the NTP accepts the published values as

sufficiently reliable for use in this evaluation.

Comparison of measured human blood 

concentrations of free bisphenol A with 

estimated concentrations in laboratory 

rodents at low doses 

More than 10 toxicokinetic and metabolism

studies have detected quantifiable levels of free

bisphenolA in the blood of adult rodents, mostly

rats, following oral administration of doses that

are considered high when compared to estimated

human daily intakes (500 – 1,000,000 μg/kg for

rodents versus <14.7 μg/kg bw/day for humans)

(3, 27, 35) (Table 1 and Table 2). These studies

were used by Vandenberg et al. (3) to estimate

circulating blood levels of free bisphenol A in

laboratory rodents at a lower oral dose of 50
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μg/kg bw/day based on the assumption of linear

proportionality between administered dose and

circulating concentration of free bisphenol A.

The estimated peak blood levels of free bisphenol A achieved in the first 30 minutes after

dosing ranged from 0.01 to 1.14 μg/L (3).

Using the estimates provided by Vandenberg et 

al. (3) for peak blood levels of free bisphenol A

at 50 μg/kg and relying on the assumption of

linear proportionality, the NTP estimated the

range of peak concentrations of free bisphenol A

at 10 μg/kg, a dose where a number of “low”

dose effects are reported, to be five times lower,

i.e., 0.002 to 0.228 μg/L. These values are 2950

to 25.9 times lower than the mean blood concentration of free bisphenol A detected in pregnant

women in Michigan (5.9±0.94 μg/L; range 0.5

to 22.4) (12).

The appropriateness of extrapolating from higher

dose studiesto predict blood levels offree bisphenolA at lower dose levels rests on the validity of

the assumption of proportionality. This assumption is warranted if, for example, blood levels

of free bisphenol A are approximately 10 times

lower following dosing with 10 mg/kg than after

dosing with 100 mg/kg. Three studies are available that used non-ELISA methods to measure

concentrations of free bisphenol A following

oral dosing with 10 and 100 mg/kg bisphenol

A in adult rats (93, 257, 258). In these studies,

the peak, or Cmax, blood concentrations of free

bisphenol A were 4.8-times (257), 22.7-times

(258), and 57-times (93) lower in rats treated

with a 10 mg/kg dose compared to rats treated

with 100 mg/kg.

Directly evaluating proportionality at lower oral

doses (<10 mg/kg) has not been possible in adult

animals because blood concentrations of free

bisphenolA are below the limits of detection for

the analytical methods employed. One strategy

that can be used to address the assumption of

proportionality at low doses is to rely on studies that have dosed young rodents because they

have higher peak blood concentrations of free

bis­phenolA compared to adults treated with the

same dose (18).Two studies have measured concentrations of free bisphenolA in young rodents

at more than one dose level (18, 92). In 3-day old

female mice orally treated with 0.035 and 0.395

mg/kg bisphenol A, Taylor et al. (92) found that

the peak blood concentration of free bisphenol

A at 0.035 mg/kg was 8.3-times lower than the

peak concentration at 0.395 mg/kg (difference

between administered does is 11.3-times). The

study by Domoradzki et al. (18) treated neonatal

rats orally with higher doses of bisphenolA than

those used by Taylor et al. (92). In 4-day old

female and male rats, the peak concentrations of

free bisphenol A were 170 to 1610-times lower

at 1 mg/kg compared to 10 mg/kg bisphenol A.

This finding, coupled with data for 21-day old

rats presented in Domoradzki et al. (18) and the

comparisons presented above fromTominaga et 

al. (258), and Pottenger et al. (93), suggest that

rodents, and presumably humans, can more efficiently metabolize lower doses of bisphenol A

compared to high doses.These data also suggest

that extrapolating from higher dose levels in the

mg/kg range may overestimate the circulating

concentrations of free bisphenol A following

administration of oral doses in the low μg/kg

range.

Any extrapolation and use of assumptions

involves some degree of uncertainty. However,

the conclusion outlined above of similar blood

levelsin the general population and in laboratory

animals at “low” doses would still hold even if

the estimated blood levels of free bisphenol A

in laboratory rodents were overestimated by a

factor of 100 or 1000, i.e., the “real” peak blood

values in laboratory animals range from 0.2 to

22.8 or 2 to 228 μg/L instead of the estimated

0.002 to 0.228 μg/L.

This possibility that blood concentrations of free

bisphenol A in humans could be significantly
43

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higher, as much as ~3000 times greater, than

the estimated peak concentrations in laboratory

animals where biological changes are observed

is a point of intense scientific controversy. In

brief, although the theoretical plausibility of

receptor­-mediated effects at “low” doses has

been described (259, 260), many scientists

expect that a compound with a significant degree

of biological “activity” at low doses would show

more profound impacts on overall toxicity at

lower doses than that observed for bisphenol A.

With bisphenol A, “low” dose developmental

effects can be observed at 0.0024 to 0.010 mg/kg

bw/day but indications of severe developmental

toxicity in rats and mice, i.e., fetal or neonatal

death are not observed except when doses are

used that are 50,000– 200,000-times higher at

≥500 mg/kg bw/day (36 – 40).

Estimated daily intake based on back 

calculating from blood and urine

Based on parameters derived from laboratory

animal studies, estimated daily intakes based

on back calculations from free bisphenol A

measured in human blood are much greater

(~500 μg/kg– 1.54 mg/kg bw/day for a 65 kg

human) (3, 249) than estimates based on any

other approach. In contrast, there is a degree of

concordance in estimates of daily intake based

on other approaches. For these reasons, the NTP

has less confidence in daily intake estimates

based on blood biomonitoring data compared

to other estimates, particularly those based on

urine biomonitoring data.

Estimates of daily bisphenol A intake in adults

based on aggregating routes of exposure fall

within the range of 0.008–1.5 μg/kg bw/day

(25, 31) (Table 1) with most estimates falling

within a range that spans one order of magnitude, 0.183–1.5 μg/kg bw/day (24–27, 30).

Daily intakes estimated from the CDC NHANES

biomonitoring data are similar and range from

0.289–0.233 μg/kg bw/day for adults aged

20–60+ years at the 95th percentile (35). The

NTP considered the possibility that the assumptions used to derive these intakes could underestimate human exposures. For estimates based

on aggregating sources of exposure, one concern is that too much emphasis has been placed

on diet as the predominant route of exposure.

For estimates based on the total concentration

of bisphenol A in urine, it is assumed that the

daily excretion of bisphenol A is a reasonable

surrogate for daily intake. Deviations from the

assumptions used to derive current estimates

could increase the daily intake estimates, but

still result in estimated intakes in the very low

μg/kg bw/day range rather than near 1 mg/kg

bw/day as predicted from the blood biomonitoring data in adult humans.

Data from an intentional dosing study conducted

byTsukioka et al. (250) provides further support

for daily intakes of <1 μg/kg.Tsukioka et al. gave

15 volunteers (12 men and 13 women) 50 μg of

bisphenol A by mouth (~ 0.83 μg/kg for a 65 kg

person) and collected urine samples for 5 hours.

The average concentration of total bis­phenol A

was 57.2 μg/L (range 26.5–80 μg/L) and free

bisphenol A was 1.13 μg/L (range 0.13–5.8

μg/L). The administered dose, ~0.83 μg/kg, and

urinary concentration of total bisphenol A, 57.2

μg/L, are ~14.8-times and 18.5-times higher,

than the estimated median intake of 0.056 μg/

kg bw/day for adults aged 20 – 39 years based

on a median urinary concentration of 3.1 μg/L

calculated by Lakind et al. (35). Extrapolating

downward for administered dose and urinary

concentrations of total bisphenol A from the

data provided by Tsukioka et al. (250) would

give values that are consistent with the daily

intake calculated by Lakind et al. (35) based on

the CDC urinary measurements (8).

Exposure Assessment Research Needs 

The NTP concurs with the CERHR Expert Panel

on Bisphenol A that more measurements in

humans are needed offree and total bis­phenolA,

its glucuronide conjugate, and other metabolite
44

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concentrations from maternal, fetal, and neonatal tissues or fluids (i.e., placenta, amniotic fluid,

breast milk, urine, serum). These data would

provide further insight into the roles of metabolism and exposure route on internal dose and

provide a firmer foundation for extrapolations of

risks to humans from the wealth of animal studies available. Available data demonstrate that a

large fraction of children and adults have detectable levels of bisphenol A, or its metabolites,

in their urine. Duplicate diet studies to identify

in detail the sources and routes of exposure

of bisphenol A would be useful. For example,

while research suggests diet is the major source

of bisphenol A for infants and young children

in the United States, the detailed analysis of

bisphenol A levels has primarily focused on

poly­carbonate baby bottle leachates and canned

food.The contributions of non-canned food and

drinking water routes of exposure for youth and

adults not occupationally-exposed to bisphenol

A remain unknown and in need of further study.

Levels of bisphenol A in residential drinking

water wells and community water sources have

not been systematically studied. Also unknown

is the impact of landfill leachates on levels of

bisphenol A in U.S. drinking well waters and

whether chlorinated congeners of bisphenol A

are found in municipal water supplies.

More research is needed to characterize the toxicokinetics of bisphenol A in developing animals

under exposure scenarios that better mimic the

low-level chronic exposures experienced by

humans. Currently, only single or “acute” dosing kinetic studies in laboratory animals are

available for predicting the metabolism and fate

of bisphenol A following long-term, daily exposure, or for comparing apparent differences in

the metabolism and fate of bisphenol A in laboratory rodents and humans. Repeated administration of many compounds has been shown to

alter the capacity of the animal to metabolize and

excrete the compound. Further characterization

of the ability of repeated exposures to bisphenol

A to change rates and extent of metabolism and

excretion in laboratory animals and humans is

a critical research need.

In addition, it is clear that there are differences

in the pharmacokinetics of bisphenolA, particularly between rats and humans, which complicate using the rat data to interpret the human

biomonitoring data. For example, the excretion

profiles of bisphenol A differ in rodents and

humans. In humans, the major route of elimination is via the urine in the form of bisphenol

A glucuronide (261). In contrast, the major

elimination routes in rodents are as bisphenol

A in the feces, as bisphenolA glucuronide in the

bile, and to a lesser extent, in the urine [reviewed

in (2)]. Also, in rats bisphenol A glucuronide

can remain in the bile and be recirculated back

to the liver (“enterohepatic circulation”). To

ad­dress these uncertainties the NTP is pursuing

studies of absorption, distribution, metabolism,

and excretion in experimental animals (rodents

and non human primates) as well as the kinetics

associated with these processes, following exposures to bisphenol A from the perinatal period

through adulthood, over a wide range of doses,

by multiple routes of administration.
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Y (2006) Toxicology. Toxicokinetics of

bisphenol A in rats, monkeys and chimpanzees by the LC-MS/MS method. 226:

208–217.

259. Lucier GW, Portier CJ, Gallo MA (1993)

Environ Health Perspect. Receptor mechanisms and dose-response models for the

effects of dioxins. 101:36– 44.

260. Welshons WV, Thayer KA, Judy BM, Taylor JA, Curran EM, vom Saal FS (2003)

Environ Health Perspect. Large effects

from small exposures. I. Mechanisms for

endocrine-disruptingchemicalswithestrogenic activity. 111:994–1006.

261. Völkel W, Bittner N, Dekant W (2005)

Drug Metab Dispos. Quantitation of bisphenol A and bisphenol A glucuronide in

biological samples by high performance

liquid chromatography-tandem massspectrometry. 33:1748–1757.
[This page intentionally left blank]
I –.1

Appendix I

A 12–member panel of scientists covering disciplines such as toxicology, epidemiology, and

medicine was recommended by the CERHR

Core Committee and approved by the Associate

Director of the National Toxicology Program.

Prior to the expert panel meeting, the panelists

critically reviewed articles from the scientific

literature, as well as a variety of other relevant

documents. Based on this material, they identified key studies and issues for discussion. At

public meetings held on March 5 – 7, 2007 and

August 6 – 8, 2007, the expert panel discussed

these studies, the adequacy of available data, and

identified data needed to improve future assessments.The expert panel reached conclusions on

whether exposure to bisphenol A might result

in adverse effects on human reproduction or

development. Panel conclusions were based on

the scientific evidence available at the time of

the public meeting. The NTP-CERHR released

the final expert panel report for public comment

on November 26, 2007 and the deadline for public comments was January 25, 2008 (Federal 

Register Vol. 72, No. 230, pp. 67730–67731,

November 30, 2007).The NTP-CERHR Expert

Panel Report on Bisphenol A is provided in

Appendix II. The expert panel report is also

available on the CERHR website (http://cerhr.

niehs.nih.gov).

Appendix I. NTP-CERHR Bisphenol A Expert Panel

NTP-CERHR Bisphenol A Expert Panel

Robert E. Chapin, Ph.D., Chair 

Pfizer, Inc.

Groton, CT

Barry S. McIntyre, Ph.D.

Schering Plough Research Institute

Summit, NJ

Jane Adams, Ph.D.

University of Massachusetts

Boston, MA

Kenneth M. Portier, Ph.D.

American Cancer Society

Atlanta, GA

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

Brown University

Providence, RI

Teresa M. Schnorr, Ph.D.

National Institute for Occupational

Safety and Health

Cincinnati, OH

L. Earl Gray, Jr., Ph.D.

U.S. Environmental Protection Agency

Research Triangle Park, NC

Sherry G. Selevan, Ph.D.

U.S. Public Health Service (Ret)

Silver Spring, MD

Simon W. Hayward, Ph.D.

Vanderbilt University Medical Center

Nashville, TN

John G. Vandenbergh, Ph.D.

North Carolina State University

Raleigh, NC

Peter S.J. Lees, Ph.D.

Johns Hopkins University

Baltimore, MD

Susan R. Woskie, Ph.D.

University of Massachusetts

Lowell, MA
Published 2008 Wiley-Liss, Inc.w Birth Defects Research (Part B) 83:157–395 (2008)

Expert Panel Report 

NTP-CERHR Expert Panel Report on the Reproductive

and Developmental Toxicity of Bisphenol A

Robert E. Chapin,1 Jane Adams,2 Kim Boekelheide,3 L. Earl Gray Jr,4 Simon W. Hayward,5 Peter S.J. Lees,6 

Barry S. McIntyre,7 Kenneth M. Portier,8 Teresa M. Schnorr,9 Sherry G. Selevan,10 John G. Vandenbergh,11 

and Susan R. Woskie12 

1

Pfizer, Inc., Groton, CT
 2

University of Massachusetts, Boston, MA
 3

Brown University, Providence, RI
 4

U.S. Environmental Protection Agency, Research Triangle Park, NC
 5

Vanderbilt University Medical Center, Nashville, TN
 6

Johns Hopkins University, Baltimore, MD
 7

Schering Plough Research Institute, Summit, NJ
 8

American Cancer Society, Atlanta, GA
 9

National Institute for Occupational Safety and Health, Cincinnati, OH
 10U.S. Public Health Service (Ret), Silver Spring, MD
 

11North Carolina State University, Raleigh, NC
 

12University of Massachusetts, Lowell, MA
 

Preface 

The National Toxicology Program (NTP)1 established 

the NTP Center for the Evaluation of Risks to Human 

Reproduction (CERHR) in June 1998. The purpose of the 

CERHR is to provide timely, unbiased, scientifically 

sound evaluations of the potential for adverse effects on 

reproduction or development resulting from human 

exposures to substances in the environment. The NTPCERHR is headquartered at NIEHS, Research Triangle 

Park, NC, and is staffed and administered by scientists 

and support personnel at NIEHS. 

Bisphenol A is a high-production volume chemical 

used in the production of epoxy resins, polyester resins, 

polysulfone resins, polyacrylate resins, polycarbonate 

plastics, and flame retardants. Polycarbonate plastics are 

used in food and drink packaging; resins are used as 

lacquers to coat metal products such as food cans, bottle 

tops, and water supply pipes. Some polymers used in 

dental sealants and tooth coatings contain bisphenol A. 

Exposure to the general population can occur through 

direct contact with bisphenol A or by exposure to food or 

drink that has been in contact with a material containing 

bisphenol A. CERHR selected bisphenol A for evaluation 

because of (1) high production volume; (2) widespread 

human exposure; (3) evidence of reproductive toxicity in 

laboratory animal studies; and (4) public concern for 

possible health effects from human exposures. 

Relevant literature on bisphenol A was identified from 

searches of the PubMed (Medline) and Toxline databases 

1

NTP is an interagency program headquartered in Research Triangle Park,

NC, at the National Institute of Environmental Health Sciences, a

component of the National Institutes of Health.

through February 2007 using the term ‘‘bisphenol’’ and 

the bisphenol A CAS RN (80-05-7). References were also 

identified from databases such as REPROTOX, HSDB, 

IRIS, and DART, from the bibliographies of the literature 

reviewed, by members of the expert panel, and in public 

comments. 

CERHR convened a 12-member, independent panel of 

government and non-government scientists to evaluate 

the scientific studies on the potential reproductive and 

developmental hazards of bisphenol A. The expert panel 

met publicly on March 5–7, 2007 and August 6–8, 2007. 

The Expert Panel Report on Bisphenol A is intended to 

(1) interpret the strength of scientific evidence that 

bisphenol A is a reproductive or developmental toxicant 

based on data from in vitro, animal, or human studies; (2) 

assess the extent of human exposures to include the 

general public, occupational groups, and other subpopulations; (3) provide objective and scientifically 

thorough assessments of the scientific evidence that 

adverse reproductive and developmental health effects 

may be associated with such exposures; and (4) identify 

knowledge gaps to help establish research and testing 

priorities to reduce uncertainties and increase confidence 

in future evaluations. This report has been reviewed by 

members of the expert panel and by CERHR staff 

scientists. Copies of this report have been provided to 

*Correspondence to: Michael D. Shelby, PhD, NIEHS EC-32, PO Box

12233, Research Triangle Park, NC 27709.

E-mail: shelby@niehs.nih.gov

Received 18 January 2008; Accepted 18 January 2008

Published online in Wiley InterScience (www.interscience.wiley.com)

DOI: 10.1002/bdrb.20147

w

This article is a U.S. Government work and, as such, is in the public domain in the United States of America.
HO 

CH3 

CH3 

OH 

158 CHAPIN ET AL. 

the CERHR Core Committee2 and will be made available 

to the public for comment. 

Following the public comment period, CERHR will 

prepare the NTP-CERHR Monograph on the Potential 

Human Reproductive and Developmental Effects of 

Bisphenol A. This monograph will include the NTP 

Brief, the Expert Panel Report, and all public comments 

received on the Expert Panel Report. The NTP-CERHR 

Monograph will be made publicly available and transmitted to appropriate health and regulatory agencies. 

Reports can be obtained from the web site (http:// 

cerhr.niehs.nih.gov) or from: Michael D. Shelby, PhD, 

NIEHS EC-32, PO Box 12233, Research Triangle Park, NC 

27709. E-mail: shelby@niehs.nih.gov 

1.0 CHEMISTRY, USE AND HUMAN 

EXPOSURE 

1.1 Chemistry 

Section 1 is based initially on secondary review 

sources. Primary study reports are addressed by the 

Expert Panel if they contain information that is highly 

relevant for determining the effect of exposure on 

developmental or reproductive toxicity or if the studies 

were released subsequent to the reviews. 

1.1.1 Nomenclature. The CAS RN for bisphenol A 

is 80-05-7. Synonyms for bisphenol A listed in Chem 

IDplus (ChemIDplus, 2006) include: 2-(4,40

-Dihydroxydiphenyl)propane; 2,2-Bis(4-hydroxyphenyl)propane; 

2,2-Bis(hydroxyphenyl)propane; 2,2-Bis(p-hydroxyphenyl)propane; 2,2-Bis-40

-hydroxyfenylpropan [Czech]; 

2,2-Di(4-hydroxyphenyl)propane; 2,2-Di(4-phenylol)propane; 4,40

-(1-Methylethylidene)bisphenol; 4,40-Bisphenol 

A; 4,40

-Dihydroxydiphenyl-2,2-propane; 4,40-Dihydroxydiphenyldimethylmethane; 4,40

-Dihydroxydiphenylpro2

The Core Committee is an advisory body consisting of scientists from

government agencies. Agencies currently represented are: Environmental

Protection Agency, Centers for Disease Control and Prevention, Food and

Drug Administration, Consumer Product Safety Commission, National

Institute for Occupational Safety and Health, and National Institute of

Environmental Health Sciences.

Prepared with the Support of CERHR Staff: NTP/NIEHS, Michael D.

Shelby, Ph.D. (Director, CERHR), Paul M.D. Foster, Ph.D. (Deputy

Director, CERHR), Kristina Thayer, Ph.D. (CERHR), Diane Spencer, M.S.

(CERHR), John Bucher, Ph.D. (Associate Director, NTP), Allen Dearry,

Ph.D. (Interim Associate Director, NTP), Mary Wolfe, Ph.D. (Director, NTP

Office of Liaison, Policy & Review), Denise Lasko (NTP Office of Liaison,

Policy & Review); Sciences International, Inc., Anthony Scialli, M.D.

(Principal Scientist), Annette Iannucci, M.S. (Toxicologist), Gloria Jahnke,

D.V.M. (Toxicologist), and Vera Jurgenson, M.S. (Research Assistant).

This report is prepared according to the Guidelines for CERHR Panel

Members established by NTP/NIEHS. The guidelines are available from the

CERHR web site (http://cerhr.niehs.nih.gov/). The format for this report

follows that of CERHR Expert Panel Reports including synopses of studies

reviewed, and an evaluation of the Strengths/Weaknesses and Utility

(Adequacy) of the study for a CERHR evaluation. Statements and

conclusions made under Strengths/Weaknesses and Utility evaluations

are those of the expert panel members and are prepared according to the

NTP/NIEHS guidelines. In addition, the report includes comments or

notes limitations of the study in the synopses. Bold, square brackets are

used to enclose such statements. As discussed in the guidelines, square

brackets are used to enclose key items of information not provided in a

publication, limitations noted in the study, conclusions that differ from

authors, and conversions or analyses of data conducted by CERHR.

The findings and conclusions of this report are those of the Expert

Panel and should not be construed to represent the views of the

National Toxicology Program. Members of this panel participated in

the evaluation of bisphenol A as independent scientists. The findings

and conclusions in this report are those of the authors and do not

necessarily represent the views of their employers.

pane; 4,40

-Isopropylidene diphenol; 4,40-Isopropylidenebisphenol; 4,40

-Isopropylidene diphenol; Biphenol A; 

Bis(4-hydroxyphenyl) dimethylmethane; Bis(4-hydroxyphenyl)dimethylmethane; Bis(4-hydroxyphenyl)propane; 

Bisferol A [Czech]; Bisphenol. Bisphenol A; DIAN; Diano; 

Dimethyl bis(p-hydroxyphenyl)methane; Dimethylbis 

(p-hydroxyphenyl)methane; Dimethylmethylene-p,p0

­

diphenol; Diphenylolpropane; Ipognox 88; Isopropylidenebis(4-hydroxybenzene); Parabis A, Phenol; (1-methylethylidene)bis-, Phenol; 4,40

-(1-methylethylidene)bis-; 

Phenol, 4,40

-dimethylmethylenedi-; Phenol, 4,40-isopropylidenedi-; Pluracol 245, Propane; 2,2-bis(p-hydroxyphenyl)-; Rikabanol; Ucar bisphenol A; Ucar bisphenol HP; 

beta,beta0

-Bis(p-hydroxyphenyl)propane; beta-Di-p-hydroxyphenylpropane; p,p0

-Bisphenol A; p,p0-Dihydroxydiphenyldimethylmethane; p,p0

-Dihydroxydiphenylpropane; p,p0

-Isopropylidenebisphenol; and p,p0-Isopropylidenediphenol. 

1.1.2 Formula and molecular mass. Bisphenol A 

has a molecular mass of 228.29 g/mol and a molecular 

formula of C15H1602 (European-Union, 2003). The structure for bisphenol A is shown in Figure 1. 

1.1.3 Chemical and physical properties. Bisphenol A is a white solid with a mild phenolic odor 

(European-Union, 2003). Physicochemical properties are 

listed in Table 1. 

1.1.4 Technical products and impurities. Purity 

of bisphenol A was reported at 99–99.8%, and common 

impurities observed were phenol and ortho and para 

isomers of bisphenol A [reviewed in (European-Union, 

2003)]. Terasaki et al. (2004) used reversed phase 

chromatography and nuclear magnetic resonance spectroscopy to characterize the composition of 5 commercial 

bisphenol A samples. The nominal purity of the samples 

was 97 or 98%. Actual purities were 95.3 to 499%. Up to 

15 contaminants were identified among which were: 4­

hydroxyacetophenone; 4,40

-(1,3-dimethylbutylidene) bisphenol; p-cumylphenol; 4-hydroxyphenyl isobutyl * methyl ketone; 2,4 -dibhydroxy-2,2-diphenylpropane; 

2,40

-dibhydroxy-2,2-diphenylpropane; 2,4-bis(4-hydroxyFig. 1. Structure for bisphenol A. 

Table 1 

Physicochemical Properties of Bisphenol Aa 

Property Value 

Odor threshold No data found 

Boiling point 2201C at 4 mm Hg; 3981C at 760 mm Hg 

Melting point 150–1571C 

Specific gravity 1.060–1.195 g/mL at 20–251C 

Solubility in water 120–300 mg/L at 20–251C 

Vapor pressure 8.7 x 10-10 –3.96 x 10-7 mm Hg at 20–251C 

Stability/reactivity No data found 

Log Kow 2.20–3.82 

Henry constant 1.0 x 10-10 atm m3

/mol 

a

Staples et al. (1998). 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 159 

cumyl)phenol; 2,3-dihydro-3-(40-hydroxyphenyl)-1,1,3­

trimethyl-1H-inden-5-ol; 2-(40

-hydroxyphenol)-2,2,4-trimethylchroman; and 4-(40

-hydroxyphenol)-2,2,4-trimethylchroman (Terasaki et al., 2005). 

No information on trade names for bisphenol A was 

located. 

1.1.5 Analytical considerations. Measurement of 

bisphenol A in environmental and biologic samples can 

be affected by contamination with bisphenol A in plastic 

laboratory ware and in reagents (Tsukioka et al., 2004; 

Vo ̈lkel et al., 2005). Accuracy is also affected by 

measurement technique, particularly at the very low 

concentrations that can now be measured. Enzymelinked immunosorbent assay (ELISA) has poor correlation with the LC-ECD method and also the different 

ELISA kits correlate poorly with each other. ELISA 

methods may overestimate bisphenol A in biologic 

samples due to lack of specificity of the antibody and 

effects of the biologic matrix (Inoue et al., 2002; Fukata 

et al., 2006). Although high performance liquid chromatography (HPLC) with ultraviolet, fluorescence, or 

electrochemical detection can be sensitive to concentrations o0.5 ng/ml (Sajiki et al., 1999; Inoue et al., 2000; 

Kuroda et al., 2003; Sun et al., 2004), these methods are 

unable to make definitive identification of bisphenol A 

or bisphenol A glucuronides, because similar retention 

times may occur for the metabolites of other endogenous 

and exogenous compounds (Vo ̈lkel et al., 2005). Use of 

LC-mass spectrometry (MS) with and without hydrolysis 

of bisphenol A glucuronide permits determination of 

free and total bisphenol A with a limit of quantification 

of 0.1 for MS (Sajiki et al., 1999) and 1 mg/L for MS/MS 

(Vo ̈lkel et al., 2005). Gas chromatography (GC)/MS has 

been used with solid phase extraction after treatment 

with glucuronidase and derivatization to measure total 

bisphenol A with a limit of detection of 0.05 mg/L for MS 

(Tan and Mohd, 2003) and 0.1 mg/L for MS/MS (Calafat 

et al., 2005). Some of the variability in studies cited in this 

and subsequent sections may be due to differences in 

measurement techniques and to contamination. Bisphenol A glucuronidate can be an unstable product that can 

be degraded in acidic and basic pH solutions and can be 

hydrolyzed to free bisphenol A at neutral pH and room 

temperature in diluted rodent urine, placental and fetal 

tissue homogenates at room temperature. However, 

conjugates in urine are stable for at least 7 days when 

stored at -41C and at least 180 days when stored at 

-701C (Waechter et al., 2007; Ye et al., 2007). 

1.2 Use and Human Exposure 

1.2.1 Production information. Bisphenol A is 

manufactured by the acid catalyzed condensation of 

phenol and acetone (SRI, 2004). 

In 1998, members of the Society of the Plastics Industry 

Bisphenol A Task Group [assumed manufacturers of 

bisphenol A] included Aristech Chemical Corporation, 

Bayer Corporation, Dow Chemical Company, and Shell 

Chemical Company (Staples et al., 1998). Current 

manufacturers of bisphenol A in the U.S. are Bayer 

MaterialScience, Dow Chemical Company, General Electric, Hexion Specialty Chemicals, and Sunoco Chemicals 

(SRI, 2004) (S. Hentges, public comments, February 2, 

2007). There are currently six bisphenol A and four 

polycarbonate plants in the U.S. (S. Hentges, personal 

communication, October 30, 2006); three of four polycarbonate plants are located within bisphenol A plants. 

In 2000, there were 13 epoxy plants in the U.S., but was 

not clear if all of the plants manufactured bisphenol 

A-containing epoxy resins. 

In mid-2004, U.S. bisphenol A production volume was 

reported at 1.024 million metric tons [B2.3 billion 

pounds] (SRI, 2004). A production volume of 7.26 billion 

g [16 million pounds] was reported for bisphenol A in 

1991 (reviewed in HSDB, 2003). United States bisphenol 

A consumption was reported at 856,000 metric tons 

[B1.9 billion pounds] in 2003 (SRI, 2004); 2003 consumption patterns included 619,000 metric tons [B1.4 

billion pounds] used in polycarbonate resins, 184,000 

metric tons [B406 million pounds] used in epoxy resins, 

and 53,000 metric tons [B117 million pounds] used in 

other applications. 

1.2.2 Use. In 1999 and 2003, it was reported that 

most bisphenol A produced in the U.S. was used in the 

manufacture of polycarbonate and epoxy resins and 

other products [reviewed in (Staples et al., 1998; SRI, 

2004)]. Polycarbonate plastics may be used in the 

manufacture of compact discs, ‘‘solid and multi wall 

sheet in glazing applications and film,’’ food containers 

(e.g., milk, water, and infant bottles), and medical devices 

[reviewed in (European-Union, 2003)]. Bisphenol A may 

have been used at one time in Europe in polyvinyl 

chloride cling film and plastic bags, but that use is 

believed to have been discontinued (European Food 

Safety Authority, 2006). Contact with drinking water may 

occur through the use of polycarbonate for water pipes 

and epoxy-phenolic resins in surface coatings of drinking 

water storage tanks [reviewed by (European Food Safety 

Authority, 2006)]. 

Polycarbonate blends have been used to manufacture 

injected molded parts utilized in alarms, mobile phone 

housings, coil cores, displays, computer parts, household 

electrical equipment, lamp fittings, and power plugs. 

Automotive and related uses for polycarbonate blends 

include light reflectors and coverings, bumpers, radiator 

and ventilation grills, safety glazing, inside lights, and 

motorcycle shields and helmets. Epoxy resins are used in 

protective coatings, structural composites, electrical 

laminates, electrical applications, and adhesives. The 

European Union (2003) reported that smaller volumes of 

bisphenol A are used in production of phenoplast, 

phenolic, and unsaturated polyester resins, epoxy can 

coatings, polyvinyl chloride (PVC) plastic, alkoxylated 

bisphenol A, thermal paper, and polyols/polyurethane. 

Other uses reported for products manufactured from 

bisphenol A included protective window glazing, building materials, optical lenses, and development of dyes 

[reviewed in (Staples et al., 1998)]. A search of the 

National Library of Medicine Household Products 

Database (NLM, 2006) revealed that bisphenol A-based 

polymers are used in coatings, adhesives, and putties 

available to the general pubic for use in automobiles, 

home maintenance and repair, and hobbies, but only 3 

epoxy products, used for crafts and hobbies, contain 

bisphenol A itself. 

Some polymers manufactured with bisphenol A are 

Food and Drug Administration (FDA)-approved for use 

in direct and indirect food additives and in dental 

materials, as reported in the Code of Federal Regulations 

(CFR) (FDA, 2006). In the CFR, bisphenol A is often 

Birth Defects Research (Part B) 83:157–395, 2008
160 CHAPIN ET AL. 

referred to as 4,40-isopropylidnediphenol. Polymers 

manufactured with bisphenol A are FDA-approved for 

use as anoxomers and in coatings, adhesives, single and 

repeated food contact surfaces, and tooth shade resin 

materials. 

The European Union (2003) noted that resins, polycarbonate plastics, and other products manufactured 

from bisphenol A can contain trace amounts of residual 

monomer and additional monomer may be generated 

during breakdown of polymer. The American Plastics 

Council reports that residual bisphenol A concentrations 

in polycarbonate plastics and epoxy resins are generally 

o50 ppm (S. Hentges, personal communication, October 

30, 2006). Polymer hydrolysis can occur at elevated 

temperature or extreme pH. An example of potential 

human exposure is migration of bisphenol A from a food 

container into the food. Exposure to bisphenol A through 

food is discussed in detail in Section 1.2.3.2. 

1.2.3 Occurrence 

1.2.3.1 Environmental fate and bisphenol A levels in 

environment: Bisphenol A may be present in the 

environment as a result of direct releases from manufacturing or processing facilities, fugitive emission during processing and handling, or release of unreacted 

monomer from products (European-Union, 2003). According to the Toxics Release Inventory database, total 

environmental release of bisphenol A in 2004 was 181,768 

pounds, with releases of 132,256 pounds to air, 3533 

pounds to water, 172 pounds to underground injection, 

and 45,807 pounds to land (TRI, 2004). 

Bisphenol A released to the atmosphere is likely 

degraded by hydroxy radicals (European-Union, 2003). 

Half-life for the reaction between bisphenol A and 

hydroxy radicals was estimated at 0.2 days. It was also 

noted that photolysis and photodegradation of bisphenol 

A in the atmosphere is possible and photo-oxidation halflives of 0.74–7.4 hr were estimated [reviewed in (Staples 

et al., 1998; European-Union, 2003)]. The European 

Union (2003) noted that because of its low volatility 

and relatively short half-life in the atmosphere, bisphenol 

A is not likely to enter the atmosphere in large amounts. 

Removal by precipitation and occurrence in rain water 

were thought likely to be negligible. Because of its short 

half-life in the atmosphere, bisphenol A is unlikely to be 

transported far from emission points. 

Based on vapor pressure and Henry constant (Table 1), 

the European Union (2003) and Staples et al. (1998) 

concluded that bisphenol A is of low volatility and not 

likely to be removed from water through volatilization. 

Both groups concluded that hydrolysis of bisphenol A in 

water is unlikely. However, there was disagreement on 

potential for photo-oxidation of bisphenol A in water. 

Based on physical and chemical properties, the European 

Union concluded that photolysis of bisphenol A in water 

is unlikely. Staples et al. (1998) noted that bisphenol A is 

able to absorb ultraviolet light, especially in a basic 

solution. Therefore, it was concluded that photolysis 

from surface water is possible, depending on conditions 

such as pH, turbidity, turbulence, and sunlight. Photooxidation half-life of bisphenol A in water was estimated 

at 66 hr to 160 days [reviewed in (Staples et al., 1998)]. 

Rapid biodegradation of bisphenol A from water was 

reported in the majority of studies reviewed by the 

European Union (2003) and Staples et al. (1998). A 

biodegradation half-life of 2.5–4 days was reported in a 

study measuring bisphenol A concentrations in surface 

waters near the receiving stream of a bisphenol A 

manufacturer [reviewed in (Staples et al., 1998)]. 

When the Staples et al. (1998) review was published, 

soil sorption constants had not been measured but were 

estimated at 314–1524. Based on such data, the European 

Union (2003) and Staples et al. (1998) concluded that 

bisphenol A adsorption to soils or sediments would be 

‘‘modest’’ or ‘‘moderate.’’ Based on data for degradation 

of bisphenol A in water, the European Union (2003) 

predicted that bisphenol A would be degraded in soil 

and estimated a half-life of 30 days for degradation of 

bisphenol A in soil. Subsequent to the Staples et al. (1998) 

and European Union (2003) reviews, a study examining 

fate of 14C-bisphenol A in soils through laboratory soil 

degradation and batch adsorption tests was released by 

Fent et al. (2003). In that study, 14C-bisphenol A was 

dissipated and not detectable in 4 different soil types 

within 3 days. Soil distribution coefficients were determined at 636–931, and based on those values, the study 

authors concluded that bisphenol A has low mobility in 

soil. The study authors concluded that bisphenol A is not 

expected to be stable, mobile, or bioavailable from soils. 

In studies reviewed by the European Union (2003) and 

Staples et al. (1998), bioconcentration factors for fish were 

measured at 3.5–68 and were found to be lower than 

values estimated from the Kow. Both groups concluded 

that potential for bioconcentration of bisphenol A is low 

in fish. Higher bioconcentration factors (134–144) were 

determined for clams [reviewed in (European-Union, 

2003)]. 

Two studies examining aggregate exposures in preschool age children in the U.S. used GC/MS to measure 

bisphenol A concentrations in environmental media 

(Wilson et al., 2003, 2006). In the first study (Wilson 

et al., 2003), bisphenol A concentrations were measured in 

air outside 2 day care centers and the homes of 9 children. 

Bisphenol A was detected in 9 of 13 outdoor air samples at 

o0.100–4.72 ng/m3 (mean concentration 5 2.53 ng/m3 at 

day care centers; 1.26 ng/m3 at home). In indoor air from 

day care centers and homes, bisphenol A was detected in 

12 of 13 samples at o0.100–29 ng/m3 (mean concentration 5 6.38 ng/m3 at day care centers; 11.8 ng/m3 at 

home). At those same locations, bisphenol A was detected 

in all of 13 samples of floor dust at means (range) of 1.52– 

1.95 (0.567–3.26) ppm (mg/g) and play area soils at means 

(range) of 0.006–0.007 (0.004–0.014) ppm (mg/g). In the 

second study (Wilson et al., 2006), bisphenol A concentrations were measured inside and outside at least 222 homes 

and 29 daycare centers. Bisphenol A was detected in 31– 

44% of outdoor air samples from each location; concentrations ranged from oLOD (0.9) to 51.5 ng/m3

. Medians 

were olimit of detection (LOD). Indoor air samples (45– 

73%) contained detectable concentrations of bisphenol A; 

concentrations were reported at oLOD (0.9)–193 ng/m3

. 

Median values were oLOD–1.82 ng/m3

. Bisphenol A was 

detected in 25–70% of dust samples; concentrations were 

reported at oLOD (20) to 707 ng/g. Median values were 

oLOD–30.8 ng/g. 

A second U.S. study used a GC/MS method to 

measure bisphenol A concentrations in dust from 1 

office building and 3 homes and in air from 1 office 

building and 1 home (Rudel et al., 2001). Bisphenol A 

was detected in 3 of 6 dust samples (reporting limit 

40.01 mg/extract) at concentrations of 0.25–0.48 mg/g 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 161 

Table 2 

Concentrations of Bisphenol A Detected in Water 

Detection Detection Concentration 

Sample type method rate (%) (mg/L) range [median] Reference 

Surface water 

German rivers GC-MS 100 0.005–0.014 [3.8] Kuch and Ballschmiter (2001) 

Louisiana, U.S. GC-MS 0 oMDL 0.1 Boyd et al. (2003) 

U.S. streams GC-MS 41.2 [0.14] max 12 Kolpin et al. (2002) 

Netherlands GC-MS 78–93 Max marine 0.33 Belfroid et al. (2002) 

Max fresh 21 

Drinking water 

Louisiana, U.S. GC-MS 0 oMDL 0.1 Boyd et al. (2003) 

Ontario, Canada GC-MS 0 oMDL 0.1 Boyd et al. (2003) 

Germany GC-MS 100 0.005–0.002 [1.1] Kuch and Ballschmiter (2001) 

Landfill leachate 

Japan GC-MS 100 740 Kawagoshi et al. (2003) 

Japan GC-MS 70% sites 1.3–17, 200 [269] Yamamoto et al. (2001) 

Sewage treatment works 

Germany GC-MS 94 0.005–0.047 [10] Kuch and Ballschmiter (2001) 

Louisiana, U.S. GC-MS 0 oMDL 0.1 Boyd et al. (2003) 

Table 3
 

Bisphenol A Concentrations in Human Breast Milk
 

Free (ng/ml) Total (ng/ml) Detection 

Source (n) Method LOD mean 7SD (range) mean7SD (range) rate (%) Reference 

Japanese (23) HPLC-Fl 0.11 ng/ml 0.6170.20 100 Sun et al. (2004) 

(0.28–0.97) 

Japanese (101) ELISA NA 3.4170.13 (1–7) 100 Kuruto-Niwa et al. 

(colostrum 3 days (2007) 

after delivery) 

United States (20) HPLC-MS/MS 0.3 ng/ml 1.3 (o0.3–6.3) 1.9 (o0.3–7.3) 60 free Ye et al. (2006) 

90 total 

Japanese (3) GC-MS 0.09 ng/g 0.46 (o0.09–0.65) 67 Otaka et al. (2003) 

U.S. (32) NA NA NA 1.4a NA Calafat et al. (2006) 

a

Estimated from a graph. 

dust. In indoor air samples collected from offices and 

residences, bisphenol A was detected in 3 of 6 samples 

(detection limit 5B0.5 ng/m3

) at concentrations of 

0.002–0.003 mg/m3

. In another study using a GC/MS 

technique, bisphenol A concentrations in indoor air from 

120 U.S. homes were below reporting limits (0.018 mg/ 

m3

) (Rudel et al., 2003). Median (range) bisphenol A 

concentration in dust in this study was 0.821 (o0.2– 

17.6) mg/g, with 86% of samples above the reporting 

limit. 

Limited information is available for bisphenol A 

concentrations in U.S. water (Table 2). In 1996 and/or 

1997, mean bisphenol A concentrations were reported at 

4–8 mg/L in surface water samples near 1 bisphenol A 

production site but bisphenol A was not detected 

(o1 mg/L) in surface water near 6 of 7 bisphenol A 

production sites in the U.S. (Staples et al., 2000). 

Bisphenol A was detected at a median concentration (in 

samples with detectable bisphenol A above the reporting 

limit of 0.09 mg/L) of 0.14 mg/L and a maximum 

concentration of 12 mg/L in 41.2% of 85 samples collected 

from U.S. streams in 1999 and 2000 (Kolpin, 2002). In 

2001 and 2002, bisphenol A was not detected (o0.001 mg/ 

L) in effluent from a wastewater treatment plant in 

Louisiana, and concentrations were not quantifiable 

[quantification limit not defined] in samples collected 

from surface waters in Louisiana and in drinking water 

at various stages of treatment at plants in Louisiana and 

Ontario, Canada (Boyd et al., 2003). In water samples 

collected in Europe and Japan from the 1970 s through 

1989, bisphenol A concentrations were r1.9 mg/L and in 

most cases were r0.12 mg/L [reviewed in (EuropeanUnion, 2003)]. 

1.2.3.2 Potential exposures from food and water: 

The European Union (2003) noted that the highest 

potential for human exposure to bisphenol A is through 

products that directly contact food. Examples of food 

contact materials that can contain bisphenol A include 

food and beverage containers with internal epoxy resin 

coatings and polycarbonate tableware and bottles, such 

as those used to feed infants. 

In addition to commercial food sources, infants 

consume breast milk. Calafat et al. (2006) reported a 

median bisphenol A concentration of B1.4 mg/L [as 

estimated from a graph] in milk from 32 women 

(Table 3). Bisphenol A was measured after enzymatic 

hydrolysis of conjugates. Ye et al. (2006) found measurable concentrations of bisphenol A in milk samples from 

18 of 20 lactating women. Free bisphenol A was found in 

samples from 12 women. The median total bisphenol 

concentration in milk was 1.1 mg/L (range: undetectable 

to 7.3 mg/L). The median free bisphenol A concentration 

was 0.4 mg/L (range: undetectable to 6.3 mg/L). Sun et al. 

(2004) used an HPLC method to measure bisphenol A 

Birth Defects Research (Part B) 83:157–395, 2008
162 CHAPIN ET AL. 

concentrations in milk from 23 healthy lactating Japanese 

women. Bisphenol A concentrations ranged from 0.28– 

0.97 mg/L, and the mean7SD concentration was reported 

at 0.6170.20 mg/L. No correlations were observed 

between bisphenol A and triglyceride concentrations in 

milk. Values from 6 milk samples were compared to 

maternal and umbilical blood samples reported previously in a study by Kuroda et al. (2003). Bisphenol A 

values were higher in milk, and the milk/serum ratio 

was reported at 1.3. Bisphenol A values in milk were 

comparable to those in umbilical cord serum. [It was not 

clear whether milk and serum samples were obtained 

from the same volunteers in the two studies.] 

Studies have measured migration of bisphenol A from 

polycarbonate infant bottles or containers into foods or 

food simulants. Results of those studies are summarized 

in Table 4. Analyses for bisphenol A were conducted by 

GC/MS or HPLC. The European Union (2003) group 

noted that in many cases bisphenol A concentrations 

were below the detection limit in food simulants. When 

bisphenol A was detected, concentrations were typically 

r50 mg/L in simulants exposed to infant bottles and 

r5 mg/kg in simulants exposed to polycarbonate tableware. An exception is one study that reported bisphenol 

A concentrations at up to B192 mg/L in a 10% ethanol 

food simulant and 654 mg/L in a corn oil simulant (Onn 

Wong et al., 2005). In the study, cut pieces of bottles were 

incubated, and the study authors acknowledged that 

bisphenol A could have migrated from the cut edges. 

[The Expert Panel notes that incubations were at 70 or 

1001C for 240 hr, representing conditions not anticipated for normal use of baby bottles.] One study 

conducted with actual infant food (formula and fruit 

juice) reported no detectable bisphenol A (Mountfort 

et al., 1997). Some studies examining the effects of 

repeated use of polycarbonate items noted increased 

leaching of bisphenol A with repeated use (Earls, 2000; 

Brede et al., 2003; CSL, 2004). It was suggested that the 

increase in bisphenol A migration was caused by damage 

to the polymer during use. Results from other reports 

suggested that leaching of bisphenol A decreased with 

repeated use, and it was speculated that available 

bisphenol A was present at the surface of the product 

and therefore removed by washing (Biles et al., 1997b; 

Table 4
 

Examination of Bisphenol A in Polycarbonate Food Contact Surfaces
 

Bisphenol A concentration in 

Sample (location) Procedure simulant Reference 

Commercially available 

infant bottles containing 

residual bisphenol A 

concentrations of 7–46 

ppm (U.S.) 

21 new and 12 used (1–2­

year-old) infant bottles 

(U.K.) 

Infant bottles with residual 

bisphenol A 

concentrations of 26 mg/ 

kg [number tested not 

indicated] (U.K.) 

6 infant feeding bottles 

(country of purchase not 

known) 

14 samples of new infant 

feeding bottles and 

tableware including a 

bowl, mug, cup, and dish 

recalled because residual 

bisphenol A and other 

phenol concentrations 

exceeded 500 ppm [mg/ 

kg] (Japan) 

Discs prepared from 

commercial food-grade 

polycarbonate resins 

Common use: bottles were boiled for 

5 min, filled with water or 10% ethanol, 

and stored at room temperature for up 

to 72 hr Worst case use: bottles were 

boiled for 5 min, filled with water or 

10% ethanol, heated to 1001C for 0.5 hr, 

cooled to room temperature, and 

refrigerated for 72 hr 

Bottles were pre-washed, steam sterilized, 

filled with boiling water or 3% glacial 

acetic acid, refrigerated at 1–51C for 

24 hr, and heated to 401C before 

sampling 

Bottles were sterilized with hypochlorite, 

in dishwasher, or by steam; filled with 

infant formula, fruit juice, or distilled 

water; microwaved for 30 sec and left to 

stand for 20 min (1 cycle); samples were 

analyzed after 3, 10, 20, or 50 cycles; 

other bottles were filled with distilled 

water and left to stand for 10 days at 

401C 

Bottles were filled with water at 261C and 

left to stand for 5 hr or filled with water 

at 951C and left to stand overnight 

Products were exposed to n-heptane, 

water, 4% acetic acid, or 20% ethanol; in 

some cases simulant was heated to 60 

or 951C; in other cases, the object was 

boiled for 5 min; analyses were usually 

conducted after a 30-min contact period 

Materials exposed to water, 10% ethanol, 

or Miglyol (fractionated coconut oil) at 

1001C for 6 hr or water, 3% acetic acid, 

ND (LOD 5 ppb [lg/L]; 

corresponding to a food 

concentration of 1.7 ppb) following 

either procedure 

ND (LOD 10 mg/L) [ppb] from new 

bottles; ND (o10 mg/L ) to 50 mg/L 

from used bottles exposed to either 

simulant [mean not given] 

ND (LOD 0.03 mg/kg) [o30 lg/kg or 

ppb] under any condition 

ND (LOD 2 ppb [lg/L]) in bottles 

filled with water at 261C and 3.1– 

55 ppb [lg/L] in bottles filled with 

water at 951C. 

Up to 40 ppb [lg/kg] from recalled 

products and ND (LOD 0.2) to 

5 mg/kg from commercially 

available products. 

ND (LOD 5 ppb [lg/L]) under all 

conditions. 

FDA (1996) 

Earls et al. 

(2000) 

Mountfort 

et al. (1997) 

Hanai (1997)a 

Kawamura 

et al. 

(1999)a,b 

Howe and 

Borodinsky 

(1998) 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 163 

Table 4
 

Continued
 

Bisphenol A concentration in 

Sample (location) Procedure simulant Reference 

(residual bisphenol A at 10% ethanol, or Migloyl at 491C for 6– 

8800 to 11,200 mg/kg) 240 hr 

from U.S. manufacturers 

2 infant bottles from Japan In three repeated tests, boiling water was Below quantification limit (LOD Sun et al. 

added to bottles; bottles were incubated 0.57 ppb [lg/L]) to mean (2000) 

at 951C for 30 min and cooled to room concentrations of 0.75 ppb before 

temperature; before repeating the test a brushing and o0.57 to 0.18 ppb 

fourth time, the bottles were scrubbed after brushing. 

with a brush 

4 new different brands of Bottles were exposed to distilled water, 1.1–2.5 ppb [lg/L]. D’Antuono 

infant bottles (Argentina) 3% acetic acid, or 15% ethanol at 801C et al. (2001) 

for 2 min or distilled water at 1001C for 

0.5 min 

12 infant bottles (Norway) Bottles were tested before washing and Mean (range) mg/L [ppb]: 0 washes: Brede et al. 

following 51 and 169 dish washings; 0.23 (0.11–0.43) 51 washes: 8.4 (3.7– (2003) 

bottles were occasionally brushed (13 17) 169 washes: 6.7 (2.5–15) 

times by second test and 23 times by 

third test) and boiled (12 times by 

second testing and 25 times by third 

testing); unwashed bottles were rinsed 

with boiling water before testing; for 

testing, bottles were filled with hot 

water and incubated at 1001C for 1 hr 

18 infant bottles (12 tested) Bottles were tested before and after 20 and Before washing: ND (LOD 1.1 ppb CSL (2004) 

(U.K.) 50 dish washings; bottles were brushed or mg/L) in 10% ethanol and ND 

after every 2 wash cycles; bottles were (LOD 0.34 ppb or mg/L) in 3% 

sterilized with boiling water, filled with acetic acid; 20 washes: ND to 

3% acetic acid, or 10% ethanol, and 4.5 ppb in 10% ethanol and ND to 

incubated at 701C for 1 hr 0.51 ppb in 3% acetic acid; 50 

washes: ND to 3.1 ppb in 10% 

ethanol and ND to 0.7 ppb in 3% 

acetic acid 

28 brands of new infant Bottles were cut and pieces were exposed ND (LOD 0.05) to 1.92 mg/in2 [o5– Onn Wong 

bottles (residual to 10% ethanol at 701C or corn oil at 192 lg/L or ppb] in 10% ethanol et al. (2005) 

bisphenol A 1001C for 8–240 hr and ND (LOD 0.05) to 6.54 mg/in2 

concentrations of o3 to [o5–654 lg/L] in corn oil over the 

141 mg/kg) 240-hr exposure period 

manufactured in Europe 

or Asia (Singapore) 

22 new infant bottles and 20 Bottles were immersed in boiling water ND in new bottles (o2.5 mg/L (LOD) FCPSA (2005) 

used (3–36 months) for 10 min before testing and filled with [ppb] in distilled water and 

bottles (Netherlands) distilled water or 3% acetic acid and o3.9 mg/L (LOD) in 3% acetic 

incubated at 40 1C for 24 hr acid) or in used bottles exposed to 

3% acetic acid; not detected to nonquantifiable (o5 mg/L) in distilled 

water from used bottles. 

New unwashed infant Bottles were exposed to water at 951C for ND (LOD 0.05 mg/L [ppb]) to Japanese 

bottles (number not 30 min 3.9 mg/L. studies 

indicated) (Japan) reviewed 

in 

Miyamoto 

and Kotake 

(2006) 

5-gallon water carboys Water was stored in the carboys for 3, 12, 0.1–0.5 mg/L [ppb] at 3 and 12 weeks Biles et al. 

or 39 weeks, temperature not indicated and. 4.6–4.7 mg/L at 39 weeksc (1997b) 

a

Reviewed by European Union (2003).
 

b

Reviewed by Haighton et al. (2002).
 

c

The authors of this study identified an error in the units reported in their study and that the correct concentrations are 1000-fold higher
 

than indicated in the article, the correct values are indicated in table above (T. Begley, email communication, August 6, 2007).
 

ND, not detected. 

Kawamura et al., 1999; Haighton et al., 2002; European exposed to products that had been recalled because of 

Union, 2003). One study (Kawamura et al., 1999) showed unacceptable residual concentrations of bisphenol A and 

higher concentrations of bisphenol A in simulants other compounds. The study by Biles et al. (1997b) 

Birth Defects Research (Part B) 83:157–395, 2008
164 CHAPIN ET AL. 

Table 5 

Surveys of Bisphenol A Concentrations in Canned Infant Formulas or Food 

Bisphenol A concentration mg/kg 

Food (no. sampled)a or mg/L Country Reference 

Infant formula (14) Mean 5 (0.1–13.2 ppb [lg/L]); when U.S. Biles et al. (1997a) FDA (1996) 

diluted with water to make 

prepared formula mean 

concentrations would be 2.5 (0.05– 

6.6) 

Infant formula (4) ND (LOD 2 mg/kg) U.K. Goodson et al. (2002) UKFSA (2001)
 

Infant formula (5) 44–113 mg/kg Taiwan Kuo and Ding (2004)
 

Infant dessert (3) 18.9–77.3 mg/kg U.K. Goodson et al. (2002)
 

Infant vegetable food (4) oLOQ (LOQ 10 mg/kg) New Zealand Thomson and Grounds (2005)
 

Infant dessert (3) oLOQ (LOQ 10 mg/kg)
 

a

Values before and after heating in can and from non-dented and dented cans; values did not differ under the various conditions and
 

were presented together.
 

ND, not detected.
 

demonstrated that infant bottles exposed to 50 or 95% 

ethanol at 651C for 240 hr leached bisphenol A at 

concentrations exceeding residual monomer concentrations, and it was suggested that hydrolysis of the 

polymer had occurred. 

High molecular weight, heat-cured bisphenol A-based 

epoxy resins are used as protective linings in cans for 

food and beverages and may be used in wine storage 

vats (European-Union, 2003). Residual bisphenol A 

monomer can migrate from the coatings to foods or 

beverages contained within cans. Studies were conducted to measure actual concentrations of bisphenol A 

in commercially available foods or to measure concentrations of bisphenol A leaching from can linings into 

food simulants. Because the actual measurement of 

bisphenol A concentrations in canned foods represents 

the most realistic situation, the CERHR review will focus 

on those data. Studies conducted with simulants will not 

be reviewed, with the exception of one study by Howe 

et al. (1998) that was considered by the FDA (1996) in 

their estimates of bisphenol A intake. 

Bisphenol A concentrations detected in infant foods 

are summarized in Table 5, and bisphenol A concentrations detected in non-infant foods are summarized in 

Table 6. With the exception of isolated cases in which 

bisphenol A concentrations were measured at up to 

B0.8 mg/kg food, most measurements were below 

0.1 mg/kg. The European Union also noted an extraction 

study conducted with an epoxy resin that is occasionally 

used to line wine vats. Based on that study, a worst-case 

scenario of 0.65 mg/L bisphenol A in wine was used. The 

European Union noted that the value represents a very 

worst-case exposure scenario but decided to use that 

number in risk estimates because no other value was 

available. [The Expert Panel notes that a study of 

bisphenol A in wine (Brenn-Struckhofova and CichnaMarkl, 2006) identified a maximum concentration of 

2.1 lg/L (Table 6).] 

In one study, empty cans were filled with soup, beef, 

evaporated milk, carrots, or 10% ethanol (Goodson et al., 

2004). The cans were then sealed, processed at 5, 20, or 

401C, and sampled at 1 or 10 days or 1, 3, or 9 months. 

Half the cans processed according to each condition were 

dented. It was determined that 80–100% of the bisphenol 

A migrated to food immediately after processing, and 

that bisphenol A concentrations did not change during 

storage or as a result of denting. The study authors 

concluded that most migration occurred during can 

processing. Boiling the cans or heating to 2301C did not 

increase migration of bisphenol A, but that finding 

appears to contrast with findings of others. Kang et al. 

(2003) examined the effects of temperature, duration of 

heating, glucose, sodium, and oil on migration of 

bisphenol A from cans. In cans filled with water, heating 

to 1211C compared to 1051C increased migration of 

bisphenol A but the duration of heating had no 

significant effect. Compared to cans filled with water, 

increased amounts of bisphenol A migrated from cans 

filled with 1–10% sodium chloride, 5–20% glucose, or 

vegetable oils and heated to 1211C. Takao et al. (2002) 

reported increased leaching of bisphenol A from cans 

into water when the cans were heated to Z801C. 

A study examining aggregate exposures of U.S. 

preschool age children measured bisphenol A concentrations in liquid food and solid food served to the children 

at home and at child care centers (Wilson et al., 2003). 

Duplicate plates of food served to nine children were 

collected over a 48-hr period. GC/MS analyses were 

conducted on four liquid food samples and four solid 

food samples from the child care center and nine liquid 

food samples and nine solid food samples from home. 

Bisphenol A was detected in all solid food samples, three 

liquid food samples from the child care center, and two 

liquid food samples from the home. Concentrations of 

bisphenol A ranged from o0.100–1.16 ng/g [lg/kg] in 

liquid foods and from 0.172–4.19 ng/g [lg/kg] in solid 

food. 

The study examining aggregate exposures of U.S. 

preschool age children was repeated with a larger sample 

and again measured bisphenol A concentrations in liquid 

food and solid food served to the children at home and at 

child care centers (Wilson et al., 2006). Bisphenol A 

concentrations were measured by GC/MS in food served 

over a 48-hr period to at least 238 children at home and 49 

children at daycare centers. Bisphenol A was detected in 

83–100% of solid food samples; concentrations were 

reported at oLOD (0.8) to 192 ng/g [lg/kg]. Sixty-nine to 

80% of liquid food contained detectable concentrations of 

bisphenol A; concentrations were reported at oLOD (0.3)– 

17.0 ng/mL in liquid food. Data were also collected for 

hand wipes of 193 children at daycare centers and 60 

children at home. Bisphenol A was detected in 94–100% of 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 165 

Table 6 

Surveys of Bisphenol A Concentrations in Canned or Bottled Foods or Food Simulants 

Bisphenol A concentration (range in mg/kg Country of 

Food (no. sampled) unless specified) purchasea Reference 

Vegetables with liquid (6) Mean (range) 16 (4–39) U.S. FDA (1996) 

Liquids from canned vegetables 4.274.1 (SD) –22.978.8 mg/can [127 Spain, U.S. Brotons et al. (1995) 

or mushrooms (10) 12–76729 mg/kg] 

Coffee (13) ND–213 [median 5 11] (LOD 2) Japan Kawamura et al. (1999) 

(reviewed in 

European-Union, 2003; 

English abstract 

available) 

Black tea (9) ND–90 [median 5o2] (LOD 2) 

Other tea (8) ND–22 [median 5 5.7] (LOD 2) 

Alcoholic beverages (10) ND except for 1 sample with 13 (LOD 2) 

Soft drinks (7) ND (LOD 2) 

Vegetables (10) 9–48 [median 5 21] U.K. Goodson et al. (2002) 

UKFSA (2001) 

Desserts (5) ND (LOD 2)–14 [median 5 10] 

Fruits (2) 19 and 38 

Pastas (5) ND–41 [median 5 11] (LOD 7) 

Meats (5) 16–422b [median 5 52] 

Fish (10) ND–44 [median 5 16.8] (LOD 2) 

Non-alcoholic or alcoholic ND except for 1 sample above LOD (LOD 2) 

beverages (11) but below LOQ (7) 

Soups (10) ND–21 [median 5o2] (LOD 2) 

Vegetables, fruits, or mushrooms ND (LOD 10)–95.3 in solid portion; ND (LOD Yoshida et al. (2001) 

(14) 0.005 ug/mL)–0.004 mg/mL in liquid 

portion; ND–11.1 mg/can [85 mg/kg] total 

Meat productsd (2) 8.6–25.7 U.K. Goodson et al. (2004) 

Pastad (1) 67.3–129.5 

Vegetables or beansc

(2) 11.3–14.4 

Soupc (1) 18.5–39.1 

Puddingc (3) 3.8–53.2 

Puddingd (1) 18.5–28.1 

Grains and potatoese 0f –75 [mean not given] Japan Miyamoto and Kotake 

(2006) 

Sugar, sweets, snackse 0f

–4 [mean not given] 

Fatse 0f 

Fruits (including canned drinks), –450 [mean not given] 0f

vegetables, mushrooms, 

seaweedse 

Seasoning and beveragese 0f

–213 [mean not given] 

Fish 9–480 [mean not given] 

Meat and eggse 12.5–602 [mean not given] 

Milk and dairy productse 0c

–6 [mean not given] 

‘‘Other’’ [not specified further]e 36–310 [mean not given] 

Canned fish (7) 1–23 [median 5 6] Japan Sajiki et al. (2007) 

Canned meat (5) 4–20 [median 5 10] 

Canned fruit (3) ND (LOD 0.2) 

Canned vegetables (13) 3–78 [median 5 15] 

Canned soup (12) 1–156 [median 5 15] 

Canned sauce (6) ND (LOD 0.2)–842 [median 5 220] 

Canned coconut milk 56–247 

Drinks in plastic containers (3) ND (LOD 0.2) to 1 [median 5 0.3] Japan Sajiki et al. (2007) 

Cookies in plastic containers (4) 1–14 [median 5 3.5] 

Soup in plastic containers (2) ND (LOD 0.2) and 3 

Fast food sandwiches (3) 3 (all values) 

Food in paper containers (16) ND (LOD 0.2)–1 [median 5o0.2] 

Fruits and vegetables (38) ND (LOQ 10)–24 [median 5o10] New Zealand Thomson and Grounds 

(2005) 

Fish (8) ND (LOQ 20)–109 [median 5o20–24] 

Soup (4) ND (LOQ 10)–16 [median 5o20] 

Sauces (4) ND (LOQ 10) –21 [median 5 16] 

Meat (6) ND (LOQ 20)–98 [median 5o20] 

Pasta (4) ND (LOQ 10) 

Dessert (2) ND (LOQ 20) 

Coconut cream (3) ND (LOQ 20)–192 [median 5 29] 

Birth Defects Research (Part B) 83:157–395, 2008
166 CHAPIN ET AL. 

Table 6 

Continued 

Bisphenol A concentration (range in mg/kg Country of 

Food (no. sampled) unless specified) purchasea Reference 

Soft drinks (4) ND (LOQ 10) 

Beverages (7) ND (LOD 0.9)–3.4 [median 5 0.4] Austria Braunrath et al. (2005) 

Vegetables (6) (only solid portion 8.5–35 [median 5 26] 

was analyzed, with the 

exception of tomatoes) 

Fruits (4) 5–24 [median 5 6.6] 

Canned fat-containing products 2.1–37.6 [median 5 20.7] 

such as soups, meats, and 

cream (9) 

Tuna (9) oND (LOQ 7.1)–102.7 [median 5 11.2] Mexico Munguı ́a-Lo ́pez et al. 

(2005) 

Beverage/beer cans exposed to ND (LOD 5) U.S. Howe et al. (1998) FDA 

10% ethanol at 1501F [65.61C] (1996) 

for 30 min and then 1201F 

[48.91C] for 10 days 

Food cans exposed to 10 or 95% ND (LOD 5)–95 (mean 5 37)g 

ethanol at 2501F [1211C] for 2 hr 

and then 1201F [48.91C] for 10 

days or at 2121F [1001C] for 

30 min and then 1201F [48.91C] 

for 10 days 

Honey (107 samples; B90% ND (LOD 2)–33.3 [median 5o2] Japan Inoue et al. (2003b) 

imported in epoxy-lined 

drums) 

Wine stored in steel, wood, or oLOQ (0.2 ng/mL) to 2.1 mg/L; mean 0.58 in Austria Brenn-Struckhofova and 

plastic vats, filled into glass samples above the LOQ Chichna-Markl (2006) 

bottles, or purchased in local 

markets (59) 

Solid food (309) ND (oB0.8 )–192 [median = 3.52–4.32] U.S. Wilson et al. (2006) 

Liquid food (287) ND (oB 0.3)–17.0 [median = 0.45–0.79] U.S. Wilson et al. (2006) 

a

Although cans were purchased in one or two countries for each study, most studies reported that cans were packaged in various
 

locations throughout North America, Europe, or Asia.
 

b

The UKFSA noted that the higher concentrations of bisphenol A detected in one meat product likely resulted from the use of bisphenol
 

A as a cross-linking agent in the resin at that time.
 

c

Values were obtained from heated and non-heated cans but presented together because it could not be determined if heating resulted in
 

differing extraction rates.
 

d

Values were determined before and after heating in can and from non-dented and dented cans; because the values did not differ under
 

the various conditions, they were presented together.
 

e

Total number of samples analyzed was not reported.
 

f

As reported by study authors; detection limits not specified.
 

g

A maximum concentration of 121 ppb reported in the first phase of the study was determined to have resulted from analytical
 

interference.
 

ND, not detected.
 

handwipe samples; concentrations ranged from oLOD 

[not defined] to 46.6 ng/cm2

. Bisphenol A was detected in 

85–89% of food preparation surface wipes from homes; 

concentrations were reported at oLOD [not defined] to 

0.357 ng/cm2

. 

A review by Miyamoto and Kotake (2006) reported 

bisphenol A concentrations of 0.011–0.086 mg/kg in noncanned foods such as fats, fruits, fish, meat, and eggs. 

However, one study used GC-MS to examine bisphenol 

A in 14 types of produce purchased in southern Italy 

(Vivacqua et al., 2003). Bisphenol A concentrations were 

below the detection limit [not reported] in 5 produce 

samples. In the remaining samples, bisphenol A was 

detected at concentrations of 0.2570.02 (SD) to 

1.1170.09 mg/kg. [These concentrations are equal to 

or higher than those found in canned foods, where the 

presumption is that the source is the epoxy liner of the 

container.] 

Bisphenol A has been found in recycled paper 

products used for food processing at 10 or more times 

the concentrations found in non-recycled paper products 

[reviewed by the (European Food Safety Authority, 

2006)]. Bisphenol A concentrations were up to 26 mg/g 

paper. Migration to food was not discussed. 

Epoxy paints are used to coat the insides of residential 

drinking water storage tanks. Bisphenol A has been 

shown to migrate from painted concrete and stainless 

metallic plates; however, a water sample from a recently 

painted reservoir showed no detectable bisphenol A 

(Romero et al., 2002). When exposed to chlorine disinfectant, bisphenol A disappears within 4 hr, but the 

chlorinated bisphenol A congeners that are formed can 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 167 

remain in solution up to 20 hr when low chlorine doses 

are used (Gallard et al., 2004). The toxicity of these 

chlorinated bisphenol A congeners is unknown; however, there is some evidence that estrogenic activity and 

receptor binding remains after chlorination (Hu et al., 

2002). 

1.2.3.3 Potential migration from dental material: Bisphenol A is used in the manufacture of materials found 

in dental sealants or composites (i.e., fillings) (EuropeanUnion, 2003). Examples of bisphenol A-derived materials 

used in dental sealants include bis-glycidyldimethacrylate and bisphenol A-dimethyl acrylate. Bisphenol A 

could potentially be present as an impurity or be released 

during degradation of the dental materials. Sealants are 

comprised of an organic matrix, while composites 

contain inorganic filler in addition to the organic matrix. 

According to the British Dental Association, filled 

composites would possibly produce lower exposure to 

bisphenol A than sealants, because they contain proportionately less resin than sealants [reviewed in (EuropeanUnion, 2003)]. During dental procedures, resin mixtures 

are applied as fluid monomers and polymerized in situ 

by ultraviolet or visible light. According to the European 

Union (2003), patients can be exposed to bisphenol A 

during the polymerization stage. 

In a review of in vitro studies examining bisphenol A 

migration from dental sealants, the European Union 

(2003) concluded that release of bisphenol A is likely to 

occur only with degradation of the parent monomer. The 

data suggested that bis-glycidyldimethacrylate does not 

degrade; therefore, release of bisphenol A is only likely to 

occur with bisphenol A-dimethyl acrylate use. In vivo 

studies measuring bisphenol A in saliva following 

sealant application were reviewed in detail by CERHR 

because they provide the most relevant human exposure 

information. 

Olea et al. (1996) measured saliva concentrations of 

bisphenol A for 1 hr before and 1 hr after application of 

50 mg bis-glycidyldimethacrylate- and bisphenol A-dimethyl acrylate-based sealant across 12 molars of 18 

patients. Concentrations of bisphenol A in saliva were 

measured by GC/MS and HPLC. Following treatment, 

saliva contained B90–931 mg bisphenol A. Based on an 

assumed saliva production rate of 0.5 mL/min, a saliva 

concentration of 3–30 mg/mL was estimated by the study 

authors. With the exception of 1 patient who was 

excluded from the study, bisphenol A was not detected 

in saliva before sealant application. 

Arenholt-Binslev (1999) measured bisphenol A in 

saliva of 8 adult patients who each had four molars 

treated with 38 mg of 1 of 2 sealants, Delton LC or Visioseal. Saliva was collected before, immediately after, and 

at 1 or 24 hr following treatment for measurement of 

bisphenol A concentrations by HPLC. Bisphenol A was 

detected at 0.3–2.8 ppm immediately after application of 

Delton LC sealant [bisphenol A-dimethyl acrylate sealant 

according to the European Union (2003)] but was not 

detected 24 hr later (detection limit 5 0.1 ppm [mg/L]). 

Bisphenol A was not detected in saliva of patients who 

received the Visio-seal sealant (bis-glycidyldimethacrylate sealant, according to the European Union). It was 

noted that saliva bisphenol A concentrations were much 

lower than those reported by Olea et al. (1996). Possible 

reasons for the inconsistencies in results between the 2 

studies were stated to be differences in the amount of 

sealant used and co-elution of compounds that could 

have confounded bisphenol A analysis. 

Fung et al. (2000) measured salivary bisphenol A 

concentrations in 40 patients treated with a dental sealant 

(Delton Opaque Light-cure Pit and Fissure Sealant) that 

was understood to contain bisphenol A-dimethyl acrylate, according to the European Union (2003). Eighteen 

patients in the low-dose group received 8 mg dental 

sealant on 1 tooth, and 22 patients in the high-dose group 

received 32 mg sealant on 4 teeth. Saliva and blood were 

collected for HPLC analysis before the procedure and at 

1 and 3 hr and 1, 3, and 5 days after the procedure. More 

details of this study are included in Section 2.1.1.1. 

Analysis of the dental sealant revealed that bisphenol A 

concentrations were below the detection limit of 5 ppb. 

At 1 hr following treatment, Bisphenol A was detected 

only in saliva samples from 3 of 18 volunteers in the lowdose group and 13 of 22 samples from volunteers in the 

high-dose group. At 3 hr post-treatment, bisphenol A was 

detected in samples from 1 of 18 volunteers in the lowdose group and 7 of 22 volunteers from the high-dose 

group. Concentrations of bisphenol A in saliva at 1 and 

3 hr following exposure were reported at 5.8–105.6 ppb 

[lg/L]. No bisphenol A was detected in saliva samples at 

24 hr after treatment or in serum samples at any time 

point. Differences in bisphenol A concentrations and the 

presence of bisphenol A in saliva of the low-dose 

compared to the high-dose group at 1 and 3 hr achieved 

statistical significance. The European Union (2003) noted 

that the concentrations of saliva bisphenol A reported by 

Fung et al. (2000) were 4250 times lower than those 

reported by Olea et al. (1996). 

Sasaki et al. (2005) used ELISA to examine salivary 

bisphenol A concentrations in 21 patients before and 

after 1 cavity was filled with 0.1 g of composite resin. The 

resins consisted of bisphenol A diglycidylether methacrylate (i.e., bis-glycidyldimethacrylate), triethylene glycol dimethacrylate, and/or urethane dimethacrylate. 

Saliva was collected before treatment, during the 5 min 

following treatment, and then immediately after gargling 

with water. Following treatment, saliva bisphenol A 

increased [from r2 to B15–100 lg/L]. Gargling reduced 

bisphenol A to near pretreatment concentrations [r5 lg/ 

L] in most patients, with the exception of 1 patient with 

the highest bisphenol A concentration [reduced from 

B100 to 18 lg/L]. [An increase in saliva bisphenol A 

concentrations was noted in 1 of 2 patients receiving a 

composite consisting solely of urethane dimethacrylate.] The study authors noted that cross-reactivity is 

possible with the ELISA technique, but that cross 

reactivity between bisphenol A diglycidylether methacrylate and triethylene glycol dimethacrylate is low. 

Therefore, the study authors thought it possible that they 

were measuring only bisphenol A. [As discussed in 

Section 1.1.5, ELISA may overestimate bisphenol A.] 

Joskow et al. (2006) examined bisphenol A in urine and 

saliva of 14 adults treated with dental sealants. The 

volunteers received either Helioseal F (n 5 5) or Delton 

LC (n 5 9) sealant. Only the Helioseal F sealant was 

noted to carry the American Dental Association Seal of 

Acceptance. Sealant was weighed before and after 

application to determine the amount applied, and the 

numbers of treated teeth were recorded. The mean 

number of teeth treated was 6/person and the mean 

total weight of sealant applied was 40.35 mg/person. In a 

Birth Defects Research (Part B) 83:157–395, 2008
168 CHAPIN ET AL. 

comparison of the two different sealants, no differences 

were reported for the number of teeth treated or amount 

of sealant applied. Saliva samples were collected before, 

immediately after, and 1 hr after sealant application. 

Urine samples were collected before and at 1 and 24 hr 

after sealant placement. A total of 14–15 saliva samples 

and 12–14 urine samples were collected at each time 

point. Samples were treated with b-glucuronidase and 

analyzed for bisphenol A concentrations using selective 

and sensitive isotope-dilution-MS-based methods. Saliva 

concentrations were highest immediately following 

treatment; mean concentrations were reported at 

42.8 ng/mL in patients treated with Delton LC and 

0.54 ng/mL in patients treated with Helioseal F. The 

highest mean urinary concentrations of bisphenol A were 

measured at 1 hr following exposure and were reported 

at 27.3 ng/mL in patients treated with Delton LC and 

7.26 ng/mL in patients receiving the Helioseal F sealant. 

The study authors noted that saliva and urine bisphenol 

A concentrations after application of Helioseal F were 

comparable to baseline concentrations. More information 

on bisphenol A concentrations in saliva and urine is 

included in Section 2, and exposure estimates are 

provided in Section 1.2.4.1.2. The study authors noted 

that saliva concentrations detected in their study were 

B1000 times lower than those reported by Olea et al. 

(1996) but were within the ranges reported by Fung et al. 

(2000) and Sasaki et al. (2005). Analytical procedures and 

use of a large amount of sealant were noted as possible 

reasons for the higher values reported by Olea et al. 

(1996). 

The European Union noted a study by Lewis et al. 

(1999) that characterized materials in 28 commercial 

resin-based composites and sealants, including those 

examined by Olea et al. (1996). HPLC and infrared 

analysis could not verify the presence of bisphenol A in 

any sealant product. Lewis et al. (1999) noted that in the 

study by Olea et al. (1996) another component in the resin 

may have been misidentified as bisphenol A because of 

difficulties with resolution. 

In their review of studies examining bisphenol A 

concentrations in saliva of patients treated with dental 

sealants, the European Union (2003) noted that the higher 

concentrations reported may have resulted from interference during analysis and thus may overestimate 

bisphenol A exposures from dental treatments. It was 

concluded that dental treatment would likely result in 

saliva bisphenol A concentrations of 0.3–3 ppm. Because 

bisphenol A was generally not detected in saliva at time 

points beyond 1 hr after treatment, it was concluded that 

bisphenol A exposure resulting from dental treatments is 

likely to be an acute event. In their 2002 position 

statement, the American Dental Association stated that 

none of the 12 dental sealants that carry the American 

Dental Association Seal release bisphenol A (American 

Dental Association, 1998). On initial analysis, one of the 

sealants was found to leach trace concentrations of 

bisphenol A, but following implementation of quality 

controls by the manufacturer, bisphenol A could no 

longer be detected in the final product. 

A study on orthodontic adhesives found no bisphenol 

A release from these materials after simulated aging 

(Eliades et al., 2007). Another study found plastic 

orthodontic brackets in water to release bisphenol A at 

0.01–0.40 mg/kg material and denture base resin in water 

to release bisphenol A at 0.01–0.09 mg/kg material 

(Suzuki et al., 2000). 

1.2.3.4 Bisphenol A concentrations measured in 

biological samples: Bisphenol A concentrations detected in human blood are summarized in Table 7. 

Goodman et al. (2006) noted that although blood 

concentration may provide information on internal dose, 

it does not allow for estimates of daily intake. It was also 

noted that in many studies in which blood concentration 

of bisphenol A was measured, sample preparation and 

analysis methods were poorly reported. Many study 

groups used an ELISA method to measure blood 

bisphenol A concentration. As discussed in Section 

1.1.5, the ELISA technique is likely to overestimate 

bisphenol A concentrations as a result of cross-reactivity 

with other substances and due to effects of biologic 

matrices (Inoue et al., 2002; Fukata et al., 2006; Goodman 

et al., 2006). 

Several studies reported concentrations of bisphenol A 

in human urine; those studies are summarized in Table 8. 

As discussed in greater detail in Section 2, the majority of 

ingested bisphenol A is excreted in urine as bisphenol A 

glucuronide after acute exposure. Smaller amounts of 

bisphenol A are metabolized to and excreted as bisphenol A sulfate. Some of the studies determined 

concentrations of parent bisphenol A before and after 

digestion with glucuronidases. With the exception of 

Fujimaki et al. (2004) who used an ELISA technique to 

measure urinary bisphenol A, other study authors used 

HPLC, GC/MS, or LC/MS. Results from 394 participants 

of the National Health and Nutrition Examination 

Survey (NHANES) III survey are included in Table 8 

(Calafat et al., 2005). Bisphenol A was detected in 95% of 

the participants, which indicated widespread exposure 

to bisphenol A in the U.S. Consistent with those findings, 

bisphenol A was detected in urine from 85 of 90 (94.4%) 

6–8-year-old girls from the U.S. (Wolff et al., 2006). In a 

review of urinary bisphenol A data, Goodman et al. 

(2006) noted that in most cases, median total urinary 

bisphenol A concentration (the sum of parent and 

conjugated bisphenol A) were B1–2 mg/L. Two studies 

(Yang et al., 2003; Mao et al., 2004) reported urinary 

bisphenol A concentrations that were orders of magnitude higher than commonly observed concentrations, 

despite the use of apparently reliable analytical techniques. Goodman et al. (2006) has suggested that reported 

hormone concentrations for the study volunteers were 

also higher than expected, indicating the possibility of 

laboratory or reporting error. The use of urinary bisphenol A concentration to estimate daily exposures 

appears in Section 1.2.4.1.2. 

In humans, bisphenol A was measured in cord blood 

and amniotic fluid, demonstrating distribution to the 

embryo or fetus. Detailed descriptions of those studies 

are also presented below. 

Engel et al. (2006) reported concentrations of bisphenol 

A in human amniotic fluid. Twenty-one samples were 

obtained during amniocentesis conducted before 20 

weeks gestation in women who were referred to a U.S. 

medical center for advanced maternal age. Bisphenol A 

concentrations in amniotic fluid were measured using LC 

with electrochemical detection. Bisphenol A was detected 

in 10% of samples at concentrations exceeding the LOD 

(0.5 mg/L). Bisphenol A concentration ranges of 0.5– 

1.96 mg/L were reported. 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 169 

Table 7 

Blood Concentrations of Bisphenol A in Adults 

Population (n) Bisphenol A mg/La,c Method Reference 

Germany 

Men (7) o0.5 HPLC-MS/MS Vo ̈lkel et al. (2005) 

Women (12) o0.5 HPLC-MS/MS Vo ̈lkel et al. (2005) 

Pregnant Caucasian women (37; 32–41 4.473.9 GC-MS Scho ̈nfelder et al. (2002a) 

weeks gestation) 

Japan 

Men (21; age 22–51) ‘‘almost all’’ o0.2 ng/ml HPLC-ECD Fukata et al. (2006) 

Men (9; age 30–50) 0.5970.21 (0.38–1.0) HPLC-MS Sajiki et al.(1999) 

Men (11) 1.4970.11 (SEM) ELISAb Takeuchi and Tsutsumi 

(2002) 

Women (31; age 22–51) ‘‘almost all’’ o0.2 ng/ml HPLC-ECD Fukata et al. (2006) 

Women (12; age 30–50) 0.3370.54 (0–1.6) HPLC-MS Sajiki et al.(1999) 

Women (14) 0.6470.10(SEM) ELISAb Takeuchi and Tsutsumi 

(2002) 

Pregnant women (37; late pregnancy) 1.470.9 ELISAb Ikezuki et al.(2002) 

Pregnant women with normal karyotype 2.24 (0.63–14.36) ELISAb Yamada et al. (2002) 

early 2nd trimester (200) 

Pregnant women with abnormal 2.97 (B0.0.7–18.5)d ELISAb Yamada et al. (2002) 

karyotype early 2nd trimester (48) 

Pregnant women (9) 0.43 (0.21–0.79) HPLC-Fl Kuroda et al. (2003) 

Infertile women (21) 0.46 (0.22–0.87) HPLC-Fl Kuroda et al. (2003) 

Women with multiple miscarriages 2.5975.23 ELISAb Sugiura-Ogasawara et al. 

(45; mean age 31.6 years) (2005) 

Healthy woman (32; mean age 32 years) 0.7770.38 ELISAb Sugiura-Ogasawara et al. 

(2005) 

Women with polycystic ovary syndrome 1.0470.10 (SEM) ELISAb Takeuchi and Tsutsumi 

(16) (2002) 

Non-obese women with polycystic 1.0570.10 (SEM) ELISAb Takeuchi et al. (2004a) 

ovarian syndrome (13; average 

age 26.5 years) 

Obese women with polycystic ovarian 1.1770.16 (SEM) ELISAb Takeuchi et al. (2004a) 

syndrome (6; average age 24.7 years) 

Non-obese women (19; average age 27.5 0.7170.09 (SEM) ELISAb Takeuchi and Tsutsumi 

years) (2002) 

Obese women (7; average age 28.8 years) 1.0470.09 (SEM) ELISAb Takeuchi et al. (2004a) 

Hyperprolactinemic women (7; average 0.8370.12 (SEM) ELISAb Takeuchi et al. (2004a) 

age 27.7 years) 

Amenorrheic women (7; average age 0.8470.10 (SEM) ELISAb Takeuchi et al. (2004a) 

25.1 years) 

Women with normal uterine 2.571.5 ELISAb Hiroi et al. (2004) 

endometrium (11; mean age 48.9 years 

Women with simple endometrium 2.972.0 ELISAb Hiroi et al. (2004) 

hyperplasia (10; mean age 48.4 years) 

Women with complex endometrium 1.470.4 ELISAb Hiroi et al. (2004) 

hyperplasia (9; mean age 48.4 years) 

Women with endometrial carcinoma 1.470.5 ELISAb Hiroi et al. (2004) 

(7; mean age 63.1 years) 

a

Mean7SD or median (range).
 b

As discussed in Section 1.1.5, ELISA may overestimate bisphenol A.
 

c

It is uncertain whether parent, conjugated, or total bisphenol A was measured.
 

d

Estimated from a graph. 

Scho ̈nfelder et al. (2002b) examined bisphenol A 

concentrations in maternal and fetal blood and compared 

bisphenol A concentrations in blood of male and female 

fetuses. In a study conducted at a German medical 

center, blood samples were obtained from 37 Caucasian 

women between 32 and 41 weeks gestation. At parturition, blood was collected from the umbilical vein after 

expulsion of the placenta. Bisphenol A concentrations in 

plasma were measured by GC/MS. Control experiments 

were conducted to verify that bisphenol A did not leach 

from collection, storage, or testing equipment. Bisphenol 

A was detected in all samples tested, and concentrations 

measured in maternal and fetal blood are summarized in 

Table 9. Mean bisphenol A concentrations were higher in 

maternal (4.473.9 [SD] mg/L) than fetal blood 

(2.972.5 mg/L). Study authors noted that in 14 cases 

fetal bisphenol A plasma concentrations exceeded those 

detected in maternal plasma. Among those 14 cases, 12 

fetuses were male. Analysis by paired t-test revealed 

significantly higher mean bisphenol A concentrations in 

Birth Defects Research (Part B) 83:157–395, 2008
170 CHAPIN ET AL. 

Table 8 

Urinary Concentrations of Bisphenol A and Metabolites in Adults or Children 

Urinary bisphenol A or metabolite concentrations as median 

(range) or mean7SEM, mg/La [detectable fraction, % 4LOD] 

LOD 

Country Study population (mg/L) Free Total Glucuronide Sulfate Reference 

U.S.	 30 urine samples from 0.3 o 0.3 2.12 1.4 0.3 Ye et al. (2005) 

demographically (o0.3–0.6) (oLODb –19.8) (oLODb (oLODb 

diverse, anonymous [10%] [97%] –19.0) –1.8) 

adult volunteers [90%] [47%] 

U.S.	 394 adult volunteers 0.1 1.28 (10–95th Calafat et al. 

(men and women; percentile: (2005) 

20–59 years old) from 0.22–5.18) 

the NHANES [95%]c 

III survey 

U.S.	 23 adults 0.5 0.47 (o1–2.24) Liu et al. (2005) 

[52%] 

U.S.	 9 girls (9 years of age) 0.5 2.4 (0.04–16) Liu et al. (2005) 

[89%] 

U.S.	 90 women (6–8 years 8.36 1.8 (o0.3–54.3) Wolff 

of age; White, Black, [85%] et al. (2006) 

Asian, or Hispanic 

ethnicity) 

Germany 7 men, 12 women 1.14 (BPA) o1.14	 o26.26 [LOQ] Vo ̈lkel 

10.1 (BPA [0%] et al. (2005)
 

monoglucuronide)
 

Korea 15 men (42.672.4 1 0.28–2.36; 0.85–9.83 0.16–11.67 oMDL Kim 

years of age)d 0.5870.14 2.8270.73 2.3470.85 –1.03; et al. (2003b) 

0.4970.27e 

Korea 15 women (43.072.7 0.28 0.068–1.65; 1.00–7.64 oMDLc –4.34 oMDL Kim 

years of age)d 0.5670.10 2.7670.54 1.0070.34 –3.40; et al. (2003b) 

1.2070.32e 

Korea 34 men, 39 women 0.012 Geometric Yang 

(mean 5 48.5 mean: 9.54 et al. (2003) 

years of age) (o0.012–586.14b

) 

[75%] 

Korea 81 men not occupationally Geometric Yang et al. 

exposed to bisphenol A mean7SD (2006) 

6.8873.72 

Korea 79 women not 0.026 Geometric Yang et al. 

occupationally mean7SD (2006) 

exposed to 5.0173.16 [97.5%] 

bisphenol A 

Japan 48 woman college 0.2 o0.2 [2%] 1.2 (0.2–19.1) Ouchi and 

students [100%] Watanabe 

(2002) 

Japan Pooled urine samples 0.12 o0.12 0.11–0.51 Brock et al. 

from at least 5 people (2001) 

Japan 23 women, 46 men; in each 0.01–0.27 Mean 5 0.81 Tsukioka 

volunteer, 2 samples per (range: et al. (2004) 

volunteer were combined 0.14–5.47) 

Japan Whole-day urine samples Mean 5 0.81 Tsukioka 

collected from 11 men (range 0.24–2.03) et al. (2004) 

and 11 women 

Japan Urine collected from 3 0.02 o0.1 0.22, 0.41, and 0.45 Kawaguchi 

volunteers [100% after et al. (2004) 

deconjugation] 

Japan Spot urine samples 1.1 o1.1 (o1.1–5.4)c Fujimaki 

collected from 56 (ELISA) [30%] et al. (2004) 

women who were 

1–9 months pregnant; 

21–43 years of age 

Japan 21 men, 31 women; 22–51 0.2 49/51 had 1.9270.27 [98%]	 Fukata 

years of age	 o0.2 et al. (2006) 

mean 0.34 

(n 5 2) [4%] 

Birth Defects Research (Part B) 83:157–395, 2008
Table 8 

Continued 

BISPHENOL A 171 

Urinary bisphenol A or metabolite concentrations as median 

(range) or mean7SEM, mg/La [detectable fraction, % 4LOD] 

Country Study population 

LOD 

(mg/L) Free Total Glucuronide Sulfate Reference 

China 10 healthy man 2.8 o2.7 –3950 Mao et al. (2004) 

volunteers; 122071380d [60%] 

21–29 years of age 

China 10 healthy woman 2.8 30–3740 Mao et al. (2004) 

volunteers; 129071220d [100%] 

21–29 years of age 

a

With the exception of the study by Fujimaki et al. (2004), which used the potentially unreliable ELISA, the studies used analytical
 

techniques based on HPLC, GC/MS, and LC/MS.
 

b

Limit of detection (LOD) for bisphenol A following digestion of conjugate was 0.3 mg/L.
 c

Samples were only digested with b-glucuronidase and do not account for bisphenol A conjugated to sulfate.
 d

Variance not indicated.
 

e

Minimum detection limit based upon free bisphenol A.
 

Table 9
 

Concentrations of Bisphenol A in Maternal and Fetal Samples
 

Study description (analytical 

Bisphenol A concentrations, mg/L median (range) or mean7SD 

Serum or plasma 

method) 

21 samples collected in women 

Maternal Fetal Amniotic fluid 

0.5 (Non-detectable 

Reference 

Engel et al. (2006) 

in the U.S. before 20 weeks o0.5–1.96) 10% of 

gestation (LC with samples detectable 

electrochemical detection) 

37 German women, 32–41 weeks 3.1 (0.3–18.9) 4.473.9 2.3 (0.2–9.2) 2.972.5 Scho ̈nfelder et al. 

gestation (GC/MS) (2002b) 

37 Japanese women in early 1.571.2 Ikezuki et al. (2002) 

pregnancy (ELISA)a 

37 Japanese women in late 1.470.9 Ikezuki et al. (2002) 

pregnancy (ELISA)a 

32 Japanese infants at delivery 2.271.8 Ikezuki et al. (2002) 

(ELISA)a 

32 Japanese amniocentesis 8.378.9 Ikezuki et al. (2002) 

samples at 15–18 weeks 

gestation (ELISA)a 

38 samples obtained at full-term 1.171.0 Ikezuki et al. (2002) 

cesarean section (ELISA)a 

200 Japanese women carrying 2.24 (0.63–14.36) 0.26 (0–5.62) Yamada et al. (2002) 

fetuses with normal karyotype 

at 16 weeks mean gestation 

(ELISA) 

48 Japanese women carrying 2.97 [B0.7–18.5]b 0 [B0–7.5]b Yamada et al. (2002) 

fetuses with abnormal 

karyotypes at a 16 weeks 

mean gestation (ELISA) 

9 sets of maternal and umbilical 0.43 (0.21–0.79) 0.64 (0.45–0.76) Kuroda et al. (2003) 

cord blood samples obtained 0.4670.2 0.6270.13 

at birth in Japanese patients 

(HPLC) 

180 Malaysian newborns (GC/ Non-detectable Tan and Mohd (2003) 

MS) (o0.05) to 4.05 88% 

of samples 

detectable 

a

As discussed in Section 1.1.5, ELISA may overestimate bisphenol A. Some samples were verified by HPLC. 

b

Estimated from a graph. 

Birth Defects Research (Part B) 83:157–395, 2008
172 CHAPIN ET AL. 

the blood of male than female fetuses (3.572.7 vs. 

1.771.5 ng/mL, P 5 0.016). Bisphenol A concentrations 

were measured in placental samples at 1.0–104.9 mg/kg. 

Ikezuki et al. (2002) measured concentrations of 

bisphenol A in serum from 30 healthy premenopausal 

women, 37 women in early pregnancy, 37 women in late 

pregnancy, and 32 umbilical cord blood samples. Concentrations of bisphenol A were also measured in 32 

samples of amniotic fluid obtained during weeks 15–18 

of gestation, 38 samples of amniotic fluid obtained at fullterm cesarean section, and 36 samples of ovarian 

follicular fluid collected during in vitro fertilization 

procedures. [It was not stated if different sample types 

were obtained from the same subjects.] An ELISA 

method was used to measure bisphenol A concentrations 

and results were verified by HPLC. The mean7SD 

concentration of bisphenol A in follicular fluid was 

reported at 2.470.8 mg/L. As summarized in Table 9 for 

maternal and fetal samples, concentrations of bisphenol 

A in follicular fluid were similar to those detected in the 

serum of fetuses and pregnant and non-pregnant women 

and in amniotic fluid collected in late pregnancy (B1– 

2 mg/L). Bisphenol A concentrations in amniotic fluid 

samples collected in early pregnancy were B5-fold 

higher than in other samples, and the difference 

achieved statistical significance (Po0.0001). Study 

authors postulated that the higher concentrations of 

bisphenol A in amniotic fluid collected during gestation 

weeks 15–18 may have resulted from immature fetal liver 

function. They noted that according to unpublished data 

from their laboratory, the percentage of glucuronidated 

bisphenol A in mid-term amniotic fluid was B34%, 

which is much lower than reported values for other 

human fluids (490%). 

Yamada et al. (2002) measured bisphenol A concentrations in maternal serum and amniotic fluid from 

Japanese women. Samples were collected between 1989 

and 1998 in women undergoing amniocentesis around 

gestation week 16. One group of samples was obtained 

from 200 women carrying fetuses with normal karyotypes, and a second group of samples was obtained from 

48 women carrying fetuses with abnormal karyotypes. 

An ELISA method was used to measure bisphenol A 

concentrations. [As discussed in Section 1.1.5, ELISA 

may overestimate bisphenol A.] Concentrations of 

bisphenol A measured in maternal plasma and amniotic 

fluid are summarized in Table 9. Median concentrations 

of bisphenol A in maternal serum (B2–3 mg/L) were 

significantly higher [B10-fold] than concentrations in 

amniotic fluid (B0–0.26 mg/L) in the groups carrying 

fetuses with normal and abnormal karyotypes. However, 

in 8 samples from women carrying fetuses with normal 

karyotypes, high concentrations (2.80–5.62 mg/L) of bisphenol A were measured in amniotic fluid. The study 

authors interpreted the data as indicating that bisphenol 

A does not accumulate in amniotic fluid in most cases 

but that accumulation is possible in some individuals. 

Bisphenol A concentrations in maternal blood were 

significantly higher [by B33%] in woman carrying 

fetuses with abnormal versus normal karyotypes. However, the study authors noted that the effect may not be 

related to bisphenol A exposure because there was no 

adjustment for maternal age, and concentrations in 

amniotic fluid did not differ between groups. In the 

group carrying fetuses with normal karyotypes, data 

obtained from 1989–1998 were summarized by year. 

Median bisphenol A concentrations in serum significantly decreased over that time from a concentration of 

5.62 mg/L detected in 1989 to 0.99 mg/L in 1998. 

Kuroda et al. (2003) used an HPLC method to measure 

bisphenol A concentrations in 9 sets of maternal and cord 

blood samples obtained from Japanese patients at the time 

of delivery. Bisphenol A concentrations were also measured in 21 sets of serum and ascitic fluid samples 

collected from sterile Japanese patients of unspecified 

sexes and ages. Results for pregnant women are summarized in Table 9. Mean7SD concentrations of bisphenol A 

were lower in maternal (0.4670.20 ppb [lg/L]) than cord 

blood (0.6270.13 ppb [lg/L]). There was a weak positive 

correlation (r 5 0.626) between bisphenol A concentrations 

in maternal and cord blood. There were no differences 

between pregnant and non-pregnant blood levels (Kuroda 

et al., 2003). Mean7SD concentrations of bisphenol A were 

higher in ascitic fluid (0.5670.19 ppb [lg/L]) than in serum 

(0.4670.20 ppb [lg/L]). The correlation between bisphenol 

A concentration in serum and ascitic fluid was relatively 

strong (r 5 0.785). 

Tan and Mohd (2003) used a GC/MS method to 

measure bisphenol A concentrations in cord blood at 

delivery in 180 patients at a Malaysian medical center. 

Bisphenol A was detected in 88% of samples. As noted in 

Table 9, concentrations ranged from o0.10–4.05 mg/L. 

Schaefer et al. (2000) measured concentrations of 

bisphenol A and other compounds in uterine endometrium of women undergoing hysterectomy for uterine 

myoma at a German medical center. Endometrial and fat 

samples were obtained between 1995–1998 from 23 

women (34–51 years old) with no occupational exposure 

to bisphenol A. Samples were handled with plastic-free 

materials and stored in glass containers. Concentrations 

of environmental chemicals were measured in samples 

by GC/MS. None of 21 fat samples had detectable 

concentrations of bisphenol A. Bisphenol A was detected 

in 1 of 23 endometrial samples; the median concentration 

was reported at o1 mg/kg wet weight, and the range was 

reported at 0–13 mg/kg. [It is not known why a median 

value and range were reported when bisphenol A was 

only detected in 1 sample.] 

As part of a study to compare an ELISA and an LC/MS 

method for biological monitoring of bisphenol A, Inoue 

et al. (2002) measured concentrations of bisphenol A in 

semen samples obtained from 41 healthy Japanese 

volunteers (18–38 years old). Analysis by the ELISA 

method indicated bisphenol A concentrations ranging 

from concentrations below the detection limit (2.0 mg/L) 

to 12.0 mg/L. The LC/MS method indicated that the 

bisphenol A concentration in all samples was o0.5 mg/L, 

the LOQ. The study authors concluded that the LC/MS 

method was more accurate and sensitive and that the 

ELISA method overestimated bisphenol A concentrations, possibly due in part to nonspecific antibody 

interactions. 

1.2.4 Human exposure 

1.2.4.1 General population exposure 

1.2.4.1.1 Estimates based on bisphenol A concentrations in

food or environment: Wilson et al. (2003) estimated 

aggregate exposures to bisphenol A in preschool aged 

children (2–5 years) from the U.S. In 1997, numerous 

chemicals were surveyed, but only bisphenol A results 

are reported here. Ten child care centers were surveyed 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 173 

Table 10 

Bisphenol A Oral Exposure Estimates by the European Uniona 

Bisphenol A intake 

Bisphenol A 

Exposure source (exposed population) Daily food intake concentration in food mg/day mg/kg bw/day 

Infant bottles 0.699 L/day milk 50 mg/L 35 8 

(1–2 month-old infant) 

Infant bottles 0.983 L/day milk 50 mg/L 50 7 

(4–6-month-old infant) 

Polycarbonate tableware 2 kg food/day 5 mg/kg 10 0.7 

(1.5–4.5-year-old child) 

Canned food 0.375 kg canned food/ 100 mg/kg 40 5 

day 

(6–12- month-old infant) 

Canned food 2 kg canned food/day 100 mg/kg 200 14 

(1.5–4.5-year-old child) 

Canned food 1.0 kg canned food/day 100 mg/kg 100 1.4 

(adult) 

Wine 0.75 L/day 650 mg/L 500 7b 

(adult) 

Canned food and wine 0.75 L/day wine 650 mg/L in wine 600 9b 

(adult) 1.0 kg canned food/day 100 mg/kg food 

a

European Union (2003).
 

b

The European Union acknowledged that exposure through wine represents a very worst-case scenario.
 

and the 2 centers with the highest and lowest overall 

concentrations of target pollutants were selected for the 

study. Both centers were located in North Carolina. Nine 

children who attended one of the child care centers 

participated in the study. Over a 48-hr period, bisphenol 

A concentrations were measured in indoor and outdoor 

air, dust, soil, and food; the ranges detected are 

summarized in Sections 1.2.3.1 and 1.2.3.2. In estimating 

exposures, absorption was considered to be 100%. 

Calculations considered ventilation rates, time spent 

indoors and outdoors, time spent at home and in day 

care, the measured weight of each child, assumed 

ingestion of dust and soil, and total weight of foods 

consumed. Mean (range) bisphenol A intake was 

estimated at 0.042981 (0.018466–0.071124) mg/kg bw/day. 

Wilson et al. (2006) conducted a second study to 

estimate aggregate exposures in 257 U.S. children 

aged 1.5–5 years. Bisphenol A was one of the 

compounds assessed in this study of homes and daycare 

centers in 6 North Carolina and 6 Ohio counties in 2000– 

2001. Over a 48-hr period, bisphenol A concentrations 

were measured in indoor and outdoor air, dust, soil, 

food, and surface and hand wipes; the ranges detected 

are summarized in Sections 1.2.3.1 and 1.2.3.2. In 

estimating exposures, absorption was considered to be 

50%. Calculations considered ventilation rates, 

time spent indoors and outdoors, time spent at home 

and in day care, the measured weight of each child, 

assumed ingestion of dust and soil, and total weight of 

foods consumed. Median (25th percentile to maximum) 

bisphenol A aggregate exposures were estimated 

at 2.56 (1.5–57.2) mg/day for children from North 

Carolina and 1.88 (1.27–48.6) mg/day in children 

from Ohio. Median (25th percentile to maximum) 

potential aggregate dose, assuming 50% absorption, 

was estimated at 0.0714 (0.0424–1.57) mg/kg bw/day in 

children from North Carolina and 0.0608 (0.0341– 

0.775) mg/kg bw/day in children from Ohio. The study 

authors noted that 99% of exposure occurred through 

dietary ingestion. 

The European Union (2003) conducted a comprehensive 

exposure estimate that considered exposures resulting from 

food and environmental sources. Oral exposure estimates 

for children and adults were reported and are summarized 

in Table 10. Estimates were based on migration studies 

conducted with polycarbonate and concentrations of 

bisphenol A measured in foods packaged in epoxy-lined 

cans. Assumptions used in exposure estimates included 

100% oral absorption and body weights of 70 kg for adults, 

14.5 kg for 1.5–4.5-year-old children, 4.5 kg for 1–2-monthold infants, 7 kg for 4–6-month-old infants, and 8.7 kg for 6– 

12-month-old infants. Estimated exposures for children 

were said to represent realistic worst-case scenarios for 

food and drink intake relative to body weight. 

The European Union (2003) also estimated human 

environmental exposure to bisphenol A from sources 

such as drinking water, fish, plants, milk, meat, and air. 

The values were apparently obtained using the European 

Union System for the Evaluation of Substances (EUSES) 

model. Total regional exposure to bisphenol A was 

estimated at 0.0178 mg/kg bw/day. The highest local 

exposure was thought to occur in the vicinity of PVCproducing plants and was estimated at 59 mg/kg bw/ 

day. Aggregate exposures in adults involving food, wine, 

and environmental sources were estimated at 9 mg/kg 

bw/day for regional scenarios and 69 mg/kg bw/day for 

worst-case local scenarios occurring near a PVC-manufacturing plant. However, it was noted in the European 

Union report that use of bisphenol A in PVC manufacture was being phased out. 

The European Union (2003) noted that exposures to 

bisphenol A through dental sealant are single and rare 

events and do not lead to repeated exposure. Therefore, 

the issue was not considered further. 

Exposures to bisphenol A from some consumer 

products were identified and characterized by the 

Birth Defects Research (Part B) 83:157–395, 2008
174 CHAPIN ET AL. 

Table 11 

Bisphenol A Exposure Estimates by the European Commissiona 

Type of food and amount Concentration of bisphenol Exposure estimate (mg/kg 

Age (body weight) consumed A in food (mg/kg) bw/day) 

0–4-month old infant (4.5 kg) 0.7 L of formula/day 10 1.6 

6–12-month old infant (8.8 kg) 0.7 L of formula/day 10 0.8 

6–12-month old infant (8.8 kg) 0.38 kg canned food/day 20 0.85 

4–6-year-old child (18 kg) 1.05 kg canned food/day 20 1.2 

Adult (60 kg) 1.05 kg canned food/day 20 0.37 

Adult (60 kg) 0.75 L wine/day 9 0.11 

a

European Commission (2002). 

European Union (2003). Products included: marine 

antifouling agents used on boats, wood varnish, wood 

fillers, and adhesives. With the exception of adhesives for 

which frequent use was thought possible, exposure to the 

other products was considered to be relatively rare. 

Exposures were estimated based on factors such as epoxy 

and residual bisphenol A concentrations, exposure time, 

area of skin exposed, and possible generation of mists 

during processes such as brushing. Inhalation exposures 

by product were estimated at 3 x 10 -4 mg for antifouling 

agents and 0.02 mg for wood varnish. Dermal exposure by 

product without protective clothing was estimated at 

29 mg for antifouling agents, 3.6 mg for wood varnish, 9 mg 

for wood filler, and 14 mg for adhesives. [Dermal 

exposure to adhesives appears to be incorrectly reported as 1 lg in Table 4.20 of the European Union 

review.] Exposure was estimated to be 1–2 orders of 

magnitude lower when protective clothing such as 

gloves was used. Assuming an absorption rate of 10%, 

dermal exposure to bisphenol A through adhesives was 

estimated at 0.02 mg/kg bw/day. 

The European Commission, 2002) reviewed the report 

by the European Union (2003) in draft and suggested 

alternate exposure estimates. Those estimates and the 

assumptions used to support those estimates are summarized in Table 11. 

Miyamoto and Kotake (2006) estimated aggregate oral 

and inhalation exposure to bisphenol A in Japanese male 

children and adults. The estimates were based on 

unpublished Japanese data. This report is the only 

known study investigating potential exposure to children 

through mouthing of toys. Mouthing times were estimated by surveying the mothers of 50 infants and 

recording 25 infants on video camera. Mean7SD daily 

mouthing times were reported at 41.7713.7 min for 

infants 0–5 months of age and 73.9732.9 min for infants 

6–11 months of age. Migration rates were estimated from 

0 mg/cm2

/min for toys that do not contain bisphenol A to 

0.0162 mg/cm2

/min, the highest value reported in the 

Japanese literature. It was assumed that most toys were 

not manufactured with polycarbonate, epoxy resins, or 

grades of PVC that contain bisphenol A. Surface area of 

toys was assumed to be 10 cm2. In estimating oral 

exposures to bisphenol A, intake from food was also 

considered. Bisphenol A concentrations measured in 

migration testing of polycarbonate bottles and food 

surveys are summarized in Section 1.2.3.2. Volume of 

food consumption and frequency of article use were 

considered in estimates of bisphenol intake through food. 

Bisphenol A concentrations in drinking water were 

considered to be 0–0.17 mg/L, and water intake was 

assumed to be 2 L/day. In estimating inhalation 

exposures, concentrations of bisphenol A were considered to range from 0–8.1 ng/m3 in indoor air and 0– 

28 ng/m3 in outdoor air. Time spent indoors and 

outdoors and breathing rates were considered. Absorption from lungs was assumed at 100%. Estimated 

exposures from mouthing of toys, food and water intake, 

and inhaled air are summarized in Table 12. 

Additional estimates of bisphenol A exposure through 

food are summarized in Table 5 and Table 6. Details of 

studies conducted by Earls et al. (2000) and Onn Wong 

et al. (2005) are presented in Section 1.2.3.2. Exposure 

estimates conducted by the FDA are described below. 

Limited details were available from the other studies that 

were presented in reviews. 

The FDA (1996) estimated bisphenol A intake in 

infants and adults resulting from exposures to epoxy 

food-can linings and polycarbonate plastics. Exposure 

estimates occurring through contact of formula with 

polycarbonate bottles were based on results of a study 

conducted by the Chemistry Methods Branch of the FDA. 

The Chemistry Methods Branch also measured concentrations of bisphenol A in 5 brands of infant formula (14 

samples total); the study is also published as Biles et al. 

(1997a). In estimating adult bisphenol A exposure 

through the consumption of canned foods, the FDA 

considered surveys conducted by the Chemistry Methods Branch, Brotons et al. (1995), and the Society of 

Plastics Industry Group. It appears that the study by the 

Society of Plastics Industry Group was later published by 

Howe et al. (1998) and included a re-analysis to correct 

some interferences observed in analytical methods. 

Exposure estimates and assumptions used to make the 

estimates are summarized in Table 13. 

Table 14 summarizes exposure estimates for aggregate 

or food exposures. Studies suggest that the majority of 

bisphenol A exposure occurs through food and that 

environmental exposures do not appear to substantially 

affect total exposure, with the possible exception of 

exposure near point sources. Table 14 includes estimates 

that CERHR believes to represent potentially realistic 

exposure scenarios and does not include data from 

extreme worst-case scenarios such as possible point– 

source exposures. 

1.2.4.1.2 Estimates based on biological monitoring: Goodman et al. (2006) noted that total urinary bisphenol A 

concentrations were useful for estimating bisphenol A 

intake. Because of extensive first-pass metabolism, little 

parent compound is systemically circulated, as discussed 

in more detail in Section 2. Because nearly 100% of an 

acute exposure to bisphenol A is excreted in urine within 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 175 

Table 12 

Average Estimated Exposure to Bisphenol A in Japanese Man Adults and Childrena 

Bisphenol A concentration Average estimated exposures in each age groupb (mg/kg bw/day) 

Exposure source (other assumptions) 

0–5 6–11 1–6 7–14 15–19 19 

months months years years years years 

Human milk Negligible 0 0 

Formula (water) 0–0.17 mg/L 0.012 0.0096 

Feeding bottle 0–3.9 mg/L 0.015 0.014 

Infant food 0–5.0 mg/kg 0.085 

Toys 0–0.0162 mg/cm2

/min 0.026 0.069 

(mean mouthing times 

of 41.7 min in 

0–5-month-olds 

and 73.9 min in 

6–11-month-olds) 

Air 0–8.1 ng/m3 in 0.0026 0.0024 0.0021 0.0017 0.0015 0.0015 

indoor air and 

0–28 ng/m3 in 

outdoor air 

(90% indoors/10% 

outdoors) 

Water 0–0.17 mg/L (intake 0.012 0.0053 0.0029 0.0027 

of 2 L/day) 

Food and drink 

Canned 0–602 mg/kg 0.38 0.21 0.20 0.29 

Non-canned 0–3 mg/kg 0.38 0.21 0.13 0.12 

Tableware 0–39.4 mg/meal/utensil 0.40 0.12 0.024 0.022 

(3 meals/day; 1–5 types 

of utensils used/meal) 

Total breast-fed: 0.028 breast-fed: 0.16 1.2 0.55 0.36 0.43 

formula-fed: 0.055 formula-fed: 0.18 

a

Miyamoto and Kotake (2006).
 

b

Assumptions for bodyweights and most media intake levels were not provided.
 

24 hr (Vo ̈lkel et al., 2002; Tsukioka et al., 2004), bisphenol 

A intake can be estimated by measuring bisphenol A in 

urine over a specified time interval. Arakawa et al. (2004) 

measured bisphenol A excretion over a 5-day period and 

reported intra- and inter-individual variability. As a 

result, caution was urged in using single time-point 

values to estimate long-term exposure. Typical daily 

intakes of bisphenol A estimated from urinary levels are 

o0.01–2.17ı ́g/kg bw/day (Table 15). A Monte Carlo 

simulation using the urine data of Tsukioka et al. (2004) 

and Arakawa et al. (2004) estimated mean exposures of 

0.028–0.049 ug/kg bw/day for males and 0.034–0.059 ug/ 

kg bw/day for females (Miyamoto and Kotake, 2006). 

Using the U.S. NHANES data and assumptions on 

excretion rates and body weight a median intake of 

0.026 ug/kg bw/day is estimated. An estimated median 

exposure based on urinary bisphenol A concentrations in 

6–8-year-old girls was 0.07 mg/kg bw/day (Wolff et al., 

2006). 

Joskow et al. (2006) used values for total bisphenol A in 

urine to estimate exposure to bisphenol A following 

dental sealant application. Urinary concentrations of 

bisphenol A are reported in Table 8. Factors or assumptions used in the exposure estimates were recovery of 

bisphenol A in urine as its glucuronide conjugate within 

24–34 hr following exposure, a 5.4-hr half-life of 

elimination for bisphenol A glucuronide, and a 1.5 L/ 

day urinary excretion volume. Estimated doses of 

bisphenol A [based on a 60-kg bw] were 49–239 mg 

[0.82–4.0 lg/kg bw] following application of Delton LC 

and 0–9.5 mg [0–0.16 lg/kg bw] following application of 

Helioseal F. The study authors stated that the estimates 

were likely low because a substantial amount of bisphenol A was potentially eliminated by collection of 

saliva samples immediately following treatment. 

1.2.4.2 Occupational exposure: Occupational exposure to bisphenol A could potentially occur during its 

manufacture, in the production of polycarbonate plastics, 

and during the manufacture or use of epoxy resins, 

powder coatings paints, or lacquers (European-Union, 

2003). Possible exposure to bisphenol A during PVC 

manufacture has been considered, but the European 

Union (2003) stated that the application was being 

phased out. According to the European Union, bisphenol 

A is generally available as granules, flakes, or pellets, 

thus reducing exposure potential. Bisphenol A is 

manufactured in closed systems, but exposure is possible 

during sampling, container filling, and plant maintenance. In the manufacture of polycarbonate, bisphenol A 

enters the plant and remains in a closed system before 

extrusion. Sampling is conducted by a closed loop 

system. Following extrusion, the polycarbonate is 

Birth Defects Research (Part B) 83:157–395, 2008
176 CHAPIN ET AL. 

Table 13 

Summaries of Studies Estimating Bisphenol A Exposures Solely from Foods 

Exposure 

estimate mg/kg 

Population Exposure source Basis and assumptions for estimates bw/day Reference 

Infants Polycarbonate 

bottles 

Infants (0–3 Polycarbonate 

months old) bottles 

Not reported Food from epoxylined cans 

Adults Cumulative 

exposures from 

food contacting 

cans and 

polycarbonate 

plastics 

Infants Cumulative 

exposures from 

food contacting 

cans and 

polycarbonate 

plastics 

Adults Canned foods 

Adults Canned foods and 

canned fish 

Adults Wine 

Hospital patients Meals served at 2 

hospitals 

Japanese adults B200 food items 

and children were collected in 

a total diet study 

Bisphenol A migration concentration of 15– 

20 mg/L; milk consumption of up to 550 

mL/day; mean body weight of 11 kg 

Mean upper-bound concentration of 

bisphenol A migration in 10% ethanol 

(0.64 mg/in2

) and in corn oil (0.43 mg/in2); 

body weights reported by National Center 

for Health Statistics, and FDA Dietary 

Exposure Guidelines with modifications 

for properties of infant formula 

Bisphenol A concentrations of 5 ppb [mg/L] 

in beverages and 37 ppb [mg/kg] in other 

foods; FDA Dietary Exposure Guidelines: 

dietary intake of 3 kg/day, body weight of 

60 kg 

22 ppb [mg/kg] bisphenol A in vegetables, 

consumption factor of 0.17 for food 

contacting polymer-coated metal, intake of 

3 kg food/bw/day, 60 kg bw, and 

insignificant contribution from 

polycarbonate 

Bisphenol A concentration of 6.6 mg/kg in 

prepared infant formula, o1.7 ppb [mg/L] 

in infant formula from polycarbonate 

bottles, consumption of 820 g food/day, 

and 4 kg infant weight 

Data from survey of canned foods and food 

intake patterns determined from surveys 

Data from survey of canned foods and food 

intake patterns determined from surveys 

Maximum bisphenol A concentration of 

2.1 ng/mL in wine, consumption of 0.75 L/ 

day, and 60 kg body weight 

Mean intake from hospital diets was 

estimated at 1.3 (0.19–3.7) mg/day; [60 kg 

body weight was assumed] 

No details 

0.75–1 Earls et al. (2000) 

15–24a Onn Wong et al. 

(2005) 

0.105	 Howe et al. (1998) 

Haighton et al. 

(2002) NAS 

(1999) 

0.183 FDA (1996) 

1.75 

Mean 5 0.0083 (0– Thomson and 

0.29) Grounds (2005) 

0.0044 for men Thomson et al. 

Z25 0.0041 for (2003) 

women Z25 

0.0048 for men 

19–24 

o0.026 BrennStruckhofova 

and CichnaMarkel (2006) 

[0.02 (0.003–0.06)]	 Miyamoto and 

Kotake (2006) 

Fujimaki et al. 

(2004) 

0.00475 for	 Miyamoto and 

children 2–6 Kotake (2006) 

years 0.00195 for 

adults 

a

The study authors acknowledged the use of aggressive migration testing conditions and conservative assumptions in calculations, thus 

leading to overestimated infant exposures. 

chopped into granules and bagged, and it is during that possible. A residual bisphenol A concentration of 

stage that exposure to residual bisphenol A (reported at 300 ppm was reported for epoxy resins, but it was noted 

r100 ppm) through dust is possible. However, it is noted that most bisphenol A was trapped within the resin 

that polycarbonate is stable and that residual bisphenol matrix. Exposure to bisphenol A during production of 

A is contained within the polymer matrix. The European epoxy paints is reported to be negligible. In the 

Union stated that exposure to bisphenol A during the manufacture of powder epoxy coatings, exposure is 

manufacture of polycarbonate items is not likely to thought possible during weighing and milling. Exposure 

exceed values observed during the manufacture of to bisphenol A during the use of powder paints has been 

polycarbonate. In the production of epoxy resin, bi- documented. 

sphenol A exposure is most likely during reactor There are no known regulatory limits for occupational 

charging, but exposure during maintenance is also exposure to bisphenol A in the U.S. In 2004, the 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 177 

Table 14 

Summary of Food or Aggregate Exposures to Bisphenol A 

Exposure 

estimate mg/kg 

Population Basis of estimates bw/daya Reference 

1–2-month-old infant Food exposure (data from migration studies of 8 European Union (2003) 

polycarbonate bottles) 

0–4-month-old infant Food exposure (data from migration studies of 1.6 European Commission 

polycarbonate bottles) (2002) 

0–5-month-old infant Aggregate exposure (based on formula, 0.055 Miyamoto and Kotake 

(formula-fed) environmental, and toy exposures) (2006) 

0–5-month-old infant Aggregate exposure (based on human milk, 0.028 Miyamoto and Kotake 

(breast fed) environmental, and toy exposures) (2006) 

4–6-month-old infant Food exposure (data from migration studies of 7 European Union (2003) 

polycarbonate bottles) 

6–11-month-old infant Aggregate exposure (based on formula, food, 0.18 Miyamoto and Kotake 

(formula-fed) environmental, and toy exposures) (2006) 

6–11-month-old infant Aggregate exposure (based on human milk, food, 0.16 Miyamoto and Kotake 

(breast-fed) environmental, and toy exposures) (2006) 

6–12-month-old infant Food exposure (data from survey of canned foods) 5 European Union (2003) 

6–12-month-old infant Food exposure (data from migration studies with 1.65 European Commission 

infant bottles and canned foods) (2002) 

Infant Food exposure (data from polycarbonate bottle 0.75–1 Earls (2000) 

leaching studies) 

Infant Food exposures (contact with cans and 1.75 FDA (1996) 

polycarbonate plastics) 

1.5–4.5-year-old child Food exposure (data from survey of canned foods 14.7 European Union (2003) 

and migration studies with polycarbonate 

tableware) 

1–6-year-old child Aggregate exposure (based on food, environmental, 1.2 Miyamoto and Kotake 

and tableware exposures) (2006) 

1.5–5 year old child Aggregate exposure (surveys of bisphenol in food, 0.06-0.07 Wilson et al. (2006) 

air, dust, soil and hand and surface wipes) (0.03–1.57) 

3–5-year-old child Aggregate exposure (surveys of bisphenol in food, 0.04 Wilson et al. (2003) 

air, dust, and soil) (0.018–0.07) 

2–6-year-old child Food exposure (collection of 200 food items) 0.004 Miyamoto and Kotake 

(2006) 

4–6-year-old child Food exposure (data from survey of canned foods) 1.2 European Commission 

(2002) 

7–14-year-old child Aggregate exposure (based on food, environmental, 0.55 Miyamoto and Kotake 

and tableware exposures) (2006) 

15–19-year-old individual Aggregate exposure (based on food, environmental, 0.36 Miyamoto and Kotake 

and tableware exposures) (2006) 

Adult, Z19 years Aggregate exposure (based on food, environmental, 0.43 Miyamoto and Kotake 

and tableware exposures) (2006) 

Adult Food exposure (data from survey of canned foods 1.4 European Union (2003) 

not including wine) 

Adult Food exposure (data from surveys of canned food) 0.37 European Commission 

(2002) 

Adult Wine exposure (data from study of epoxy-lined wine 0.11 European Commission 

drums) (2002) 

Adult Wine exposure (data from wine samples) o0.026 Brenn-Struckhofova and 

Cichna-Markel (2006) 

Adult Food exposure (from contact with epoxy-lined cans 0.183 FDA (1996) 

and polycarbonate) 

Adults Food exposure (survey of canned foods) 0.008 Thomson and Grounds 

(2005) 

Adult Food exposure (collection of 200 food items) 0.002 Miyamoto and Kotake 

(2006) 

a

Estimates involving extreme worst case scenarios and Japanese data with very limited information were not included in this table. 

American Industrial Hygiene Association proposed a value is consistent with the time weighted average 

workplace environmental exposure level (WEEL) of (TWA) exposure limits established in Germany and the 

5 mg/m3 for bisphenol A. The draft WEEL was based Netherlands (European-Union, 2003). 

on irritation observed in an inhalation toxicity study The European Union (2003) summarized occupational 

(American Industrial Hygiene Association, 2004). The exposure data for bisphenol A in Europe and the U.S. 

Birth Defects Research (Part B) 83:157–395, 2008
178 CHAPIN ET AL. 

Table 15 

Estimates of Bisphenol A Intakes Based on Urinary Excretion 

Mean or median (range) 

of estimated intake 

Population Basis for estimates mg/kg bw/daya Reference 

22 Japanese adults Mean excretion of 1.68 mg/day (0.48–4.5 mg/day) 0.028 (0.008–0.075) Tsukioka (2004) 

36 Japanese male Median excretion of 1.2 mg/day (o0.21–14 mg/ 0.02 (o0.0035–0.23) Arakawa et al. (2004) 

students day) 

5 Japanese males Median excretion of 1.3 mg/day (o0.58–13 mg/ 0.022 (o0.01–0.22) Arakawa et al. (2004) 

day) over a 5-day period 

Data from Tsukioka Monte Carlo simulations Mean exposure: 0.028– Miyamoto and Kotake 

(2004) and Arakawa 0.049 in men and (2006) 

et al. (2004) 0.034–0.059 in women; 

low exposures (5th 

percentile) 0.021–0.037 

in men and 0.025– 

0.044 in women; high 

exposures (95th 

percentile): 0.037– 

0.064 in men and 

0.043–0.075 in women 

56 pregnant Japanese Bisphenol A concentration in one spot sample o0.04 (o0.006–0.16)b Fujimaki et al. (2004) 

females was normalized to creatinine and exposure 

was estimated using average creatinine and 

urine volume excretion rates, which resulted in 

a median intake of o2 mg/day (o0.3–7.9 mg/ 

day) 

48 Japanese female Authors estimated bisphenol A intake of 0.6– 0.01–1.2 based on study Ouchi and Watanabe 

college students 71.4 mg/day, based on a median bisphenol A author assumptions (2002) 

concentration of 0.77 ng/mg (0.1–11.9 ng/mg) [0.015 (0.002–0.24) 

creatinine in a spot urine sample, assumed based on a 100% 

creatinine excretion of 1200 mg/day and that urinary excretion 

20% of the dose is excreted in urine. [CERHR rate] 

recalculated values using a 100% urinary 

excretion rate that is consistent with human 

data] 

7 males and 12 females All measurements oLOD of 1.14 lg/L Based on 2 L urine Vo ̈lkel et al. (2005) 

without intentional excreted and 60 kg 

exposure adult exposure 

o0.038 

394 participants in the Median (10th–95th percentile) 1.32 (0.23–7.95) mg [median 5 0.026; 10th– Calafat et al. (2005) 

NHANES III survey bisphenol A/g creatinine in a spot urine 95th percentile: 0.005­

(U.S.) sample; [assumed 100% urinary excretion of 0.159] 

bisphenol A in 24 hr and creatinine excretion 

of 1200 mg/day] 

90 girls, 6–8-years-old Median (range) 1.8 mg/L (o0.3–54.3) [assumed [0.07 (o0.012–2.17)] Wolff et al. (2006) 

(U.S.) 100% urinary excretion of bisphenol A in 

24 hr; 1 L per day; 25 kg body weight ] 

a

Consistent with estimates conducted by Goodman et al. (2006), body weights of 60 kg were assumed, unless otherwise indicated. 

b

A 50-kg body weight was assumed. 

Only measured data for bisphenol A are summarized in 

this report. The European Union stated that the values 

reported did not account for the effects of personal 

protective equipment in order to avoid difficulties in 

attempting to quantify protection provided. TWA bisphenol A concentrations measured in occupational 

settings are summarized in Table 16. The limited number 

of values reported indicated that bisphenol A concentrations were below 5 mg/m3

. Bisphenol A exposures 

(41 mg/m3

) were observed in spraying of powdered 

bisphenol A-containing coatings, bisphenol A manufacture and manufacture of epoxy resins. The highest daily 

average exposures were observed in the manufacture of 

bisphenol A. There is limited information on short-term 

exposure to bisphenol A. In manufacture of bisphenol A 

one facility reported short-term task exposures from 

0.13–9.5 mg/m3 (European-Union, 2003). 

Data for powder paint use summarized in Table 16 

were obtained from a NIOSH Health Hazard Evaluation 

conducted at a company that manufactured fan and 

ventilation equipment (NIOSH, 1979). In Plant 1 of the 

company, parts were coated with an epoxy-based 

powder paint by dipping. At Plant 2, an epoxy-based 

powder was applied to parts via electrostatic spraying. 

As evident in the data in Table 16, exposures were higher 

at the plant utilizing electrostatic spraying. Monitoring 

for bisphenol A was discussed in 2 other NIOSH Health 

Hazard Evaluation reports. In those reports, bisphenol A 

was not detected in a plant where an epoxy resin coating 

was used in the manufacture of electronic resistors 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 179 

Table 16 

TWA Measurements of Bisphenol A in the Workplace 

8-hr TWA (mg/m3

) 

Industry or activity Location/year No. of samples Sample type mean (range)b 

Bisphenol A 

manufacture 

Various U.S./not specified Not specified Bisphenol A NS (Not detected to 2.6) 

Filling big bags Europe/1998 3 Inhalable bisphenol A 0.81 (0.21-1.79) 

Filling silo tankers Europe/1998 3 Inhalable bisphenol A 0.89 (o0.5-1.61) 

Various tasks Europe/1998 8 Inhalable bisphenol A 0.3 (0.13-0.62) 

Plant operator Europe/not specified 7 Inhalable bisphenol A NS (0.021-1.04) 

Maintenance Europe/not specified 3 Inhalable bisphenol A NS (0.52-1.35) 

Maintenance Europe/1998–2000 8 Bisphenol A NS (o0.05–0.62) 

Charging big bags Europe/1996–1997 5 Inhalable bisphenol A 0.35 (0.02–0.93) 

Plant operator Europe/not specified 13 Bisphenol A 0.61 (0.02–2.13) 

Maintenance Europe/not specified 2 Bisphenol A 1.06 (0.4–2.08) 

operator 

Epoxy resin 

manufacture 

Loading/unloading U.S./1970-mid 1990s 26 Bisphenol A 0.18 (o0.1-0.99) 

Bagging/palletizing U.S./1970-mid 1990s 37 Bisphenol A 0.25 (o0.1-2.8) 

Process operators U.S./1970-mid 1990s 25 Bisphenol A 0.26 (o0.1-1.1) 

Equipment U.S./1970-mid 1990s 6 Bisphenol A o0.1 

technician 

Maintenance U.S./1970-mid 1990s 2 Bisphenol A 0.8 (0.37-1.2) 

Bisphenol A Use 

Powder paint usea U.S./B1979 7 (3 personal and 4 area Bisphenol A (plant 1) 0.005 (0.004–0.006) 

samples) 

21 (15 personal and 6 Bisphenol A (plant 2) 0.175 (0.001–1.063) 

area samples) 

a

NIOSH (1979). Other data are from the European Union (2003). 

b

Range given representing different occupational activities. 

NS, not specified. 

(NIOSH, 1984) or in a plant where an epoxy resin coating 

was applied to steam turbine generators (NIOSH, 1985). 

Rudel et al. (2001) used a GC/MS technique to measure 

bisphenol A concentrations at one United States workplace where plastics were melted and glued; a concentration of 0.208 mg/m3 was reported. 

[Bisphenol A exposures in U.S. powder paint workers were estimated at B0.1–100 lg/kg bw/day based on 

TWA exposures of 0.001–1.063 mg/m3

, an inhalation 

factor of 0.29 m3

/kg day (USEPA, 1988), 100% absorption from the respiratory system, and 8 hr worked per 

day.] 

No information was located for dermal exposure to 

bisphenol A in occupational settings. Using their 

Estimation and Assessment of Substance Exposure 

model, the European Union (2003) estimated that dermal 

exposure of workers to bisphenol A was unlikely to 

exceed 5 mg/cm2

/day. It was noted that the highest 

potential exposure to bisphenol A would occur during 

bag filling and maintenance work. 

One study provided information on biological monitoring of bisphenol A in workers exposed to an epoxy 

compound. In 3 Japanese plants, exposed workers 

included 42 men who sprayed an epoxy hardening agent 

consisting of a mixture of bisphenol A diglycidyl ether 

(10–30%), toluene (0–30%), xylene (0–20%), 2-ethoxyethanol (0–20%), 2-butoxyethanol (0–20%), and methyl isobutyl 

ketone (0–30%) (Hanaoka et al., 2002). The workers wore 

‘‘protection devices’’ during spraying. Controls consisted 

of 42 male assembly workers from the same plants who 

did not use bisphenol A diglycidyl ether. In 1999, urine 

samples were collected periodically, treated with bglucuronidase, and examined for bisphenol A by HPLC. 

Urinary bisphenol A concentrations were significantly 

higher in exposed workers (median: 1.06 mmol/mol 

creatinine [2.14 lg/g creatinine]; range: o0.05 pmol to 

11.2 mmol/mol creatinine [o0.1 pg to 22.6 lg/g creatinine]) 

compared to controls (median: 0.52 mmol/mol creatinine 

[1.05 lg/g creatinine]; range: o0.05 pmol to 11.0 mmol/mol 

creatinine [o0.1 pg to 22.2 lg/g creatinine]). The difference of the averages was reported as 2.5 mmol/mol 

creatinine [5.05 lg/g creatinine] (95% confidence interval 

[CI] 1.4–4.7 mmol/mol creatinine [2.8–9.5]). Bisphenol A 

was not detected in three exposed workers and one 

control. [Assuming excretion of 1200 mg/day creatinine 

(Ouchi and Watanabe, 2002), mean (ranges) of bisphenol 

excretion in urine were 2.57 lg/day (o0.12 pg to 27.1 lg/ 

day) in exposed workers and 1.26 lg/day (o0.12 pg to 

26.6 lg/day) in unexposed workers. With an assumed 

body weight of 60 kg, bisphenol A occupational intake 

was estimated at 0.043 lg/kg bw/day (o0.002 pg to 

0.45 lg/kg bw/day) in exposed workers and 0.021 lg/kg 

bw/day (o0.002 pg to 0.44 lg/kg bw/day) in unexposed 

workers.] 

1.3 Utility of Data 

Numerous studies reported bisphenol A concentrations in canned foods and infant formula. Experiments 

examined potential concentrations of bisphenol A 

Birth Defects Research (Part B) 83:157–395, 2008
180 CHAPIN ET AL. 

Table 17 

Maximum Reported Bisphenol A Concentrations in U.S. Ambient Air and Dust Samples 

Sample Bisphenol A concentration Reference 

Outdoor air o52 ng/m3 Monthly average 0.12–1.2 ng/m3 Wilson et al. (2003, 2006); Matsumoto et al. (2005) 

Indoor air r193 ng/m3 Wilson et al. (2003, 2006); Rudel et al. (2001, 2003) 

Indoor dust r17.6 mg/g Wilson et al. (2003, 2006); Rudel et al. (2001, 2003) 

Drinking water o0.1 (MDL) o0.005 Boyd et al. (2003); Kuch and Ballschmiter (2001) 

Table 18
 

Maximum Reported Bisphenol A Concentrations Measured in Foods or Food Simulants
 

Exposure source Bisphenol A concentration Table reference 

Polycarbonate infant bottles r55 mg/L (o5 mg/L in U.S. study) Table 4 

Polycarbonate tableware r5 mg/kg Table 4 

Canned infant formulas r113 mg/L (o6.6 mg mg/kg in U.S. study of water mixed Table 5 

formula; o13 mg/kg in U.S. formula concentrate) 

Canned infant foods r77.3 mg/kg 

Canned foods r842 mg/kg (r39 mg/kg in U.S. studies) Table 6 

resulting from leaching of bisphenol A from polycarbonate bottles under a variety of conditions. There minimal 

data available for bisphenol A concentrations in drinking 

water but these show concentrations below the limit of 

detection. Bisphenol A has been detected in surface 

waters and solid waste landfill leachates. Bisphenol A 

has been detected in indoor dust samples and indoor and 

outdoor air samples. Data for occupational exposure to 

bisphenol A in the U.S. are very limited. Only 2 studies 

reported TWA exposures to bisphenol A in U.S. workers. 

Several estimates of human bisphenol A exposure were 

developed using bisphenol A concentrations measured 

in food and the environment. Although very limited for 

U.S. populations, there are data reporting bisphenol A 

concentrations in urine, breast milk, and amniotic fluid, 

but none for blood or fetal blood. Exposure estimates 

have been derived from urinary bisphenol A concentrations in multiple studies. 

1.4 Summary of Human Exposure 

In 1999 and 2003, it was reported that most bisphenol 

A produced in the U.S. was used in the manufacture of 

polycarbonate and epoxy resins and other products 

[reviewed in (Staples et al., 1998; SRI, 2004)]. Polycarbonate plastics are used in various consumer products and 

the products most likely to contribute to human exposure 

are polycarbonate food containers (e.g., milk, water, and 

infant bottles). Epoxy resins are used in protective 

coatings. Food cans lined with epoxy resin are a potential 

source of human exposure. Some polymers manufactured with bisphenol A are FDA-approved for use in 

direct and indirect food additives and in dental materials 

(FDA, 2006). Resins, polycarbonate plastics, and other 

products manufactured from bisphenol A can contain 

trace amounts of residual monomer and additional 

monomer may be generated during breakdown of the 

polymer (European-Union, 2003). 

Bisphenol A may be present in the environment as a 

result of direct releases from manufacturing or processing facilities, fugitive emissions during processing 

and handling, or release of unreacted monomer 

from products (European-Union, 2003). Because of its 

low volatility and relatively short half-life in the 

atmosphere, bisphenol A is unlikely to be present in 

the atmosphere in high concentrations (EuropeanUnion, 2003). A study of 222 homes and 29 day care 

centers found bisphenol A in 31–44% of outdoor air 

samples with concentrations of oLOD (0.9) to 51.5 ng/ 

m3 (Wilson et al., 2006). Rapid biodegradation of 

bisphenol A in water was reported in the majority of 

studies reviewed by the European Union (2003) and 

Staples et al. (1998). Drinking water concentrations of 

bisphenol A at Louisiana and Detroit Michigan water 

treatment plants were below the limit of detection 

(o0.1 ng/L). Chlorinated congeners of bisphenol A 

resulting from chlorination of water may be degraded 

less rapidly (Gallard et al., 2004). Bisphenol A is not 

expected to be stable, mobile, or bioavailable from soils 

(Fent et al., 2003). A study of 222 homes and 29 day care 

centers found bisphenol A in 25–70% of indoor dust 

samples with concentrations of oLOD (20) to 707 ng/g 

(Wilson et al., 2006). The potential for bioconcentration of 

bisphenol A in fish is low (Staples et al., 1998; EuropeanUnion, 2003). Table 17 summarizes concentrations of 

bisphenol A detected in environmental samples and 

drinking water. 

The highest potential for human exposure to 

bisphenol A is through products that directly contact 

food such as food and beverage containers with 

internal epoxy resin coatings and polycarbonate tableware and bottles, such as those used to feed infants 

(European-Union, 2003). Studies examining the extraction of bisphenol A from polycarbonate bottles or 

tableware into food simulants are summarized in 

Table 4. Studies measuring bisphenol A concentrations 

in canned infant foods are summarized in Table 5 and 

studies measuring bisphenol A concentrations in 

canned food are summarized in Table 6. Table 18 

summarizes the general findings from all the food 

contact–material studies. Bisphenol A concentrations 

were measured in canned foods produced and purchased from various countries. 

Table 19 summarizes BPA concentrations reported in 

human body fluids. Measurement of bisphenol A 

concentrations are affected by measurement technique, 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 181 

Table 19 

Maximum Reported Biological Measures of Bisphenol A Concentrations in Humans 

Biological medium Population Concentration free BPAa (mg/L) Total BPAa (mg/L) Reference 

Urine	 Adult r2.36 (o0.6 in U.S. study) r3950 (o19.8 U.S. studies) Table 8 

Children o54 (2 U.S. studies) 

Blood	 General oLOD (0.5) oLOD (0.5) Table 7 

Infertile women o0.87 Table 7 

Women o1.6 Table 7 

Men o1 Table 7 

Fetal o9.2 Table 9 

Breast milk Women o6.3 (U.S.) o7.3 Table 3 

Amniotic fluid Fetus o1.96 (U.S) Table 9 

Semen Adult o0.5 Inoue et al.(2002) 

Saliva after dental sealant Adult o2800 Arenholt-Bindslev 

et al. (1999) 

a

Measurements by HPLC, GC/MS, and LC/MS only. 

Table 20 

Summary of Reported Human Dose Estimates 

Exposure source Population BPA mg/kg bw/dayNotes	 Source 

Estimates based on intake 

Formula Infant 1.6–8 8 assumes 700 ml formula with 50 mg/L Table 14 

Formula Infant 1.0 Assumes 4.5 kg, 700 ml formula with Expert Panel 

6.6 mg/L from U.S. canned formula 

Breast milk Infant 1.0 Assumes 4.5 kg, 700 ml with 6.3 mg/L Expert Panel 

from breast milk 

Food	 Infant 1.65–5 5 assumes 0.375 kg canned food at 100 mg/kg Table 14
 

Child 0.00475–1.2 1.2 assumes 1 kg canned food at 20 mg/kg Table 14
 

Adult 0.00195–1.4 1.4 assumes 1 kg canned food at 100 mg/kg Table 14
 

Aggregate	 Infant (formula) 0.055–0.18 Assumes 0–0.17 mg/L in formula Table 14
 

Infant (breast milk) 0.028–0.16 Assumes 0 exposure from breast milk Table 14
 

Child 0.042981–14.7 14.7 assumes 2 kg canned food at 100 mg/kg Table 14
 

Adult 0.36–0.43 Assumes 0–602 mg/kg in canned food Table 14
 

Occupational Adult 0.043–100 EPA and 

Expert Panel 

Estimates based on urinary metabolites 

Aggregate	 Child 0.07 (2.17) Median (max) U.S. 6–8-year-old girls Table 15 

Adult 0.026 Median NHANES Table 15 

Adult 0.66 Assume max 19.8 mg/L from U.S., Ye et al. (2005) 

2 L urine/day, 60 kg 

particularly at the very low concentrations that can now 

be measured. ELISA has poor correlation with the LCECD method and also the different ELISA kits correlate 

poorly with each other. ELISA methods may overestimate bisphenol A in biologic samples due to lack of 

specificity of the antibody and effects of the biologic 

matrix (Inoue et al., 2002; Fukata et al., 2006). In addition, 

contamination from labware and reagents or sample 

degradation during storage can impact the accuracy of 

measurements. [The panel therefore finds the greatest 

utility in studies that use sensitive and specific 

analytical methods for biological samples (LC-MS or 

GC-MS) and report quality control measures for 

sample handling and analysis.] 

Table 20 summarizes food and/or aggregate exposure 

estimates calculated from bisphenol A concentrations in 

food, environmental and toy exposures along with 

estimates of consumption and body weights. It was 

noted that dietary sources account for 99% of exposure 

(Wilson et al., 2006). Metabolite-based estimates of 

bisphenol A used urinary concentrations along with 

estimates of urinary and/or creatinine excretion, and 

body weight. 

Dental sealant exposure to bisphenol A occurs primarily with use of dental sealants bisphenol A dimethylacylate. This exposure is considered an acute and infrequent 

event with little relevance to estimating general population exposures. 

Very limited information is available for bisphenol A 

exposure in the U.S. workplace. Data obtained from the 

U.S. and Europe indicate highest potential exposures 

during spraying of powdered bisphenol A-containing 

coatings and during tank filling, plant operation 

activities, and maintenance work in plants where bisphenol A is manufactured. (European-Union, 2003). 

One study measured total urinary bisphenol A in 

Japanese workers who sprayed an epoxy compound 

(Hanaoka et al., 2002). 

Birth Defects Research (Part B) 83:157–395, 2008
182 CHAPIN ET AL. 

2.0 GENERAL TOXICOLOGY AND 

BIOLOGICAL EFFECTS 

As discussed in Section 1.4, the quantified amount of 

free bisphenol A present in biological samples may be 

affected by contamination with bisphenol A in plastic 

laboratory ware and in reagents (Tsukioka et al., 2004; 

Vo ̈lkel et al., 2005). In addition, the accuracy may also be 

affected by measurement technique, particularly at the 

very low concentrations that can now be measured. 

ELISA have the potential to overestimate bisphenol A in 

biologic samples due to lack of specificity of the antibody 

and effects of the biologic matrix (Inoue et al., 2002; 

Fukata et al., 2006). High performance liquid chromatography (HPLC) with ultraviolet, fluorescence, or electrochemical detection is unable to make definitive 

identification of bisphenol A or bisphenol A glucuronides, because similar retention times may occur for the 

metabolites of other endogenous and exogenous compounds (Vo ̈lkel et al., 2005). Use of LC-tandem mass 

spectrometry (MS/MS) with and without hydrolysis of 

bisphenol A glucuronide permits determination of free 

and total bisphenol A with a limit of quantification of 

1 mg/L (Vo ̈lkel et al., 2005). Gas chromatography (GC)/ 

MS/MS has been used with solid phase extraction after 

treatment with glucuronidase and derivatization to 

measure total bisphenol A with a limit of detection of 

0.1 mg/L (Calafat et al., 2005). Bisphenol A glucuronidate 

has been shown to be unstable and can be hydrolyzed to 

free bisphenol A at neutral pH and room temperature in 

diluted urine of rats and in rat placental and fetal tissue 

homogenates at room temperature. Bisphenol A glucuronide can also be hydrolyzed and in some cases 

degraded to unknown components either in acidic or 

basic pH solutions of diluted urine, adding another 

potential source of error in the measurement of sample 

levels of bisphenol A and its conjugates (Waechter et al., 

2007). These considerations taken together, suggest that it 

is possible that free bisphenol A concentrations measured in biological samples may be overestimated. 

2.1 Toxicokinetics and Metabolism 

The studies presented in this section demonstrate that 

bisphenol A is absorbed in humans and experimental 

animals following oral exposure. In humans and experimental animals, most of the dose is present in blood as 

the main metabolite, bisphenol A glucuronide, and 

smaller percentages are present as the parent compound. 

Bisphenol A and its metabolites are widely distributed in 

humans and animals. More than 90% of unmetabolized 

bisphenol A is reportedly bound to plasma protein. 

Bisphenol A is distributed to fetal fluids in humans and 

experimental animals, and a limited number of studies in 

humans demonstrate fetal concentrations of bisphenol A 

within an order of magnitude of concentrations in 

maternal blood. None of the studies detected bisphenol 

A glucuronide in fetal fluids. Transfer of bisphenol A to 

milk was demonstrated in humans and experimental 

animals. One study in humans reported bisphenol A in 

milk at concentrations exceeding maternal blood concentrations. In humans and experimental animals, most 

of a bisphenol A dose is metabolized to bisphenol A 

glucuronide before absorption. Studies in humans and 

experimental animals demonstrated that glucuronidation 

of bisphenol A can occur in the liver, and one study in 

rats demonstrated that bisphenol A is glucuronidated 

upon passage through the intestine. Bisphenol A 

glucuronide is excreted in the bile of rats, and enterohepatic cycling is thought to occur in rats but not 

humans. In humans, most of a bisphenol A dose is 

eliminated through urine as bisphenol A glucuronide. In 

rats, bisphenol A is eliminated through feces as bisphenol A and in urine as bisphenol A glucuronide. 

2.1.1 Humans. Human toxicokinetics studies that 

were judged potentially important to interpret developmental and reproductive toxicity were reviewed in full. 

These studies include reports of potential exposure of 

fetuses during pregnancy or of infants through human 

milk and reports of toxicokinetics or metabolism following low-dose exposure of humans. Information from 

secondary sources was included if the information was 

not considered to be critical to the interpretation of 

developmental and reproductive toxicity data. 

2.1.1.1 Absorption: Two studies described here examined oral absorption of bisphenol A from dental 

sealants, and one study examined in vitro dermal 

absorption. Bisphenol A (as parent or the monoglucuronide) is absorbed in humans as indicated by the 

detection of bisphenol A (and metabolites) in blood from 

the general population (Section 1) and in maternal and 

fetal fluids (Table 9). 

Fung et al. (2000) examined the toxicokinetics of 

bisphenol A leaching from dental sealant. Volunteers 

included 18 men and 22 non-pregnant women (20–55 

years of age) who did not have dental disease, existing 

composite resin restorations or pit and fissure sealants, 

or a history of resin exposure. Volunteers were treated 

with a widely used commercial dental sealant (Delton 

Opaque Light-cure Pit and Fissure Sealant). Components 

of the sealant were analyzed by HPLC. The low-dose 

group (n 5 7 men, 11 women) received 8 mg dental sealant 

on 1 tooth, and the high-dose group (11 men, 11 women) 

received 32 mg sealant on 4 teeth. Saliva and blood 

samples were collected before the procedure and at 1 and 

3 hr and 1, 3, and 5 days after the procedure. Blood and 

saliva were analyzed by HPLC. Statistical analyses of data 

were conducted by nonparametric test, Wilcoxon signed 

rank test, and w2 test. Analysis of the dental sealant 

revealed that bisphenol A concentrations were below the 

detection limit of 5 ppb. At 1 hr following treatment, 

bisphenol A was detected in samples from 3 of 18 

volunteers in the low-dose group and 13 of 22 samples 

from volunteers in the high-dose group. At 3-hr posttreatment, bisphenol A was detected in samples from 1 of 

18 volunteers in the low-dose group and 7 of 22 volunteers 

in the high-dose group. Concentrations of bisphenol A in 

saliva at 1 and 3 hr following exposure were reported at 

5.8–105.6 ppb [lg/L]. No bisphenol A was detected in 

saliva samples at 24 hr or in serum samples at any time 

point. Differences between the low-dose and high-dose 

groups in bisphenol A saliva concentrations and in the 

proportion of bisphenol A-positive saliva samples at 1 and 

3 hr achieved statistical significance. In the high-dose 

group, a significant difference in ‘‘readings’’ was observed 

between 1 and 3 hr. [The data as presented did not 

illustrate possible quantitative differences in saliva 

bisphenol A concentrations from the 2 dose groups or 

at different sampling times.] 

Joskow et al. (2006) examined bisphenol A in urine and 

saliva of 14 adults (19–42 years old) treated with dental 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 183 

Table 21 

Saliva and Urinary Concentrations of Total Bisphenol A in Adults Receiving Dental Sealantsa 

Mean7SD bisphenol A concentration (ng/mL)b 

Collection time Both sealants Delton LC Helioseal F 

Saliva 

Pretreatment 0.3070.17 0.3470.19 0.2270.03 

Immediately after treatment 26.5730.7 42.8728.9 0.5470.45 

1 hr post-treatment 5.12710.7 7.86712.73 0.2170.03 

Urine (creatinine-adjusted) 

Pretreatment 2.4171.24 2.671.4 2.1270.93 

1 hr post-treatment 20.1733.1 27.3739.1 7.26713.5 

24 hr post-treatment 5.1473.96 7.3473.81 2.0671.04 

a

Joskow et al. (2006). 

b

Samples were treated with b-glucuronidase. 

sealants. Excluded from the study were individuals with 

resin-based materials on their teeth, smokers, users of 

antihistamines, and patients with Gilbert syndrome. The 

volunteers received either Helioseal F (n 5 5) or Delton 

LC (n 5 9) sealant. Sealant was weighed before and after 

application to determine the amount applied, and the 

number of treated teeth was recorded. The mean number 

of teeth treated was 6/person and the mean total weight 

of sealant applied was 40.35 mg/person. In a comparison 

of the 2 sealants, no differences were reported for 

number of teeth treated or amount of sealant applied. 

Saliva samples were collected before treatment, immediately after, and at 1 hr following sealant application. 

Urine samples were collected before treatment and at 1 

and 24 hr following sealant placement. A total of 14–15 

saliva samples and 12–14 urine samples were collected at 

each time point. Samples were treated with b-glucuronidase and analyzed for bisphenol A concentrations 

using selective and sensitive isotope-dilution-MS-based 

methods. Table 21 summarizes changes in saliva and 

bisphenol A concentrations. Immediately and at 1 hr after 

sealant application, salivary concentrations of bisphenol 

A compared to baseline were significantly higher in the 

patients who received the Delton LC sealant. Bisphenol 

A concentrations in saliva increased 484-fold following 

application of the Delton LC sealant. Urinary concentrations of bisphenol A were increased 1 hr following 

application of the Delton LC sealant. Concentrations of 

bisphenol A in saliva and urine following application of 

Helioseal F were reported to be similar to baseline. 

The European Union (2003) reviewed unpublished 

preliminary data from a human dermal absorption study. 

Skin samples obtained from 3 human donors (6 samples/ 

donor/dose) were exposed to 5 or 50 mg/cm2 (3.18 or 

31.8 mg/mL) 14C-bisphenol A in ethanol vehicle. Following evaporation of the vehicle, bisphenol A was 

resuspended in artificial sweat. Radioactivity was measured in receptor fluid at various time intervals over a 24­

hr period. Radioactivity was measured in the stratum 

corneum and ‘‘lower’’ skin layer at 24 hr. Authors of the 

European Union report noted that tritiated water was not 

used as a marker for skin integrity. However, based on 

the patterns of results, they concluded that skin integrity 

was likely lost after 4–8 hr. The European Union authors 

therefore concluded that the only reliable data from the 

study were those for the cumulative percentage of the 

dose in receptor fluid at 8 hr, which was reported at 0.57– 

1.22% at 5 mg/cm2 and 0.491–0.835% at 50 mg/cm2

. 

Because radioactivity in skin was not measured at 8 hr, 

the percentage of the applied dose remaining on skin and 

available for future absorption could not be determined. 

Based on ratios of receptor fluid concentrations and 

lower skin levels (1:2 to 1:8) at 24 hr, and assuming that 

the higher ratio applies to skin at 8 hr, the authors of the 

European Union report predicted that 10% of the dose 

would be present in ‘‘lower’’ skin layers. Therefore, 

dermal absorption of bisphenol A was estimated at 10%. 

2.1.1.2 Distribution: In humans, bisphenol A was 

measured in cord blood and amniotic fluid, demonstrating distribution to the embryo or fetus. Studies reporting 

bisphenol A concentrations in fetal and/or maternal 

compartments are summarized in Table 9. Detailed 

descriptions of those studies are also presented below. 

Engel et al. (2006) reported concentrations of bisphenol 

A in human amniotic fluid. Twenty-one samples were 

obtained during amniocentesis conducted before 20 

weeks gestation in women who were referred to a U.S. 

medical center for advanced maternal age. Bisphenol A 

concentrations in amniotic fluid were measured using LC 

with electrochemical detection. Bisphenol A was detected 

in 10% of samples at concentrations exceeding the LOD 

(0.5 mg/L). Bisphenol A concentration ranges of 0.5– 

1.96 mg/L were reported. 

Scho ̈nfelder et al. (2002b) examined bisphenol A 

concentrations in maternal and fetal blood and compared 

bisphenol A concentrations in blood of male and female 

fetuses. In a study conducted at a German medical 

center, blood samples were obtained from 37 Caucasian 

women between 32–41 weeks gestation. At parturition, 

blood was collected from the umbilical vein after 

expulsion of the placenta. Bisphenol A concentrations 

in plasma were measured by GC/MS. Control experiments were conducted to verify that bisphenol A did not 

leach from collection, storage, or testing equipment. 

Bisphenol A was detected in all samples tested, and 

concentrations measured in maternal and fetal blood are 

summarized in Table 9. Mean bisphenol A concentrations 

were higher in maternal (4.473.9 [SD] mg/L) than fetal 

blood (2.972.5 mg/L). Study authors noted that in 14 

cases fetal bisphenol A plasma concentrations exceeded 

those detected in maternal plasma. Among those 14 

cases, 12 fetuses were male. Analysis by paired 

t-test showed significantly higher mean bisphenol 

A concentrations in the blood of male than female 

Birth Defects Research (Part B) 83:157–395, 2008
184 CHAPIN ET AL. 

fetuses (3.572.7 vs. 1.771.5 ng/mL, P 5 0.016). Bisphenol A concentrations were measured in placenta samples 

at 1.0–104.9 mg/kg. 

Ikezuki et al. (2002) measured concentrations of 

bisphenol A in serum from 30 healthy premenopausal 

women, 37 women in early pregnancy, 37 women in late 

pregnancy, and 32 umbilical cord blood samples. Concentrations of bisphenol A were also measured in 32 

samples of amniotic fluid obtained during weeks 15–18 

of gestation, 38 samples of amniotic fluid obtained at fullterm cesarean section, and 36 samples of ovarian 

follicular fluid collected during in vitro fertilization 

procedures. [It was not stated if different sample types 

were obtained from the same subjects.] An ELISA 

method was used to measure bisphenol A concentrations 

and results were verified by HPLC. The mean7SD 

concentration of bisphenol A in follicular fluid was 

reported at 2.470.8 mg/L. As summarized in Table 7 for 

nonpregnant women and Table 9 for maternal and fetal 

samples, concentrations of bisphenol A in follicular fluid 

were similar to those detected in the serum of fetuses 

and pregnant and non-pregnant women and in amniotic 

fluid collected in late pregnancy (B1–2 mg/L). Bisphenol 

A concentrations in amniotic fluid samples collected in 

early pregnancy were B5-fold higher than in other 

samples, and the difference achieved statistical significance (Po0.0001). Study authors postulated that the 

higher concentrations of bisphenol A in amniotic fluid 

collected during gestation weeks 15–18 may have 

resulted from immature fetal liver function. They noted 

that according to unpublished data from their laboratory, 

the percentage of glucuronidated bisphenol A in midterm amniotic fluid was B34%, which is much lower 

than reported values for other human fluids (490%). 

Yamada et al. (2002) measured bisphenol A concentrations in maternal serum and amniotic fluid from 

Japanese women. Samples were collected between 

1989–1998 in women undergoing amniocentesis around 

gestation week 16. One group of samples was obtained 

from 200 women carrying fetuses with normal karyotypes, and a second group of samples was obtained from 

48 women carrying fetuses with abnormal karyotypes. 

An ELISA method was used to measure bisphenol A 

concentrations. [As discussed in Section 1.1.5, ELISA 

may overestimate bisphenol A.] Concentrations of 

bisphenol A measured in maternal plasma and amniotic 

fluid are summarized in Table 9. Median concentrations 

of bisphenol A in maternal serum (B2–3 mg/L) were 

significantly higher [B10-fold] than concentrations in 

amniotic fluid (B0–0.26 mg/L) in the groups carrying 

fetuses with normal and abnormal karyotypes. However, 

in 8 samples from women carrying fetuses with normal 

karyotypes, high concentrations (2.80–5.62 mg/L) of 

bisphenol A were measured in amniotic fluid. The 

study authors interpreted the data as indicating that 

bisphenol A does not accumulate in amniotic fluid in 

most cases but accumulation is possible in some 

individuals. Bisphenol A concentrations in maternal 

blood were significantly higher [by B33%] in woman 

carrying fetuses with abnormal versus normal karyotypes. However, the study authors noted that the 

effect may not be related to bisphenol A exposure 

because there was no adjustment for maternal age, and 

concentrations in amniotic fluid did not differ between 

groups. In the group carrying fetuses with normal 

karyotypes, data obtained from 1989–1998 were summarized by year. Median bisphenol A concentrations in 

serum significantly decreased over that time from a 

concentration of 5.62 mg/L detected in 1989 to 0.99 mg/L 

in 1998. 

Kuroda et al. (2003) used an HPLC method to measure 

bisphenol A concentrations in 9 sets of maternal and cord 

blood samples obtained from Japanese patients at the 

time of delivery. Bisphenol A concentrations were also 

measured in 21 sets of serum and ascitic fluid samples 

collected from sterile Japanese patients of unspecified 

sexes and ages. Results for pregnant women are 

summarized in Table 9. Mean7SD concentrations of 

bisphenol A were lower in maternal (0.4670.20 ppb [lg/ 

L]) than cord blood (0.6270.13 ppb [lg/L]). There was a 

weak positive correlation (r 5 0.626) between bisphenol 

A concentrations in maternal and cord blood. Concentrations of bisphenol A in the blood of sterile patients are 

summarized Table 7. There were no differences between 

pregnant and non-pregnant blood levels (Kuroda et al., 

2003). Mean7SD concentrations of bisphenol A were 

higher in ascitic fluid (0.5670.19 ppb [lg/L]) than in 

serum (0.4670.20 ppb [lg/L]). The correlation between 

bisphenol A concentration in serum and ascitic fluid was 

relatively strong (r 5 0.785). 

Tan and Mohd (2003) used a GC/MS method to 

measure bisphenol A concentrations in cord blood at 

delivery in 180 patients at a Malaysian medical center. 

Bisphenol A was detected in 88% of samples. As noted in 

Table 9 concentrations ranged from o0.10–4.05 mg/L. 

Calafat et al. (2006) reported a median bisphenol A 

concentration of B1.4 mg/L [as estimated from a graph] 

in milk from 32 women. Bisphenol A was measured after 

enzymatic hydrolysis of conjugates. Ye et al. (2006) found 

measurable milk concentrations of bisphenol A in 

samples from 18 of 20 lactating women. Free bisphenol 

A was found in samples from 12 women. The median 

total bisphenol concentration in milk was 1.1 mg/L 

(range: undetectable to 7.3 mg/L). The median free 

bisphenol A concentration was 0.4 mg/L (range: undetectable to 6.3 mg/L). 

Sun et al. (2004) used an HPLC method to measure 

bisphenol A concentrations in milk from 23 healthy 

lactating Japanese women. Bisphenol A concentrations 

ranged from 0.28–0.97 mg/L, and the mean7SD concentration was reported at 0.6170.20 mg/L. No correlations 

were observed between bisphenol A and triglyceride 

concentrations in milk. Values from six milk samples 

were compared to maternal and umbilical blood samples 

previously reported in a study by Kuroda et al. (2003). 

Bisphenol A values were higher in milk, and the milk/ 

serum ratio was reported at 1.3. Bisphenol A values in 

milk were comparable to those in umbilical cord serum. 

[It was not clear whether milk and serum samples were 

obtained from the same volunteers in the two studies.] 

Schaefer et al. (2000) measured concentrations of 

bisphenol A and other compounds in uterine endometrium of women undergoing hysterectomy for uterine 

myoma at a German medical center. Endometrial and fat 

samples were obtained between 1995–1998 from 23 

women (34–51 years old) with no occupational exposure. 

Samples were handled with plastic-free materials and 

stored in glass containers. Concentrations of environmental chemicals were measured in samples by GC/MS. 

None of 21 fat samples had detectable concentrations of 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 185 

bisphenol A. Bisphenol A was detected in 1 of 23 

endometrial samples; the median concentration was 

reported at o1 mg/kg wet weight, and the range was 

reported at 0–13 mg/kg. [It is not known why a median 

value and range were reported when bisphenol A was 

only detected in 1 sample.] 

As part of a study to compare an ELISA and an LC/MS 

method for biological monitoring of bisphenol A, Inoue 

et al. (2002) measured concentrations of bisphenol A in 

semen samples obtained from 41 healthy Japanese 

volunteers (18–38 years old). Analysis by the ELISA 

method indicated bisphenol A concentrations ranging 

from concentrations below the detection limit (2.0 mg/L) 

to 12.0 mg/L. The LC/MS method indicated that 

the bisphenol A concentration in all samples was 

o0.5 mg/L, the LOQ. The study authors concluded that 

the LC/MS method was more accurate and sensitive and 

that the ELISA method overestimated bisphenol A 

concentrations, possibly due in part to nonspecific antibody interactions. 

2.1.1.3 Metabolism: Vo ̈lkel et al. (2005) measured 

bisphenol A and metabolite concentrations in human 

urine following exposure to a low bisphenol A dose. The 

human volunteers consisted of 3 healthy females (25–32 

years old) and 3 healthy males (37–49 years old) who were 

asked to refrain from alcohol and medicine intake for 2 

days before and during the study. Volunteers received 

25 mg D16-bisphenol A in drinking water [0.00028– 

0.00063 mg/kg bw based on reported body weights],a 

dose reported to represent a worst-case human exposure. 

Urine samples were collected at 0, 1, 3, 5, and 7 hr 

following exposure. Analyses for D16-bisphenol A and 

D16-bisphenol A-glucuronide were conducted by LC/MS 

and HPLC. Recovery of D16-bisphenol A-glucuronide in 

urine within 5 hr of dosing was 85% of dose in males and 

75% of dose in females. Analysis following treatment of 

urine with glucuronidase resulted in recovery of 97% of 

the dose in males and 84% of the dose in females. The 

highest concentrations of bisphenol A glucuronide in 

urine were measured at 1 hr (221–611 pmol [50–139 ng 

bisphenol A eq]/mg creatinine) and 3 hr (117–345 pmol 

[27–79 ng bisphenol A eq]/mg creatinine) following 

exposure. Elimination half-life was estimated at 4 hr. 

Bisphenol A concentrations exceeding the detection limit 

were detected in only 2 urine samples at concentrations of 

B10 pmol [2 ng]/mg creatinine. 

Vo ̈lkel et al. (2002) examined toxicokinetics and 

metabolism of bisphenol A in humans administered a 

low dose. Volunteers in this study consisted of 3 healthy 

females (24–31 years of age) and 6 healthy males (28–54 

years of age) who were non- or occasional smokers; 

volunteers were asked to refrain from alcohol and 

medicine intake for 2 days before and during the study. 

In two different studies, D16-bisphenol A was orally 

administered to volunteers via gelatin capsules at a dose 

of 5 mg (0.054–0.090 mg/kg bw). The dose was reported 

to be B10-fold higher than the estimated human 

exposure level of 0.6 mg/day. In the first study, urine 

samples were collected at 6-hr intervals until 42 hr 

following exposure and blood samples were collected 

at 4-hr intervals until 32 hr following exposure in 3 males 

and 3 females. In a second, more detailed study 

conducted in 4 of the male volunteers, blood samples 

were collected at 30–60-min intervals until 381 min 

following exposure. Samples were analyzed by GC/MS 

and LC/MS. In the first study, a terminal half-life 

of 5.3 hr was reported for D16-bisphenol A glucuronide 

clearance from blood. The half-life for urinary 

elimination was reported at 5.4 hr. D16-Bisphenol A 

glucuronide concentrations in plasma and urine fell 

below LOD at 24–34 hr post-dosing. Complete urinary 

recovery (100%) was reported for the D16-bisphenol 

A glucuronide. In the second study, maximum 

plasma concentration of D16-bisphenol A glucuronide 

(B800 pmol [183 ng bisphenol A eq]/mL) was obtained 

80 min after oral administration. The half-life for 

initial decline in plasma was reported at 89 min. 

Free D16-bisphenol A was not detected in plasma. 

According to study authors, the study demonstrated 

rapid absorption of bisphenol A from the gastrointestinal 

tract, conjugation with glucuronic acid in the liver, and 

rapid elimination of the glucuronide in urine. Study 

authors noted that the rapid and complete excretion of 

bisphenol A glucuronide in urine suggested that in 

contrast to rats, enterohepatic circulation did not occur in 

humans. 

Table 8 in Section 1 provides information on bisphenol 

A and metabolites detected in human urine. A study 

conducted in the U.S. used an HPLC method to examine 

30 urine samples collected from a demographically 

diverse adult population in 2000–2004 (Ye et al., 2005). 

Mean urinary compound composition was 9.5% bisphenol 

A, 69.5% bisphenol A glucuronide, and 21% bisphenol A 

sulfate conjugate. A study conducted in Korea used an 

HPLC method to examine urine collected from 15 men 

(mean age 5 42.6 years) and 15 women (mean age 5 43.0 

years) (Kim et al., 2003b). Sex-related differences were 

observed for urinary metabolic profiles. Mean urinary 

compound composition in men was reported at 29.1% 

bisphenol A, 66.2% bisphenol A glucuronide, and 4.78% 

bisphenol sulfate conjugate. The urinary metabolite 

profile in females was 33.4% bisphenol A, 33.1% bisphenol 

A glucuronide, and 33.5% bisphenol A sulfate conjugate. 

The study authors concluded that women had a greater 

ability for sulfation than men. 

2.1.1.4 Excretion: As discussed in greater detail in 

Section 2.1.1.3, two studies in which human volunteers 

were administered low doses of D16-bisphenol A 

(B0.00028–0.090 mg/kg bw) demonstrated that most of 

the dose (85–100%) was eliminated through urine (Vo ̈lkel 

et al., 2002, 2005). In those studies, the half-lives for 

urinary elimination were reported at 4–5.4 hr. As discussed in more detail in Section 2.1.1.3, examination of 

human urine samples revealed that bisphenol A glucuronide and sulfate conjugates are present at higher 

concentrations than is the parent compound (Kim et al., 

2003b; Ye et al., 2005). 

2.1.2 Experimental animal. Original animal studies that were potentially important for the interpretation of developmental and reproductive toxicity were 

reviewed thoroughly. Examples included: 

*	 Studies examining toxicokinetics or metabolism in 

pregnant or lactating animals; 

*	 Studies examining toxicokinetic difference observed 

with different doses or exposure routes; 

*	 Studies looking at age-related differences in toxicokinetics or metabolism; and 

*	 Studies in non-rodent species such as primates. 

Birth Defects Research (Part B) 83:157–395, 2008
186 CHAPIN ET AL. 

Secondary sources were utilized for general information not considered critical to the interpretation of 

developmental and reproductive toxicity data. 

2.1.2.1 Absorption: In rats orally exposed to bisphenol A at doses r100 mg/kg bw, maximum bisphenol A concentrations (Cmax) were generally 

measured in plasma within 0.083–0.75 hr following 

exposure (Pottenger et al., 2000; Takahashi and Oishi, 

2000; Yoo et al., 2001; Domoradzki et al., 2004; Negishi 

et al., 2004b). At doses of 1 or 10 mg/kg bw, time to 

maximum bisphenol A concentration (Tmax) in plasma 

was longer in postnatal day (PND) 21 rats (1.5–3 hr) than 

in PND 4 and 7 rats (0.25–0.75 hr) (Domoradzki et al., 

2004). In a limited number of studies in which rats were 

subcutaneously (s.c.) dosed with up to 100 mg/kg bw 

bisphenol A, time (0.5–4 hours) to reach Cmax was longer 

than with oral dosing, although the findings were not 

always consistent (Pottenger et al., 2000; Negishi et al., 

2004b). In one study, Tmax was comparable in oral and 

intraperitoneal (i.p.) dosing of rats (Pottenger et al., 2000). 

Another study reported that Cmax was attained at 0.7 hr 

in monkeys orally exposed to 10 or 100 mg/kg bw 

bisphenol A and at 0.5 hr in chimpanzees orally exposed 

to 10 mg/kg bw bisphenol A (Negishi et al., 2004b). In 

the same study, a longer Tmax (2 hr) was observed 

following exposure of monkeys and chimpanzees to the 

same doses by s.c. injection compared to oral intake. 

Additional details for these studies are presented below. 

As discussed in greater detail in Section 2.1.2.3, 

bisphenol A is glucuronidated in the liver and intestine, 

and most of the dose is absorbed as bisphenol A 

glucuronide following oral exposure of rats (Domoradzki 

et al., 2004). In ovariectomized rats gavaged with 

bisphenol A, bioavailability of bisphenol A was reported 

at 16.4% at a 10 mg/kg bw dose and 5.6% at a 100 mg/kg 

bw dose (Upmeier et al., 2000). The findings are fairly 

consistent with a second study in which maximum 

plasma values of free bisphenol A represented low 

percentages [o2–8%] of the total radioactive dose in rats 

orally administered bisphenol A at 10 or 100 mg/kg bw 

(Pottenger et al., 2000); maximum values of free bisphenol A represented higher percentages of the radioactive dose in rats given 10 or 100 mg/kg bw s.c. [64–82% 

free bisphenol A] or i.p. [19–54%] (Pottenger et al., 2000). 

Percentages of parent bisphenol A in blood were also 

higher in monkeys exposed intravenously (i.v.; 5–29%) 

than orally (0–1%) (Kurebayashi et al., 2002). Similarly, 

HPLC analysis of plasma conducted 1 hr following s.c. or 

gavage dosing of 4 female 21-day-old Sprague–Dawley 

rats/group with bisphenol A revealed higher bisphenol 

A plasma concentrations with s.c. than with gavage 

dosing (Table 22) (Yamasaki et al., 2000). One study in 

male and female rats gavaged with 10 mg/kg bw 

bisphenol A demonstrated higher plasma concentrations 

of bisphenol A in immature animals than in adults (10.2– 

48.3 mg/g [mg/L] plasma at 4 days of age; 1.1–1.4 mg/g 

[mg/L] plasma at 7 days of age; 0.2 mg/g [mg/L] plasma 

at 21 days of age; and 0.024–0.063 mg/g [mg/L] plasma in 

adulthood) (Domoradzki et al., 2004). 

A review by the European Union (2003) noted that in 

the study by Pottenger et al. (2000), fecal excretion 

represented the highest proportion of the eliminated 

dose (74–83% in males and 52–72% in females) following 

oral or parenteral exposure of rats to 10 or 100 mg/kg bw 

bisphenol A. The authors of the European Union report 

Table 22 

Plasma Bisphenol A Concentrations in 21-Day-Old Rats 

at 1 Hr Following Oral Gavage or S.C. Dosinga 

Plasma concentration, mg/L 

Dose, mg/kg bw Injection (s.c.) Oral gavage 

0 (sesame oil vehicle) Not detected Not detected 

8 94.6758.0 Not examined 

40 886.3756.4 Not detected 

160 29487768.8 198.8788.2 

800 Not examined 2879.072328.3 

Values presented as mean7SD. a

Yamasaki et al. (2000). 

therefore concluded that absorption [assumed to be of 

the radioactive dose] is likely extensive following oral 

intake. Adding to the proof of extensive oral absorption 

is the observation that 450% of fecal elimination 

occurred at 24 hr post-dosing, a time period beyond the 

average gastrointestinal transit time of 12–18 hr for rats. 

Possible explanations provided for the detection of 

parent compound in feces were cleavage of conjugates 

within intestines and enterohepatic circulation. 

2.1.2.2 Distribution 

2.1.2.2.1 Pregnant or lactating animals: Information on 

distribution in pregnant or lactating rats is presented first 

followed by other species. Studies including oral 

exposures are summarized before those with parenteral 

exposures. 

Takahashi and Oishi (2000) examined disposition and 

placental transfer of bisphenol A in F344 rats. Rats were 

orally administered 1000 mg/kg bw bisphenol A (495% 

purity) in propylene glycol on gestation day (GD) 18 (GD 

0 5 day of vaginal plug). Rats were killed at various time 

points between 10 min and 48 hr after bisphenol A 

dosing. At each time point, 2–6 dams and 8–12 fetuses 

obtained from 2–3 dams were analyzed. Blood was 

collected from dams and kidneys, livers, and fetuses 

were removed for measurement of bisphenol A concentrations by HPLC. Results are summarized in Table 23. 

Study authors noted the rapid appearance of bisphenol A 

in maternal blood and organs and in fetuses. Concentrations of bisphenol A at 6 hr following dosing were 2% of 

peak concentrations in maternal blood and 5% of peak 

concentrations in fetuses. It was noted that in fetuses, 

area under the time-concentration curve (AUC) was 

higher and mean retention time, variance of retention 

time, and terminal half-life were longer than in maternal 

blood. 

Dormoradzki et al. (2003) examined metabolism, 

toxicokinetics, and embryo-fetal distribution of bisphenol 

A in rats during 3 different gestation stages. Sprague– 

Dawley rats were gavaged with bisphenol A (99.7% 

purity)/radiolabeled 14C-bisphenol A (98.8% radiochemical purity) at 10 mg/kg bw. Bisphenol A was administered to 1 group of non-pregnant rats and 3 different 

groups of pregnant rats on GD 6 (early gestation), 14 

(mid gestation), or 17 (late gestation). GD 0 was defined 

as the day that sperm or a vaginal plug were detected. 

Blood, urine, and feces were collected at multiple time 

points between 0.25 and 96 hr post-dosing. It appears 

that most and possibly all samples were pooled. Four rats 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 187 

in each group were killed at 96 hr post-dosing. Maternal 

organs, 6 embryos or fetuses/dam (when possible), and 

placentas were collected. Samples were analyzed for 

radioactivity and bisphenol A and/or bisphenol A 

glucuronide by HPLC/liquid scintillation spectrometry. 

In all groups, 90–94% of radioactivity was recovered. 

Elimination of bisphenol A and its metabolites is 

discussed in Section 2.1.2.4. At 96 hr following dosing, 

low percentages of the dose were present in carcass (B1– 

6%) and tissues such as brain, fat, liver, kidney, ovary, 

uterus, and skin. The only quantifiable data in placentas 

and fetuses at 96 hr were obtained in the GD 17 group, 

and those samples contained 0.01–0.07% of the bisphenol 

A dose. Standard deviations for maternal and fetal 

tissues generally exceeded 50% of the mean. Study 

authors concluded that disposition of radioactivity was 

similar in pregnant and non-pregnant rats. 

Toxicokinetic data obtained from plasma profiles are 

summarized in Table 24. The authors stated that there 

was high inter-animal variability. The presence of two 

Cmax values was noted by the authors, and they stated 

that it was the result of enterohepatic circulation of 

radioactivity. Bisphenol A was not quantifiable in most 

plasma samples. Because bisphenol A glucuronide 

represented most (B95–99%) of the radioactivity, plasma 

profiles for that metabolite were nearly identical to 

profiles for radioactivity. 

Table 23
 

Toxicokinetic Endpoints for Bisphenol A in Rats Dosed
 

With 1000 mg/kg bw Bisphenol A on GD 18a
 

Maternal tissue 

Endpoint Blood Liver Kidney Fetus 

Cmax, mg/L 14.7 171 36.2 9.22 

Tmax, min 20 20 20 20 

AUC, mg hr/L 13.1 700 84.0 22.6 

Mean retention time, hr 10.6 29.3 12.0 20.0 

Variance in retention 203 657 227 419 

time, hr squared 

Half-life, hr 

From 20–40 min 0.0952 0.178 0.245 0.55 

From 40 min–6 hr 2.58 1.75 2.98 1.60 

From 6–48 hr 4.65 No data No data 173 

a

Takahashi and Oishi (2000). 

A second study was conducted by Dormoradzki et al. 

(2003) to measure bisphenol A and bisphenol A 

glucuronide concentrations in maternal and fetal tissues. 

Rats were gavaged with radiolabeled bisphenol A at 

10 mg/kg bw on GD 11, 13, or 16. Blood was collected 

over a 24-hr period. Five rats/group/time period were 

killed at 0.25, 12, and 96 hr post-dosing. Maternal blood 

and organs, yolk sacs/placentas, and embryos/fetuses 

were removed for measurement of bisphenol A and 

bisphenol A glucuronide. Yolk sacs/placentas and 

fetuses were pooled at most time periods. Results are 

summarized in Table 25. 

At 0.25 hr following dosing, bisphenol A glucuronide 

concentrations in maternal plasma were similar in 

groups dosed on GD 11 and 13 but concentrations were 

1.7–2 times higher in the group dosed on GD 16. At 12 hr 

post-dosing in all exposure groups, bisphenol A glucuronide concentrations in maternal plasma were reduced 7­

to 11-fold from values observed at 0.25 hr. Levels of 

radioactivity in plasma were not sufficient for analysis at 

96 hr post-dosing. Bisphenol A was detected in maternal 

plasma at 0.25 hr post-dosing in rats that were exposed to 

a higher radioactive concentration (0.5 mCi compared to 

0.2 mCi) on GD 16; bisphenol A concentrations were 

26.5-fold lower than bisphenol A glucuronide 

concentrations. 

In animals dosed on GD 11, bisphenol A glucuronide 

was only detected in yolk sac/placenta at 0.25 hr postdosing and the concentration was B17 times lower than 

the concentration detected in maternal blood for the 

same time period. With dosing on GD 11, bisphenol A 

glucuronide was not detected in embryos and bisphenol 

A was not detected in yolk sac/placenta or embryos. In 

animals dosed on GD 13, bisphenol A glucuronide was 

detected in yolk sac/placenta at 0.25 and 12 hr postdosing and concentrations were 9–24-fold lower than 

those detected in maternal plasma for the same time 

period. Bisphenol A was also detected in yolk sac/ 

placenta at 0.25 and 12 hr after dosing on GD 13 and 

concentrations were similar to those detected in the 

blood of 2 dams. A lower concentration of bisphenol A 

was detected in embryos of dams at 0.25 hr following 

dosing on GD 13, and bisphenol A was the only moiety 

detected in embryos. Following dosing on GD 16, 

bisphenol A glucuronide and bisphenol A were detected 

in yolk sac/placenta at 0.25 and 12 hr post-dosing. 

Concentrations of bisphenol A glucuronide in yolk sac/ 

Table 24
 

Toxicokinetic Data for Radioactivity in Pregnant and Non-Pregnant Rats Gavaged With 10 mg/kg bw 14C-bisphenol Aa
 

Endpoint Non-pregnant GD 6–10 GD 14–18 GD 17–21 

Cmax1, mg eq/L 0.716 0.370 0.482 1.006 

Tmax1, hr 0.25 0.25 0.25 0.25 

Cmax2, mg eq/L 0.171 0.336 0.211 0.278 

Tmax2, hr 18 12 24 12 

Time to non-quantifiable level, hr 72 Not determined 72 96 

AUC 14C, mg-eq � hr/L 6.1 12.4 7.1 10.2 

Bisphenol A glucuronide, 5.8 12.3 6.8 9.7 

mg-eq � hr/L 

Percent as bisphenol 95.1 99.2 95.8 95.1 

A glucuronide 

a

Dormoradzki et al. (2003). 

Birth Defects Research (Part B) 83:157–395, 2008
188 CHAPIN ET AL. 

Table 25 

Maternal and Fetal Concentrations of Bisphenol A Following Gavage Dosing of Dams With 10 mg/kg bw Bisphenol Aa 

Bisphenol A concentration, mg/L or mg/kg 

Maternal plasma Yolk sac/placenta Embryo/fetus 

Exposure Glucuronide Parent Glucuronide Parent Glucuronide Parent 

GD 11, 0.2 mCi 

0.25 hr 1.06070.258 0.041 0.062 oLODb oLOD oLOD 

12 hr 0.09970.036 oLOD oLOD oLOD oLOD oLOD 

96 hr NA NA oLOD oLOD oLOD oLOD 

GD 13, 0.2 mCi 

0.25 hr 0.86870.189 0.078 0.036 0.019 oLOD 0.013 

12 hr 0.11770.033 0.008 0.013 0.009 oLOD oLOD 

96 hr Not analyzed due to insufficient radioactivity 

GD 16, 0.2 mCi 

0.25 hr 1.76870.783 0.485, 0.129c 0.22370.104 0.16670.069 0.031, 0.009c 0.122, 0.020c 

12 hr 0.17470.045 oLOD 0.02570.005 0.03470.002 NA NA 

96 hr Not analyzed due to insufficient radioactivity 0.016 0.008 

GD 16, 0.5 mCi 

0.25 hr 1.69970.501 0.06470.025 0.34270.104 0.09570.031 0.01370.008 0.01870.011 

Data expressed as mean7SD or single values for individual or pooled data.
 a

Dormoradzki et al. (2003).
 

b

Limit of detection (LOD) for bisphenol A reported at 0.005–0.029.
 

c

Detected only in two animals at the concentrations listed.
 

placenta were 7- to 8-fold lower than concentrations 

detected in maternal plasma. From 0.25 to 12 hr, 

concentrations of bisphenol A decreased 4.9-fold and 

concentrations of bisphenol A glucuronide decreased 9­

fold. Mean concentrations of bisphenol A in yolk/sac 

placenta following exposure on GD 16 were similar to the 

blood concentration detected in 1 of 2 dams. 

In yolk sac/placenta and fetuses of dams dosed with a 

higher level of radioactivity (0.5 mCi) on GD 16, 

bisphenol A glucuronide and bisphenol A were detected 

at 0.25 hr following dosing. Compared to concentrations 

detected in placenta, fetal concentrations of bisphenol A 

glucuronide were B26-fold lower and bisphenol A 

concentrations were 5-fold lower. Bisphenol A concentrations were lower than bisphenol A glucuronide 

concentrations by 3.6-fold in yolk sac/placenta and by 

0.7-fold in fetuses. Study authors concluded that there is 

no selective affinity for bisphenol A or bisphenol A 

glucuronide by the yolk sac/placenta or embryo/fetus. 

Kurebayashi et al. (2005) examined distribution of 

radioactivity in pregnant and lactating rats dosed with 

14C-bisphenol A. Pregnant rats were orally dosed with 

0.5 mg/kg bw 14C-bisphenol A on GD 12, 15, or 18. The 

rats were killed at 30 min or 24 hr following dosing 

(n 5 1/time period) and examined by whole-body radioluminography. Study authors noted that the distribution of label was nearly identical in dams at each 

gestation time point. At 30 min following dosing, the 

concentration of radioactivity in dam blood was B31– 

43 mg bisphenol A eq/L. The highest concentration of 

radioactivity was detected in maternal liver (B219– 

317 mg bisphenol A eq/kg) and kidney (B138–270 mg 

bisphenol A eq/kg); concentrations in other tissues 

(lung, ovary, placenta, skin, and uterus) were B10-fold 

lower. Fetuses, fetal membranes, and yolk sacs did not 

contain quantifiable levels of radioactivity at 30 min 

following maternal exposure at any gestation time point. 

At 24 hr following exposure of dams, radioactivity 

concentrations in blood (B4–11 mg bisphenol A eq/L) 

were nearly 3–10-fold lower than values obtained at 

30 min following exposure. Levels of radioactivity 

remained highest in liver. At 24 hr following exposure, 

radioactivity was only detected in fetuses and fetal 

tissues from dams dosed on GD 18. Radioactivity levels 

in fetuses or fetal tissues compared to maternal blood 

were B30% in fetuses, nearly equal in fetal membranes, 

and B5-fold higher in yolk sacs. Study authors concluded that there was limited distribution of radiolabel to 

fetuses. 

In another study by Kurebayashi et al. (2005), a 

lactating rat was orally dosed with 0.5 mg/kg bw 14Cbisphenol A on PND 11 and caged with 5 neonatal rats 

for 24 hours. One male and one female neonatal rat were 

killed at the end of the 24-hr period and examined by 

whole-body radioluminography. The 3 remaining neonates were caged for 24 hr with a dam that was not 

exposed to bisphenol A. One male and one female 

neonate were then killed and examined by whole-body 

radioluminography. In pups killed immediately after 

being nursed by the lactating dam exposed to 14Cbisphenol A, most of the radioactivity was detected in 

intestinal contents (B30–46 mg bisphenol A eq/kg) and 

lower levels were found in gastric contents and urinary 

bladder (o10 mg bisphenol A eq/kg). After being nursed 

for 24 hr by a dam that was not exposed to bisphenol A, 

radioactivity was only detected in intestinal contents and 

the level was B20–40% of that measured in pups 

examined immediately after being nursed by dams 

receiving 14C-bisphenol A. 

An additional 3 lactating dams were dosed with 

0.5 mg/kg bw 14C-bisphenol A on PND 11 for examination of radioactivity in plasma and milk over a 48-hr 

period. Table 26 summarizes toxicokinetic endpoints for 

radioactivity in milk and plasma. Study authors concluded that there was significant secretion of 14Cassociated radioactivity into milk. 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 189 

Table 26
 

Toxicokinetic Endpoints for Radioactivity in Lactating
 

Rats Orally Administered 0.5 mg/kg bw 14C-Bisphenol A
 

on PND 11a
 

Endpoint Milk Maternal plasma 

Cmax, mg-eq/L 4.46 27.2 

Tmax, hr 8 4 

Elimination half-life, hr 26 31 

AUC (0–48 hr), mg-eq � hr/L) 156 689 

a

Kurebayashi et al. (2005). 

Miyakoda et al. (1999) examined placental transfer of 

bisphenol A in rats. Wistar rats were administered an 

oral dose of bisphenol A (99% purity) at 10 mg/kg bw on 

GD 19. Blood was collected and fetuses were removed at 

1, 3, and 24 hr following dosing. Bisphenol A concentrations were measured in plasma and fetuses by GC/MS. 

[A statement in Figure 3 of the study indicated that 

values were the means of 5 or 7 experiments; it is 

possible the authors meant that 5 or 7 dams were 

dosed.] Concentrations of bisphenol A peaked in 

maternal plasma and fetuses within 1 hr of dosing, with 

bisphenol A concentrations measured at B34 ppb [lg/L] 

in maternal plasma and 11 ppb [lg/kg] in fetuses. At 3 hr 

after dosing, bisphenol A concentrations were B10% of 

peak concentrations in maternal plasma and 40% of peak 

concentrations in fetuses. At 24 hr post-dosing, bisphenol 

A concentrations in fetuses were detected at 70% of peak 

value and concentrations in fetuses were more than twice 

the concentrations in maternal plasma. Study authors 

concluded that bisphenol A is rapidly transferred to the 

fetus and tends to remain longer in fetuses than in 

maternal blood. 

Snyder et al. (2000) examined the toxicokinetics of 

bisphenol A in lactating rats. On PND 14, lactating CD 

rats were gavaged with 100 mg/kg bw 14C-bisphenol A. 

Milk, blood, and organs were collected from 2–4 dams/ 

group at 1, 8, 24, or 26 hr after dosing. [While the text 

indicates collection of samples at 26 hr, Table 3 of the 

study indicates collection at 24 hr. The collection time 

reported in the study table was used when there were 

discrepancies between text and table.] Animals were 

injected with oxytocin before milk collection. Radioactivity in pup carcasses was measured at 2, 4, 6, and 

24 hr following exposure of dams; 8–16 pups/time 

period were examined [pup data does not appear to be 

analyzed by litter]. Samples were analyzed by scintillation counting, HPLC, and/or nuclear magnetic resonance. At 1 and 8 hr following exposure, the highest 

percentage of the radioactive dose was detected in 

intestine with contents (75–83%). Among the other 

organs examined, the highest percentage of the radioactive dose was detected in liver (0.38–0.74%) and much 

lower percentages were detected in kidney and lung 

(r0.02%). Low percentages of the radioactive dose were 

also detected in milk (r0.0020%), blood (B0.006%), 

plasma (B0.01%), and fat (r0.004%). Compared to 

earlier time periods, radioactivity levels were lower at 

24 hr post-dosing (26% of the dose detected in intestine 

and contents), but distribution was similar. At all 3 

sampling time points, radioactivity levels were highest in 

plasma 4 blood 4 milk. The major radioactivity peak in 

plasma was represented by bisphenol A glucuronide at 1, 

8, and 26 hr following exposure. Bisphenol A glucuronide also represented the major radioactive peak 

detected in milk. Radioactivity levels in pups amounted 

to o0.01% of the maternal dose. Radioactivity levels in 

pups tended to increase over time. From 2–24 hr 

following exposure, mean7SD radioactivity levels rose 

from 44724 to 78711 mg bisphenol A eq/pup. 

Yoshida et al. (2004) compared bisphenol A concentrations in rats and their offspring during the lactation 

period. The main focus of the study was developmental 

toxicity, which is discussed in Section 3.2.3.2. In the 

distribution study, Donryu rats (12–19/group) were 

gavaged with bisphenol A at 0 (carboxymethylcellulose 

solution), 0.006, or 6 mg/kg bw/day from GD 2 to the 

day before weaning (21 days post-delivery). Bisphenol A 

concentrations were measured in maternal and pup 

serum, milk, and pup liver by GC/MS on PND 10, 14, 

and/or 21. Milk samples were obtained from pup 

stomachs. Pup serum and liver samples were pooled. 

Two to six dams/litter were examined in each dose 

group and time period. Samples of tap water, drinking 

water from plastic containers, and feed were measured 

for bisphenol A content by HPLC. Bisphenol A was not 

detected in fresh tap water but was detected at B3 mg/L 

following storage of that water in plastic containers. 

Bisphenol A concentration in feed was B40 mg/kg. 

Results for maternal and fetal tissues are summarized 

in Table 27. Bisphenol A concentrations in the serum of 

high-dose-dams were significantly elevated compared to 

the control group on PND 21. No other significant 

differences were observed in bisphenol A concentrations 

in samples between treated and control groups. 

Kim and Huang (2003) used an HPLC method to 

measure bisphenol A concentrations in rat dams and 

their offspring. Dams were gavaged with bisphenol A 

(499.7% purity) at doses of 0 (corn oil vehicle), 0.002, 

0.020, 0.200, 2, or 20 mg/kg bw/day on GD 7–17. Dams 

and offspring were killed at 21 days following parturition, and serum was collected for measurement of 

bisphenol A. Development effects observed in this study 

are summarized in Section 3.2.1.1. Bisphenol A was not 

detected in the serum of dams at the two lowest doses. 

Respective concentrations of bisphenol A in the serum of 

dams at the 3 highest doses were 0.900, 0.987, and 

1.00 mg/L. In offspring, bisphenol A was not detected in 

serum at the 3 lowest doses. At the 2 highest doses, the 

respective concentrations of bisphenol A in offspring 

were 0.69 and 0.74 mg/L in males and 0.71 and 0.82 mg/ 

L in females. 

Shin et al. (2002) examined elimination of bisphenol A 

from maternal–fetal compartments of rats. On 1 day 

between GD 17 and 19, four Sprague–Dawley rats were 

i.v. injected with 2 mg/kg bw bisphenol A. Amniotic 

fluid, placenta, and fetuses were collected at multiple 

intervals between 5 min and 8 hr following injection. 

Bisphenol A concentrations in samples were measured 

by HPLC. Transfer rate constants and clearance rates 

were determined using a five-compartment model 

consisting of maternal central, maternal tissue, placental, 

fetal, and amniotic fluid compartments. Toxicokinetic 

findings are summarized in Moors et al. (2006) evaluated 

the kinetics of bisphenol A in pregnant rats on GD 18 

after a single i.v. dose of 10 mg/kg bw. Unconjugated 

bisphenol A represented almost 80% of total bisphenol A 

Birth Defects Research (Part B) 83:157–395, 2008
190 CHAPIN ET AL. 

Table 27 

Bisphenol A Concentrations in Maternal and Pup Samples During Lactation in Rats Gavaged With Bisphenol Aa 

Dose group, mg/kg bw/day 

0 0.006 6 

Sample Time of analysis Sex Bisphenol A concentration, ppb [mg/L or mg/kg] 

Damb 

Serum PND 21 370 470 1174 

Milk PND 10 2879 8721 873 

PND 14 255778 20577 185750 

Pupc 

Serum PND 10 Female 4 10 23 

Male 15 5 7 

PND 14 Female 5 4 3 

Male 4 5 4 

PND 21 Female 9 3 9 

Male 14 9 20 

Liver PND 10 Female 13 12 17 

Male 9 9 14 

Pupc 

Liver PND 14 Female 22 100 18 

Male 45 14 16 

PND 21 Female 60 70 37 

Male 69 9 60 

a

Yoshida et al. (2004).
 

b

Values are presented as mean7SD.
 c

Pup samples were pooled.
 

Table 28 

Toxicokinetic Endpoints for Bisphenol A in Pregnant Rats iv Dosed With 2 mg/kg bw Bisphenol Aa 

Endpoint 

Compartment 

Maternal serum Placenta Fetus Amniotic fluid 

AUC, mg � hr/L 

Elimination half-life, hr 

Mean residence time, hr 

Cmax, mg/L 

Tmax, hr 

905.57275.8 

2.570.9 

3.071.1 

927.37194.3 

No data 

40097962.7 

2.270.8 

2.070.5 

1399.27323.7 

0.170.1 

1964.77678.5 

2.270.8 

3.070.9 

7947360.6 

0.670.3 

180.47102.0 

3.973.1 

5.674.7 

75.1759.7 

0.370.2 

Values presented as mean7SD. a

Shin et al. (2002). 

Table 29 

Intercompartmental Transfer and Clearances in Pregnant Rats Following Intravenous Bisphenol Aa 

Compartment Transfer rate constant, hr -1 Clearance rate mL/min 

Maternal central to maternal tissue 

Maternal tissue to maternal central 

Maternal central to placental 

Placental to maternal central 

Placental to fetal 

Fetal to placental 

Fetal to amniotic fluid 

Fetal 

Amniotic fluid to fetal 

Amniotic fluid to placental 

Placental to amniotic fluid 

Maternal central 

3.472.6 

1.771.3 

0.770.5 

23.6714.7 

46.4729.2 

22.8728.0 

0.0000170.00002 

0.006270.0044 

14.0721.0 

7.976.7 

1.071.3 

0.970.6 

38.2726.5 

50.2736.7 

8.375.4 

2.271.3 

4.172.1 

7.676.0 

0.0000170.00001 

0.002470.0015 

0.871.1 

0.770.7 

0.170.1 

9.775.3 

Values presented as mean7SD. a

Shin et al. (2002). 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 191 

Table 30 

Toxicokinetic Endpoints in Lactating Rats Infused With Bisphenol Aa 

Endpoint 

Bisp

0.13 

henol A infusion rate, mg

0.27 

/hr 

0.54 

Systemic clearance, mL/min/kg 119.2723.8 142.4745.3 154.1744.6 

Steady state serum bisphenol A concentration, ng/mL 66.1715.5 120.0734.7 217.1765.0 

Steady state milk bisphenol A concentration, ng/mL 173.1743.3 317.47154.4 493.97142.2 

Milk/serum ratio 2.770.9 2.671.2 2.470.6 

Data presented as mean7SD. a

Yoo et al. (2001). 

5 min after injection, 50% of total bisphenol A 20 min 

after injection, and B10% of total bisphenol A 6 hr after 

the injection. The half-life of free bisphenol A in the 

dam’s blood was 0.34 hr, and the half-life of total 

bisphenol A was 0.58 hr. Bisphenol A in fetal tissues 

peaked 20–30 min after maternal injection at 4.0 mg/kg in 

placenta, 3.4 mg/kg in fetal liver, and 2.4 mg/kg in 

remaining fetal tissues. Peak maternal blood bisphenol A 

had been 3.8 mg/L shortly after injection. 

Rapid distribution of bisphenol A was observed in 

placenta, fetus, and amniotic fluid. Bisphenol A concentrations in placenta and fetus remained higher than those 

in maternal serum over most of the sampling period. 

Amniotic fluid contained the lowest concentration of 

bisphenol A. Decay curves in amniotic fluid, fetus, and 

placenta paralleled decay curves in maternal serum. 

Transfer rate constants and clearance rates are summarized in Table 29. Transfer rate constants were greater in 

the direction of amniotic fluid to fetus or placenta than in 

the opposite direction. The elimination rate constant and 

clearance rate from the fetal compartment were much 

lower than for the maternal central compartment. The 

clearance rate from placenta to fetus was higher than 

clearance rate from fetus to placenta. The authors 

calculated that 65.4% of the bisphenol A dose was 

delivered to the fetus, 33.2% to the maternal central 

compartment, and 1.4% to amniotic fluid. According to 

the study authors, the low transfer rate from the fetal to 

amniotic compartment suggested minimal fetal excretion 

of unchanged bisphenol A through urine and feces into 

the amniotic fluid. They also noted that the small fetal 

compartment transfer constant compared to the relative 

fetal–placental transfer constant indicated minimal metabolism by the fetus. Authors estimated that 100% of 

bisphenol A was eliminated from the fetus via the 

placental route and concluded that fetal elimination 

represents 0.05% of total elimination from the maternal– 

fetal unit. 

Moors et al. (2006) evaluated the kinetics of bisphenol 

A in pregnant rats on GD 18 after a single i.v. dose of 

10 mg/kg bw. Unconjugated bisphenol A represented 

almost 80% of total bisphenol A 5 min after injection, 50% 

of total bisphenol A 20 min after injection, and B10% of 

total bisphenol A 6 hr after the injection. The half-life of 

free bisphenol A in the dam’s blood was 0.34 hr, and the 

half-life of total bisphenol A was 0.58 hr. Bisphenol A in 

fetal tissues peaked 20–30 min after maternal injection at 

4.0 mg/kg in placenta, 3.4 mg/kg in fetal liver, and 

2.4 mg/kg in remaining fetal tissues. Peak maternal 

blood bisphenol A had been 3.8 mg/L shortly after 

injection. 

Yoo et al. (2001) examined mammary excretion of 

bisphenol A in rats. At 4–6 days postpartum, 4–6 

lactating female Sprague–Dawley rats/group were i.v. 

injected with bisphenol A at 0.47, 0.94, or 1.88 mg/kg bw 

and then infused with bisphenol A over a 4-hr period at 

rates of 0.13, 0.27, or 0.54 mg/hour. Blood samples were 

collected at 2, 3, and 4 hr, and milk was collected at 4 hr 

following initiation of infusion. Before collection of milk, 

rats were injected with oxytocin to increase milk 

production. HPLC was used to measure bisphenol A 

concentrations in serum. Differences in data for mean 

systemic clearance were analyzed by analysis of variance 

(ANOVA). Results are summarized in Table 30. The 

study authors noted extensive excretion of bisphenol A 

into milk, with milk concentrations exceeding serum 

concentrations. No significant differences were reported 

for systemic clearance rates between the 3 doses. Steady 

state concentrations of bisphenol A in maternal serum 

and milk increased linearly according to dose. 

Kabuto et al. (2004) reported bisphenol A concentrations in mice indirectly exposed to bisphenol A during 

gestation and lactation. The focus of the study was 

oxidative stress; more details are presented in Section 

3.2.7. Six ICR mouse dams were given drinking water 

containing 1% ethanol vehicle or bisphenol A at 5 or 

10 mg/L. [Based on the reported water intake of 5 mL/ 

day and an assumed body weight of 0.02 kg (USEPA, 

1988), it is estimated that bisphenol A intakes in mice at 

the start of pregnancy were 0.0013 and 0.0025 mg/kg bw/ 

day.] Mice gave birth about 3 weeks following mating 

and pups were housed with dams for 4 weeks. [Based on 

an assumed body weight of 0.0085 kg and assumed 

water intake rate of 0.003 L/day (USEPA, 1988), it is 

estimated that intake of bisphenol A in weanling males 

was 0.0018 and 0.0035 mg/kg bw/day.] At 4 weeks of age, 

male pups were killed and a GC/MS technique was used 

to measure bisphenol A concentrations in brain, kidney, 

liver, and testis in an unspecified number of control pups 

and in four pups from the 10 mg/L group. Study authors 

reported that they could not detect bisphenol A in control 

pups. In pups from the 10 mg/L group, the highest 

concentration of bisphenol A was detected in kidney 

(B24 mg/kg wet weight), followed by testis (B20 mg/kg 

wet weight), brain (B18 mg/kg wet weight), and liver 

(B11 mg/kg wet weight). 

Zalko et al. (2003) examined metabolism and distribution of bisphenol A in pregnant CD-1 mice. A series of 

studies was conducted in which mice were treated with 

3

H-bisphenol A (499.9% purity)/unlabeled bisphenol A 

(499% purity). Mice were exposed to different regimens; 

biological samples examined included blood, liver, fat, 

Birth Defects Research (Part B) 83:157–395, 2008
192 CHAPIN ET AL. 

Table 31
 

Qualitative Analysis of Maternal and Fetal Tissues Following Injection of Mice With 0.025 mg/kg bw Radiolabeled
 

Bisphenol A on GD 17a
 

Bisphenol A-associated compound detected 

Hydroxylated glucuronide Double glucuronide Metabolite Fb Glucuronide Parent Others 

Hr after dose 11.5 ng/g % 17.5 ng/g % 24.0 ng/g % 25.0 ng/g % 33.5 ng/g % % 

Maternal plasma 

0.5 0.0770.01 3 0.1170.02 4 0.1170.02 4 1.0170.19 39 1.0670.19 41 9 

2 0.0270.01 2 0.0370.01 4 0.0370.01 4 0.5570.14 63 0.1570.04 17 10 

24 0.0470.04 20 0 0 0.1370.05 65 0 15 

Placenta 

0.5 0 0 0.4670.48 2 5.5074.24 25 15.98712.02 72 1 

2 0.0370.02 1 0.0470.03 1 0.3770.07 7 3.1372.34 62 1.3270.95 26 3 

24 0.0570.04 5 0.0470.02 4 0.6470.19 59 0.2170.22 19 0.0670.04 6 6 

Fetus 

0.5 0.0570.03 1 0.0470.04 0 0.4670.27 5 3.8372.65 44 4.2072.16 49 1 

2 0.0270.02 1 0.0170.02 0 0.3770.22 13 1.9370.45 66 0.4870.55 16 3 

24 0.0170.01 1 0 0.1170.07 13 0.5170.12 60 0.1370.16 15 2 

Amniotic fluid 

0.5 0.1070.14 1 0.1970.14 2 0.0970.13 1 8.1776.55 83 0.9070.89 9 4 

2 0.0670.03 1 0.0770.03 1 0.2670.15 5 4.8274.81 88 0.1070.07 2 2 

24 0.1370.05 8 0.0170.02 1 0.3770.09 24 0.7070.13 44 0.0370.03 2 20 

Maternal liver 

0.5 0.1270.12 0 0.1870.24 0 6.2271.75 18 12.9072.81 37 10.8572.77 31 12 

2 0.0870.08 1 0.7770.25 8 2.1670.91 20 4.9571.82 45 1.5170.97 13 13 

24 0.1670.14 2 0.3570.13 7 0.9970.42 16 2.5671.62 36 1.7271.18 23 17 

Data presented as mean7SD
 a

Zalko et al. (2003).
 

b

Most likely bisphenol A glucuronide conjugated to acetylated galactosamine or glucosamine.
 

gall bladder, uterus, ovaries, digestive tract and contents, 

urine, and feces. In the first exposure scenario, mice were 

s.c. injected with 0.025 mg/kg bw labeled/unlabeled 

bisphenol A on GD 17; three animals/time period were 

examined at 0.5, 2, and 24 hr following dosing. In the 

second exposure scenario, 2 mice/group were s.c. 

injected with 50 mg/kg bw bisphenol A on GD 17 and 

killed 24 hr following dosing. In the third scenario, 3 nonpregnant female mice/group were ‘‘force-fed’’ a single 

oral dose of 0.025 mg/kg bw bisphenol A; urine and feces 

were collected over 24 hr, and animals were killed at 

24 hr. Biological samples were analyzed by scintillation 

analysis, HPLC, MS, and/or nuclear magnetic resonance. 

In pregnant mice injected with 0.025 mg/kg bw/day 

bisphenol A and examined 24 hr later, 85.68% of the 

radioactivity was recovered. The highest percentages of 

radioactivity were detected in the digestive tract and 

contents (B45%) and feces (B21%). Less radioactivity 

was detected in the litter (B4%), liver (B2%), bile 

(B2%), urine (B6%), and carcass (B3%). Blood, ovaries, 

uterus, placenta, amniotic fluid, fat, and cage washes 

each contained o1% of the radioactive dose. At 0.5 hr 

following dosing, levels of radioactivity were highest in 

uterus 4 liver 4 placenta 4 fetus 4 amniotic fluid 4

ovaries 4 carcass 4 blood. Radioactivity levels in tissues 

were lower by 24 hr following exposure. [Compared to 

radioactive levels detected in tissues at 24 hr, levels 

detected at 0.5 hr were B12-fold higher in uterus, 3-fold 

higher in liver, 8-fold higher in placenta, 3.5-fold 

higher in fetuses, 2-fold higher in amniotic fluid, and 

3.5-fold higher in ovaries.] The only information 

provided for mice s.c. dosed with 50 mg/kg bw 

bisphenol A and examined 24 hr later was for radioactivity levels in organs; the highest levels (pg/g) were 

detected in uterus 4 blood 4 ovary 4 carcass 4 liver. 

Study authors stated that distribution of radioactivity 

was comparable in mice treated with 50 and 0.025 mg/kg 

bw bisphenol A. In the mice orally dosed with 0.025 mg/ 

kg bw bisphenol A and examined 24 hr later, levels of 

radioactivity in blood, ovaries, and uterus were reported 

to be significantly lower [by B1–2 orders of magnitude] 

than levels in animals exposed by s.c. injection, but the 

level in the liver was not significantly different. There 

was significantly more residue in mouse carcass after 

oral than s.c. dosing (B2.5 fold) (A. Soto, personal 

communication, March 2, 2007). No qualitative differences in metabolites were observed following oral or s.c. 

exposure. [Data were not shown by study authors.] 

Distribution of parent compound and metabolites detected in maternal and fetal tissues is summarized in 

Table 31. Further discussion on metabolites is included in 

Section 2.1.2.3. 

Uchida et al. (2002) examined distribution of bisphenol 

A in pregnant mice and monkeys. On GD 17 (GD 0 5 day 

of vaginal plug), ICR mice were s.c. injected with 

bisphenol A 100 mg/kg bw in sesame oil vehicle. More 

than 3 mice/time point were killed at various points 

between 0.5–24 hr following injection. An untreated 

control group consisted of 6 mice. [Data were not 

presented for controls.] Maternal and fetal serum and 

organs were collected. Among organs collected were fetal 

uteri and testes, which were pooled. On GD 150, 2 

Japanese monkeys (Macaca fuscata) were s.c. injected with 

50 mg bisphenol A/kg bw and at 1 hr following injection, 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 193 

Table 32 

Toxicokinetic Values for Bisphenol A in Rats Following Gavage Dosing With 1 or 10 mg/kg bwa 

Age at exposure and sex 

PND 4 PND 7 PND 21 Adult 

Endpoint Male Female Male Female Male Female Male Female 

Bisphenol A dose: 1 mg/kg bw 

Tmax, hr 0.25 0.25 0.25 0.25 3 3 

Cmax, mg/L 0.03 0.06 0.04 0.08 0.005 0.006 

Half-life, hr 7.2 7.3 21.8 8.8 

AUC, mg � hr/L 0.2 0.1 0.1 0.1 

Bisphenol A dose: 10 mg/kg bw 

Tmax, hr 0.25 0.25 0.25 0.25 1.5 1.5 0.25 0.75 

Cmax, mg/L 48.3 10.2 1.1 1.4 0.2 0.2 0.024 0.063 

Half-life, hr 17 6.7 11.4 8.5 4.3 6.6 ‘‘0’’ ‘‘0’’ 

AUC, mg � hr/L 23.1 7.2 1.9 1.7 1.1 1 ‘‘0’’ ‘‘0’’ 

Ratio of value at 10 to 1 mg/kg bw/day 

Cmax 1610 170 27.5 17.5 

AUC 115.2 72 19 17 

Data missing from table cells were not determined. 

a

Domoradzki et al. (2004). 

fetuses were removed by cesarean section. Two untreated 

fetuses were used as controls. Maternal and fetal serum 

and organs, not including reproductive organs, were 

collected from monkeys. Bisphenol A concentrations were 

measured by GC/MS in mouse and monkey samples. 

In mice, bisphenol A was detected within 0.5 hr of 

exposure in all tissues examined, including placenta, 

maternal and fetal serum, liver, and brain, and fetal 

uterus, and testis. Bisphenol A concentrations were 

higher in fetal than maternal serum and liver. [Peak 

concentrations were observed within 0.5–1 hr in most 

tissues, with the exception of fetal brain (2 hr), and 

concentrations remained elevated for 1–6 hr, depending 

on tissue. More than one peak was observed in fetal 

serum, uterus, and testis.] In exposed monkeys, bisphenol A was found at the highest concentrations (15.6– 

72.50 mg/kg) in fetal heart, intestine, liver, spleen, 

kidney, thymus, muscle, cerebrum, pons, and cerebellum; bisphenol A concentrations in the same organs from 

control monkeys were measured at 3.70–22.80 mg/kg. 

Lower concentrations of bisphenol A were detected in 

umbilical cord and maternal and fetal serum of the 

exposed group (1.70–6.10 mg/kg) and control group 

(0.02–0.25 mg/kg). The study authors stated that the 

most likely source of bisphenol A in control monkeys 

was the feed, which was found to contain bisphenol A. 

The study authors concluded that the placental barrier 

does not protect the fetus from bisphenol A exposure. 

Halldin et al. (2001) examined distribution of bisphenol A in quail eggs or hens. After injection of 

fertilized quail egg yolk sacs with 67 mg/g 14C-bisphenol 

A egg on incubation day 3, o1% of radioactivity was 

detected in embryos at incubation day 6 or 9. A similar 

finding was reported for diethylstilbestrol. At incubation 

day 6, no specific localization was observed in the 

embryo but in 10- and 15-day-old embryos a high 

amount of radioactivity was observed in liver and bile. 

[Low transfer of labeled bisphenol A to the egg was 

reported after oral or i.v. dosing of quail hens (with 

apparently 105 lg bisphenol A), but concentrations in 

eggs were not quantified by study authors.] 

2.1.2.2.2 Non-pregnant and non-lactating animals: Domoradzki et al. (2004), examined the effects of dose and 

age on toxicokinetics and metabolism of bisphenol A in 

rats. Neonatal and adult male and female Sprague– 

Dawley rats were gavaged with 14C-bisphenol A (B99% 

radiochemical purity)/non-radiolabeled bisphenol A 

(99.7% purity). Three neonatal rats/age/sex/time period 

were dosed on PND 4, 7, and 21 with 1 or 10 mg/kg bw 

bisphenol A. Adult rats (11 weeks old) [number treated 

not specified] were dosed with 10 mg/kg bw bisphenol 

A. Blood samples were collected at various time points 

from 0.25–24 hr post-dosing in neonatal rats and from 

0.25–96 hr in adult rats. Plasma samples were pooled on 

PND 4. Immature rats were killed at 24 hr post-dosing, 

and adult rats were killed at 96 hr post-dosing. Brain, 

liver, kidneys, skin, and reproductive organs were 

collected from neonatal rats. Levels of radioactivity, 

bisphenol A, and/or metabolites were analyzed in blood 

and tissue samples using HPLC and liquid scintillation 

spectrometry. 

In neonatal and adult rats, radioactivity levels in 

plasma generally peaked within 0.25–0.75 hr. With the 

exception of 0.25 hr post-dosing on PND 4, when plasma 

radioactivity levels were B4-fold higher in males than 

females, plasma radioactivity levels were generally 

similar in male and female rats. At 24 hr post-dosing, 

plasma radioactivity levels were 4–100 times lower in all 

groups of neonatal rats. Trends were noted for decreasing 

radioactivity levels with increasing age. Information 

related to dose- and age-related effects on metabolism 

is presented in Section 2.1.2.3. 

Toxicokinetic values for bisphenol A are listed in 

Table 32. Cmax and AUC values for bisphenol A 

decreased with increasing age, especially following 

dosing with 10 mg/kg bw. Bisphenol A concentrations 

were lower in adults than neonates. No patterns were 

observed for half-lives, and the authors stated that values 

in neonates may not have been reliable because bisphenol A concentrations were near the LOD at the end 

of the 24-hr observation period. Ratios of Cmax and AUC 

values for the 10 and 1 mg/kg bw doses were different at 

Birth Defects Research (Part B) 83:157–395, 2008
194 CHAPIN ET AL. 

Table 33
 

Toxicokinetic Values for Bisphenol A Glucuronide in Rats Following Gavage Dosing With 1 or 10 mg/kg bw
 

Bisphenol Aa
 

Age at exposure and sex 

PND 4 PND 7 PND 21 Adult 

Endpoint Male Female Male Female Male Female Male Female 

Bisphenol A dose: 1 mg/kg bw 

Tmax, hr 0.75 0.75 0.75 0.25 0.25 0.25 

Cmax, mg/L 1.3 1.5 2 1.1 0.8 0.8 

Half-life, hr 26.1 24.2 6.6 6.4 4.2 4.1 

AUC, mg � hr/L 9 9.6 7.7 7.7 4.1 3.3 

AUCBPA-glucuronide/AUCBPA 45 96 77 77 

Bisphenol A dose: 10 mg/kg bw 

Tmax, hr 1.5 1.5 1.5 0.75 0.75 0.75 0.25 0.25 

Cmax, mg/L 13.1 6.3 6.6 10.3 10.4 7.8 0.6 0.7 

Half-life, hr 7.3 9.8 9.1 8.4 4.4 4.4 22.5 10.8 

AUC, mg � hr/L 80 50.3 58.9 60.9 60.3 56.1 31.5 9.8 

AUCBPA-glucuronide/AUCBPA 3.5 7 31 36 55 56 

Ratio of value at 10 to 1 mg/kg bw/day 

Cmax 10.1 4.2 3.3 9.4 13 9.8 

AUC 8.9 5.2 7.6 7.9 14.7 17 

Data missing from table cells were not determined. 

a

Domoradzki et al. (2004). 

each age and generally decreased with age. Plasma 

bisphenol A concentrations were very low in adults 

dosed with 10 mg/kg bw; therefore, few data were 

available. 

Toxicokinetic values for bisphenol A glucuronide are 

listed in Table 33. Peak plasma concentrations of bisphenol A glucuronide were 9–22 times higher in 

neonates than adult rats dosed with 10 mg/kg bw 

bisphenol A. AUC values for bisphenol A glucuronide 

were also higher in neonates than adults [B2–6 times 

higher]. In neonates dosed with 1 mg/kg bw, AUC 

values and elimination half-lives for bisphenol A 

glucuronide decreased with age. Ratios of Cmax and 

AUC values for the 10 and 1 mg/kg bw doses were 

nearly proportional. In adults dosed with 10 mg/kg bw, 

bisphenol A glucuronide concentrations peaked at 

0.25 hr and secondary peaks were observed at 18 and 

24 hr. In neonates dosed with 10 mg/kg bw, concentrations of bisphenol A glucuronide peaked at 0.75–1.5 hr 

and then bisphenol A glucuronide was eliminated in an 

apparently monophasic manner. Half-lives of elimination 

were shorter in neonates compared to adults. In neonatal 

rats, the bisphenol A glucuronide represented 94–100% 

of the 1 mg/kg bw dose and 71–97% of the 10 mg/kg 

bw/day dose. In adult rats, B100% of the dose was 

represented by bisphenol A glucuronide. 

Half-life and AUC data for bisphenol A-derived 

radioactivity in organs of neonatal rats are summarized 

in Table 34. Radioactivity was distributed to all organs 

and dose-related increases were observed. The study 

authors noted lower concentrations in brain than in other 

tissues. [Levels of radioactivity in reproductive organs 

compared to those in plasma varied at each evaluation 

period but were usually within the same or one order of 

magnitude lower.] With the exception of males dosed 

with 10 mg/kg bw bisphenol A, half-lives decreased with 

age. There were some disproportionate increases in ratios 

of AUC at 10 and 1 mg/kg bw. 

The study authors concluded: 

*	 Metabolism of bisphenol A to its glucuronide conjugate occurred as early as PND 4 in rats; 

*	 Dose-dependent differences occurred in neonatal rats, 

as noted by a larger fraction of the lower dose being 

metabolized to the glucuronide; and 

*	 There were no major sex differences in metabolism or 

toxicokinetics of bisphenol A. 

Pottenger et al. (2000) examined the effects of dose and 

route on metabolism and toxicokinetics of bisphenol A in 

rats. Information focusing on toxicokinetics is summarized primarily in this section, while metabolic data are 

summarized primarily in Section 2.1.2.3. Adult male and 

female F344 rats were dosed with 14C-bisphenol A (99.3% 

radiochemical purity)/non-radiolabeled bisphenol A 

(99.7% purity) at doses of 10 or 100 mg/kg bw by oral 

gavage or i.p. or s.c. injection. Blood was collected at 

multiple time points between 0.083 and 168 hr postdosing, and excreta were collected for 7 days. Animals 

were killed 7 days post-dosing. Blood, brain, gonads, 

kidneys, liver, fat, skin, uterus, and carcass were 

analyzed by liquid scintillation counting and HPLC. 

Some samples were analyzed by HPLC/electrospray 

ionization/MS. 

Toxicokinetic endpoints for bisphenol A in blood are 

summarized in Table 35. Study authors noted that 

concentration-time profiles of bisphenol were dependent 

on dose, exposure route, and sex. The longest Tmax was 

observed with s.c. dosing. Cmax and AUC values were 

lowest following oral administration. Time to nonquantifiable concentrations of bisphenol A was longest 

following s.c. exposure. The only sex-related difference 

was a higher Cmax value in females than males following 

oral dosing. In most cases, bisphenol A toxicokinetics 

were linear across doses within the same administration 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 195 

Table 34 

Distribution of Radioactivity to Tissues at 24 Hr Following Dosing With Radiolabeled Bisphenol Aa 

PND 4 PND 7 PND 21 

AUC 

AUC mg � AUC ratio Half- AUC mg � ratio of AUC mg � AUC ratio 

Tissue Half-life hr hr/kg of doses life hr hr/kg doses Half-life hr hr/kg of doses 

Females, 1 mg/kg bw 

Brain 11.7 0.4 6.7 0.2 3.6 0.1 

Liver 18 7.5 7.9 7.1 3.6 2.9 

Kidney 18.1 9.4 7.3 9.5 5.0 3.0 

Ovary 11.7 7.3 6.0 3.5 3.7 0.9 

Uterus 7.4 8.3 6.2 3.0 3.4 1.0 

Carcass 11.2 22.2 10.0 16.6 4.0 8.3 

Plasma 19.5 9.4 6.4 7.8 3.6 3.5 

Females, 10 mg/kg bw 

Brain 7.2 3.3 8.3 8.0 2.5 12.5 4.9 1.7 17.0 

Liver 11.1 44.8 6.0 10.0 59.6 8.4 4.5 39.1 13.5 

Kidney 15.2 43.9 4.7 8.6 66.6 7.0 5.3 36.5 12.2 

Ovary 6.5 136.2 18.7 5.0 69.7 19.9 3.6 21.1 23.4 

Uterus 15.2 127.0 15.3 4.8 108.5 36.2 3.4 30.6 30.6 

Carcass 6.6 112.8 5.1 7.0 130.7 7.9 4.8 100.9 12.2 

Plasma 9.2 61.0 6.5 8.1 67.0 8.6 3.7 59.0 16.9 

Males, 1 mg/kg bw 

Brain 14.1 0.3 6.0 0.3 3.4 0.1 

Liver 19.7 6.1 6.6 7.3 3.7 3.2 

Kidney 19.3 8.5 7.0 8.6 4.6 3.4 

Testis 10.3 3.4 5.7 2.0 3.4 0.8 

Carcass 11.1 22.2 9.0 17.3 4.1 9.0 

Plasma 24.0 9.2 6.6 7.7 3.4 4.2 

Males, 10 mg/kg bw 

Brain 3.1 4.7 15.7 8.0 2.9 9.7 4.7 1.7 17.0 

Liver 11.6 48.4 7.9 11.8 62.0 8.5 5.1 40.9 12.8 

Kidney 5.4 68.9 8.1 9.8 59.6 6.9 6.9 30.4 8.9 

Testes 5.8 36.8 10.8 7.6 22.1 11.1 5.2 8.1 10.1 

Carcass 8.3 111.7 5.0 8.6 135.5 7.8 4.8 95.2 10.6 

Plasma 6.9 113.0 12.3 9.9 69.0 9.0 4.0 62.0 14.8 

a

Domoradzki et al. (2004). 

Table 35 

Toxicokinetic Endpoints for Bisphenol A in Blood Following Dosing of Rats by Gavage or Injectiona 

Exposure route and doses (mg/kg bw) 

Endpoint 10 oral 100 oral 10 i.p. 100 i.p. 10 s.c. 100 s.c. 

Males 

Tmax, hr N/A 0.083 0.5 0.25 0.75 0.5 c Cmax, mg/L, hrb 0.2270.09 0.6970.08 9.771.27 0.3970.16 5.1970.98 

Time to non-quantifiable 0.083 0.75 8 12 18 24 

concentration, hr 

AUC, mg � hr/L 0.1 1.1 16.4 2.6 24.5 

Females 

Tmax, hr 0.25 0.25 0.25 0.25 4 0.75 

Cmax, mg/L, hrb 0.0470.03 2.2971.82 0.8770.15 13.1374.13 0.3470.06 3.9770.6 

Time to non-quantifiable 1 24 72 48 72 

concentration, hr 

AUC, mg � hr/L 0.42 4.4 1.4 26.2 3.1 31.5 

Missing values were not determined.
 

a

Pottenger et al. (2000).
 

b

Mean7SD.
 c

Non-quantifiable (0.01 mg/g at 10 mg/kg bw and 0.1 mg/g at 100 mg/kg bw).
 

route, as noted by approximate proportionate increases summarized in Table 36. Concentrations of radioactivity 

in Cmax and AUC values from the low to the high-dose. were dependent on exposure route and to a lesser extent, 

Toxicokinetics data for radioactivity in plasma are dose and sex. AUC values for radioactivity were lowest 

Birth Defects Research (Part B) 83:157–395, 2008
196 CHAPIN ET AL. 

Table 36 

Toxicokinetics for Radioactivity Following Dosing of Rats with Bisphenol A Through Different Exposure Routesa 

Exposure route and doses (mg/kg bw) 

Endpoint 10 oral 100 oral 10 i.p. 100 i.p. 10 s.c. 100 s.c. 

Males 

Tmax, hr 0.25 0.25 0.5 0.25 1 0.75 

Cmax, mg eq/L, hr 0.7370.22 3.9271.93 1.2670.09 29.3711.7 0.6170.24 6.3370.43 

Time to non-quantifiable 72 72 96 96 96 144 

concentration, hr 

AUC, mg-eq � hr/L 8.1 66.5 16.9 170 15.5 218 

Females 

Tmax, hr 0.083 0.25 0.25 0.5 0.75 0.75 

Cmax, mg eq/L, hr 1.8270.66 28.3378.64 2.2770.19 67.8177.33 0.5270.06 5.6670.95 

Time to non-quantifiable 72 72 72 120 120 168 

concentration, hr 

AUC, mg-eq � hr/L 9.54 94.9 15.3 247 21.6 297 

a

Pottenger et al. (2000). 

Table 37
 

Toxicokinetic Values for Bisphenol A in Adult Rats
 

Exposed to Bisphenol A Through the Intravenous or Oral
 

Routea
 

Bisphenol A dosing 

0.1 mg/kg 10 mg/kg 

Endpoint bw, i.v. bw, gavage 

Distribution half-life, min 6.171.3 

Terminal elimination 0.970.3 21.377.4 

half-life, hr 

AUC, mg � hr/L 16.173.2 85.6733.7 

Systemic clearance, 107.9728.7 

mL/min/kg 

Steady-state volume of 5.672.4 

distribution, L/kg 

Cmax, mg/L 14.7710.9 

Tmax, hr 0.270.2 

Apparent volume of 427372007.3 

distribution, L/kg 

Oral clearance, mL/min/kg 2352.17944.7 

Absolute oral bioavailability, % 5.372.1 

Data presented as mean7SD. a

Yoo et al. (2001). 

following oral exposure. Time to non-quantifiable concentration was longest following s.c. dosing. For most 

groups, Cmax and AUC values were proportionate across 

doses within the same exposure route. A second part of 

the study examined metabolites and is summarized in 

Section 2.1.2.3. 

Upmeier et al. (2000) examined toxicokinetics in rats 

exposed to bisphenol A through the oral or i.v. route. 

Ovariectomized DA/Han rats (130–150 g bw) were 

exposed to bisphenol A by i.v. injection with 10 mg/kg 

bw or oral gavage with 10 or 100 mg/kg bw. Blood was 

collected from treated rats at multiple time points until 

2 hr following i.v. dosing and 3 hr following oral dosing. 

Three to five rats were sampled during each time period. 

To reduce stress, only some of the rats were sampled at 

each time point. In control animals, blood was collected 

2 hr following dosing with vehicle. Bisphenol A 

concentrations in plasma were measured by GC/MS. 

Dosing with 10 mg/kg bw i.v. resulted in a maximum 

plasma concentration of 15,000 mg/L bisphenol A. Concentrations decreased to 700 mg/L within 1 hr, 100 mg/L 

within 2 hr, and non-detectable concentrations by 24 hr 

followingexposure. The apparent final elimination halflife was estimated at 38.5 hr. In rats gavaged with 10 mg/ 

kg bw, an initial maximum blood concentration of 30 mg/ 

L was obtained at 1.5 hr. A decrease in bisphenol A blood 

concentration at 2.5 hr was followed by a second peak of 

40 mg/L at 6 hr, leading study authors to conclude that 

enterohepatic cycling was occurring. The same patterns 

of bisphenol A concentrations in blood were observed 

following gavage dosing with 100 mg/kg bw. Peak 

concentrations were observed at 30 min (150 mg/L) and 

3 hr (134 mg/L) following exposure. According to the 

study authors, the differences in peak concentrations 

observed between the two doses suggested lower 

bioavailability at the high-dose than at the low dose. 

Oral bioavailability of bisphenol A was estimated at 

16.4% at the low dose and 5.6% at the high-dose. 

Yoo et al. (2001) examined toxicokinetics of a low i.v. 

dose and a higher gavage dose of bisphenol A in male 

rats. Five adult male Sprague–Dawley rats/group were 

administered bisphenol A by i.v. injection at a dose of 

0.1 mg/kg bw or by gavage at a dose of 10 mg/kg bw. 

Multiple blood samples were collected until 3 hr following i.v. dosing and 24 hr following gavage dosing. HPLC 

was used to measure bisphenol A concentrations in 

serum. Route-specific differences in mean systemic 

clearance were analyzed by Student t-test. Results are 

summarized in Table 37. The study authors noted biexponential decay of serum bisphenol A concentrations 

following i.v. dosing, significantly longer elimination 

half-life with oral than i.v. exposure, and low oral 

bioavailability of bisphenol A. 

Kurebayashi et al. (2003) conducted a series of studies 

to examine toxicokinetics and metabolism of bisphenol A 

in adult F344N rats exposed through the oral or i.v. route. 

In these studies, radioactivity levels were measured by 

scintillation counting. Bisphenol A or its metabolites 

were quantified by HPLC, electrospray ionization/MS, 

or nuclear magnetic resonance. As discussed in greater 

detail in Section 2.1.2.4, fecal excretion was the main 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 197 

route of elimination for radioactivity following oral or i.v. 

dosing of rats with 0.1 mg/kg bw 14C-bisphenol A. A 

study describing biliary excretion and metabolites in bile 

is summarized in Section 2.1.2.3. Toxicokinetic endpoints 

were determined in a study in which blood was drawn 

from 3 male rats/group at various time points between 

0.25–48 hr following oral gavage or i.v. dosing with 

0.1 mg/kg bw bisphenol A. Results of the study are 

summarized in Table 38. Rapid absorption of radioactivity was observed following oral dosing. AUC values 

were significantly lower for oral than i.v. dosing. In a 

another study, rats were administered 14C-bisphenol A 

by i.v. injection and blood was collected 30 min later for 

determination of blood/plasma distribution and protein 

binding. At a blood radioactivity level of 80 nM [18 lg 

Table 38
 

Toxicokinetic Endpoints for 14C-Bisphenol A-Derived
 

Radioactivity in Rats Exposed to 0.1 mg/kg bw 14CBisphenol A Through the Oral or I.V. Routea
 

Endpoint I.V. exposure Oral exposure 

Tmax, hr 0.3870.10 

Cmax, mg-eq/L 5.570.3 

Half-life-a, hr 0.5970.09 No data 

Half-life-b, hr 39.572.1 44.574.1 

Absorbance rate, hr -1 3.671.0 

Volume of distribution, L/kg 27.070.7 No data 

Total body clearance. L/hr/kg 0.52270.011 0.54470.049 

Mean residence time, hr 51.772.4 No data 

AUC, mg-eq � hr/L 

0–6 hr 33.971.6 18.470.7b 

0–24 hr 79.373.3 60.077.1b 

0–48 hr 11874 102713b 

0–N 19274 185716 

Oral bioavailabilityc 

0–6 hr 0.54 

0–24 hr 0.76 

0–48 hr 0.86 

0–N 0.97 

Data presented as mean7SD. Missing values are not applicable
 

or were not reported.
 

a

Kurebayashi et al. (2003).
 

b

P o 0.05 compared to i.v. exposure.
 c

Variances not reported.
 

bisphenol A eq/L], preferential distribution to plasma 

was observed, with the blood/plasma ratio reported at 

0.67. At radioactivity levels of 6–31 mg-eq/L (27–135 nM), 

plasma protein binding was reported at 95.4%. Additional studies reviewed by Teeguarden et al. (2005) 

reported plasma protein binding of bisphenol A at 

B90–95%. An additional study by Kurebayashi et al. 

(2003) compared metabolic patterns and excretion 

following exposure to a higher bisphenol A dose; that 

study is discussed in Section 2.1.2.3. 

Kurebayashi et al. (2005) administered 14C-bisphenol A 

to adult male and female F344 rats (3/dose/sex) at doses 

of 0.020, 0.1, or 0.5 mg/kg bw orally or 0.1 or 0.5 mg/kg 

bw by i.v. injection. Plasma samples were analyzed for 

radioactivity over a 72-hr period to determine toxicokinetic endpoints. Results are summarized in Table 39. 

Study authors noted that the AUC was almost linearly 

correlated with dose. Several peaks were observed with 

oral or i.v. exposure, indicating enterohepatic cycling, 

according to the study authors. Study authors noted that 

substantially lower AUC values in females than in males 

following oral exposure could have resulted from lower 

absorption and/or a higher elimination rate. Distribution 

of radioactivity was evaluated 0.5, 24, and 72 hr following oral administration of 0.1 mg/kg bw bisphenol A to 

adult male and female Wistar rats (3/sex/time point). At 

0.5 hr following exposure, most of the radioactivity 

(B12–51 mg bisphenol A eq/kg) was found in kidney 

and liver. [A large amount of radioactivity was also 

reported for intestinal contents, but those data were not 

shown by the study authors.]. Lower amounts of 

radioactivity (B2–7 mg bisphenol A eq/kg or L) were 

detected in adrenal gland, blood, lung, pituitary gland, 

skin, and thyroid gland of both sexes; uterus; and bone 

marrow, brown fat, and mandibular gland of males. In 

males,omg bisphenol A eq/kg was detected in skeletal 

muscle and testis. Radioactivity was non-quantifiable in 

brain and eye of both sexes; epididymis, prostate gland, 

and heart of males; and bone marrow, brown fat, skeletal 

muscle, and mandibular gland of females. At Z24 hr 

following exposure, radioactivity was detected primarily 

in only kidney, liver, and intestinal contents, with the 

exception of B3 mg bisphenol A eq/L detected in blood 

of males at 24 hr following dosing. Study authors noted 

that elimination of radioactivity from some tissues 

Table 39
 

Toxicokinetic Endpoints for Plasma Radioactivity in Rats Dosed With 14C Bisphenol Aa
 

Route and dose (mg/kg bw) 

Oral I.V. 

Endpoints 20 100 500 100 500 

Males 

Elimination half-life, hr 78752 1873 2173 1972 2173 

AUC, mg-eq � hr/L 3676 178744 6637164 266746 865797 

Apparent absorption, % 82 81 60 

Females 

Elimination half-life, hr 2077 22713 1878 1373 1672 

AUC, mg-eq � hr/L 1475 99719 500743 190745 1029781 

Apparent absorption, % 35 50 50 

Data presented as mean7SD. a

Kurebayashi et al. (2005). 

Birth Defects Research (Part B) 83:157–395, 2008
198 CHAPIN ET AL. 

appeared to occur more rapidly in females than in males. 

Distribution in pregnant animals was also examined and 

is described in Section 2.1.2.2.1. 

Kabuto et al. (2003) reported distribution of bisphenol 

A in mice. Male ICR mice were i.p. dosed with bisphenol 

A at 0, 25, or 50 mg/kg bw/day for 5 days and killed 6 hr 

following the last dose. Bisphenol A concentrations in 

tissues of animals from the high-dose group were 

determined by GC/MS. In mice of the high-dose group, 

the highest concentrations of bisphenol A were detected 

in kidney (B2.02 mg/kg wet weight) and body fat 

(B1.25 mg/kg wet weight). Lower concentrations of 

bisphenol A (r0.42 mg/kg wet weight or mg/L) were 

detected in brain, lung, liver, testis, and plasma. 

Kurebayashi et al. (2002) examined the toxicokinetics of 

a low bisphenol A dose in Cynomolgus monkeys 

following gavage or i.v. dosing. Three adult male and 

female monkeys were dosed with 0.1 mg/kg bw 14CTable 40
 

Toxicokinetic Endpoints for Radioactivity in Male and
 

Female Cynomolgus Monkeys Exposed to 14C-Bisphenol
 

A Through IV Injection or by Gavagea
 

Endpoint Male Female 

Intravenous exposure 

AUC, mg-eq � hr/L 377785 382796 

Volume of distribution, L/kg 1.5870.11 1.8270.41 

Half-life, hr 13.572.6 14.772.1 

Total body clearance, L/hr/kg 0.2770.05 0.2870.08 

Mean residence time, hr 5.9370.91 6.6870.72 

Oral exposure 

AUC, mg-eq � hr/L 265774 244721 

Tmax, hr 1.0070.87 0.3370.14 

Cmax. mg-eq/L 104785 107737 

Half-life. hr 9.6372.74 9.8072.15 

Bioavailability 0.7070.16 0.6670.13 

[Mean7SD assumed based on data presentations elsewhere in 

this study.] 

a

Kurebayashi et al. (2002). 

bisphenol A (99% radiochemical purity)/non-radiolabeled 

bisphenol A [purity not reported]. Monkeys were dosed 

by i.v. injection on Day 1 of the study and by gavage on 

Day 15 of the study. Urine and feces were collected for 7 

days post-dosing. Blood samples were collected at various 

time points from 0.083–72 hr following i.v. dosing and for 

0.25–71 hr after oral dosing. Binding to plasma protein 

was determined at some time points over 0.25–4 hr. 

Samples were analyzed by liquid scintillation counting 

and HPLC. Following oral or i.v. exposure, the percentage 

of radioactivity recovered in excreta and cage washes was 

81–88% over a 1-week period. As discussed in greater 

detail in Section 2.1.2.4, most of the radioactivity was 

excreted in urine and very little was excreted in feces. 

Toxicokinetic endpoints are summarized in Table 40. 

Based on the toxicokinetic values, study authors concluded that absorption of bisphenol A following oral 

exposure was rapid and high, and terminal elimination 

half-lives of bisphenol A/metabolites were longer following i.v. than oral exposure. As discussed in more detail in 

Section 2.1.2.3, glucuronide compounds were the major 

metabolites detected in urine, and higher percentages of 

the radioactive dose in plasma were represented by 

bisphenol A following i.v. than oral dosing. 

Negishi et al. (2004b) compared toxicokinetics of 

bisphenol A in female F344/N rats, Cynomolgus 

monkeys, and Western chimpanzees. Bisphenol A was 

administered by oral gavage and s.c. injection at doses of 

10 or 100 mg/kg bw/day to rats and monkeys and 

10 mg/kg bw to chimpanzees. Three rats/dose/time 

point were killed before and at various times between 0.5 

and 24 hr following bisphenol A administration. Three 

monkeys/group and 2 chimpanzees were first exposed 

orally and 1 week later by s.c. injection. In monkeys, 

blood samples were drawn before and at various times 

from 0.5–24 hr after dosing. In chimpanzees, blood was 

drawn before and at multiple time points between 0.25– 

24 hr following dosing. Bisphenol A was measured in 

serum by ELISA, and toxicokinetics endpoints were 

determined. Results are summarized in Table 41. The 

study authors noted that the bioavailability of bisphenol 

Table 41
 

Toxicokinetic Endpoints for Bisphenol A by ELISA in Rats, Monkeys, and Chimpanzeesa
 

10 mg/kg bw 100 mg/kg bw 

Endpoints Oral S.C. Oral S.C. 

Rat (data presented as mean7SD) 

Cmax, mg/L 8727164 5807398 34397679 

Tmax, hr 1.0 0.5 1.0 

AUC0–4 h, mg � hr/L 19127262 5067313 931472634 

AUC0–24 h, mg � hr/L 33777334 13537462 23,00176387 

Monkey (data presented as mean7SD) 

Cmax, mg/L 27977920 57,93471902 57327525 10,85173915 

Tmax, hr 0.770.2 2.070.0 0.770.2 2.070.0 

AUC0–4 h, mg � hr/L 32097536 15,31675856 14,74772495 48,010711,641 

AUC0–24 h, mg � hr/L 32477587 39,040710,738 52,59578951 189,627721,790 

Chimpanzee (data presented for 2 animals) 

Cmax, mg/L 325; 96 2058; 1026 Dose not administered 

Tmax, hr 0.5; 0.5 2.0; 2.0 

AUC0–4 h, mg � hr/L 491; 235 5658; 3109 

AUC0–24 h, mg � hr/L 1167; 813 21,141; 12,492 

Data were not reported in cases where table cells are empty. 

a

Negishi et al. (2004b). 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 199 

Table 42 

Toxicokinetic Endpoints for Bisphenol A by LC-MS/MS in Rats, Monkeys, and Chimpanzeesa 

10 mg/kg bw 100 mg/kg bw 

Endpoints Oral S.C. Oral S.C. 

Rat (data presented as mean7SD) 

Cmax, mg/L 2.171.6 746780 47.5710.6 26317439 

Tmax, hr 0.770.3 0.870.3 0.570.0 1.270.8 

t1/2, hr not calculated 3.270.7 not calculated 4.570.7 

AUC0–4 h, mg � hr/L 4.2b 15427200 43.279.7 692671071 

AUC0–24 h, mg � hr/L 7.2b 19777182 3507294 15,57672263 

Monkey (data presented as mean7SD) 

Cmax, mg/L 11.572.2 421373319 28.673.9 701073045 

Tmax, hr 1.070.9 1.770.6 3.371.2 2.771.2 

t1/2, hr 8.973.0 3.870.8 4.570.7 12.973.6 

AUC0–4 h, mg � hr/L 21.476.1 882874309 85.3718.6 19,98177567 

AUC0–24 h, mg � hr/L 42.577.3 18,85573870 350713 79,796721,750 

Chimpanzee (data presented as mean for 2 animals) 

Cmax, mg/L 5.5 703 Dose not administered 

Tmax, hr 0.8 1.0 

t1/2, hr 6.8 4.2 

AUC0–4 h, mg � hr/L 13.3 2148 

AUC0–24 h, mg � hr/L 33.1 6000 

a

Tominaga et al. (2006). 

b

1 or 2 animals. 

was lowest in ratsochimpanzeesomonkeys following 

exposure through either route. In most cases, bisphenol 

A was not detected in rat serum following oral administration of the 10 mg/kg bw dose. In all species, higher 

bioavailability was observed with s.c. than oral dosing. 

In a subsequent report (Tominaga et al., 2006), these 

authors noted that ELISA may overestimate bisphenol A 

concentrations due to non-specific binding. They reported measurements by LC-MS/MS in animals evaluated using the same study design [possibly the same 

specimens reported previously]. These results are 

summarized in Table 42. The authors proposed that 

primates, including humans, may completely glucuronidate orally-administered bisphenol A on its first pass 

through the liver and excrete it in the urine whereas 

bisphenol A remains in the rat for a more extended 

period due to enterohepatic recirculation. They suggested that the rat may not be a good model for human 

bisphenol A kinetics. 

2.1.2.3 Metabolism: Information is arranged in this 

section according to species. In rats, study summaries are 

arranged in order of those providing general or routespecific information on metabolites, specifics on organs 

or enzyme isoforms involved in metabolism, and 

pregnancy-, sex-, or age-related effects on metabolism. 

Pottenger et al. (2000) examined the effects of dose and 

route on toxicokinetics of bisphenol A in rats. Disposition 

of bisphenol A and its metabolites in urine and feces is 

described in this section, while results of the toxicokinetics 

study are described in Section 2.1.2.2. Five adult F344 

rats/sex/group were dosed with 14C-bisphenol 

A (99.3% radiochemical purity)/non-radiolabeled bisphenol A (99.7% purity) at doses of 10 or 100 mg/kg bw by 

oral gavage or i.p. or s.c. injection. Excreta were collected 

for 7 days. Samples were analyzed by HPLC or HPLC/ 

electrospray ionization/MS. The percentage of radioactivity recovered from all groups was 84–98%. Fecal 

elimination represented the largest percentage of 

radioactivity in all exposure groups (52–83%). Eight peaks 

were identified in feces, and the largest peak (representing 

86–93% of radioactivity) was for unchanged bisphenol A. 

Elimination of radioactivity through urine was B2-fold 

higher in females (21–34%) than males (13–16%) in all 

dose groups. Fourteen different peaks were identified in 

urine. It was estimated that radioactivity in urine was 

represented by bisphenol A monoglucuronide (57–87%), 

bisphenol A (3–12%), and bisphenol A sulfate (2–7%). 

Some differences were noted for retention of radioactivity 

following dosing by gavage (0.03–0.26%), i.p. injection 

(0.65–0.85%), and s.c. injection (1.03–1.29%). 

Metabolites associated with bisphenol A exposure 

were examined in a second study by Pottenger et al. 

(2000). Three rats/sex/dose/route/time point were 

dosed with 14C-bisphenol A/non-radiolabeled bisphenol 

A at 10 or 100 mg/kg bw by oral gavage or i.p. or s.c. 

injection. Rats were killed at 2 different time points 

following dosing, Tmax, and the time when bisphenol A 

concentrations were no longer quantifiable. Times at 

which rats were killed were determined by data obtained 

during the first study. Plasma samples were pooled at 

each time period and examined by HPLC or HPLC/ 

electrospray ionization/MS. Qualitative and quantitative 

differences were observed for parent compound and 

metabolites in plasma following exposure through 

different routes. Following oral exposure, bisphenol A 

glucuronide was the most abundant compound detected 

in plasma at both time periods (Cmax and time when 

parent compound was not quantifiable) and represented 

68–100% of total radioactivity. Following i.p. or s.c. 

exposure, unmetabolized bisphenol A was the most 

abundant compound at Tmax; levels of radioactivity 

represented by unmetabolized bisphenol A were 27– 

51% following i.p. exposure and 65–76% following s.c. 

exposure. Only 2–8% of radioactivity was represented by 

bisphenol A following oral exposure. Some compounds 

observed following i.p. or s.c. exposure were not 

Birth Defects Research (Part B) 83:157–395, 2008
200 CHAPIN ET AL. 

Table 43
 

Biliary Excretion in Male and Female Rats Exposed to
 

0.1 mg/kg bw 14C-Bisphenol A Through the Oral or
 

Intravenous Routea
 

Male Female 

Parameters I.V. Oral I.V. Oral 

Biliary excretion, % 

0–2 hr 

0–4 hr 

0–6 hr 

Radioactivity in bile 

represented by glucuronide, % 

Dose excreted as glucuronide 

in bile, % 

48 

61 

66 

84 

55 

32 

44 

50 

86 

43 

35 

50 

58 

87 

50 

28 

39 

45 

88 

40 

a

Kurebayashi et al. (2003). 

observed following oral exposure. A compound tentatively identified as a sulfate conjugate was observed 

following i.p. exposure and represented a small portion 

of radioactivity. An unresolved peak of 3 compounds 

was observed following i.p. or s.c. exposure, at the time 

when parent compound was not quantifiable and 

represented that major percent of radioactivity for that 

time point. Three additional unidentified, minor peaks 

were observed following i.p. or s.c. but not oral exposure. 

The major sex differences observed were higher Cmax 

values for bisphenol A and bisphenol A glucuronide in 

females than males, especially following i.p. administration. A review by the European Union (2003) noted that 

the substantially higher concentrations of parent compound with i.p. and s.c. compared to oral exposure 

indicated the occurrence of first-pass metabolism following oral intake. 

Elsby et al. (2001) examined bisphenol A metabolism 

by rat hepatocytes. In the hepatocyte metabolism study, 

hepatocytes were isolated from livers of adult female 

Wistar rats and incubated in dimethyl sulfoxide (DMSO) 

vehicle or bisphenol A 100 or 500 mM [23 or 114 mg/L] for 

2 hr. Metabolites were identified by HPLC or LC/MS. 

Data were obtained from 4 experiments conducted in 

duplicate. At both concentrations, the major metabolite 

was identified as bisphenol A glucuronide, which was 

the only metabolite identified following incubation with 

100 mM bisphenol A. Two additional minor metabolites 

identified at the 500 mM concentration included 5-hydroxy-bisphenol A-sulfate and bisphenol A sulfate. Another 

part of the study comparing metabolism of bisphenol A 

by rat and human metabolites is discussed in Section 

2.1.1.3. Another study (Pritchett et al., 2002) comparing 

metabolism of bisphenol A in humans, rats, and mice is 

also summarized in Section 2.1.1.3. 

In neonatal rats gavaged with 1 or 10 mg/kg bw 14C-bisphenol A on PND 4, 7, and 21 and adult rats 

gavaged with 10 mg/kg bw bisphenol A, the major 

compounds detected in plasma were bisphenol A 

glucuronide and bisphenol A (Domoradzki et al., 2004). 

Up to 13 radioactive peaks were identified in neonatal 

rats dosed with 10 mg/kg bw and 2 were identified in 

neonates dosed with 1 mg/kg bw/day. At the 10 mg/kg 

bw dose, the concentration of bisphenol A glucuronide 

detected in plasma increased with age. Metabolic 

profiles were generally similar in males and 

females. The study authors noted that metabolism of 

bisphenol A to its glucuronide conjugate occurs as 

early as PND 4 in rats. However, age-dependent 

differences were observed in neonatal rats, as noted by 

a larger fraction of the lower dose being metabolized to 

the glucuronide. More details from this study are 

included in Section 2.1.2.2. 

Kurebayashi et al. (2005) used a thin layer chromatography technique to examine metabolite profiles in 

blood, urine, and feces of 3 male rats orally dosed with 

0.5 mg/kg bw 14C-bisphenol A. [The procedure did not 

identify metabolites.] Parent bisphenol A represented 

B2% of the dose in plasma at 0.25 and 6 hr post-dosing 

and B0.3% of the dose at 24 hr after exposure. 

Unmetabolized bisphenol A represented 1.6% of compounds in urine and 77.2% of compounds in feces 

collected over a 24-hr period. Free bisphenol A represented 47.1% of compounds in urine following bglucuronidase hydrolysis of urine, and there was an 

almost equivalent decrease in a metabolite the study 

authors identified as ‘‘M2.’’ Therefore, the study authors 

stated that M2 was most likely bisphenol A glucuronide. 

M2 was the major metabolite identified in plasma (B74– 

77%) and urine (B40%). 

The European Union (2003) reviewed studies by 

Atkinson and Roy ([1995a,b) that reported two major and 

several minor adducts in DNA obtained from the liver of 

CD-1 rats dosed orally or i.p. with 200 mg/kg bw bisphenol 

A. Chromatographic mobility of the two major adducts was 

the same as that observed when bisphenol A was incubated 

with purified DNA and a peroxidase or microsomal P450 

activation system. The profile closely matched that of 

adducts formed with the interaction between bisphenol Oquinone and purified rat DNA deoxyguanosine 30

-monophosphate. Formation of the adduct appeared to be 

inhibited by known inhibiters of cytochrome P (CYP) 450. 

It was concluded that bisphenol A is possibly metabolized 

to bisphenol O-quinone by CYP450. 

Biliary excretion of bisphenol A and its metabolites 

following oral or i.v. dosing with bisphenol A was 

examined by Kurebayashi et al. (2003). Bile ducts of 3 

rats/sex/group were cannulated, and the rats were 

dosed with 0.1 mg/kg bw 14C-bisphenol A (499% 

radiochemical purity) in phosphate buffer vehicle by 

oral gavage or i.v. injection. Biliary fluid was collected 

every 2 hr over a 6-hr period to determine percent total 

biliary excretion and percent of dose represented by 

bisphenol A glucuronide. Results are summarized in 

Table 43. The study authors noted that the importance of 

biliary excretion following oral or i.v. dosing. 14Cbisphenol A-glucuronide was the predominant metabolite in bile. 

In another study by Kurebayashi et al. (2003), biliary, 

fecal, and urinary metabolites were examined in male 

rats gavaged with 100 mg/kg bw bisphenol A or D16­

bisphenol A in corn oil. Bile was collected over an 18-hr 

period, and urine and feces were collected over a 72-hr 

period. The primary metabolite detected in urine was 

bisphenol A glucuronide, which represented 6.5% of the 

dose. Lower percentages of the dose (r1.1%) were 

present in urine as bisphenol A and bisphenol A sulfate. 

In feces, the primary compound detected was bisphenol 

A, which represented 61% of the dose. No glucuronide or 

sulfate conjugated metabolites of bisphenol A were 

detected in feces. Most of the dose in bile consisted of 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 201 

bisphenol A glucuronide (41% of the dose). Bisphenol A 

represented 0.3% of the dose in bile. The study authors 

noted that as with oral or i.v. exposure to a smaller dose, 

feces was the main route of elimination for bisphenol A 

and bile was the main elimination route for bisphenol A 

glucuronide. 

A study by Yokota et al. (1999) examined the hepatic 

isoform of uridine diphosphate glucuronosyltransferase 

(UDPGT) involved in the metabolism of bisphenol A and 

distribution of the enzyme in organs of Wistar rats. Using 

yeast cells genetically engineered to express single rat 

UDPGT enzymes, it was determined that UGT2B1 was 

the only isoform capable of glucuronidating bisphenol A. 

Microsomal UDPGT activity toward bisphenol A was 

demonstrated in liver, kidney, and testis, but activity was 

minimal in lung and brain. [Minimal activity was also 

observed for intestine]. Northern blot analyses revealed 

high expression of UGTB1 only in liver. It was demonstrated that 65% of glucuronidation activity was absorbed by binding with anti-UGTB1, indicating that 

additional isoforms are likely involved in glucuronidation of bisphenol A. 

The intestine was determined to play a role in the 

metabolism of bisphenol A in rats. Nine-week-old male 

Sprague–Dawley rats were orally administered 0.1 mL of 

a solution containing 50 g/L bisphenol A [5 mg total or 

B17 mg/kg bw assuming a body weight of B0.3 kg 

(USEPA, 1988)] (Sakamoto et al., 2002). Rats were killed 

at multiple time intervals between 15 min and 12 hr 

following exposure. The small intestine was removed 

and separated into upper and lower portions. Intestinal 

contents were removed from each section. Bisphenol A 

and metabolite concentrations were measured by HPLC. 

Activities and expression of b-glucuronidase were 

determined. A large amount of bisphenol A glucuronide 

was detected in the upper and lower portions of the 

small intestine, and a large amount of free bisphenol A 

was detected in the cecum. Less bisphenol A was 

detected in colon and feces. The observations lead the 

study authors to conclude that free bisphenol A 

generated in the cecum as a result of deconjugation 

was reabsorbed in the colon. The presence of large 

amounts of bisphenol A glucuronide in the small 

intestine at 12 hr following exposure suggested that 

bisphenol A was reabsorbed in the colon and re-excreted 

as the glucuronide. As determined in an assay using pnitrophenol-b-d-glucuronide as a substrate, B70% of 

total b-glucuronidase activity was present in the cecum 

and 30% in the colon. Western blot analysis revealed a 

large amount of bacterial b-glucuronidase protein in 

cecum and colon contents. 

Glucuronidation and absorption of bisphenol A in rat 

intestine were studied by Inoue et al. (2003a). Intestines 

were obtained from 8-week-old male Sprague–Dawley 

rats, and the small intestine was divided into 4 sections. 

Small intestine and colon were everted and exposed to 40 

mL of a solution containing bisphenol A at 10, 50, or 

100 mM [2.3, 11, or 23 mg/L, resulting in delivery of 91, 

456, or 913 lg bisphenol A to the everted intestine]. 

Every 20 min during a 60-min time period, reaction 

products were collected from serosal and mucosal sides 

and analyzed by HPLC. Optimal glucuronidation was 

observed at 50 mM [11 mg/L]. At 60 min following 

exposure to 50 mM bisphenol A, B37% of bisphenol A 

was absorbed by the small intestine and B83% was 

glucuronidated. Approximately 74.7% of the glucuronide 

was excreted on the mucosal side and B25.3% transported to the serosal side of small intestine. Slightly 

greater absorption of bisphenol A in the colon (48.6%) 

compared to the proximal jejunum (37.5%) was observed 

at 60 min following exposure to the 50 mM solution. 

Transport of both bisphenol A and bisphenol A glucuronide to the serosal side of intestine increased distally 

and was greatest in the colon. Minimal mucosal excretion 

was observed in the colon. 

Inoue et al. (2004) compared glucuronidation of 

bisphenol A in pregnant, non-pregnant, and male rats. 

Livers of 4 male and non-pregnant Sprague–Dawley 

rats/group were perfused via the portal vein for 1 hr 

with solutions containing bisphenol A at 10 or 50 mM [2.3 

or 11 mg/L]. The total amount of bisphenol A infused into 

livers was 1.5 or 7.5 mmol [0.34 or 1.7 mg]. On GD 20 or 

21, livers of 4 pregnant Sprague–Dawley rats were 

perfused for 1 hr with 10 mM [2.3 mg/L] bisphenol A. At 

the start of perfusion, excreted bile and perfusate in the 

vein were collected every 5 min for 1 hr. Samples were 

analyzed by HPLC. Statistical analyses were conducted 

by Student t-test and ANOVA. Bisphenol A glucuronidation in the liver was 59% in male rats and 84% in nonpregnant female rats perfused with the 10 mM solution. 

The glucuronide was excreted primarily through bile in 

both males and females, but a significantly higher 

amount was excreted through bile in non-pregnant 

females than in males. The total amount of glucuronide 

excreted into bile and vein was B1.4-fold higher in 

females than males following perfusion with the 10 mM 

[2.3 mg/L] solution. At the 50 mM [11 mg/L] concentration, 

bisphenol A glucuronidated within liver was 66% in 

males and 91% in females. In males the glucuronide was 

excreted mainly in bile, and in females, a higher amount 

of glucuronide was excreted in the vein. In livers of 

pregnant rats perfused with the 10 mM [2.3 mg/L] solution, 69% of bisphenol A was glucuronidated in the liver. 

Percentages of glucuronide excretion were 54.5% through 

bile and 45.5% through the vein in pregnant rats. In a 

comparison of pregnant rats and non-pregnant rats 

perfused with 10 mM [2.3 mg/L] bisphenol A, biliary 

excretion in pregnant rats was half that observed in 

non-pregnant rats, and venous excretion in pregnant rats 

was 3-fold higher than in non-pregnant rats. To determine the pathway of bisphenol A glucuronide excretion, 

livers of 4 male Eisai hyperbilirubinemic rats, a strain 

deficient in multidrug resistance-associated protein, 

were perfused with 50 mM [11 mg/L] bisphenol A. During 

and after perfusion, nearly all of the bisphenol A was 

excreted into the vein, thus indicating that multidrug 

resistance-associated protein mediates biliary excretion 

of bisphenol A glucuronide. The study authors concluded that bisphenol A is highly glucuronidated and 

excreted into bile using a multidrug resistance-associated 

protein-dependent mechanism, and that venous excretion increases and biliary excretion decreases during 

pregnancy. 

Miyakoda et al. (2000) examined the production of 

bisphenol A glucuronide in fetal and adult rats. Bisphenol A was orally administered at 10 mg/kg bw to 

pregnant Wistar rats on GD 19 and to 10-week-old adult 

male Wistar rats. [The number of animals exposed was 

not reported. In some legends for study figures, it was 

stated that the data were from 4 experiments, 

Birth Defects Research (Part B) 83:157–395, 2008
202 CHAPIN ET AL. 

suggesting that 4 pregnant rats and adult males may 

have been exposed.] Fetuses were removed at 1 hr 

following dosing. Blood was drawn and testes were 

removed from adult males at 1, 3, and 8 hr following 

dosing. GC/MS was used to measure bisphenol A 

concentrations in 19 fetuses and in testis of adult rats 

before and following homogenization with b-glucuronidase. In fetal extracts, there were no differences in 

bisphenol A concentrations before or after treatment with 

b-glucuronidase, suggesting that bisphenol A glucuronide was not present at detectable concentrations. The 

study authors noted the possibility that bisphenol A 

glucuronide was not transferred from dams to fetuses 

and stated that glucuronidation by the rat fetus is 

unlikely. At 1 hr following dosing of adult male rats, 

90% of bisphenol A was detected as glucuronide in 

plasma and testis. Bisphenol A glucuronide concentrations gradually decreased and bisphenol A concentrations increased slightly in testis over the 8-hr sampling 

period. In plasma, bisphenol A-glucuronide decreased to 

55% of the maximum observed concentration at 3 hr 

following dosing and increased to 100% of maximum 

observed concentration at 8 hr following dosing. Based 

on concentrations of bisphenol A glucuronide in testis 

and blood (40 ppb [lg/kg] and 600 ppb [lg/L]) at 8 hr, the 

study authors concluded that bisphenol A glucuronide 

passage through the testicular barrier was unlikely. It 

was thought that bisphenol A passed through the 

testicular barrier, was converted to the glucuronide 

within the testis, and was then gradually released 

following digestion of the glucuronide by bglucuronidase. 

Matsumoto et al. (2002), studied developmental 

changes in expression and activity of the UDPGT isoform 

UGT2B toward bisphenol A in Wistar rats. Activity 

toward other compounds was also examined but this 

summary focuses on bisphenol A. Microsomes were 

prepared from livers of fetuses, neonates on PND 3, 7, 14, 

and 21, and pregnant rats on GD 10, 15, and 19. Activity 

toward the bisphenol A substrate was measured using an 

HPLC method. Expression of UGT2B1 protein was 

examined by Western blot and messenger ribonucleic 

acid (mRNA) expression was examined by Northern 

blot. Little to no UGT2B activity toward bisphenol A was 

detected in microsomes of fetuses. Activity increased 

linearly following birth and reached adult concentrations 

by PND 21. [No data on UGT2B activity for nonpregnant adult rats were shown and it was not clear if 

activity in adults was examined in this study.] The same 

developmental patterns were observed for expression of 

UGT2B1 protein and mRNA. Activity and protein 

expression of UGT2B1 were also found to be reduced 

in pregnant rats. 

The European Union (2003) reviewed an unpublished 

study by Sipes that compared clearance of bisphenol A 

by hepatic microsome from fetal (n 5 8/sex), immature 

(n 5 4/sex), and adult (n 5 4) rats. The clearance rate in 

microsomes from male and female GD 19 rat fetuses (0.7– 

09 mL/min/mg) was lower than clearance rates in 

microsomes from 4-day-old males and females (1.2–2.6 

mL/min/mg), 21-day-old males and females (2.4–2.7 

mL/min/mg), and their dams (2.6 mL/min/mg). The 

European Union concluded that clearance rate was lower 

in fetuses but reached adult concentrations by 4 days of 

age. 

In a qualitative study of bisphenol A metabolites in 

pregnant mice injected with 0.025 mg/kg bw bisphenol 

A, 10 radioactive peaks were observed in urine by Zalko 

et al. (2003). The major metabolites detected in urine 

were bisphenol A glucuronide and a hydroxylated 

bisphenol A glucuronide. Unchanged bisphenol A was 

the major compound detected in feces (495%). Bisphenol A glucuronide represented 490% of the 

compounds detected in bile. Additional compounds 

detected in urine, feces, digestive tract, or liver included 

a double glucuronide of bisphenol A and sulfate 

conjugates. Unchanged bisphenol A, bisphenol A glucuronide, and ‘‘metabolite F’’ (disaccharide conjugate of 

BPA) were the major compounds detected in all tissues. 

[Authors state that formation of glucuronic acid 

conjugate of BPA, several double conjugates, and 

conjugated methoxylated compounds, demonstrate the 

formation of potentially reactive intermediates.] The 

most abundant compound in all tissues was bisphenol A 

glucuronide, except in placenta where bisphenol A and 

metabolite F were the major compounds detected. 

Concentrations of bisphenol A decreased rapidly in all 

tissues. It was determined that metabolite F was most 

likely bisphenol A glucuronide conjugated to acetylated 

galactosamine or glucosamine. Distribution of bisphenol 

A and its metabolites in maternal and fetal tissues in 

summarized in Table 31. Additional details of this study 

are included in Section 2.1.2.2. 

Jaeg et al. (2004) reported metabolites observed 

following incubation of CD-1 mouse liver microsomes 

or S9 fractions with bisphenol A at 20–500 mM [4.6– 

114 mg/L]. The metabolites included isopropyl-hydroxyphenol, bisphenol A glutathione conjugate, glutythionylphenol, glutathionyl 4-isopropylphenol, 2,2-bis-(4-hydroxyphenyl)1-propanol, 5-hydroxy bisphenol A, and bisphenol A dimers. It was postulated that bisphenol Aortho-quinone, produced from 5-hydroxy bisphenol A 

(catechol), may be the reactive intermediate leading to 

the formation of these metabolites. 

Kurebayashi et al., (2002) examined metabolism of 

bisphenol A in monkeys. Three adult male and female 

Cynomolgus monkeys were dosed with 0.1 mg/kg bw 

14C-bisphenol A/non-radiolabeled bisphenol A by i.v. 

injection on Study Day 1 and by gavage on Study Day 15 

(Kurebayashi et al., 2002). Additional details of the study 

are included in Section 2.1.2.2. Up to five peaks were 

identified in urine. Analysis by radio-HPLC suggested 

that the major peaks in both sexes treated by either 

exposure route were mono- and diglucuronides. Five 

peaks were identified in plasma, and some differences 

were noted in comparisons of i.v. to oral exposure. In the 

2 hr following dosing, most of the radioactivity in plasma 

was represented by bisphenol A glucuronide after i.v. 

dosing (57–82%) and oral dosing (89–100%). The percentage of radioactivity represented by unchanged bisphenol A was higher following i.v. (5–29%) than oral (0–1%) 

dosing. 

Kang et al. (2006) reviewed studies that provided some 

information about metabolism of bisphenol A in fish and 

birds. One study reported bisphenol A sulfate and 

bisphenol A glucuronide as the major metabolites 

detected in zebra fish exposed to bisphenol A. A second 

study conducted in carp reported an increase in UDPGT 

activity for bisphenol A in microsomes and metabolism 

of bisphenol A to bisphenol A glucuronide in intestine. In 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 203 

Table 44 

Excretion of Radioactivity Following Oral or Intravenous
 

Dosing of Rats With 0.1 mg/kg bw 14C-Bisphenol Aa
 

Percent radioactive dose excreted 

Time post-dosing, hr Urine Feces Total 

Oral 

0–24 6.371.1 49.372.1 55.772.8 

24–48 3.871.0 32.372.1 36.173.0 

Total 10.171.6 81.673.7 91.875.0 

Intravenous 

0–24 8.471.8 46.271.8 54.673.4 

24–48 4.170.9 31.471.5 35.471.8 

Total 12.570.9 77.671.8 90.172.7 

Values presented as mean7SD. a

Kurebayashi et al. (2003). 

quail embryos, metabolism and excretion of bisphenol A 

was reported, but specific metabolites were not indicated. Another study reported that 14C-bisphenol A 

administered orally or i.v. to laying quail was rapidly 

removed via bile and excreted through feces. 

2.1.2.4 Elimination: Elimination of bisphenol A and 

its metabolites was examined in Sprague–Dawley rats 

that were gavaged with bisphenol A and 14C-bisphenol A 

at 10 mg/kg bw (Domoradzki et al., 2003). One group of 

rats was not pregnant, and three additional groups were 

treated on either GD 6 (early gestation), 14 (mid 

gestation), or 17 (late gestation). More details of this 

study are available in Section 2.1.2.2. Most of the 

radioactivity (65–78%) was eliminated in feces. Elimination in urine accounted for 14–22% of the dose, and 

considerable variability for urinary elimination among 

animals was evident by the large standard deviations, 

which were 50% of means. The authors stated that 

bisphenol A glucuronide represented 62–70% of radioactivity in urine and bisphenol A represented 19–23% of 

radioactivity in urine [data were not shown by authors]. 

Nine peaks were identified in urine. In feces, 83–89% of 

radioactivity was represented by bisphenol A and 2–3% 

was represented by bisphenol A glucuronide; 7 peaks 

were identified in feces. The study authors concluded 

that urinary elimination and fecal elimination of radioactivity were similar in pregnant and non-pregnant rats. 

Difference in excretion following oral or i.v. exposure 

of rats to a low bisphenol A dose was examined by 

Kurebayashi et al. (2003). Three male rats/group were 

exposed to 0.1 mg/kg bw 14C-bisphenol A (499% 

radiochemical purity) in phosphate buffer vehicle by 

oral gavage or i.v. injection. Radioactivity levels were 

measured in urine and feces, which were collected over a 

48-hr period. Additional details of the study are included 

in Section 2.1.2.2. Results of that study are summarized in 

Table 44. With both oral and i.v. dosing, fecal excretion 

was the main route of elimination. 

Kurebayashi et al. (2005) examined elimination of 

radioactivity in 3 adult male and female F344 rats that 

were orally dosed with 0.1 mg/kg bw 14C-bisphenol A. 

Urine and feces were collected over a 168-hr period and 

analyzed by liquid scintillation counting. Total radioactivity excreted in urine and feces over the 168-hr period 

was B98% in males and females. In male rats, B10% was 

excreted in urine and B88% was excreted in feces. 

Female rats excreted B34% of the radioactivity in urine 

and B64% in feces. [The majority of radioactivity, 

B90%, was excreted over 48 hr by males and 72 hr by 

females.] 

Snyder et al. (2000) compared toxicokinetics of bisphenol A in CD and F344 rats. Four CD and F344 rats 

were gavaged with 100 mg/kg bw 14C-bisphenol A in 

propylene glycol vehicle. Disposition of radioactivity in 

urine, feces, and carcass was examined over a 144-hr 

period. Samples were analyzed by scintillation counting, 

HPLC, or nuclear magnetic resonance. Data were 

analyzed by ArcSin transformation of the square root of 

the mean and using two-sample t-test. Recovery of 

radioactivity was 93% in both strains. The highest 

concentrations of radioactivity were detected in feces 

(70% of dose in CD rat and 50% of dose in F344 rats) 

followed by urine (21% of dose in CD rat and 42% of 

dose in F344 rats). The percentages of the dose excreted 

in urine and feces differed significantly by strain. Much 

lower percentages of radioactivity were detected in the 

carcass (B1%). Bisphenol A glucuronide, representing 

81–89% of the dose, was the major urinary metabolite 

detected in both strains. A much lower percentage (2.2– 

10%) of the dose was represented by urinary bisphenol 

A. 

Kim et al. (2002b) reported urinary excretion of 

bisphenol A in 4-week-old male F344 rats given bisphenol A in drinking water at 0 (ethanol vehicle), 0.1, 1, 

10, or 100 ppm (equivalent to 0.011, 0.116, 1.094, or 

11.846 mg/kg bw/day) for 13 weeks. Urine samples were 

collected for 24 hr following administration of the last 

dose and analyzed by HPLC before and after digestion 

with b-glucuronidase. The focus of the study was male 

reproductive toxicity; the study is described in detail in 

Section 4.2.2.1. Bisphenol A was not detected in the urine 

of rats from the control and 2 lowest dose groups. [At the 

2 highest doses, free bisphenol A represented 60 and 

30% of the total urinary bisphenol A concentrations.] 

In rats exposed to 10 or 100 mg/kg bw/day 14Cbisphenol A through the oral, i.p., or s.c. routes, fecal 

elimination represented the highest percentage of radioactivity in all exposure groups (52–83%) (Pottenger et al., 

2000). Elimination of radioactivity through urine was 

B2-fold higher in females (21–34%) than males (13–16%) 

in all dose groups. Additional details of this study are 

included in Section 2.1.2.3. 

Elimination of bisphenol A and metabolites was 

examined in 3 adult male and female Cynomolgus 

monkeys dosed with 0.1 mg/kg bw 14C-bisphenol A/ 

non-radiolabeled bisphenol A by i.v. injection on Study 

Day 1 and by gavage on Study Day 15 (Kurebayashi 

et al., 2002). Additional details of the study are included 

in Section 2.1.2.2. Following oral or i.v. exposure, the 

percentage of radioactivity recovered in excreta and cage 

washes was 81–88% over a 1-week period. Most of the 

radioactivity was recovered in urine (combination of 

urine and cage washes), with most of the radioactivity 

excreted in urine within 12 hr and essentially all of the 

dose excreted within 24 hr following treatment. Percentages of radioactive doses recovered in urine within 1 

week after dosing were B79–86% following i.v. dosing 

and 82–85% following oral dosing. Much smaller 

amounts were recovered in feces during the week 

following i.v. or oral exposure (B2–3%). The study 

authors concluded that because fecal excretion was very 

Birth Defects Research (Part B) 83:157–395, 2008
204 CHAPIN ET AL. 

low following oral exposure, absorption was considered 

to be complete. The authors also noted that there were no 

obvious route or sex differences in excretion of radioactivity. The study authors concluded that terminal 

elimination half-lives were longer following i.v. than oral 

exposure. A limited amount of information was presented for the fast phase, defined as the 2 hr following i.v. 

injection. Fast-phase elimination half-life of bisphenol A 

following i.v. exposure was significantly lower in females 

(0.39 hr) than males (0.57 hr). There were no sex-related 

differences in fast-phase half-life for bisphenol A 

glucuronide (0.79–0.82 hr) or total radioactivity (0.61– 

0.67 hr). 

2.1.3 Comparison of humans and experimental 

animals. Studies comparing toxicokinetics and metabolism of bisphenol A in humans and laboratory animals 

were reviewed and are summarized below. In most cases 

the data were from original sources, but information 

from secondary sources was included if the information 

was not new or critical to the evaluation of developmental or reproductive toxicity. 

Elsby et al. (2001) compared bisphenol A metabolism 

by rat and human microsomes. Microsomes were 

obtained from 8 immature Wistar rats (21–25 days old) 

and histologically normal livers from 4 male (25–57 years 

old) and 4 female (35–65 years old) Caucasian donors 

who were killed in accidents. Human microsomes were 

pooled according to sex of the donor. Glucuronidation 

Table 45
 

Glucuronidation Kinetics in Microsomes From Immature
 

Rats and Adult Humansa
 

Sex/species 

Vmax, nmol/minute/ 

mg protein Km, mM 

Male/human 

Female/human 

Female/immature rat 

5.970.4 

5.270.3 

31.678.1 

77.578.3 

66.377.5 

27.071.2 

Data presented as mean7SEM. a

Elsby et al. (Elsby et al., 2001). 

was examined following exposure of microsomes to 

bisphenol A concentrations of 0–1000 mM [0–228 mg/L] 

for 30 min with human microsomes and 10 min with rat 

microsomes. Metabolites were identified by HPLC or 

LC/MS. Data were obtained from 4 experiments conducted in duplicate. Data were analyzed by Mann– 

Whitney test. Maximum velocity (Vmax) and the rate 

constant (Km) values are summarized in Table 45. The 

study authors reported a significant difference between 

the Vmax for glucuronidation in immature rats and 

humans. No sex-related difference was reported for 

glucuronidation by human microsomes. As a result of 

less extensive glucuronidation by human than rat 

microsomes, the study authors noted that estrogen target 

tissues in humans may receive higher exposure to 

bisphenol A than tissues of immature female rats used 

in estrogenicity studies. Lastly, oxidation of bisphenol A 

by female rat or human microsomes was examined 

following incubation with 200 mM [46 mg/L] bisphenol A 

and NADPH. The only metabolite identified was 5­

hydroxybisphenol A. 

The European Union (2003) reviewed a series of 

studies by Sipes that compared metabolism of bisphenol 

A in microsomes from male and female humans (15 

pooled samples/sex and 3–5 individual samples/sex), 

rats (4/sex), and mice (4/sex). It was concluded that the 

studies generally agreed with the findings of Elsby et al. 

(2001). Clearance rates (Vmax/Km) in human microsomes 

(0.4–0.9 mL/min/mg for pooled samples and 0.3–0.5 

mL/min/mg in individual samples) were lower than 

those observed in rats (1.0–1.7 mL/min/mg) and mice 

(1.3–3.0 mL/min/mg). 

Pritchett et al. (2002) compared metabolism of bisphenol A in hepatocyte cultures from humans, rats, and 

mice. Cell cultures were prepared from adult male and 

female F344 rats, Sprague–Dawley rats, and CF1 mice. 

Human hepatocyte cultures were obtained from 3 

females and 2 males. [No information was provided 

about the age of human donors.] Cells were exposed to 14C-bisphenol A (99.3% purity)/bisphenol A (499% 

purity) in a DMSO vehicle. In a cytotoxicity assessment, 

Table 46
 

Metabolites Obtained From Incubation of Human, Rat, and Mouse Hepatocyte Cultures With 20 mM [4.6 mg/L]
 

Bisphenol Aa
 

Percentage of parent compound or metabolites 

Sex and species Glucuronide/sulfate Sulfate Glucuronide Bisphenol A 

Human samples 

Female–1 4 0 93 0 

Female–2 2 0 84 2 

Female–3 43 2 55 0 

Male–1 1 0 85 0 

Male–2 0 7.5 75 0 

Rodent samples 

Male F344 rat 70 0 30 0 

Female F344 rat 10 0 86 0 

Male Sprague–Dawley rat 30 2 58 0 

Female Sprague–Dawley rat 0 0 100 0 

Male CF1 Mouse 0 0 100 0 

Female CF1 mouse 0 0 93 0 

Human cells were incubated for 3 hr, and animal cells were incubated for 6 hr. 

a

Pritchett et al. (2002). 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 205 

lactate dehydrogenase activity was measured in rat cells 

following incubation for 18 hr in 5–100 mM [1.1–23 mg/L] 

bisphenol A, and cytotoxicity was observed at Z75 mM 

bisphenol A. Bisphenol A concentrations tested and 

times of exposure were 5–20 mM [1.1–4.6 mg/L] for up to 

6 hr in time-dependent metabolism studies and 2.5– 

30 mM [0.57–6.8 mg/L] for 10 min in concentration-dependent metabolism studies. Metabolites in cell media were 

analyzed by HPLC and LC-MS/MS. 

Analysis of media from human hepatocytes incubated 

with bisphenol A indicated that the major metabolite was 

bisphenol A glucuronide, and compounds found at 

lower concentrations were bisphenol A glucuronide/ 

sulfate diconjugate, and bisphenol A sulfate conjugate. 

Table 46 summarizes percentages of each type of 

metabolite detected in media following incubation with 

Table 47
 

Rates of Bisphenol A Glucuronide Formation Following
 

Incubation of Human, Rat, and Mouse Hepatocytes With
 

Bisphenol Aa
 

Vmax, nmol/min/ Hepatic 

0.5 x 106 capacity, 

Species and sex hepatocytes mmol/hrb 

Human female 0.27 8000 

F344 rat female 0.46 46.5 

F344 rat male 0.36 61.8 

Sprague Dawley female 0.39 54.5 

Sprague Dawley male 0.45 79.9 

CF1 mouse female 0.50 13.8 

CF1 mouse male 0.82 23.6 

a

Pritchett et al. (2002).
 

b

Hepatic capacity was estimated by multiplying Vmax by total
 

numbers of hepatic cells in vivo.
 

Table 48
 

Toxicokinetic Endpoints for Bisphenol A in Mice, Rats,
 

Rabbits, and Dogs Intravenously Dosed With 2 mg/kg
 

bw Bisphenol Aa
 

Endpoint Mouseb Rat Rabbit Dog 

Systemic 

clearance, L/hr 

Volume of 

0.3 

0.1 

1.970.4 

1.370.4 

12.674.9 

7.172.3 

27.178.0 

20.075.4 

distribution, L 

Half-life, min 39.9 37.6712.8 40.8717.1 43.7721.9 

Data are presented as mean7SD.
 a

Cho et al. (2002).
 

b

Variances not reported.
 

20 mM [4.6 mg/L] bisphenol A for 3 hr in human cells and 

6 hr in rodent cells. In cells from all sexes and species 

except male F344 rats, bisphenol A glucuronide was the 

major metabolite detected. The glucuronide/sulfate 

diconjugate was the major metabolite detected in cells 

from male F344 rats. In concentration-dependent studies 

conducted in F344 rat hepatocytes, a biphasic curve was 

obtained following a 10-min incubation, with a Vmax of 

0.36 nmol/min at bisphenol A concentrations of 20– 

30 mM [4.6–6.8 mg/L] and a Vmax of B0.15 nmol/min at 

bisphenol A concentrations of 2.5–10 nM [0.57–2.3 mg/L]. 

Table 47 summarizes the higher Vmax values obtained 

with cells from human, rat, and mouse livers. Total 

hepatic capacity was determined by multiplying Vmax by 

total number of hepatocytes/liver in vivo. [The only 

graphical data presented were for male F344 rats]. The 

authors noted that Vmax values were highest in mice4rats4humans. However, when adjusted for total hepatocyte number in vivo, the values were predicted to be 

highest in humans 4 rats 4 mice. 

Data from Pritchett et al. (2002) appeared to be 

included in a series of unpublished studies by Sipes that 

were reviewed by the European Union (2003). In their 

review, the European Union noted that metabolic 

patterns appear to be similar in humans, rats, and mice. 

It was stated that the biphasic kinetic profile indicated 

involvement of a high-affinity glucuronidase enzyme at 

low concentrations and a high-capacity enzyme at high 

concentrations. In the interpretation of kinetic profiles in 

humans and experimental animals, the authors of the 

European Union report noted that the study calculations 

did not consider in vivo conditions such as varying 

metabolic capacity of hepatic cells, relationship of hepatic 

size to body size, and possibly important physiological 

endpoints such as blood flow. In addition, it was 

noted that calculations were based on limited data that 

did not address inter-individual variability in enzyme 

expression. 

Cho et al. (2002) examined toxicokinetics of bisphenol 

A in mouse, rat, rabbit, and dog and used that 

information to predict toxicokinetic values in humans. 

Bisphenol A was administered by i.v. injection at 2 mg/ 

kg bw to 5 male ICR mice and at 1 mg/kg bw to 7 male 

Sprague–Dawley rats, 7 male New Zealand White 

rabbits, and 5 male beagle dogs. Blood samples were 

drawn before dosing and at multiple time points 

between 2 min and 6 hr following injection. Serum 

bisphenol A concentrations were measured by HPLC. 

Toxicokinetic endpoints in animals are summarized in 

Table 48. The study authors noted that clearance and 

volume of distribution increased with increasing animal 

weight but that terminal half-life remained relatively 

constant across the different species. Simple allometric 

Table 49
 

Predicted Bisphenol A Toxicokinetic Endpoints in Humans Based on Results From Experimental Animal Studiesa
 

Prediction method 

Endpoint Allometric scaling Kallynochrons Apolysichrons Dienetichrons 

Systemic clearance, L/hr 127.1 123 120.7 46.0 

Volume of distribution, L 125.3 229.7 138.0 149.3 

Half-life, min 43.6 110.4 67.8 196.2 

a

Cho et al. (2002). 

Birth Defects Research (Part B) 83:157–395, 2008
206 CHAPIN ET AL. 

scaling and species-invariant time methods were used to 

predict values for a 70-kg human, and those values are 

summarized in Table 49. Regression analyses of estimates 

using the species-invariant time methods demonstrated 

that data from the 4 animal species were superimposable 

(r 5 0.94–0.949). 

Teeguarden et al. (2005) developed a physiologically 

based pharmacokinetic (PBPK) model for bisphenol A. 

Rat toxicokinetic data for the model were obtained from 

the studies by Pottenger et al. (2000) and Upmeier et al. 

(2000). Human toxicokinetic data were obtained from the 

study by Vo ̈lkel et al. (2002). The model was developed to 

simulate blood and uterine concentrations of bisphenol A 

following exposure of humans through relevant routes. 

Correlations were determined for simulated bisphenol A 

binding to uterine receptors and increases in uterine wet 

weight, as determined by an unpublished study by 

Twomey. Although intestinal metabolism of bisphenol A 

to the glucuronide metabolite had been demonstrated 

recently, the model attributed bisphenol A metabolism 

entirely to the liver. Plasma protein binding was 

considered in both the rat and human model. The model 

accurately simulated plasma bisphenol A glucuronide 

concentrations in humans orally administered 5 mg 

bisphenol A, with the exception of underpredicting 

bisphenol A glucuronide concentrations at the 24–48-hr 

period following exposures. Cumulative urinary elimination of bisphenol A glucuronide in human males and 

females was simulated accurately. Less accurate simulations were observed for toxicokinetics in orally exposed 

rats, and the study authors indicated that a likely cause 

was oversimplification of the rat gastrointestinal compartment. Comparisons in metabolic clearance rates for 

i.v. and oral exposure suggested significant intestinal 

glucuronidation of bisphenol A. Enterohepatic recirculation strongly affected terminal elimination in rats but not 

humans. Consideration of bound versus unbound bisphenol A was found to be important in simulating 

occupancy of the estrogen receptor (ER) and uterine 

weight response. No increase in uterine weight was 

reported with simulated receptor occupancy of B1–15%. 

An increase in uterine weight was reported with B25% 

receptor occupancy, and doubling of uterine weight was 

reported with 63% receptor occupancy. 

Shin et al. (2004) developed a PBPK model to predict 

the tissue distribution (lung, liver, spleen, kidneys, heart, 

testes, muscle, brain, adipose tissue, stomach, and small 

intestine) and blood pharmacokinetics of bisphenol A in 

rats and humans. The model was based on experimentally determined steady state blood-to-serum and tissueto-blood partition ratios and does not include parameters 

to account for elimination via glucuronidation or 

differences in metabolism between rats and humans 

(e.g., enterohepatic circulation). Predicted concentrationtime profiles were then compared to actual rat toxicokinetic data and to a profile for a simulated 70-kg human. 

Rat toxicokinetic information was obtained by administering multiple i.v. injections of bisphenol A (0.5 mg/kg) 

to adult male rats to achieve steady state. Bisphenol A 

concentrations were determined by a modified HPLC 

method with fluorescence detection. The authors noted 

good agreement between predicted and observed 

concentration-time profiles for blood and all tissues but 

did not present any statistical analysis or evaluate 

the performance of alternative models in order to 

Table 50
 

LD50 s for Bisphenol A
 

Species Exposure route LD50 (mg/kg bw) 

Rat Oral 3300–4100a 

5000b 

3250c 

Inhalation 4170 mg/m3b 

Mouse Oral 4100–5200a 

2400c 

Intraperitoneally 150c 

Guinea pig Oral 4000c 

Rabbit Oral 2230b,c 

Dermal 42000b 

3 mL/kgc 

a

National Toxicology Program (NTP, 1982). 

b

Reviewed by the European Union (2003). 

c

Reviewed in ChemIDplus (2006). 

establish goodness of fit. Based on the figures presented 

in the article, the PBPK model appeared to more 

accurately predict concentrations of bisphenol A in some 

tissues (e.g., blood, lung, and liver) better than others 

such as the small intestine and adipose tissue. The model 

was then applied to predict blood and tissue levels of 

bisphenol A in a 70 kg human after single i.v. injection 

(5-mg dose) and multiple oral administrations to 

steady state (100-mg doses every 24 hr). Tissue volumes 

and blood flow rates for a 70 kg human were taken 

from the literature. The authors concluded that 

simulated steady-state human blood levels (0.9–1.6 ng/ 

ml) were comparable to blood levels of bisphenol A 

reported in the literature (1.49 ng/ml). In addition, the 

authors noted the similarity of predicted toxicokinetic 

endpoints obtained from their PBPK model to those 

predicted by Cho et al. (2002) based on simple allometric 

scaling on rat data. 

2.2 General Toxicity, Estrogenicity, and 

Androgenicity 

This section includes information on general toxicity as 

well as information on estrogenicity and androgenicity; 

however, results of estrogenicity and androgenicity 

testing are not considered a priori evidence of toxicity. 

2.2.1 General toxicity. The European Union (2003) 

reported there were no adequate studies for assessing 

acute toxicity of bisphenol A in humans. 

In an acute toxicity study in rats orally dosed with 

bisphenol A at Z2000 mg/kg bw, clinical signs included 

lethargy, prostration, hunched posture, and piloerection 

[reviewed by (European-Union, 2003)]. Gross signs in 

animals that died included pale livers and hemorrhage in 

the gastrointestinal tract. In a study in which male and 

female rats were subjected to whole body inhalation 

exposure to 170 mg/m3 bisphenol A dust for 6 hr, there 

were no gross signs of toxicity [reviewed by (EuropeanUnion, 2003)]. Effects observed in the respiratory tract at 

2 but not 14 days following exposure included slight 

inflammation of nasal epithelium and slight ulceration of 

the oronasal duct. LD50 reported in studies with oral, 

dermal, inhalation, or i.p. exposure are summarized in 

Table 50. The European Union (2003) concluded that 

bisphenol A is of low acute toxicity through all exposure 

routes relevant to humans. 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 207 

The European Union (2003) noted limited anecdotal 

data reporting skin, eye, and respiratory tract irritation in 

workers exposed to bisphenol A, but concluded that the 

reports were of uncertain reliability. It was noted that a 

recent, well-conducted study in rabbits demonstrated 

that bisphenol A is not a skin irritant. Other studies 

conducted in rabbits demonstrated eye irritation and 

damage, and it was concluded the bisphenol A can 

potentially cause serious eye damage. Slight respiratory 

tract inflammation occurred in rats inhaling Z50 mg/m3 

bisphenol A, and it was concluded that bisphenol A had 

limited potential for respiratory irritation. Based on the 

results of the studies described above, the European 

Union concluded that bisphenol A is not corrosive. 

The European Union (2003) reviewed studies examining possible sensitization reactions in humans exposed to 

products containing bisphenol A, and those studies 

reported mixed results. In studies reporting positive 

findings, it was unclear if bisphenol A or epoxy resins 

were the cause of hypersensitivity. Cross-sensitization 

responses in individuals exposed to compounds similar 

to bisphenol A were also reported. Animal studies were 

determined unreliable for assessing sensitization. Based 

on the results of human studies, it was concluded that 

bisphenol A may have potential for sensitization in 

individuals exposed to resins. Human studies suggested 

that bisphenol A can induce dermal photosensitization 

responses. Photosensitization studies in mice resulted in 

reproducible positive results. Mechanistic studies in mice 

suggested that sensitization occurs through an immunemediated process. The overall conclusion of the European Union was that it was somewhat unclear if 

bisphenol A induces orthodox skin sensitization, photosensitization, or responses in individuals previously 

sensitized to another substance, such as epoxy resins. 

No information was available on potential respiratory 

sensitization by bisphenol A. 

The European Union (2003) summarized systemic 

toxicity reported in subchronic, chronic, and reproductive toxicity studies of rats, mice, and dogs. CERHR also 

reviewed the studies that examined reproductive organs, 

and those studies are summarized in detail in the 

appropriate section of this report. A relevant study by 

Yamasaki et al. (2002a) was published subsequent to the 

European Union review and was reviewed in detail by 

CERHR. 

In studies reviewed by the European Union (2003) and 

in a study by Yamasaki et al. (2002a), rats were orally 

exposed to bisphenol A for periods of 28 days to 2 years. 

Cecal enlargement occurring at doses Z25 mg/kg bw/ 

day was the effect observed most frequently in those 

studies but was not considered toxicologically significant 

by the European Union. Histological alteration in the 

cecum consisting of mucosal hyperplasia was only 

reported in one study at doses Z200 mg/kg bw/day. 

Histopathological changes in liver and kidney were 

reported at doses Z500 mg/kg bw/day. The changes in 

liver were characterized by prominent hepatocyte nuclei 

or inflammation. Histopathology in kidney was characterized by renal tubule degeneration or necrosis. 

Testicular toxicity (degeneration of seminiferous tubules 

and arrested spermatogenesis) was observed in 1 study 

at doses Z235 mg/kg bw/day. 

The European Union (2003) found subchronic and 

chronic studies conducted by the NTP (NTP, 1982) to be 

the only reliable studies for assessing systemic toxicity in 

mice orally exposed to bisphenol A. The liver was found to 

be the target organ of toxicity, with multinucleated giant 

hepatocytes observed in male mice exposed to Z120 mg/kg 

bw/day and female mice exposed to 650 mg/kg bw/day. 

In a 90-day dietary study in dogs reviewed by the 

European Union (2003), an increase in relative liver weight 

with no accompanying histopathological alterations was 

found to be the only effect at doses Z270 mg/kg bw/day. 

This finding was considered by the European Union to be 

of doubtful toxicological significance. 

In a subchronic inhalation exposure study in rats 

reviewed by the European Union (2003), cecal enlargement as a result of distention by food was observed at 

Z50 mg/m3

. Also observed at Z50 mg/m3 were slight 

hyperplasia and inflammation of epithelium in the 

anterior nasal cavity. 

A limited number of repeat-dose systemic toxicity 

studies were summarized in detail by CERHR because 

they included examination of reproductive organs. Those 

studies are summarized in detail below. 

NTP (1982), conducted acute, subacute, and subchronic bisphenol A toxicity studies in F344 rats and B6C3F1 

mice. Animals were randomly assigned to treatment 

groups. Purity of bisphenol A was o98.2%. Concentration and stability of bisphenol A in feed were verified. In 

acute studies, single doses of bisphenol A in a 1.5% 

acacia vehicle were administered by gavage to 5 rats/ 

group/sex at doses of 2150, 3160, 4640, or 6810 mg/kg 

bw/day and 5 mice/group/sex at 1470, 2150, 3160, 4640, 

6810, or 10,000 mg/kg bw. LD50 values for that study are 

summarized in Table 50. 

In a 14-day repeat dose study, survival and body 

weight gain were evaluated in 5 rats and mice/sex/ 

group that were fed diets containing bisphenol A at 0, 

500, 1000, 2500, 5000, or 10,000 ppm. Survival was 

unaffected by treatment. Weight gain was reduced by 

60% or more in male rats exposed to Z2500 ppm and 

40% or more in female rats exposed to Z5000 ppm 

bisphenol A. Survival and weight gain in mice were not 

affected by bisphenol A exposure. 

In subchronic studies, 10 rats and mice/sex/group 

were exposed to bisphenol A in diet for 13 weeks. 

Dietary doses were 0, 250, 500, 1000, 2000, or 4000 ppm 

for rats and 0, 5000, 10,000, 15,000, 20,000, or 25,000 ppm 

for mice. A review by the European Union (2003) 

estimated bisphenol A intake at 0, 25, 50, 100, 200, and 

400 mg/kg bw/day for rats, 0, 600, 1200, 1800, 2400, and 

3000 mg/kg bw in male mice, and 0, 650, 1300, 1950, 

2600, and 3250 mg/kg bw/day in female mice. Animals 

were observed and weighed during the study and killed 

and necropsied on Day 91 of the study. [Histopathological evaluations were conducted but it was not clear if 

all dose groups and all animals/dose group were 

examined. There was no mention of statistical analyses.] In rats, the only deaths occurred in 2/10 males of 

the 1000 ppm group. Weight gain was reduced by 18% or 

more in male rats and 10% or more in female rats 

exposed to Z1000 ppm. There were no effects on feed 

intake. Hyaline masses in the bladder lumen were not 

observed in control male rats but were observed in 5 of 

10 males exposed to 250 ppm, 3 of 10 exposed to 

500 ppm, 3 of 10 exposed to 1000 ppm, 6 of 10 exposed 

to 2000 ppm, and 4 of 10 exposed to 4000 ppm. Cecal 

enlargement, which was observed in rats at a rate of 

Birth Defects Research (Part B) 83:157–395, 2008
208 CHAPIN ET AL. 

60–100% in each dose group with the exception of 

females exposed to 250 ppm was considered to be 

treatment-related. No histological alterations were observed in the cecum. Death in mice was limited to 2 of 10 

females in the 5000 ppm group. Weight gain was reduced 

by at least 14% in male mice exposed to Z15,000 ppm. 

Non-dose-related decreases in weight gain of 17% or 

more occurred in female mice of all dose groups. A doserelated increase in multinucleated giant hepatocytes was 

observed in all dose groups of male mice; the only 

incidence data reported for multinucleated giant hepatocytes were 0 of 10 female controls and 9 of 10 male mice 

of the 25,000 ppm group. [A complete set of data for 

histopathological findings was not presented for rats or 

mice.] 

Yamasaki et al. (2002a) examined the effects of 

bisphenol A exposure on male and female CD rats in a 

study conducted according to Good Laboratory Practices 

(GLP). [Because this study included a number of 

reproductive organ and hormone endpoints, it is also 

discussed in Sections 4.2.1.1 and 4.2.2.1.] Rats were fed a 

commercial diet (MF Oriental Yeast Co.) and housed in 

stainless steel wire-mesh cages. Rats were groups 

according to body weight and then randomly assigned 

to treatment groups. Ten 7-week-old rats/sex/group 

were gavaged with bisphenol A at 0 (olive oil vehicle), 

40, 200, or 1000 mg/kg bw/day for 28 days. Due to the 

death of 1 animal exhibiting clinical signs in the 

1000 mg/kg bw/day group, the high-dose was reduced 

to 600 mg/kg bw/day on Study Day 8. In an additional 

study, rats were exposed to ethinyl estradiol at 0, 10, 50, 

or 200 mg/kg bw/day for 28 days. Endpoints examined 

during the study were clinical signs, body weight gain, 

and food intake. Estrous cyclicity was examined in 

females for 2 weeks beginning on Study Day 15. Males 

were killed on Study Day 29 and females were killed in 

diestrus on Study Day 30, 31, or 32. Hematology and 

clinical chemistry endpoints were assessed, and blood 

hormone concentrations were measured by immunoassay systems. Sperm motility and viability were evaluated. Organs, including those of the reproductive system, 

were weighed and subjected to histopathological evaluation. With the exception of the testis and epididymis, 

which were fixed in Bouin solution, the organs were 

fixed in 10% neutral buffered formalin. Statistical 

analyses included Bartlett test for homogeneity of 

variance, ANOVA, Dunnett test, and/or Kruskall–Wallis 

test. 

One female and 3 males from the high-dose group 

died; clinical signs observed in those animals included 

soft stools, decreased mobility, reduced respiration rate, 

and decreased body temperature. Soft stools were also 

observed in surviving males and females of the mid- and 

high-dose groups. Results of the study are summarized 

in Table 51. Terminal body weights were lower in females 

of the mid- and high-dose groups and males of the highdose group. During the first week of study, food intake 

was decreased in both sexes of the mid- and high-dose 

group. [Data were not shown by study authors.] As 

noted in Table 51, some alterations in hematological and 

clinical chemistry endpoints were observed, mainly at 

the high-dose. [Data were not shown by study authors.] 

There were no treatment-related abnormalities in sperm 

or alterations in blood concentrations of thyroid hormones, follicle stimulating hormone (FSH), luteinizing 

Table 51
 

Toxicological Effects in Rats Gavaged With Bisphenol A
 

for 28 Daysa
 

Bisphenol A dose 

(mg/kg bw/day) 

Endpoint 

40 200 600–1000c 

Males 

Terminal body weight 

Relative testes weight 

Relative ventral prostate 

weight 

Relative adrenal weight 

Feed intakeb 

Prothrombin timeb 

Glutamic-oxaloacetic 

transaminaseb 

Triglycerideb 

Alkaline phosphataseb 

g-Glutamyl transpeptidaseb 

Chlorideb 

Renal tubular degeneration 

and necrosis 

Forestomach squamous 

epithelial cell hyperplasia 

Lacteal dilatation in 

duodenum 

Lacteal dilation in jejunum 

Mucosal hyperplasia in cecum 

Mucosal hyperplasia in colon 

Adrenal cortical vacuolization 

Females 

Terminal body weight 

Relative thyroid weight 

Relative liver weight 

Relative heart weight 

Feed intakeb 

Hemoglobin and 

hematocrit valuesb 

Cholinesteraseb 

Glutamic-oxaloacetic 

transaminaseb 

Albumin and albumin: 

globulin ratsb 

Diestrus Z4 days 

Prominent hepatocyte nuclei 

Renal tubular degeneration 

and necrosis 

Forestomach squamous 

epithelial cell hyperplasia 

Lacteal dilatation in duodenum 

Mucosal hyperplasia in cecum 

Adrenal cortical vacuolization 

a

Yamasaki et al. (2002a).
 

b

Data were not shown by study authors.
 

c

The dose was 1000 mg/kg bw/day at the beginning of the
 

study, but was decreased to 600 mg/kg bw/day in the second
 

week of the study due to excessive toxicity.
 

m,k Statistically significant increase, decrease compared to
 

controls; 2 no statistically significant effects compared to
 

controls.
 

hormone (LH), 17b-estradiol, prolactin, or testosterone. 

Number of females with diestrus lasting 4 or more days 

was increased in the high-dose group. Changes in 

relative organ weights [assumed to be relative to body 

2 2

2 2

2 2

2 2

2 k

2 2

2 m

2 2

2 2

2 2

2 2

0/10 0/10 

0/10 0/10 

0/10 10/10 

0/10 0/10 

0/10 3/10 

0/10 2/10 

0/10 0/10 

2 k 7% 

2 2

2 2

2 k 9% 

2 k

2 2

2 k

2 2

2 2

0/10 0/10 

0/10 0/10 

0/10 0/10 

0/10 0/10 

0/10 7/10 

0/10 6/10 

0/10 0/10 

k 17% 

m 21% 

k 28% 

m 19% 

k

m

m

k

m

m

m

7/7 

6/7 

2/7 

2/7 

6/7 

7/7 

3/7 

k 5%
 

m 22%
 

m 10%
 

k 15%
 

k 

k 

k

m

k

3/9 

4/9 

9/9 

5/9 

6/9 

4/9 

3/9 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 209 

weight] included decreased heart weight in females from 

the mid- and high-dose groups. At the high-dose, there 

were decreases in relative weight of ventral prostate and 

increases in relative weights of testis and adrenals in 

males and thyroid and liver in females. Gross signs 

observed in animals that died included enlarged kidney, 

elevated mucosa in the forestomach, and atrophied 

spleen and thymus. In surviving animals, the cecum 

was enlarged in the mid- and high-dose group and 

forestomach mucosa was elevated in the high-dose 

group. As described in more detail in Table 51, histopathological alterations were observed in the intestine, 

cecum, and colon of males and intestine and cecum of 

females in the mid- and high-dose groups. Additional 

histopathological alterations were observed in the highdose group in the kidney, forestomach, and adrenals of 

males and females and livers of females. 

Male rats from the mid- and high-dose ethinyl 

estradiol groups experienced decreased prostate, seminal 

vesicle, and pituitary weights, increased testis weight, 

and histopathological alterations in prostate, seminal 

vesicle, mammary gland, and testis. Females from the 

mid- and high-dose ethinyl estradiol group experienced 

alterations in estrous cyclicity. Females from the 

high-dose group experienced decreased ovary weight, 

increased uterine weight, and histopathological changes 

in ovary, uterus, and vagina. 

General Electric (1984) conducted a subchronic toxicity 

study in Beagle dogs orally dosed with bisphenol A 

[purity not reported]. Dogs weighing 6.5–13.4 kg were 

housed in metal metabolism cages and fed Purina Dog 

Chow. During a 90-day period, 4 dogs/sex/group were 

given feed containing bisphenol A at 0, 1000, 3000, or 

9000 ppm. The European Union (2003) estimated bisphenol A intake at 0, 28, 74, or 261 mg/kg bw/day in 

males and 0, 31, 87, or 286 mg/kg bw/day in females. 

Dogs were observed for body weight gain, food, intake, 

and clinical signs. Ophthalmoscopic examination was 

conducted before and following the treatment period. 

Hematology, clinical chemistry, and urinalysis evaluations were conducted before treatment and at 1, 2, and 3 

months into the study. Dogs were killed at the end of the 

treatment period. Organs were weighed and fixed in 10% 

neutral buffered formalin. Histopathological evaluations 

were conducted in organs from the control and high-dose 

groups; prostate, uterus, testis, and ovary were among 

organs evaluated. [Procedures for statistical analyses 

were not described.] No treatment-related clinical signs 

(conducted monthly), ophthalmological changes, or 

death were observed during the study. Bisphenol A 

treatment did not affect body weight gain or food intake. 

There were no treatment-related effects on hematology, 

biochemistry, or urinalysis. Relative liver weight was 

significantly increased [by 18% in males and 26% in 

females] in the high-dose group, and the study authors 

considered the effect to be treatment-related. No treatment-related gross or histopathological lesions were 

observed in the high-dose group. 

Nitschke et al. (1988) conducted a subchronic inhalation toxicity test with bisphenol A in F344 rats. Rats were 

fed Purina Certified Rodent Chow 5002 and housed in 

stainless steel wire cages. At 7 weeks of age, rats were 

stratified according to body weight and randomly 

assigned to treatment groups. Thirty rats/sex/group 

received whole-body exposures to polycarbonate grade 

bisphenol A dust (99.7% purity) at 0, 10, 50, or 150 mg/ 

m3 for 6 hr/day, 5 days/week, for 13 weeks. Mass 

median aerodynamic diameter of bisphenol A dust was 

measured at r5.2 microns. Stability and concentrations 

of bisphenol A were verified. Rats were observed for 

clinical signs, body weight gain, and food intake. Ten 

rats/sex/group in each time period were killed and 

necropsied on the day following and at 4 and 12 weeks 

following exposure. At each necropsy period, hematological and clinical chemistry endpoints were examined. 

The lungs, brain, kidneys, and testes were weighed. 

Numerous organs were preserved in 10% phosphatebuffered formalin. In most cases, histological examinations were conducted in organs from the control and 

high-dose groups. Respiratory organs and organs with 

lesions or signs of toxicity were histologically examined 

at all dose levels. Included among organs undergoing 

histopathological examination immediately after the 

exposure period were the epididymis, mammary gland, 

ovary, oviduct, prostate, seminal vesicles, testis, uterus, 

and vagina. No reproductive organs were examined 

following the recovery periods. Statistical analyses 

included Bartlett’s test, ANOVA, Dunnett test, Wilcoxon 

Rank-Sum test, and Bonferroni correction for multiple 

comparisons. Gross pathology and histopathology data 

did not appear to have been statistically analyzed. 

During the exposure period, a reddish material around 

the nose (most likely porphyrin) was observed in 2–10 of 

10 animals/sex in the 50 and 150 mg/m3 groups. Perineal 

soiling was observed in 2 of 10 females in the 10 mg/m3 

group and 9–10 of 10 animals/sex in the 50 and 150 mg/ 

m3 groups. Decreased body weight gain during treatment was observed in males from all dose groups and 

females in the 50 and 150 mg/m3 groups. Immediately 

following the treatment period, terminal body weights 

were reduced by B5% in males and B11% in females 

from the 150 mg/m3 group. [Body weights were B4% 

lower in males from the 50 mg/m3 group.] No differences in feed intake were observed at this or any other 

time period in the study. The only hematological effect 

observed was slightly increased hemoglobin in males 

exposed to 10 mg/m3

, but the study authors did not 

consider the effect to be biologically significant. Clinical 

chemistry observations in the 150 mg/m3 group included 

decreased serum glutamic pyruvic transaminase activity, 

serum glutamic oxaloacetic transaminase activity, and 

glucose in males and decreased total protein and 

albumin and increased alkaline phosphatase activity in 

females. Alkaline phosphatase activity was also increased in females exposed to 50 mg/m3

. The study 

authors did not consider any of the clinical chemistry 

changes to be biologically [toxicologically] significant. 

Absolute liver weight was decreased in males exposed to 

Z10 and 150 mg/m3

, and relative brain weight was 

increased in females exposed to Z50 mg/m3

. Additional 

organ weight changes observed in females from the 

150 mg/m3 group included decreased absolute liver and 

kidney weights and increased relative lung weights. 

Because the organ weight changes were not associated 

with microscopic changes in organs, the study authors 

concluded that the effects reflected decreases in body 

weight and were not toxicologically significant. Cecal 

size was increased as a result of distention by food in all 

(10/dose/sex) males and females exposed to Z50 mg/ 

m3

, and the effect was considered to be treatmentBirth Defects Research (Part B) 83:157–395, 2008
210 CHAPIN ET AL. 

related. No histopathological alterations were observed 

for cecal wall morphology. Hemolyzed blood was 

observed in the stomachs of three to seven of 10 

males/group exposed to 50 and 150 mg/m3

, but there 

were no signs of histopathological alterations in the 

gastrointestinal tract. Slight histopathological alterations, 

consisting of hyperplasia in stratified squamous and 

ciliated epithelium lining and inflammation of submucosal tissues was observed in the anterior nasal cavities 

of all (10/dose/sex) males and females exposed to 

Z50 mg/m3

. Slight-to-moderate hyperplasia of goblet 

cells was also observed in the lateral nasal wall. No other 

treatment-related histopathological alterations were observed, including in reproductive organs. 

During the 4-week recovery period, body weights 

remained lower in males and females of the 50 and 

150 mg/m3 groups At 4 weeks following exposure, 

terminal body weights of males and females in the 

150 mg/m3 group were B6% lower than control values. 

A decrease in white blood cell count in females from the 

10 and 150 mg/m3 groups was the only hematological 

effect observed. The clinical chemistry effects that were 

somewhat consistent with effects observed immediately 

following treatment were increased alkaline phosphatase 

activity in females exposed to 10 and 150 mg/m3 and 

decreased serum glutamic pyruvic activity transaminase 

activity in females exposed to 150 mg/m3

; the study 

authors did not consider the clinical chemistry changes 

to be treatment-related. The study authors concluded 

that an increase in relative brain weight in males of the 

150 mg/m3 group was related to decreased body weights 

in those animals. Enlarged cecal size was observed in 5 of 

10 males of the 150 mg/m3 group, a decreased incidence 

compared to the period immediately following treatment. Nasal histopathology was observed in the 150 mg/ 

m3 but was reduced in magnitude and severity compared to rats observed immediately following exposure. 

In rats examined following 12 weeks of recovery, body 

weights of males in the 150 mg/m3 group remained 

lower than controls, and terminal body weight was 

decreased by B6%. An increase in white blood cell 

counts but not differential counts was observed in male 

rats of the 10 and 150 mg/m3 group. The only clinical 

chemistry finding consistent with earlier observations 

was decreased total protein and globulin in females from 

the 150 mg/m3 group, but the study authors did not 

consider the effect to be biologically significant. Organ 

weight changes in the 150 mg/m3 group included 

decreased absolute kidney and lung weights in males 

and decreased absolute and relative kidney weights in 

females. No histopathological alterations were observed 

in kidney or lung. No other gross or histopathological 

alterations were observed, including cecal enlargement 

and nasal histopathology, which were observed at earlier 

time periods. 

2.2.2 Estrogenicity. The first identification of bisphenol A as an estrogen has been attributed to Dodds 

and Lawson (1936), who reported that 100 mg injected by 

an unspecified route twice daily for 3 days resulted in 

maintenance of 5 of 5 rats in vaginal estrus for 40 days. 

The estrogenicity of bisphenol A has since been 

evaluated using several different kinds of assays. In 

vitro studies are summarized in Table 52, and in vivo 

studies are summarized in Table 53 using comparisons 

with 17b-estradiol, ethinyl estradiol, diethylstilbestrol, 

and, in one study, estrone. There is considerable 

variability in the results of these studies with the 

estrogenic potency of bisphenol A ranging over about 8 

orders of magnitude, but similar means (Fig. 2). 

The most common method of comparing potency is to 

test responses over a range of concentrations and to 

compare the concentrations producing the half-maximal 

(or other fractional) response of the comparator estrogen. 

An alternative is to compare the magnitude of the 

response at an equimolar concentration of the 2 estrogens. The difference in these two methods is illustrated 

in Figure 3. An example of the difference in potency 

estimations according to comparison method is the study 

of Vivacqua et al. (2003), in which the fold-increase in 

reporter activity for an estrogen-responsive gene was 

compared over a range of concentrations for bisphenol A 

and for 17b-estradiol. This study’s Figure 3 presents 

curves analogous to Figure 3, but also presents a bar 

graph comparing response of the reporter at a 10 -7 M 

concentration of each estrogen. Based on the halfmaximal response to 17b-estradiol, bisphenol-A 

appeared 1000 times less potent than 17b-estradiol, 

but based on the fold-difference in reporter activity 

at 10 -7 M, bisphenol A was about half as potent. 

Data for other estrogenicity comparisons in this study 

and in many other studies are presented only using bar 

graphs comparing responses at the same molar concentrations of the 2 estrogens, thereby overestimating the 

estrogenic potency of bisphenol A compared to studies in 

which comparisons are based on the half-maximal 

response. 

Competitive binding assays, which evaluate the concentration at which bisphenol A displaces labeled 17bestradiol from ER, are summarized in the top part of 

Table 52. The receptor binding of bisphenol A in these 

assays varies over 3 orders of magnitude. Bisphenol A 

competes for human ER binding at molar concentrations 

20–10,000 times that of the native ligand. When bisphenol 

A binding to ERa and ERb was compared in the same 

study, 3 reports found little difference by receptor 

subtype (Kuiper et al., 1998; Paris et al., 2002; Takayanagi 

et al., 2006), and 3 studies found binding to ERb to be 4, 

10, 47, and 254 times greater than binding to ERa

(Routledge et al., 2000; Matthews et al., 2001; Seidlova ́-

Wuttke et al., 2004, 2005; Takemura et al., 2005). Yeast 

reporter systems, which reflect activation of post-receptor pathways, show less variability; these studies show 

bisphenol A activity to be 10,000–26,000 times less than 

that of 17b-estradiol. 

Some variability in estimating bisphenol A potency 

appears to be due to differences between laboratories. 

Andersen et al. (1999a) reported results from 3 laboratories that evaluated the proliferative response of MCF-7 

breast cancer cells to bisphenol A. The laboratories, 

which were in the U.S., Spain, and Denmark, were sent 

samples of the same stock of bisphenol A, 17b-estradiol, 

and MCF-7 cells. Procedures were similar in the labs, 

although two different counting methods were used. The 

bisphenol A potencies relative to 17b-estradiol were 

5 x 10 -7

,3 x 10 -6, and 1 x10 -5. Laboratory variability 

may underlie some of the large differences in cell-based 

assays for ER activation; in those studies bisphenol A 

molar potency compared to 17b-estradiol were reported 

to vary by over 7 orders of magnitude (Table 52). Another 

explanation for this wide range of reported values is the 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 211 

Table 52 

In Vitro Estrogenicity Testing of Bisphenol A 

Endpoint Molar potency relative to 17b-estradiol Reference 

Binding assays 

Frog liver cytosol binding [1.4 x 10 -3

] Lutz and Kloas (1999) 

Carp liver cytosol binding [1.3 x 10 -3

] Segner et al. (2003) 

Rainbow trout ER binding 5.8 x 10 -5 Olsen et al. (2005) 

Rainbow trout ER binding 2.1 x10 -3 Matthews et al. (2000) 

Anole ER binding 1.3 x 10 -3 Matthews et al. (2000) 

Chicken ER binding 4.4 x 10 -4 Matthews et al. (2000) 

Mouse ERa binding 8.6 x 10 -5 Matthews et al. (2000) 

Mouse uterine cytosol binding [1.2 x 10 -4

] Matthews et al. (2001) 

Rabbit uterine ER binding [1.3 x 10 -5

] Andersen et al. (1999a) 

Rat uterine cytosol binding B5 x 10 -4 Krishnan et al. (1993) 

Rat uterine cytosol binding 8 x 10 -5 Blair et al. (2000) 

Rat uterine cytosol binding 1–2 x 10 -4 Kim et al. (2001a) 

Rat ERa binding [2.5 x 10 -4

] Strunck et al. (2000) 

ER binding in rat lactotroph 1–10 x 10 -5 Chun and Gorski (2000) 

Rat ERa binding 5 x 10 -4 Kuiper et al. (1997) 

Rat ERb binding 3.3 x 10 -4 Kuiper et al. (1997) 

Rat uterine ERa and b binding 6.2 x 10 -5 Washington et al. (2001) 

Rat uterine Type II estrogen-binding site 4 x 10 -3 Washington et al. (2001) 

ER binding in MCF-7 lysates 1 x10 -2 Dodge et al. (1996) 

Human ERa binding 4 x 10 -4 Bolger et al. (1998) 

Human ERa binding 1 x10 -4 Kuiper et al. (1998) 

Human ERb binding 1 x10 -4 Kuiper et al. (1998) 

Human ER binding 5.6 x 10 -4 Perez et al. (1998) 

Human ER binding [1.3 x 10 -4

] Andersen et al. (1999a) 

ER binding in ECC-1 cells 3 x 10 -3 Bergeron et al. (1999) 

Human ERa binding 8 x 10 -5 Matthews et al. (2000) 

Human ERa binding [2.5 x 10 -3 diethylstilbestrol] Nakagawa and Suzuki (2001) 

Human ERa binding 7.3 x 10 -4 Routledge et al. (2000) 

Human ERb binding 7.5 x 10 -3 Routledge et al. (2000) 

Human ER binding [7.1 x10 -5

] Sheeler et al. (2000) 

Human ERa binding [8 x 10 -5

] Matthews et al. (2001) 

Human ERb binding [3.8 x 10 -3

] Matthews et al. (2001) 

Human ERa binding 5 x 10 -2 Paris et al. (2002) 

Human ERb binding 4 x 10 -2 Paris et al. (2002) 

Human ER binding [3 x 10 -4

] Stroheker et al. (2004) 

Human ERa binding [2.4 x 10 -4

] Seidlova ́-Wuttke et al. (2005) 

Human ERb binding [2.8 x 10 -2

] Seidlova ́-Wuttke et al. (2004) 

Human ERa binding [1.1 x10 -4

] Takemura et al. (2005) 

Human ERb binding [4.4 x 10 -4

] Takemura et al. (2005) 

Human ER binding 3.15 x 10 -3 Olsen et al. (2005) 

ERa binding [9.4 x 10 -4

] Takayanagi et al. (2006) 

ERb binding [9.6 x 10 -4

] Takayanagi et al. (2006) 

Recombinant yeast reporter systems 

Human ER activation 5 x 10 -5 Coldham et al. (1997) 

Human ER activation 6.7 x 10 -5 Gaido et al. (1997) 

Human ER activation [2.5 x 10 -5

] Harris et al. (1997) 

Human ER activation [4–8 x 10 -5

] Andersen et al. (1999a) 

Human ER activation [3.9 x 10 -5

] Sheeler et al. (2000) 

Human ER activation B1 x10 -4 Sohoni and Sumpter (1998) 

Human ER activation 3.7 x 10 -5 Metcalfe et al. (2001) 

ERa activation 6.2 x 10 -5 Silva et al. (2002) 

ERa activation [1 x10 -4

] Nishihara et al. (2000) 

ERa activation [B1 x10 -4

] Beresford et al. (2000) 

Human ERa [3.3 x 10 -5

] Rajapakse et al. (2001) 

Human ERa, no microsomes [5.5 x 10 -5

] Elsby et al. (2001) 

Human ERa, human liver microsomes [6.6 x 10 -6

] Elsby et al. (2001) 

ER activation B10 -5 Chen et al. (2002) 

Human ER activation [8.1 x10 -5

] Segner et al. (2003) 

Human ER activation 9 x 10 -5 Li et al. (2004) 

ERa activation [4 x 10 -5

] Singleton et al. (2006) 

Human ERa, with denatured rat S9 [2.4 x 10 -6

] Yoshihara et al. (2004) 

Human ERa, with active rat S9 [9.2 x 10 -6

] Yoshihara et al. (2004) 

Human ERa, with denatured mouse S9 [3.0 x 10 -6

] Yoshihara et al. (2004) 

Human ERa, with active mouse S9 [7.8 x 10 -6

] Yoshihara et al. (2004) 

Birth Defects Research (Part B) 83:157–395, 2008
212 CHAPIN ET AL. 

Table 52 

Continued 

Endpoint Molar potency relative to 17b-estradiol Reference 

Human ERa, with denatured monkey S9 [2.4 x 10 -6

] Yoshihara et al. (2004)
 

Human ERa, with active monkey S9 [6.0 x 10 -6

] Yoshihara et al. (2004)
 

Human ERa, with denatured human S9 [2.2 x 10 -6

] Yoshihara et al. (2004)
 

Human ERa, with active human S9 [4.6 x 10 -6

] Yoshihara et al. (2004)
 

Human ERa activity [2.3 x 10 -5

] Terasaki et al. (2005)
 

Medaka ERa activity [3.3 x 10 -4

] Terasaki et al. (2005)
 

‘‘Estrogenic activity’’ 3.4 x 10 -5 Kawagoshi et al. (2003)
 

ERa activation [2.3 x 10 -4

] Singleton et al. (2006)
 

Fish ERa activation 4.1 x10 -4 Fu et al. (2007)
 

Fish ERb2 activation 3.2 x 10 -5 Fu et al. (2007)
 

Other cell-based recombinant reporter systems 

ER activation in trout gonad cell line 5.4 x 10 -3 Ackerman et al. (2002) 

Mouse ERa in HeLa cells [o1 x10 -5

] Ranhotra and Teng (2005) 

Mouse ERb in HeLa cells [B1 x10 -2

] Ranhotra and Teng, 2005 

HepG2 cells, human ERa [3.0 x 10 -3

] Snyder et al. (2000) 

HepG2 cells, human ERb [1.1 x10 -2

] Snyder et al. (2000) 

Rat ERa in HeLa cells [1.6 x 10 -7

] Yamasaki et al. (2002b) 

ER activation in HeLa cells [8.8 x 10 -4

] Takahashi et al. (2004) 

ERa activation in HeLa cells [2.5 x 10 -2

] Hiroi et al. (1999) 

ERb activation in HeLa cells [2.3 x 10 -2

] Hiroi et al. (1999) 

ERa activation in HeLa cells [6.1 x10 -1

] Vivacqua et al. (2003) 

ERb activation in HeLa cells [5.6 x 10 -1

] Vivacqua et al. (2003) 

ERa activation in HeLa cells [7.7 x 10 -1

] Recchia et al. (2004) 

ERb activation in HeLa cells [1.2] Recchia et al. (2004) 

ERa activation in T47D cells [6.2–7.9 x 10 -1

] Recchia et al. (2004) 

Proliferation in T47D cells [6.6 x 10 -1

] Recchia et al. (2004) 

Human ER in hepatoma cells [3 x 10 -2

] Gould et al. (1998) 

Human ERa, human embryonal kidney [4.8 x 10 -3

] Kurosawa et al. (2002) 

Human ERb, human embryonal kidney [4.6 x 10 -3

] Kurosawa et al. (2002) 

Human ERa, endometrial carcinoma [5.4 x 10 -3

] Kurosawa et al. (2002) 

Human ERb, endometrial carcinoma [4.9 x 10 -3

] Kurosawa et al. (2002) 

Human ERa, osteosarcoma [7.3 x 10 -3

] Kurosawa et al. (2002) 

Human ERb, osteosarcoma [7.7 x 10 -3

] Kurosawa et al. (2002) 

Human ERa, human hepatoma cells [2.7 x 10 -1

] Gaido et al. (2000) 

Human ERb, human hepatoma cells [1.8 x 10 -1

] Gaido et al. (2000) 

Human ERa, 239HEK cells 2 x 10 -4 diethylstilbestrol Lemmen et al. (2004) 

Human ERb, 239HEK cells 7 x 10 -4 diethylstilbestrol Lemmen et al. (2004) 

Human ERa, endometrial carcinoma [6.1 x10 -3

] Singleton et al. (2006) 

MCF-7 cells 

G6PD activity [1 x10 -1

] Kim et al. (2003a) 

Expression of proteins [1 x10 -3

] Perez et al. (1998) 

Progesterone receptor mRNA Not increased at 10 -6 Ma Diel et al. (2002) 

Androgen receptor mRNA Not decreased at 10 -6 Ma Diel et al. (2002) 

Progesterone receptor B2 x 10 -4 Krishnan et al. (1993) 

ER binding, serum-free 3.3 x 10 -4 Samuelsen et al. (2001) 

ER binding, 100% human serum 1.7 x 10 -4 Samuelsen et al. (2001) 

ER binding 3.2 x 10 -3 Olsen et al. (2003) 

ER activation [1.4 x 10 -5

] Kitamura et al. (2005) 

ERa expression [7.5 x 10 -5

] Matthews et al. (2001) 

ERb expression [1.8 x 10 -4

] Matthews et al. (2001) 

ERa activation [4.7–6.9 x 10 -1

] Vivacqua et al. (2003) 

ERa activation [5.5–6.7 x 10 -1

] Recchia et al. (2004) 

pS2 induction [1.8 x 10 -6

] Leffers et al. (2001) 

ER production [7 x 10 -8

] Olsen et al. (2003) 

Progesterone receptor production [6.8 x 10 -8

] Olsen et al. (2003) 

pS2 production [10 -7

] Olsen et al. (2003) 

pS2 mRNA [1.1] Vivacqua et al. (2003) 

pS2 mRNA [8.9 x 10 -1

] Recchia et al. (2004) 

Cathepsin D mRNA [8.2 x 10 -1

] Recchia et al. (2004) 

Transcription of human telomerase reverse transcriptase [B10 -2

] Takahashi et al. (2004) 

Proliferation [3.8 x 10 -4

] Krishnan et al. (1993) 

Proliferation 1 x10 -3 Brotons et al. (1995) 

Proliferation 1 x10 -4 Soto et al. (1997) 

Proliferation [B1 x10 -3

] Dodge et al. (1996) 

Proliferation [1 x10 -4

] Perez et al. (1998) 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 213 

Table 52
 

Continued
 

Endpoint Molar potency relative to 17b-estradiol Reference 

Proliferation [9.8 x 10 -4

] Schafer et al. (1999) 

Proliferation (3 different laboratories) 5–100 x 10 -7 Andersen et al. (1999a) 

Proliferation 6 x 10 -5 Ko ̈rner et al. (2000) 

Proliferation 3 x 10 -5 Kim et al. (2001a) 

Proliferation [2.5 x 10 -6

] Suzuki et al. (2001) 

Proliferation 2 x 10 -5 Samuelsen et al. (2001) 

Proliferation [9.2 x 10 -4

] Nakagawa and Suzuki (2001) 

Proliferation [B1 x10 -3

] Shimizu et al. (2002) 

Proliferation [7 x 10 -9

] Diel et al. (2002) 

Proliferation 1.6 x 10 -5 Olsen et al. (2003) 

Proliferation [4.5–5 x 10 -1

] Vivacqua et al. (2003) 

Proliferation [1.1 x10 -4

] Stroheker et al. (2004) 

Proliferation [6 x 10 -1

] Recchia et al. (2004) 

Proliferation 2 x 10 -5 Olsen et al. (2005) 

Proliferation, with denatured rat S9 [6.5 x 10 -5

] Yoshihara et al (2001) 

Proliferation, with active rat S9 [3.4 x 10 -4

] Yoshihara et al (2001) 

Rat pituitary cells 

Proliferation 1–10 x 10 -6 Chun and Gorski (2000) 

Proliferation [B8.4 x 10 -3

] Steinmetz et al. (1997) 

Prolactin release 1 x10 -5 Chun and Gorski (2000) 

Prolactin release (GH3 cell) [6 x 10 -3

] Steinmetz et al. (1997) 

Prolactin release (F344 pituitary) 2–10 x 10 -4 Steinmetz et al. (1997) 

Prolactin gene expression [B1 x10 -3

] Steinmetz et al. (1997) 

Rat uterine adenocarcinoma cells 

Induction of complement C3 mRNA [8 x 10 -3

] Strunck et al. (2000) 

Human uterine adenocarcinoma cells 

Progesterone receptor mRNA/protein [B1 x10 -2

] Bergeron et al. (1999) 

Proliferation No effect at 10 -5 M Bergeron et al. (1999) 

Vitellogenin production, fish hepatocytes 

Carp 1 x10 -4 Smeets et al. (1999) 

Carp [3.1 x10 -3

] Segner et al. (2003) 

Carp [1 x10 -5

] Letcher et al. (2005) 

Carp [3 x 10 -4

] Rankouhi et al. (2002) 

Trout 2 x 10 -5 Shilling and Williams (2000) 

Trout [8 x 10 -4

] Segner et al. (2003) 

Trout 2.9 x 10 -5 Olsen et al. (2005) 

Frog hepatocytes 

Vitellogenin mRNA expression [B1 x10 -3

] Kloas et al. (1999) 

Vitellogenin production No effect at 100 mM Rankouhi et al. (2004) 

ER mRNA expression B10 -2 Lutz et al. (2005) 

a

Progesterone receptor was increased and androgen receptor was decreased by 17b-estradiol 10 -10 M. 

difference in defining relative potency in some assays, as 

discussed above. [According to a study author, the wide 

variability in relative bisphenol A potency was due to a 

wide fluctuation in the 17b-estradiol dose at which halfmaximal proliferation was achieved (0.1–70 pM) (A. Soto, 

personal communication, March 2, 2007).] 

A study using ERa- and ERb-reporting systems in 3 

human cell lines found that bisphenol A had a small 

antagonistic effect on ERa activation in the presence of 

17b-estradiol in human embryonal kidney and endometrial carcinoma cells (Kurosawa et al., 2002). There were 

no significant interactions between bisphenol A and 17bestradiol on ERa activation in human osteosarcoma cells 

or on ERb activation in any tested cell type. By contrast, a 

study using a recombinant yeast assay for ERa activation 

found 17b-estradiol and bisphenol A to have additive 

effects (Rajapakse et al., 2001), and a study using MCF-7 

cell proliferation found 17b-estradiol and bisphenol A to 

have synergistic effects (Suzuki et al., 2001). 

The data in Table 52 are applicable only to unconjugated bisphenol A. Estrogenic activity has not been 

identified for bisphenol A glucuronide (Matthews et al., 

2001) or sulfate (Shimizu et al., 2002). 

In vivo tests (Table 53) have been conducted principally in rats and mice. Most endpoints in these studies 

involved the uterus, and effects on uterine weight in 

immature or ovariectomized animals are the uterine 

endpoints reported most commonly. The potency of 

bisphenol A in increasing uterine weight varies over B4 

orders of magnitude. Some of this variation may be 

related to the short half-life of bisphenol A. Uterotrophic 

evaluations are typically performed 24 hr after the last 

dose of the test agent is administered. Laws et al. (2000) 

showed no significant effect of bisphenol A at doses 

r400 mg/kg bw/day given orally on uterine wet weight 

assessed 24 hr after administering the last dose. When 

assessed 6 hr after the last oral dose, bisphenol A 

200 mg/kg bw/day increased uterine wet weight to 

B2.5 times the control [estimated from a graph], which 

was about the same as the increase produced by 

administering 17b-estradiol 0.005 mg/kg bw/day sc. 

Increase in uterine weight in the first 6 hr after treatment 

Birth Defects Research (Part B) 83:157–395, 2008
214 CHAPIN ET AL. 

Table 53 

In Vivo Estrogenicity Tests of Bisphenol A 

Model and exposure Husbandrya Endpoint Molar potency/comparatorb Reference 

Rat uterus 

Adult ovariectomized 

Sprague–Dawley, 

gavage x 4 days 

Immature Sprague– 

Dawley, bisphenol A 

given ‘‘orally’’ x 3 

days; 17b-estradiol 

i.p. x 3 days 

Adult ovariectomized 

Crl:CD BR, gavage x 4 

days 

Adult ovariectomized 

F344, i.p. x 1 

Adult ovariectomized 

F344 or Sprague– 

Dawley, silastic 

implant x 3 days 

Juvenile 

ovariectomized DA/ 

Han, Wistar, or 

Sprague–Dawley, 

gavage x 3 days 

Immature Alpk:AP, 

s.c. x 3 days 

Immature Alpk:AP, 

gavage x 3 days 

Immature Long–Evans, 

gavage x 3 days 

Adult ovariectomized 

Long–Evans 

Juvenile 

ovariectomized DA/ 

Han, gavage x 3 days 

TD89222 diet, 

metal cage 

Not indicated 

Purina 5002 diet, 

steel cage 

Not indicated 

Not indicated 

Not indicated 

RM3 diet, wire 

cage 

RM3 diet, wire 

cage 

Purina 5001 diet 

Purina 5001 diet 

Ssniff R-10 diet 

Uterine wet weight 

Uterine weight 

Progesterone receptor 

Peroxidase activity 

Uterine weight 

Stromal cell proliferation 

cfos expression 

Uterine wet weight: 

F344 

Sprague–Dawley 

Uterine cell height: 

F344 

Sprague–Dawley 

Uterine wet weight: 

DA/Han 

Wistar 

Sprague–Dawley 

Uterine epithelium 

Vaginal epithelium 

Clusterin mRNA 

Uterine wet weight 

Uterine dry weight 

Uterine wet weight 

Uterine dry weight 

Uterine wet weight 6 hr 

after dosing 

Uterine wet weight 24 hr 

after dosing 

Uterine wet weight 

Uterine wet weight 

relative to bw 

Expression of:

Androgen receptor 

ER

Progesterone receptor 

Complement C3 

Clusterine 

Glyceraldehyde 

phosphate 

dehydrogenase 

[3.9 x 10 -3

]/ethinyl estradiol 

Not affected by bisphenol A at up to 

150 mg/kg bw/day; 17b-estradiol 

was positive at 0.005 mg/day 

[B0.089 mg/kg bw/day] 

[5.9 x 10 -3

]/17b-estradiol 

[7.6 x 10 -3

]/17b-estradiol 

[3.5 x 10 -5

]/17b-estradiol 

[4.1 x10 -5

]/17b-estradiol 

[2.1 x10 -4

]/17b-estradiol 

[8.2 x 10 -3

]/17b-estradiol 

[6.0 x 10 -3

]/17b-estradiol 

[1.1 x10 -2

]/17b-estradiol 

[9.2 x 10 -3

]/17b-estradiol 

[1.8 x 10 -5

]/ethinyl estradiol 

No response to 200 mg/kg/d 

[1.7 x 10 -5

]/ethinyl estradiol 

No response to 200 mg/kg/day 

No response to 200 mg/kg/day 

No response to 200 mg/kg/day 

[2.6–2.7 x 10 -5

]/diethylstilbestrol 

[2.5–3.0 x 10 -5

]/diethylstilbestrol 

[2.3–3.1 x10 -5

]/diethylstilbestrol 

[2.7–3.6 x 10 -5

]/diethylstilbestrol 

[1.4 x 10 -5

]/17b-estradiol 

No effect at bisphenol A at r400 mg/ 

kg bw/day 

No effect of bisphenol A at r100 mg/ 

kg bw/day 

[1.2 x 10 -5

]/ethinyl estradiol 

[3.9 x 10 -4

]/ethinyl estradiol 

[1.9 x 10 -4

]/ethinyl estradiol 

bisphenol A and ethinyl estradiol 

produced opposite effects 

[2.2 x 10 -5

]/ethinyl estradiol 

No bisphenol A effect at 200 mg/kg 

bw/day; ethinyl estradiol showed 

an effect at 0.1 mg/kg bw/day 

Dodge et al. 

(1996) 

Gould et al. 

(1998) 

Cook et al. (1997) 

Steinmetz et al. 

(1998) 

Steinmetz et al. 

(1998) 

Diel et al. (2004) 

Ashby and 

Tinwell (1998) 

Laws et al. (2000) 

Laws et al. (2000) 

Diel et al. (2000) 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 215 

Table 53
 

Continued
 

Model and exposure Husbandrya Endpoint Molar potency/comparatorb Reference 

Adult ovariectomized 

Alpk:ApfSD, s.c. x 3 

days 

Immature Crj:CD (SD), 

s.c. x 3 days 

Immature Crj:CD (SD), 

gavage x 3 days 

Adult ovariectomized 

Wistar, s.c. x 7 days 

Adult ovariectomized 

Sprague–Dawley, 

exposed in drinking 

water x 3 days 

Adult ovariectomized 

Sprague–Dawley, 

s.c. x 3 days 

Immature Alpk:ApfSD, 

s.c. x 3 days 

Immature Alpk:ApfSD, 

gavage x 3 days 

Immature Sprague– 

Dawley, s.c. x 3 days 

Immature Crj:CD (SD), 

s.c. x 3 days 

Immature Sprague– 

Dawley, s.c. x 3 days 

Pubertal Sprague– 

Dawley, gavage PND 

22–42/43 

Pregnant Sprague– 

Dawley, s.c. bisphenol 

A on GD 17–19 (17bestradiol s.c. x 1) 

Pregnant Sprague– 

Dawley, s.c. bisphenol 

A on GD 17–19 (17bestradiol s.c. x 1) 

Lactating Sprague– 

Dawley, s.c. bisphenol 

A x 5 days (17bestradiol s.c. x 1) 

Not indicated 

MF diet, steel 

cage 

MF diet, steel 

cage 

Not indicated 

Glass water 

bottles, plastic 

cage (negative 

E-Screen of 

ethanol cage 

washes) 

PMI Certified 

Rodent Diet, 

polycarbonate 

cage, elm 

bedding 

RM1 diet 

RM1 diet 

Soy-free diet, 

polycarbonate 

cage 

MF diet, steel 

cage 

Soy-free diet, 

polycarbonate 

cage, corncob 

bedding 

Purina 5002 diet, 

polycarbonate 

cage, chip 

bedding 

Soy-free diet, 

polycarbonate 

cage 

Soy-free diet, 

polycarbonate 

cage 

Soy-free diet 

Uterine wet weight 

Uterine dry weight 

Wet and blotted uterine 

weight 

Wet and blotted uterine 

weight 

Blotted uterine weight 

Uterine wet weight 

Uterine wet weight 

Uterine dry weight 

Uterine wet weight 

Uterine dry weight 

Uterine wet weight 

Uterine dry weight 

Uterine wet weight 

Calbindin D9k expression 

ERa expression 

Uterine wet weight 

Blotted uterine weight 

Epithelial cell height 

Blotted uterine weight 

Vaginal opening 

Maternal uterine weight 

Maternal uterine 

calbindin D9k protein 

Maternal uterine 

calbindin D9k mRNA 

calbindin D9k protein 

[1.7 x 10 -4

]/17b-estradiol 

[1.8 x 10 -4

]/17b-estradiol 

Effect noted at Z8 mg/kg bw/day 

bisphenol A/no comparator 

Effect noted at Z160 mg/kg bw/day 

bisphenol A/no comparator 

Increased relative weight compared to 

placebo at Z11 mg/kg bw/day; 

uterus reached 83% of weight of 

sham-ovariectomized control at 

bisphenol A dose of 250 mg/kg bw/ 

day. 

No effect of bisphenol A at up to 

16.9 mg/kg bw/day; estrone
 

positive at 0.12 mg/kg bw/day
 

[1.7 x 10 -6

]/17b-estradiol 

[2.3 x 10 -6

]/17b-estradiol 

[2.9 x 10 -4

]/17b-estradiol 

No effect of bisphenol A at 800 mg/kg 

bw/day; 17b-estradiol positive at 

0.4 mg/kg bw/day 

[2.3–5.5 x 10 -4

]/17b-estradiol 

[2.4–7.1 x10 -4

]/17b-estradiol 

No effect of bisphenol A at 

r1000 mg/kg bw/day; 17bestradiol was positive at 0.04 mg/kg 

bw/day 

[8.4 x 10 -6

]/17b-estradiol 

[3.4 x 10 -5

]/17b-estradiol 

[5.1 x10 -5

]/ethinyl estradiol 

[8 x 10 -7

]/ethinyl estradiol 

[1.2 x 10 -6

]/ethinyl estradiol 

Absolute organ weight decreased with 

increase dose (400 and 600 mg/kg 

bw/day); no effect on relative organ 

weight 

No effect at 400 and 600 mg/kg bw/ 

day 

[1.8 x 10 -5

]/17b-estradiol 

[1.7 x 10 -5

]/17b-estradiol 

[2.2 x 10 -5

]/17b-estradiol 

[6.9 x 10 -5

]/17b-estradiol 

Ashby et al. 

(2000) 

Yamasaki et al. 

(2000) 

Goloubkova 

et al. (2000) 

Rubin et al. 

(2001) 

Kim et al. 

(2001a) 

Matthews et al. 

(2001) 

An et al. (2002) 

Yamasaki et al. 

(2002b) 

Wade et al. 

(2003) 

George et al. 

(2003) 

Hong et al. 

(2003) 

Hong et al. 

(2003) 

Hong et al. 

(2004) 

Birth Defects Research (Part B) 83:157–395, 2008
216 CHAPIN ET AL. 

Table 53 

Continued 

Model and exposure Husbandrya Endpoint Molar potency/comparatorb Reference 

Immature and adult 

ovariectomized Wistar, 

gavage x 4 days 

Immature Sprague– 

Dawley, s.c. x 3 days 

Immature Sprague– 

Dawley, s.c. x 3 days 

Immature Alpk:ApfSD, 

gavage x 3 days 

Immature AP, s.c. x 3 

days 

Adult ovariectomized 

Sprague–Dawley, 

diet x 3 months 

Immature Sprague– 

Dawley, s.c. x 3 days 

Adult ovariectomized 

Crj:CD (SD), s.c. x 3 

days 

Adult Holzman, 

progesterone-treated to 

delay implantation, 

given test agent s.c. on 

GD 7 

Rat vagina 

Adult ovariectomized 

F344, i.p. x 1 

Adult ovariectomized 

Long Evans, bisphenol 

A by gavage x 11 days; 

17b-estradiol by s.c. 

Long–Evans treated 

PND 21–35 by gavage 

Adult ovariectomized 

F344 and Sprague– 

Dawley, i.p. x 1 

AO4C diet, wire 

cage 

Soy-free feed, 

polycarbonate 

cage 

Shinchon diet 

RM1 diet, 

polycarbonate 

cage 

RM1 diet, 

polypropylene 

cages, 

sawdust and 

shredded 

paper bedding 

Phytoestrogenfree diet 

PMI Certified 

Rodent Diet 

Estrogen-free 

NIH-07PLD 

diet, 

aluminum 

cage, paper 

bedding 

Unspecified 

Purina rodent 

chow, plastic 

cage, pine 

shavings 

Not indicated 

Purina 5001 diet 

Purina 5001 diet 

Not indicated 

Uterine wet and dry 

weight 

Calbindin D9k protein 

Uterine wet weight 

Uterine wet weight 

relative to bw 

Glutathione peroxidase 

activity 

Blotted uterine weight 

Expression of:

Progesterone receptor A 

Progesterone receptor B 

Complement C3 

Lipocalcin 

Uterine wet weight 

Uterine dry weight 

Uterine weight, 

endometrial thickness, 

ERa, ERb expression 

Complement C3 

expression 

Uterine wet weight 

Uterine dry weight 

Uterine wet weight, 

relative to bw 

Blotted uterine weight, 

relative to bw 

Implantation 

BrdU labeling 

cfos expression 

Vaginal cytology 

Vaginal opening 

BrdU labeling 

No effect in either model of bisphenol 

A at r200 mg/kg bw/day/17bestradiol positive at 0.025– 

0.035 mg/kg bw/day 

[5.1 x10 -5

]/17b-estradiol 

[1.5 x 10 -6

]/17b-estradiol 

[1.3 x 10 -6

]/17b-estradiol 

[4.2 x 10 -3

]/17b-estradiol 

[2.5 x 10 -4

]/17b-estradiol 

[3.8 x 10 -4

]/17b-estradiol 

[4.2 x 10 -4

]/17b-estradiol 

[1.8 x 10 -4

]/17b-estradiol 

[2.3 x 10 -4

]/17b-estradiol 

[1.0 x 10 -6

]/ethinyl estradiol 

[1.2 x 10 -6

]/ethinyl estradiol 

No bisphenol A effect at 0.37 mg/kg 

bw/day; estradiol benzoate positive 

control 

Bisphenol A and estradiol benzoate 

produced opposite effects 

[4.5 x 10 -7

]/ethinyl estradiol 

[4.9 x 10 -7

]/ethinyl estradiol 

[2.1 x10 -5

]/17b-estradiol 

[1.7 x 10 -6

]/17b-estradiol 

[4–34 x 10 -6

]/estrone 

Increased at bisphenol A dose of 37.5 

but not 18.5 mg/kg bw/no 

comparator 

[1.3 x 10 -4

]/17b-estradiol 

No effect at bisphenol A dose of 

100 mg/kg bw/day; 17b-estradiol 

0.005 mg/kg bw/day resulted in 

persistent estrus 

No effect at bisphenol A dose 

r400 mg/kg bw/day; ethinyl 

estradiol was active at 0.01 mg/kg 

bw/day 

F344: [4.5 x 10 -6

]/17b-estradiol 

Sprague–Dawley: [1.4 x 10 -6

]/17bestradiol 

Stroheker et al. 

(2003) 

An et al. (2003) 

Kim et al. 

(2003a) 

Ashby and 

Odum (2004) 

Tinwell and 

Ashby (2004) 

Seidlova ́-Wuttke 

et al. (2004) 

Kim et al. (2005) 

Koda et al. (2005) 

Cummings and 

Laws (2000) 

Steinmetz et al. 

(1998) 

Laws et al. (2000) 

Laws et al. (2000) 

Long et al. (2000) 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 217 

Table 53
 

Continued
 

Model and exposure Husbandrya Endpoint Molar potency/comparatorb Reference 

Immature Wistar, 

gavage x 4 days 

Adult ovariectomized 

Wistar, gavage x 4 days 

Immature Sprague– 

Dawley, s.c. x 3 days 

Other rat organs 

Ovariectomized 

Sprague–Dawley, daily 

gavage for 5 weeks 

Adult ovariectomized 

Sprague–Dawley, 

treated in feed 

Adult ovariectomized 

Sprague–Dawley and 

F344, by s.c. 

implant x 3 days 

Adult ovariectomized 

Wistar, s.c. x 7 days 

Mouse uterus 

Immature CFLP, 

s.c. x 3 days 

Adult ovariectomized 

CD-1, s.c. x 1 

Juvenile-adult 

ovariectomized 

B6C3F1, s.c. x 4 days 

Papaconstantinou et al. 

(2000) 

Juvenile-adult 

ovariectomized 

B6C3F1, s.c. x 4 days 

Papaconstantinou et al. 

(2001) 

Juvenile-adult 

ovariectomized 

B6C3F1, s.c. x 4 days 

Papaconstantinou et al. 

(2002) 

Juvenile-adult
 

ovariectomized
 

B6C3F1, s.c. x 1
 

Papaconstantinou et al. 

(2003) 

Adult ovariectomized 

transgenic ER-reporter, 

s.c. x 1 

AO4C diet, wire 

cage 

AO4C diet, wire 

cage 

PMI Certified 

Rodent Diet 

TD89222 diet, 

metal cage 

Phytoestrogenfree diet 

Not indicated 

Not indicated 

Not indicated 

Not indicated 

Purina 5001, 

polypropylene 

cage, chip 

bedding 

Purina 5001, 

polypropylene 

cage, cellulose 

fiber bedding 

Purina 5001, 

polypropylene 

cage, cellulose 

fiber bedding 

Purina 5001, 

polypropylene 

cage, cellulose 

fiber bedding 

Purina 5001, 

polystyrene 

cage 

Vaginal cornification 

Vaginal cornification 

Vaginal weight 

Prevention of bone 

mineral density decline 

Prevention of bone 

mineral density decline 

Serum prolactin 

Pituitary weight 

Serum prolactin 

Relative uterine weight 

IGF1 expression 

Uterine wet weight 

Endothelial proliferation 

Induction of grp94 

Induction of hsp72 

Induction of hsp90 

Uterine weight 

Induction of hsp90a

Induction of grp24 

Blotted uterine weight, 

6 hr after dose 

Blotted uterine weight, 

12 hr after dose 

Uterine wet weight 

ER activation 

[3.8 x 10 -4

]/17b-estradiol 

No effect at bisphenol A dose of 

100 mg/kg bw/day; 17b-estradiol 

was positive at 0.1 mg/kg bw/day 

[5.3 x 10 -7

]/ethinyl estradiol 

No effect at bisphenol A dose up to 

10 mg/kg bw/day; no standard 

estrogen comparator 

No effect at bisphenol A dose r

370 mg/kg bw/day; estradiol 

benzoate was effective at 1.18 mg/ 

kg bw/day 

F344: [1.7 x 10 -2

]/17b-estradiol 

Sprague Dawley: no effect of 

bisphenol A at 40–45 mg/day or 17bestradiol at 1.2–1.5 mg/day 

Increased compared to vehicle control 

at 128 but not 78 mg/kg bw/day 

Increased compared to vehicle control 

at 128 mg/kg bw/day 

No response at up to 0.5 mg [B50 mg/ 

kg bw/day] 

[8.4 x 10 -4

]/17b-estradiol 

[2.3 x 10 -5

]/17b-estradiol 

[6.9 x 10 -6

]/17b-estradiol 

[2.4 x 10 -5

]/17b-estradiol 

[3.5 x 10 -6

]/17b-estradiol 

[5.3 x 10 -6

]/17b-estradiol 

[5.3 x 10 -6

]/17b-estradiol 

[1.2 x 10 -5

]/17b-estradiol 

[8.4 x 10 -6

]/17b-estradiol 

[8.4 x 10 -6

]/17b-estradiol 

[4.2 x 10 -6

]/17b-estradiol 

[2.9 x 10 -5

]/diethylstilbestrol 

[1.0 x 10 -4

]/diethylstilbestrol 

Stroheker et al. 

(2003) 

Kim et al. (2005) 

Dodge et al. 

(1996) 

Seidlova ́-Wuttke 

et al. (2004) 

Steinmetz et al. 

(1997) 

Goloubkova 

et al. (2000) 

Coldham et al. 

(1997) 

Klotz et al. (2000) 

Nagel et al. 

(2001) 

Nagel et al. 

(2001) 

Birth Defects Research (Part B) 83:157–395, 2008
218 

Table 53 

Continued 

CHAPIN ET AL. 

Model and exposure Husbandrya Endpoint Molar potency/comparatorb Reference 

Immature AP, s.c. x 3 RM1 diet, plastic Blotted uterine weight [2.3 x 10 -5

]/diethylstilbestrol in 4 of 8 Tinwell and 

days cage, sawdust trials; other trials showed no effect Joiner (2000) 

and shredded at bisphenol doses up to 300 mg/kg 

paper bedding bw/day 

Immature AP, RM1 diet, plastic Blotted uterine weight No effect at bisphenol A doses up to Tinwell and 

gavage x 3 days cage, sawdust 300 mg/kg bw/day; Joiner (2000) 

and shredded diethylstilbestrol produced 

paper bedding response at 10 mg/kg bw/day 

Immature CD-1, RM1 diet Lactoferrin expression No effect at bisphenol A doses up to Mehmood et al. 

s.c. x 3 days 1000 mg/kg bw/day; (2000) 

diethylstilbestrol showed effect at 

0.1 mg/kg bw/day 

Uterine weight, BrdU No effect at bisphenol A doses up to 

incorporation, 100 mg/kg bw/day; 

peroxidase production diethylstilbestrol showed effect at 

1–5 mg/kg bw/day 

Immature CD-1, s.c. RMH 3000 diet, Uterine wet weight [1.6 x 10 -5

]/17b-estradiol Markey et al. 

minipump x 3 days cage, and (2001b) 

bedding 

estrogennegative by EScreen 

Epithelial cell height [3.8 x 10 -5

]/17b-estradiol 

Lactoferrin expression [3.9 x 10 -5

]/17b-estradiol 

Ovariectomized adult Not indicated Relative uterine to body [3.6–74 x 10 -5

]/17b-estradiol Kitamura et al. 

B6C3F1, i.p. x 3 days weight (2005) 

Ovariectomized adult Economy Increased uterine B1 x10 -4

/17b-estradiol Milligan et al. 

Swiss, s.c. x 1 Rodent vascular permeability (1998) 

Maintenance 

diet 

Other mouse organs 

Juvenile-adult NMF diet Uterine and ovarian Dietary bisphenol A (0.1%) exerted Toda et al. (2002) 

aromatase knock-out, histology, bone mineral estrogenic effects. Mean7SD serum 

diet x 4 months density bisphenol A 84.378.7 mg/L. No 

comparator estrogen was used for 

these endpoints 

Fish 

Immature rainbow Plasma vitellogenin [3 x 10 -4

]/17b-estradiol Christiansen 

trout, injected et al. (1997) 

Juvenile rainbow trout, Plasma vitellogenin [5.6 x 10 -3

]/17b-estradiol Andersen et al. 

injected (1999a) 

Juvenile rainbow trout, Plasma vitellogenin [B8.4 x 10 -5

]/17b-estradiol Lindholst et al. 

exposed in water (2000) 

Male medaka, exposed Plasma vitellogenin [1.4 x 10 -4

]/ethinyl estradiol Chikae et al. 

in feed (2003) 

Male medaka, exposed Hepatic vitellogenin and [8.4 x 10 -6

]/17b-estradiol Yamaguchi et al. 

in water ERa mRNA (2005) 

Male killfish, injected Plasma vitellogenin [2.7 x 10 -4

]/17b-estradiol Pait and Nelson 

(2003) 

Male zebrafish, Plasma vitellogenin [B0.2]/ethinyl estradiol Van den Belt 

juvenile rainbow trout, et al.(2003) 

exposed in water 

Invertebrates 

Mud snail, exposed in New embryo production [1.5 x 10 -4

]/ethinyl estradiol Jobling et al. 

water (2004) 

Ramshorn snail, Egg production Increased (EC10 13.9 ng/L); blocked by Oehlmann et al. 

exposed in water Faslodex and tamoxifen. No (2006) 

comparison to reference estrogen 

a

Husbandry information for rodent studies includes caging and bedding materials and diet when indicated by the authors. 

b

Estimates include comparison of administered dose, magnitude of effect, and molecular weight. 

represents fluid inbibition and not true tissue growth. A 42/43 with bisphenol A by gavage at 400 or 600 mg/kg
 

dose-related decrease in blotted uterine weight and body bw/day (George et al., 2003).
 

weight, with no effect on weight-adjusted uterine For studies showing an increase in uterine weight after
 

weight, was shown in pubertal rats treated on PND 22– bisphenol A treatment, dose route affects response;
 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 219 

Fig. 2. In vitro estrogenic potency (log10) in ER a and b binding and transcriptional assays and estrogen-dependent cell proliferation 

assays) distributions of bisphenol A and estrogen responses in vivo in rats, mice and fish. Each data point represents one bisphenol A 

study in which bisphenol A was compared to a reference estrogen in rats, mice, fish, or in vitro. Data summarized from Table 52 and 

Table 53, midrange values used when a range is given in the table. response

half maximal response 

comparator estrogen 

test estrogen 

a 

b 

10 9 8 7 6 5

log molar concentration 

Fig. 3. Alternative approaches to comparing estrogenic potency. 

In this example, the half-maximal response to the comparator 

estrogen occurs at 10 -8 M. A similar response occurs with the 

test estrogen at 10 -5 M, suggesting a 1000-fold difference in 

potency. If the magnitudes of response at equimolar concentrations are compared, the apparent potency may be much 

different. The response to the test estrogen at 10 -7 M (a) 

is about half the response to the comparator estrogen at 10 -7 M 

(a1b). 

bisphenol A given by gavage increased uterine weight by 

approximately 25% while the same dose given s.c. 

increased uterine weight by approximately 170% (Laws 

et al., 2000). A greater response by the s.c. than oral route 

was also shown by Yamasaki et al. (2000) and Kanno 

et al. (2003b) in the OECD multilaboratory study who 

showed a lowest effective bisphenol A dose of 8 mg/kg 

bw/day by the s.c. route and 160 mg/kg bw/day by the 

oral route. The greater activity per unit dose of s.c. 

than oral bisphenol A is due presumably to glucuronidation of the orally administered compound with consequent loss of estrogenicity (Matthews et al., 2001). 

A few studies could not confirm the greater effect of 

s.c. compared to oral bisphenol A on uterine 

weight. Ashby and Tinwell (1998) concluded that the 

magnitude of uterine weight response was similar 

for s.c. and oral routes. [The Expert Panel notes a 

greater numerical magnitude of response after s.c. than 

oral exposure in most of the experiments reviewed in 

this report, and that statistical comparison of the dose 

routes was not reported.] Matthews et al. (2001) found a 

similar increase in uterine weight in rats given s.c. or oral 

bisphenol A at 800 mg/kg bw/day. 

Nagel et al. ([1997, 1999) noted that 17b-estradiol is 

extensively protein-bound in vivo and bisphenol A is 

minimally protein-bound. A recent study indicated more 

extensive binding of bisphenol A to plasma binding 

proteins (Teeguarden et al., 2005). Nagel suggested that 

estrogenicity of BPA (as well as other steroid hormones) 

can be predicted more accurately in rats by considering 

the free fraction of a chemical in human serum. [The 

Expert Panel notes that Figure 2 does not suggest that 

bisphenol A is more potent than 17b-estradiol in vivo 

than in vitro. The developmental effects of bisphenol A 

in the prostate are discussed in Section 3.2.] 

Inter-strain variability in rats has been evaluated as a 

source of variability in estrogenicity assays. Inspection of 

Table 53 does not suggest large sensitivity differences 

between Sprague–Dawley, Wistar, and Long-Evans rats. 

Greater sensitivity of F344 than Sprague–Dawley rats has 

been shown with respect to uterine weight and epithelial 

cell height (Steinmetz et al., 1998), where 17b-estradioladjusted potencies differed by 20–37% between the 

strains. BrDu labeling of vaginal epithelium was 3 times 

greater in F344 than Sprague–Dawley rats in another 

study (Long et al., 2000), and a third study (Steinmetz 

et al., 1997) showed that both bisphenol A and 17bestradiol increase serum prolactin in ovariectomized 

F344 but not ovariectomized Sprague–Dawley rats. Diel 

et al. (2004) evaluated estrogenic response to bisphenol A 

Birth Defects Research (Part B) 83:157–395, 2008
6.0 

5.5 PCNA labeling 

Multiple of control value 5.0 

4.5 

3.0 

2.5 Vaginal opening PND 26 

2.0 Epithelial cell height 

1.5 Uterine wet weight

1.0 Body weight 

0.5 

0.0 

0.1 1.0 10.0 100.0 

Bisphenol A dose, mg/kg bw/day 

220 CHAPIN ET AL. 

Table 54
 

Differences Between Laboratories in Rat Uterotrophic
 

Assay With Bisphenol Aa 

Laboratory Rat strain 

Lowest effective dose 

level (mg/kg bw/day) 

Immature,	 

gavage x 3 days 

2 

7 

12 

13 

Immature, 

s.c. x 3 days
 

2 

6 

7 

8 

12 

13 

15 

18 

20 

21 

Adult, s.c.	 

x 3 days 

2 

6 

7 

8 

12 

Adult, s.c.	 

x 7 days 

2 

7 

CD(SD)IGS 

CD(SD)IGS 

CD(SD)IGS BR 

Wistar 

CD(SD)IGS 

CD(SD)IGS BR 

CD(SD)IGS 

Alpk:ApfSD 

CD(SD)IGS BR 

Wistar 

Wistar 

Sprague–Dawley 

Sprague–Dawley 

CD(SD) BR 

CD(SD)IGS 

CD(SD)IGS BR 

CD(SD)IGS 

Alpk:ApfSD 

CD(SD)IGS BR 

CD(SD)IGS 

CD(SD)IGS 

200 

10 

x
 

x
 

x
 

10 

10 

375 

x 

x 

100 

x
 

x
 

x
 

100 

x 

x 

x 

x 

x 

100 

x 

x 

600 

x 

300 

x
 

x
 

x
 

300 

300 

1000 

x 

600 

x
 

600 

600 

1000 

1000 

1000 

a

Kanno et al. (2003b). 

in juvenile ovariectomized DA/Han, Sprague–Dawley, 

and Wistar rats. After 3 days of treatment with bisphenol 

A 200 mg/kg bw/day, there were small statistically 

significant increases in uterine weight in DA/Han and 

Sprague–Dawley rats but not in Wistar rats. There were 

no alterations in uterine or vaginal epithelium or in 

uterine clusterin mRNA expression in any of the strains 

after bisphenol A treatment. 

Inter-laboratory variation in the uterotrophic assay 

was evaluated by the Organization for Economic 

Cooperation and Development (OECD) (Kanno et al., 

2003b). Coded chemicals, including bisphenol A, were 

sent to up to 212 different laboratories. Four assay 

protocols were evaluated including oral treatment of 

intact immature rats for 3 days, s.c. treatment of intact 

immature rats for 3 days, s.c. treatment of ovariectomized 6–8-week-old rats for 3 days, and s.c. treatment of 

ovariectomized 6–8-week-old rats for 7 days. Not all 

laboratories used all protocols or tested all compounds. 

Rat strains and suppliers were not standardized across 

laboratories. Comparisons were made between labs 

based on the lowest dose level at which body weightadjusted blotted uterine weight was significantly different from the control. Results are summarized in Table 54. 

The lowest effective dose of bisphenol A was uniformly 

identified for the assays performed in ovariectomized 

adults. Assays performed in immature animals varied in 

identification of the lowest effective bisphenol A dose 

Fig. 4. Dose–response curves for endpoints of estrogenic activity 

in s.c.-dosed mice. On pair-wise testing, body weight was 

increased at 0.5 mg/kg bw/day and decreased at 100 mg/kg 

bw/day; vaginal opening was advanced at 0.1 and 100 mg/kg 

bw/day; epithelial cell height was increased at 5, 75, and 

100 mg/kg bw/day; PCNA labeling was increased at 75 and 

100 mg/kg bw/day; and uterine wet weight was increased at 

100 mg/kg bw/day. Data from Markey et al. (2001b). 

level. There was no apparent effect of strain on 

sensitivity of the uterotrophic response in immature 

(intact or castrate) or adult female rats. 

Intra-laboratory variability has been noted for the 

bisphenol A uterotrophic assay in immature mice 

(Tinwell and Joiner, 2000). Of 8 studies performed over 

a 2-year period at s.c. bisphenol A dose levels up to 200 

or 300 mg/kg bw/day, 4 showed a significant increase in 

uterine weight at 200 mg/kg bw/day. The other 4 

studies, including the 2studies that went to 300 mg/kg 

bw/day, showed no effect of bisphenol A treatment on 

uterine weight despite the expected response to diethylstilbestrol. Study authors noted that reducing the 

permissible body weight of the mice selected for study 

resulted in lower and less variable control uterine 

weights and greater likelihood of bisphenol A effect 

(Tinwell and Joiner, 2000; Ashby et al., 2004). [The Expert 

Panel notes that these studies all used high s.c. doses of 

bisphenol A.] 

Markey et al. (2001b) proposed that the rodent 

uterotrophic assay is relatively insensitive to the estrogenic effects of bisphenol A. These authors treated 

immature CD-1 mice with bisphenol A in s.c. minipumps 

and evaluated uterine weight, relative area of uterine 

compartments, epithelial height, expression of lactoferrin 

and proliferating cell nuclear antigen (PCNA), and 

induction of vaginal opening. Dose–response curves for 

the endpoints that showed significant changes from 

control are illustrated in Figure 4. The study authors also 

noted that significant alterations in some endpoints were 

observed at much lower doses (0.1 mg/kg bw/day for 

vaginal opening and 5 mg/kg bw/day for epithelial cell 

height), giving rise to a U-shaped dose–response curve. 

[The assertions of some investigators notwithstanding, 

the Expert Panel notes that oral bisphenol A does not 

consistently produce robust estrogenic responses and, 

when seen, estrogenic effects after oral treatment occur 

at high-dose levels.] 

Transgenic reporter mice have permitted in vivo 

identification of activation of the estrogen response 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 221 

element. Eight hr after i.p. injection on GD 13.5 of wildtype dams carrying transgenic fetuses, luciferase reporter 

activity was increased for bisphenol A 1 and 10 mg/kg bw 

(Lemmen et al., 2004). The luciferase response after 

bisphenol A was about 50% of that after a similar dose 

of estradiol dipropionate and B25% of that after a 10-fold 

higher dose of diethylstilbestrol [estimated from a graph]. 

Use of an in vitro reporter system showed bisphenol A 

potency to be 3–4 orders of magnitude less than that of 

diethylstilbestrol (Table 52). The authors concluded that 

the in vivo estrogenic potency of bisphenol A may be 

greater than predicted by in vitro assays. 

Nagel et al. (2001) developed a transgenic mouse with 

a thymidine kinase-lacZ reporter linked to 3 copies of the 

vitellogenin estrogen response element. This model 

showed an increase in ER activity after a single s.c. 

bisphenol A dose of 25 mg/kg bw (P 5 0.052), with 

further increases in activity after 0.8 and 25 mg/kg bw. 

Uterine weight was only increased at the 25 mg/kg bw 

dose level. Normalized to the diethylstilbestrol response, 

uterine weight response to bisphenol A 25 mg/kg bw 

was less than one-third the response in ER activity 

[estimated from a graph]. 

Gene expression profiles have been performed to 

compare the presumably ER-mediated response to bisphenol A with the response to reference ER agonists. 

Naciff et al. (2002) evaluated expression in the uteri and 

ovaries of Sprague–Dawley fetuses after s.c. dosing of 

dams on GD 11–20 with ethinyl estradiol 0, 0.5, 1, or 

10 mg/kg bw/day or bisphenol A 0, 5, 50, or 400 mg/kg 

bw/day. The high-dose of both compounds induced 

nipples and areolae in male and female fetuses. There 

were 366 genes in which expression was altered by 

ethinyl estradiol and 397 genes in which expression was 

altered by bisphenol A. Expression of 66 genes was 

changed in the same direction with high-doses of ethinyl 

estradiol, bisphenol A, and genistein (which was also 

tested in this model). Of the 40 genes with at least a 1.8­

fold change in expression, 17 responded similarly to 

ethinyl estradiol and bisphenol A. The authors identified 

50 mg/kg bw/day as the lowest dose level at which 

estrogen-like gene expression activity could be identified, which is lower than the 400–800 mg/kg bw/day 

dose range at which uterotrophic activity is typically 

reported in rats (Ashby and Tinwell, 1998). 

Terasaka et al. (2006) used expression of 120 estrogenresponsive genes (based on previous work) in MCF-7 cells 

to compare the profiles of bisphenol A and 17b-estradiol. 

Response was highly correlated (R 5 0.92) between the 2 

compounds. Another gene array study (Singleton et al., 

2004) used MCF-7 cells that had lost ER and were reengineered to express ERa. Among 40 estrogen-responsive 

genes, 12 responded to both bisphenol A and 17bestradiol, 9 responded only to bisphenol A, and 19 

responded only to 17b-estradiol. In the ER-deficient 

MCF-7 cell line from which these cells had been 

engineered, 1 gene responded to both bisphenol A and 

17b-estradiol and 14 responded to bisphenol A alone, 

suggesting ER-independent activity. The same group 

reported the response of an additional 31 genes, associated 

with growth and development, from the same chip 

(Singleton et al., 2006). In the ERa-containing cells, 5 of 

these genes showed regulation with both 17b-estradiol 

and bisphenol A, 13 were regulated only by bisphenol A, 

and 13 were regulated by only 17b-estradiol. 

Table 55
 

Bisphenol A Receptor Binding and Recruitment of Co-


Activator Proteinsa
 

Activity relative to 17b-estradiol 

Assay ERa ERb

Receptor binding 7.3 x 10 -4 7.5 x 10 -3
 

TIF2 recruitment o 1 x 10 -6 5 x 10 -4
 

SRC-1a recruitment 3 x 10 -4 2 x 10 -4
 

a

Routledge et al. (2000). 

Differences in the estrogenic activity of bisphenol A 

and reference estrogens may be due to differences in 

recruiting by the liganded receptor of co-regulatory 

proteins. Singleton et al. (2006) used a co-regulatorindependent yeast reporter system to evaluate the 

estrogenicity of bisphenol A and 17b-estradiol. Bisphenol 

A activity was more than 3 orders of magnitude less than 

17b-estradiol in the yeast system, compared to about a 2­

order-of-magnitude difference in an MCF-7 cell assay, 

leading the authors to postulate that mammalian coactivators may be involved in enhancing bisphenol A 

activity. In a comparison of ER binding and co-activator 

recruitment, Routledge et al. (2000) showed bisphenol A 

to bind the receptor more avidly than the liganded 

receptor recruited 2 co-activator proteins, normalized to 

17b-estradiol (Table 55). 

The classical ERs are receptors that, when bound, 

produce their activity through alterations in genomic 

transcription. In contrast, a membrane-bound ER has 

been described in murine pancreatic islet cells (Nadal 

et al., 2000, 2004; Quesada et al., 2002; Alonso-Magdalena 

et al., 2005). This membrane-bound receptor regulates 

calcium channels and modulates insulin and glucagon 

release. Bisphenol A has been shown to activate this 

receptor in vitro at a concentration of 1 nM, which is 

similar to the active concentration of diethylstilbestrol 

(Nadal et al., 2000; Alonso-Magdalena et al., 2005). 

Treatment of mice with bisphenol A or 17b-estradiol 

s.c. at 10 mg/kg bw acutely or daily for 4 days resulted in 

decreased plasma glucose and increased insulin (AlonsoMagdalena et al., 2006). By contrast, Adachi et al. (2005) 

reported that exposure of rat pancreatic islets to 0.1– 

1 mg/L [0.4–4.4 nM] bisphenol A did not alter insulin 

secretion over a 1-hr period. Exposure of islets to 

bisphenol A 10 mg/L [44 nM] for 24 hr increased insulin 

release. This response was prevented by actinomycin D 

and by ICI 182,780, supporting the conclusion that 

bisphenol A insulin release occurs through interaction 

with the cytoplasmic ER rather than the membranebound receptor. 

A membrane-bound ERa in the pituitary could be 

related to regulation of the release of stored prolactin in 

response to estrogens, a non-genomic response mediated 

by calcium influx. Using a rat prolactinoma cell line, 

bisphenol A was shown to promote calcium influx and 

release prolactin over a concentration range similar to 

that for 17b-estradiol (Wozniak et al., 2005; Watson et al., 

2007). The response to bisphenol was bimodal, with 

maximal responses at concentrations of 10 -12 and 10 -8 M 

and little-to-no response at intermediate concentrations. 

Calcium influx in MCF-7 cells has been shown to occur 

Birth Defects Research (Part B) 83:157–395, 2008
222 CHAPIN ET AL. 

Table 56 

Anti-Androgenicity Studies of Bisphenol A in Cells Transfected With Androgen Receptor Reporter 

Reference androgen Bisphenol A median inhibitory 

Cell type concentration (nM) concentration (IC50) mM [mg/L] Reference 

Human prostate R1881 0.1 7 [1.6] Paris et al. (2002) 

adenocarcinoma 

Chinese hamster ovary R1881 0.1 19.6 [4.5] Roy et al. (2005) 

Yeast Testosterone 10 1.8 [0.4] Lee et al. (2003a) 

Yeast Dihydrotestosterone 1.25 2 [0.5] Sohoni and Sumpter (1998) a 

Monkey kidney Dihydrotestosterone 1 0.746 [0.2] Xu et al. (2005) 

Monkey kidney Dihydrotestosterone 1 2.14 [0.5] Sun et al. (2006) 

Mouse fibroblast Dihydrotestosterone 0.01 4.3 [1.0] Kitamura et al. (2005) 

Human hepatoma Dihydrotestosterone 100 No anti-androgenic activity Gaido et al. (2000) 

a

Estimated from a graph. 

rapidly after exposure to bisphenol and 17b-estradiol 

concentrations of 10 -10 M through a non-ER-mediated 

mechanism (Walsh et al., 2005). 

Recently, bisphenol A was identified as competitor to 

17b-estradiol for binding to the GPR30 receptor; a novel 

seven-transmembrane receptor that mediates nongenomic estrogen actions to upregulate adenylyl cyclase 

and MAPK activities (Thomas and Dong, 2006). Similar 

to findings reported previously with nuclear estrogen 

receptors and membrane estrogen receptors, bisphenol A 

was identified as a relatively effective competitor of 17bestradiol binding, with relative binding affinities of 2.8% 

that of the natural estradiol ligand and an IC50 of 

630 x 10-9 M. Bisphenol A, at a concentration of 

200 nM significantly increased cAMP levels in transfected cells 30 min after compound addition. 

Bisphenol A has been found to bind estrogen-related 

receptor g, a nuclear receptor with no known natural 

ligand that shows little affinity for 17b-estradiol (Takayanagi et al., 2006). Estrogen-receptor g demonstrates 

high constitutive activity that is maintained by bisphenol 

A in the presence of 4-hydroxytamoxifen, which otherwise blocks nuclear ER activity. This observation led to 

the suggestion that bisphenol A may maintain estrogenrelated receptor g activity in the presence of a yet-to-beidentified natural antagonist and that cross talk between 

the estrogen-related receptor and ER systems could be 

responsible for the estrogenic activity of bisphenol A in 

spite of low binding affinity for ERa and b (Takayanagi 

et al., 2006). 

In addition to the studies reviewed for this section, 

there are studies in which the putative estrogenicity of 

environmental samples or synthetic products were 

evaluated using one or another assay. For example, Olea 

et al. (1996) evaluated resin-based dental composites in 

an MCF-7 culture system. The response of the system 

was attributed to the bisphenol and its methacrylate 

detected in the composites, but bisphenol A was not 

specifically tested. These articles were not reviewed for 

this section. 

2.2.3 Androgen activity. Transfected cell-based 

assays have not identified bisphenol A as having 

androgenic activity (Sohoni and Sumpter, 1998; Gaido 

et al., 2000; Kitamura et al., 2005; Xu et al., 2005). 

However, bisphenol A is mitogenic in cultured human 

prostate carcinoma cells at a concentration of 1 nM 

(Wetherill, 2002). Based on stimulated cell growth in this 

system, the potency of bisphenol A is about 5% that of 

dihydrotestosterone [estimated from a graph]. This 

bisphenol A activity was shown to be mediated by 

interaction with a mutant tumor-derived androgen 

receptor called AR-T877A. Anti-androgenic activity has 

been demonstrated using cells transfected with androgen 

receptor reporting systems (Table 56). The anti-androgenic activity of bisphenol A is expressed as the 

concentration needed to halve the androgen reporter 

response to a reference androgen. Studies in transfected 

cells have shown that bisphenol A interferes with the 

binding of dihydrotestosterone to the androgen receptor, 

interferes with translocation of the liganded receptor to 

the nucleus, and prevents transactivation at the androgen-response element (Lee et al., 2003a). 

Kim et al. (2002a) conducted a Hershberger assay to 

determine the effects of bisphenol A exposure on 

reproductive organs of rats. Sprague–Dawley rats were 

fed PMI Certified Rodent LabDiet and housed in 

polycarbonate cages. No information was provided 

about bedding materials. One experiment was conducted 

to determine the optimum dose and age for observing 

testosterone exposure effects. In a second experiment, 10 

rats/group rats were castrated at 5 weeks of age and 7 

days later gavaged with bisphenol A (99% purity) at 

doses of 0 (ethanol/corn oil vehicle) 10, 100, or 1000 mg/ 

kg bw/day for 7 days. A second group of castrated 6­

week-old males rats was gavaged with bisphenol A at 0, 

50, 100, 250, or 500 mg/kg bw/day for 7 days. In a third 

experiment, 10 castrated 6-week-old rats/group were 

treated with 0.4 mg/kg bw/day testosterone by s.c. 

injection in addition to gavaged bisphenol A at 50, 100, 

250, or 500 mg/kg bw/day or flutamide at 1, 5, 10, or 

25 mg/kg bw/day for 7 days. A positive control group 

was given 0.4 mg/kg bw/day testosterone for 7 days. 

[There is some confusion in the article regarding ages at 

castration and start of treatment. For the first group of 

bisphenol A-treated rats, it is reported that rats were 

castrated at 5 weeks of age and treated at 6 weeks of 

age. For the other groups of bisphenol A-treated rats, 

the Methods section reported that treatment began at 6 

weeks of age, but tables in the Results section 

indicated that rats were castrated at 6 weeks of age.] 

During the study, clinical signs were observed and body 

weights were measured. Blood was collected and rats 

were killed B24 hr after administration of the last 

dose. Accessory reproductive organs were removed 

and weighed. Serum luteinizing hormone (LH) 

and testosterone concentrations were measured by 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 223 

radioimmunoassay (RIA). Statistical analyses included 

Bartlett test, analysis of covariance (ANCOVA), Dunnett 

test, and Bonferroni test. Exposure to bisphenol A did not 

affect weights of the ventral prostate, seminal vesicles, 

glans penis, or levator ani plus bulbocavernosus muscle; 

or serum concentrations of LH or testosterone. Testosterone increased the weights of accessory reproductive 

organs. Flutamide increased serum LH concentrations 

and inhibited testosterone-induced increases in accessory 

reproductive organ weights. Study authors concluded 

that bisphenol A did not exhibit androgenic or antiandrogenic effects in rats. 

Yamasaki et al. (2003) conducted a Hershberger assay 

in rats exposed to bisphenol A or 1 of 29 other chemicals. 

In this study, which was conducted according to GLP, 

animals were housed in stainless steel wire-mesh cages. 

Assuming these males were fed the same diets as rats 

used in an uterotrophic assay also described in this study, 

they received MF Oriental Yeast feed. Rats were 

randomly assigned to treatment groups. Beginning at 

56 days of age and continuing for 10 days, 6 castrated 

male Brl Han: WIST Jcl (GALAS) rats/group were 

administered bisphenol A by stomach tube at doses of 

0 (olive oil vehicle), 50, 200, or 600 mg/kg bw/day. An 

additional group of rats was administered the same 

vehicle and doses of bisphenol A in addition to 0.2 mg/ 

kg bw/day testosterone propionate by s.c. injection. Dose 

selection was based on results of preliminary studies. A 

positive control group was given 10 mg/kg bw/day 

flutamide in addition to 0.2 mg/kg bw/day testosterone 

propionate. Rats were killed 24 hr after receiving the final 

dose. Ventral prostate with fluid, seminal vesicles with 

fluid, bulbocavernosus/levator ani muscle, glans penis, 

and Cowper gland were collected and weighed. Data 

were analyzed by Student t-test. Bisphenol A did not 

affect body weight and there were no clinical signs of 

toxicity. The only statistically significant effect on relative 

organ weight was a [24%] increase in glans penis weight 

in rats given 600 mg/kg bw/day bisphenol A without coadministration of testosterone. In contrast, rats treated 

with flutamide plus testosterone propionate experienced 

increases in weights of ventral prostate, seminal vesicle, 

bulbocavernosus/levator ani muscle, glans penis, and 

Cowper gland. [Absolute organ weights were not 

reported. It is assumed but was not stated that relative 

weights were based on body weight.] Study authors 

noted that because glans penis weights were variable in 

control rats and weights of other accessory reproductive 

organs were not affected, bisphenol A could not be 

clearly determined to have androgen agonistic 

properties. 

Nishino et al. (2006) performed a Hershberger assay in 

Wistar rats. At 2 weeks of age, rats were given ssniffR 10 

diet and housed in Makrolon cages with ssniff bedding. 

Seven days after orchiectomy, rats were placed in groups 

of 13 [randomization not discussed] and treated orally 

[gavage assumed] with bisphenol A [purity not indicated] in propylene glycol at 0, 3, 50, 200, or 500 mg/kg 

bw/day for 7 days or s.c. with testosterone propionate 

1 mg/kg bw. Another group was given oral bisphenol A 

500 mg/kg bw/day and flutamide 3 mg/kg bw/day. 

Rats were killed by decapitation after treatment. Seminal 

vesicles and prostates were weighed and fixed in 4% 

neutral buffered paraformaldehyde. Immunohistochemical evaluation of androgen receptor, PCNA, and MIB-5 

was performed. Epithelial cell height and duct luminal 

area were determined morphometrically. Review by the 

Expert Panel indicated that this study was inadequate 

due to methodological issues. 

2.3 Genetic Toxicity 

Assessment of mutagenicity associated with bisphenol 

A was based primarily on reviews by the European 

Union (2003) and Haighton et al. (2002). CERHR 

summarized a limited number of studies that were not 

included in reviews. Results of in vitro genetic toxicity 

testing are summarized in Table 57, and results of in vivo 

genetic toxicity tests are summarized in Table 58. 

The European Union (2003) noted that bisphenol A 

demonstrated aneugenic potential and micronuclei formation in in vitro tests without metabolic activation. 

However, there was no evidence of micronuclei formation in an in vivo mouse study. Other studies demonstrated disruption of microtubule formation and the 

presence of DNA adducts. In the studies reviewed by the 

European Union, there was no evidence of gene mutations or structural chromosomal aberrations in in vitro 

tests and negative results were obtained in a dominant 

lethal test in rats; however, the European Union noted 

several limitations for those studies. Based on their 

review of genotoxicity data and the lack of significant 

tumors reported in animal studies, the European Union 

(2003) concluded that bisphenol A does not appear to 

have significant mutagenicity potential in vivo. Because 

aneugenic potential was apparently observed only in in 

vitro tests, it was judged to be of no concern. The 

relevance of DNA adduct formation was unclear, but 

based on weight of evidence, i.e., negative findings for 

gene mutation and clastogenicity in cultured mammalian 

cells, DNA adduct formation was thought unlikely to be 

of concern for humans. 

Haighton et al. (2002) concluded that results of 

standardized and validated genetic toxicity tests 

demonstrated the lack of mutagenic and genotoxic 

activity of bisphenol A in vivo. Studies demonstrating 

disrupted microtubule formation or DNA adduct 

formation were noted, but because the studies used 

high-doses, they were judged to be of limited relevance. 

The lack of activity in an in vivo micronucleus assay in 

mice was said to confirm negative results observed in in 

vivo tests. Lastly, it was concluded that bisphenol A 

(parent) had no structural features that suggested 

mutagenic activity. 

Subsequent to the release of the European Union (2003) 

and Haighton et al. (2002) reviews, Hunt et al. (2003), 

published a study examining meiotic aneuploidy potential of bisphenol A in female mice. In 1998, a large 

increase in background rate of congression failure (from 

1–2 to 40%) and in aneuploidy (from 0.7 to 5.8%) was 

observed in the study authors’ laboratory. The increase 

was found to coincide with damage to polycarbonate 

caging material. Removal of the most damaged cages and 

change to polysulfone cages resulted in decreased background rates of congression failure. Intentionally damaging polycarbonate cages and water bottles resulted in 

increased rates of congression failure. As noted in 

Table 58, congression failure rates were increased in 

juvenile female mice orally exposed to Z20 mg/kg bw/ 

day bisphenol A for 6–8 days or 20 mg/kg bw/day for 7 

Birth Defects Research (Part B) 83:157–395, 2008
224 Table 57 In Vitro Genetic Toxicity Studies of Bisphenol A

Concentration Cell	 Endpoint Results Reference

Haworth et al. (1983)a,b 

Schweikl et al. (1998)a,b 

Takahata et al. (1990)a,b Dean and Brooks

(1978)a,b

JETOC (1996)a,b

Masuda et al. (2005) 

Schweikl et al. (1998)a,b 

Myhr and Caspary

(1991)a,b

Honma et al. (1999)a,b; 

Moore et al. (1999)a,b 

Tsutsui et al. (1998)a,b

Takahashi et al. (2001)

Dean and Brooks

(1978)a,b

Iso et al. (2006) Ivett et al. (1989)a,b;

Tennant et al. (1986,1987)b

Hilliard et al. (1998)a

Galloway et al. (1998)a 

Tsutsui et al. (1998)a,b 

Dean and Brooks

(1978)b

Tsutsui et al. (1998)a,b 

Salmonella typhimurium strains TA98, TA100, Mutagenicity

TA1535, TA1537 

Salmonella typhimurium strains TA97a, TA98, Mutagenicity

TA100, TA102 

Salmonella typhimurium strains TA97, TA98, Mutagenicity

TA100, TA102 

Salmonella typhimurium strain TA1538 and Mutagenicity

Escherichia coli strains

WP2 and WP2uvrA 

Salmonella typhimurium strains TA98, TA100, Mutagenicity

TA1535, TA1537,and Escherichia coli strain WP2uvrA 

Salmonella typhimurium strains TA98 and TA100 Mutagenicity Chinese hamster V79 cells, hprt locus Mutagenicity Mouse lymphoma L5178Y cells, tk1/- locus Mutagenicity 

Mouse lymphoma L5178Y cells, tk1/- locus Mutagenicity Syrian hamster embryo cells (Na1/K1 ATPase Mutagenicity

and hprt loci) 

Human RSa cells Mutagenicity Saccharomyces cerevisiae strain JDI Mutagenicity 

MCF-7 cells DNA damage (assessed

by comet assay) 

MDA-MB-231 cells DNA damage (assessed

by comet assay) 

Chinese hamster ovary (CHO) cells Chromosomal

aberration 

CHO cells, clone WBL Chromosomal

aberration 

CHO cells, clone WBL Chromosomal

aberration 

Syrian hamster embryo cells Chromosomal

aberration 

Epithelial-type rat liver cell line (RL1) Chromosomal

aberration 

Syrian hamster embryo cells Aneuploidy/

polyploidy 

3.3–333.3 mg/plate, with and without metabolic

activation

50–500 mg/plate, with and without metabolic

activation 

r5000 mg/plate with and without metabolic

activation 

r1000 mg/mL, with and without metabolic

activation

5–1250 mg/plate, with and without metabolic

activation

1 mM [228 mg/L], with and without metabolic

activation

0.1–0.2 mM [23–46 mg/L], without metabolic

activation

5–60 mg/L without metabolic activation, 25–

200 mg/Lwith metabolic activation, or 5–60 mg/L withandwithout metabolic activationd

Concentrations not specified, with and without

metabolic activation 

25–200 mM [5.7–46 mg/L], without metabolic

activation 

10	 -8–10 -5M [0.002–2 mg/L], without metabolic

activation 

r500 mg/L, with and without metabolic

activation 

10–8–10 -4M [0.002–23 mg/L], without metabolic

activation 

10 -4M [23 mg/L], without metabolic activation 

20–40 mg/L, without metabolic activation and

30–50 mg/Lwith metabolic activation 

350–450 mM [80–103 mg/L], without metabolic

activationand 

r250 mM [57 mg/L] with metabolic

activation

400 and 450 mM [91 and 103 mg/L], without 

metabolic activation 

25–200 mM [5.7–46 mg/L], without metabolic

activation

10–30 mg/L 25–200 mM [5.7–46 mg/L], without metabolic

activation 

NegativeNegativeNegativeNegativeNegative

Negative

Negative

Negative (results questioned due to

possible inability to count smallcolonies)

Inconclusive without and negative with

metabolic activation 

Negative

m at all doses 

Negative

m at 

Z10 -6M [0.2 mg/L] 

mNegative (inconsistent m at high dose

with metabolic activation) 

Positive at 

Z400 mM [91.3 mg/L]

without metabolic activationc

;

negative with metabolic activation 

Positivec

NegativeNegative 

CHAPIN ET AL. 

Birth Defects Research (Part B) 83:157–395, 2008 
225 

0.8–25 mg/L, without metabolic activation and

30–50 mg/mL,with metabolic activation 

0.2–0.5 mM or nMd [46–114 mg/L or mg/L] 

10	 -9–10 -5M [0.0002–2 mg/L], without 

metabolic activation 

Not specified, but stated to cover range of

cytotoxicity

25–200 mM [5.7–46 mg/L], without metabolic

activation 

r50 mg/L for 24 hr; 

r30 mg/L for 7 days,

withoutmetabolic activation 

2–60 mg/L 50–200 mM [11.5–46 mg/L], without metabolic

activation

1000 mg presence of peroxidase and hydrogen

peroxide 

10–100 mM [2.3–23 mg/L], metabolic activation

unknown

50–200 mM [11.5–46 mg/L], no metabolic

activation

20–200 mM [4.6–46 mg/L], metabolic activation

unknown

200 mM [46 mg/L], without metabolic activation;

100 mM

[23 mg/L] for metaphase arrest assay 

100–200 mM [23–46 mg/L], without metabolic

activation

10 or 30 mM [2.3 or 6.9 mg/L] 0.05–0.4 mg/L 

CHO cells Rat hepatocytes Human RSa cells A31-1-13 clone of BALB/c-3T3 cells Syrian hamster embryo cells Syrian hamster embryo cells Syrian hamster embryo cells Syrian hamster embryo cells Purified rat DNA Bovine brain microtubule protein Bovine brain microtubule protein Bovine brain microtubule protein Chinese hamster V79 cells Chinese hamster V79 cells Oocytes from Balb/c mice Oocytes from MF1 mice 

Sister chromatid

exchange

DNA strand breaks Unscheduled DNA

synthesis

Transformation Transformation Transformation Transformation DNA adduct formation DNA adduct formation 

Inhibited microtubule

polymerization

Inhibited microtubule

polymerization

Inhibited microtubule

polymerization

Aneuploidogenic

potential asassessed bymicronucleiformation,microtubule assay,and metaphase arrest 

Aneuploidogenic

potential as assessedby micronucleiformation

Meiotic spindle

formation

Aneuploidy 

Inconclusive (non-dose-related m in 

aneuploidy at 

Z50 mM [11 mg/L])

e; 

apparently positivef 

Negative (one small m was not 

reproducible) 

Negative (m noted but scored as

negative by study authors due toexcessive cytotoxicity) 

m at 10 -6 M [0.2 mg/L] and k at 10 -7 

mg/L] [0.02 and 10 -5M [2 mg/L] 

NegativePositive at 

Z50 mM [11.4 mg/L] (nondose-related m)

e; equivocalf 

NegativeNegativePositive at 

Z50 mM [11 mg/L] (doserelated m) 

Positive

PositivePositive (dose-related) Positive (EC50 5 150 mM [34 mg/L]) 

PositivePositiveCentrosomes and spindles

disorganized 

No hyper haploidy but m diploid

metaphase II oocytes at 0.2 mg/L 

Ivett et al. (1989)a,b

Tennant et al. (1986)b 

Storer et al. (1996)a,b

Takahashi et al. (2001)

Matthews et al. (1993)a 

Tsutsui et al. (1998,

2000)a,b

LeBoeuf et al. (1996)a 

Jones et al. (1988)b

Tsutsui et al. (1998)a,b 

Atkinson and Roy

(1995a)

Metzler and Pfeiffer

(1995)a

Pfeiffer et al. (1996)b 

Pfeiffer et al. (1997)a,b 

Pfeiffer et al. (1997)a,b 

Ochi (1999)a,b

Can et al. (2005) Pacchierotti et al. (2007) 

Birth Defects Research (Part B) 83:157–395, 2008 

BISPHENOL A 

aReviewed by Haighton et al. (2002).
bReviewed by European-Union (2003).
cAccording to the Haighton et al. (2002) review, positive results occurred at cytotoxic concentrations.
dDiscrepancies noted between information presented by Haighton et al. (2002) and European-Union (2003).
eConclusion by Haighton et al. (2002).
fConclusion by European-Union (2003).


m,k increase, decrease.
 
226 CHAPIN ET AL. 

Table 58 

In Vivo Genetic Toxicity Studies of Bisphenol A 

Species and sex Dose (route) Cells Endpoint Results Reference 

Male rat 85 mg/kg bw/day Germ Dominant lethality Negative Bond et al. (1980)a,b 

for 5 days (i.p.) (abstract only) 

Male rat 200 mg/kg bw (i.p.) DNA Adduct formation Positive Atkinson and 

and 200 mg/kg bw Roy (1995b) 

for 4, 8, 12, or 16 days (oral) 

Male and female 500–2000 mg/kg bw (oral) Bone marrow Micronuclei Negative Gudi and Krsmanovic 

mouse (1999)a

; Shell Oil 

Co. (1999)b 

Male mouse 1 mmol/kg bw [228 mg/kg Peripheral Micronuclei Negative Masuda et al. (2005) 

bw] (oral) blood 

reticulocyte 

20–22-day-old 0.02–0.100 mg/kg bw/day Oocyte Congression failure Positive at all doses; Hunt et al. (2003) 

female mouse (oral) for 6–8 days or 0.02 mg/ statistically 

kg bw for 3, 5, or 7 days significant 

with 7-day 

exposure 

Pregnant mouse 0.4 mg/day s.c. pellet Oocyte Evaluation of pachytene Incomplete synapsis, Susiarjo et al. (2007) 

GD 11.5–18.5 [B20 mg/kg bw/day] fetal oocyte and end-to-end 

of ploidy in oocytes association 

and 2-cell of sister 

embryos from chromatids, 

adults that mhyperploidy 

were exposed 

in utero 

Female mouse 0.2 or 20 mg/kg bw acutely Oocyte Aneuploidy Negative Pacchierotti et al. 

or daily for 7 days or (2007) 

0.4 mg/L in drinking water 

for 7 weeks 

Male (102/ 0.002–0.2 mg/kg bw for Spermatocyte Meiotic delay Negative Pacchierotti et al. 

ElxC3H/El) 6 days (oral) and aneuploidy (2007) 

F1 mouse 

Drosophila 10,000 ppm (oral) Offspring Sex-linked recessive Negative Foureman et al. 

melanogaster lethal test (1994)a,b 

Turbot 50 ppb in aquarium Erythrocyte Micronuclei Positive Bolognesi et al. 

water for 2 weeks (2006) 

a

Reviewed by Haighton et al. (2002). 

b

Reviewed by European-Union (2003). 

days. The study authors concluded that bisphenol A was 

a potential meiotic aneugen. 

In a follow-up study (Susiarjo et al., 2007), pregnant 

C57Bl/6 mice on GD 11.5 were implanted with s.c. pellets 

designed to release bisphenol A 0 or 0.4 mg/day. [The 

authors assume a 20 g bw, giving an estimated dose 

level of 20 lg/kg bw/day.] Oocytes from GD 18.5 female 

fetuses showed an increase in pachytene synaptic 

abnormalities including incomplete synapsis and endto-end associations of sister chromatids. There was also 

paradoxically an increase in recombinant foci in pachytene oocytes of bisphenol A-exposed females. Some 

female offspring of bisphenol A-treated dams were 

fostered to untreated dams. Eggs or 2-cell embryos from 

these female offspring at 4–5 weeks of age showed an 

increase in hyperploidy. Pachytene oocyte abnormalities 

similar to those identified in fetuses exposed to bisphenol A were seen in oocytes obtained from ERb

knock-out mice, suggesting to the authors that bisphenol 

A may exert adverse effects on meiosis by blocking ERb. 

In response to the study of Hunt et al. (2003), 

Pacchierotti et al. (2007) investigated the aneugenic 

effects of bisphenol A in mouse somatic and germ cells. 

C57Bl/6 female mice were superovulated using pregnant 

mare serum and hCG after which they were gavaged 

with bisphenol A 0.2 or 20 mg/kg bw. Metaphase II 

oocytes were collected after 17 hr and evaluated using Cbanding. Additional female mice were gavaged with 

bisphenol A 0.04 mg/kg bw/day for 7 days or were 

given bisphenol A in drinking water at a concentration of 

0.4 mg/L for 7 weeks. These mice were superovulated at 

the end of the 7-day or 7-week treatment period and 

housed overnight with untreated males. Females without 

vaginal plugs were killed for evaluation of oocytes by Cbanding. Females with vaginal plugs were treated with 

colchicine to prevent the first embryonic cleavage, and 

zygotes were collected the next morning for evaluation 

by C-banding. There were no bisphenol A effects on 

induction of aneuploidy. There was a statistically 

significant increase in premature centromere separation 

in the group treated for 7 weeks, but there was no effect 

of bisphenol A treatment on the proportion of zygotes 

with structural or numeric chromosome changes. Male 

mice were treated with bromodeoxyuridine 8 days before 

being treated with bisphenol A 0.2 mg/kg bw/day for 6 

days. Evaluation of sperm after 21–25 days did not show 

a significant mitotic delay in spermatocytes. Additional 

male mice were given bisphenol A orally at doses of 0, 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 227 

0.002, 0.02, and 2 mg/kg bw/day for 6 days. Epididymal 

sperm were collected 22 days after the end of bisphenol 

A treatment and multicolor fluorescent in situ hybridization was used to evaluate decondensed sperm for 

aneuploidy. Sperm count was decreased by bisphenol 

A 0.002 mg/kg bw/day, but there was no increase in the 

frequency of hyperhaploidy or diploidy. Bisphenol A was 

negative in a bone marrow micronucleus test at dose 

levels up to 2 mg/kg/day for 2 days. 

2.4. Carcinogenicity 

No human data examining the carcinogenicity of 

bisphenol A were identified. 

NTP (1982) and Huff (2001) examined carcinogenicity 

of bisphenol A in F344 rats and B6C3F1 mice. Animals 

were randomly assigned to treatment groups. Bisphenol 

A (o98.2% purity) was administered through feed for 

103 weeks to 50 rats/sex/dose at 0, 1000, or 2000 ppm, 50 

male mice/group at 0, 1000, or 5000 ppm, and 50 female 

mice/group at 0, 5000, or 10,000 ppm. NTP estimated 

mean bisphenol A intakes of 74 and 148 mg/kg bw/day 

for male rats and 74 and 135 mg/kg bw/day for female 

rats. [Data on bisphenol A intake, food intake, and 

body weights were not provided for mice.] Using 

default values, the European Union (2003) estimated 

bisphenol A intakes of 120 and 600 mg/kg bw/day in 

male mice and 650 and 1300 mg/kg bw/day in female 

mice. Concentration and stability of bisphenol A in feed 

were verified. Body weights and clinical signs were 

observed during the study. Following the exposure 

period, animals were killed and necropsied. Organs, 

including seminal vesicle, prostate, testis, ovary, and 

uterus, were preserved in 10% neutral buffered formalin 

and examined histologically. Statistical analyses included 

Cox and Tarone methods, 1-tailed Fisher exact test, 

Bonferroni inequality criterion, Cochran-Armitage test, 

and life table methods for linear trend. 

In rats, body weights of males and females from both 

dose groups were lower than controls throughout the 

study. Feed intake was decreased in females of both dose 

groups beginning at Week 12. No adverse effects on 

survival were observed. There were no non-neoplastic 

lesions [including in male and female reproductive 

organs] that appeared to be treatment-related. The 

incidence of leukemia was increased in males (13 of 50, 

12 of 50, and 23 of 50 in control and each respective dose 

group) and females (7 of 50, 13 of 50, and 12 of 50). In 

males the trend for leukemia was significant by CochranArmitage test, but statistical significance was not shown 

by life table analysis for trend or incidence in the highdose group, according to the unpublished version of the 

study. The published version of the study indicated 

statistical significance at the high-dose. Statistical significance was not attained for leukemia incidence in 

female rats. An increased incidence of testicular interstitial cell tumors (35 of 49, 48 of 50, 46 of 49) was 

statistically significant in both dose groups. An increased 

incidence of mammary fibroadenomas in males of the 

high-dose group (0 of 50, 0 of 50, and 4 of 50) achieved 

statistical significance for trend by Cochran-Armitage 

test but not by Fisher exact test. In bisphenol A groups, 

there were decreased incidences of adrenal pheochromocytomas in males, adrenal cortical adenomas in 

females, and uterine endometrial stromal polyps. The 

NTP concluded that none of the increases in tumor 

incidence in rats was clearly associated with bisphenol A 

exposure. 

In mice, body weights were lower in high-dose males 

and in females of both dose groups. Feed intake could 

not be accurately determined because of spillage. Bisphenol A did not affect the survival of mice. Incidence 

of multinucleated hepatocellular giant cells was increased in treated males (1 of 49, 41 of 49, and 41 of 

50). [A review of the data indicated no increases in 

incidence of non-neoplastic lesions in the reproductive 

organs of male or female mice.] The incidence of 

leukemia or lymphoma in male mice by dose group (2 

of 49, 9 of 50, and 5 of 50) was not statistically significant. 

The published version of the report indicated an 

increasing trend for lymphoma. The linear trend for 

increased pituitary chromophobe carcinomas in male 

mice (0 of 37, 0 of 36, 3 of 42) was reported to be 

statistically significant by Cochran-Armitage test but 

statistical significance was not shown by Fisher exact 

test. The study authors concluded that none of the 

increases in tumor incidence in mice could be unequivocally associated with bisphenol A exposure. 

NTP concluded that under the conditions of this study, 

there was no convincing evidence the bisphenol A was 

carcinogenic in F344 rats or B6C3F1 mice. However, 

study authors stated that there was suggestive evidence 

of increased cancer in the hematopoietic system based on 

marginally significant increases in leukemia in male rats, 

non-statistically significant increases in leukemia in 

female rats, and a marginally significant increase in 

combined incidence of lymphoma and leukemia in male 

mice. A statistically significant increase in testicular 

interstitial cell tumors in aging F344 rats was also 

considered suggestive evidence of carcinogenesis. The 

effect was not considered conclusive evidence because of 

the high incidence of the testicular neoplasm in aging 

F344 rats (88% incidence in historical controls). 

The NTP study was reviewed by the European Union 

(2003) and Haighton et al. (2002). For increases in 

leukemia, mammary gland fibroadenoma, and Leydig 

cell tumors in male rats, both groups noted the lack of 

statistical significance using the appropriate analyses and 

the common occurrence of these tumor types in F344 rats. 

The European Union (2003) concluded, ‘‘Overall, all of 

these [tumor] findings in rats and mice are not considered 

toxicologically significant. Consequently, it is concluded 

that bisphenol A was not carcinogenic in this study in both 

species.’’ Haighton et al. (2002) concluded, ‘‘Overall, the 

results of this bioassay did not provide any compelling 

evidence to indicate that [bisphenol A] was carcinogenic 

in F344 rats or in B6C3F1 mice.’’ Based on the experimental animal data, the European Union concluded that 

‘‘ythe evidence suggests that bisphenol A does not have 

carcinogenic potential.’’ Using a weight of evidence 

approach, Haighton et al. (2002) concluded that bisphenol 

A was not likely to be carcinogenic to humans. This 

conclusion was based on NTP study results; lack of 

activity at noncytotoxic concentrations in both in vitro 

genetic toxicity tests and in an in vivo mouse micronucleus test; and data from metabolism studies that show 

rapid glucuronidation and no formation of possibly 

reactive intermediates, with the possible exception of 

reactive intermediates potentially generated as a result of 

saturated detoxification pathways at high-doses. 

Birth Defects Research (Part B) 83:157–395, 2008
228 CHAPIN ET AL. 

Table 59 

Development of UDPGT Activity in Humansa 

UDPGT activity, nmol/min/mg 

protein 

Age Bilirubin Testosterone 1-Napthol 

30 weeks gestation 0.05 0 0.56 

30 weeks gestation 0.4; 1 0.14; 0.85 3.0; 1.8 

with 10 weeks survival 

Full-term infants 0.0770.04 0.1070.06 0.7570.68 

surviving 1–10 days 

(n 5 7) 

Full-term infants 0.6470.32 0.1270.05 2.471.1 

surviving 8–15 

weeks (n 5 6) 

Full-term infants 0.9971.1 0.0970.06 3.672.1 

surviving 22–55 

weeks (n 5 5) 

Adult males (n 5 3) 0.7670.43 0.4670.61 7.272.2 

a

Coughtrie et al. (1988).
 

Data presented as individual values or mean7SD.
 

2.5 Potentially Susceptible Subpopulations 

As noted in Section 2.1.1.3, one pathway of bisphenol 

A metabolism in humans and experimental animals is 

glucuronidation. Studies in experimental animals demonstrated that both the intestine and liver can 

glucuronidate bisphenol A. UGT2B1 was identified as 

the isoform involved in bisphenol A glucuronidation in 

rat liver (Yokota et al., 1999). The UDPGT isoform 

involved in human intestinal glucuronidation of bisphenol A is not known to have been identified. Despite 

uncertain isoform identification, studies in humans and 

experimental animals demonstrate developmental 

changes in expression of activities of several UDPGT 

isoforms that potentially affect bisphenol A metabolism. 

Coughtrie et al. (1988) examined the ontogeny of 

UDPGT activity in human liver microsome samples 

obtained postmortem from adults and premature or 

full-term infants. Results of this analysis are listed in 

Table 59. Activities for isoenzymes catalyzing glucuronidation of bilirubin, testosterone, and 1-napthol were very 

low at birth in premature and full-term infants. Activities 

increased with age for the isoenzymes catalyzing 

glucuronidation of bilirubin (B80% of adult levels by 

8–15 weeks of age) and 1-naphthol (B30% of adult levels 

at 8–15 weeks of age). During the first 55 weeks of life, no 

consistent increase in activity was noted for the isoenzyme catalyzing glucuronidation of testosterone. 

Using an immunoblot technique with antibodies developed toward liver testosterone/4-nitrophenol and kidney naphthol/bilirubin, 1 immunoreactive protein was 

observed in microsomes of 18- and 27-week-old fetuses 

and 3 immunoreactive proteins were observed in microsomes of full-term infants. Most isoenzymes present in 

adults were observed in infants within 3 months of age at 

levels B25% those of adults. 

Strassburg et al. (2002) used a reverse transcript (RT)­

polymerized chain reaction (PCR) technique to examine 

developmental changes in expression for 13 UDPGT

genes in liver samples obtained from 16 pediatric 

patients undergoing liver transplant for extrahepatic 

biliary atresia (6–24 months old) and 12 adults 

undergoing liver transplant for carcinoma (25–75 years). 

Changes in gene expression were also assessed in hepatic 

RNA samples for two 20-week-old fetuses. No transcripts for UDPGT were detected in samples from 20­

week-old fetuses. In infant and adult livers, transcripts 

were detected for UGT1A1, UGT1A3, UGT1A4, UGT1A6, 

UGT1A9, UGT2B4, UGT2B7, UGT2B10, and UGT2B15; 

there were no age-related differences in expression. 

Expression of UGT1A9 and UGT2B4 mRNA was lower 

in the pediatric samples. Western blot analyses of protein 

expression for UGT1A1, UGT1A6, and UGT2B7 were 

consistent with findings for mRNA expression. Activities 

toward 18 specific substrates were assessed in microsomes. In 13–24-month-old children compared to adults, 

glucuronidation activity was lower for ibuprofen (24­

fold), amitriptyline (16-fold), 4-tert-butylphenol (40-fold), 

estrone (15-fold), and buprenorphine (12-fold). 

Cappiello et al. (2000) compared uridine 50

-diphosphoglucuronic acid concentrations in livers and kidneys 

of human fetuses and adults and in placenta. In adults 

undergoing surgery, liver samples were obtained from 1 

man and 4 women (23–72 years of age) and kidney 

samples were obtained from 1 woman and 4 men (55–63 

years of age). Fetal livers and kidneys were obtained 

from 5 fetuses legally aborted between 16 and 25 weeks 

gestation. Five placenta samples were obtained on 

delivery at 17–25 weeks gestation. Compared to adults, 

fetal uridine 50

-diphosphoglucuronic acid concentrations 

were 5-fold lower in liver and 1.5-fold lower in kidney. 

Concentrations of uridine 50

-diphosphoglucuronic acid 

in placenta were 3–4-fold lower than in fetal liver. Based 

on these findings, study authors concluded that glucuronidation is potentially limited in the human fetus. 

As noted in Sections 2.1.2.2 and 2.1.2.3, rat fetuses 

appear to have no or low ability to glucuronidate 

bisphenol A (Miyakoda et al., 2000; Matsumoto et al., 

2002; Domoradzki et al., 2003). Although rats glucuronidate bisphenol A at birth, glucuronidation capacity 

appears to increase with age (Matsumoto et al., 2002; 

European-Union, 2003; Domoradzki et al., 2004). 

Some possible interindividual or sex-related differences in the ability to produce the bisphenol A sulfate 

conjugate were identified in a limited number of human 

studies. As discussed in more detail in Section 2.1.1.3 and 

shown in Table 8, higher amounts of urinary bisphenol A 

sulfate were detected in 15 adult women than in 15 adult 

males (Kim et al., 2003b). In a study examining bisphenol 

A metabolism by human hepatocytes, an B10-fold 

higher concentration of a bisphenol A glucuronide/ 

sulfate conjugate was observed in the sample from 1 

female than in samples from 2 other females and 2 males 

(Pritchett et al., 2002). 

Yang et al. (2003) examined the effects of polymorphisms in sulfotransferase enzymes on urinary excretion of 

total bisphenol A (conjugated and free) in Korean 

volunteers. Urinary bisphenol A concentrations were 

measured by HPLC and a PCR method was used to * determine sulfotransferase genotype. The SULT1A1 1 

allele was reported to have greater enzyme activity than * the SULT1A1 2 enzyme and it was expected that * individuals with the SULT1A1 1 allele would be able to 

rapidly eliminate bisphenol A. However, no significant 

differences in urinary bisphenol A concentrations were * observed between 57 individuals with the SULT1A1 1 

allele (geometric mean7SD 5 10.1078.71 mg/L) and 15 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 229 

* individuals with the SULT1A1 2 enzyme (6.3178.91 mg/ 

L). Adjustment for possible bisphenol A exposure 

through vinyl wrap use also did not result in significant 

differences between the 2 groups. The study authors 

concluded that additional enzymes involved in bisphenol A metabolism should be studied to determine 

possible sensitivity differences. 

One animal study demonstrated sex-related differences in sulfation. Male versus female Sprague–Dawley 

and F344 rats were found to produce higher amounts of a 

bisphenol A glucuronide/sulfate conjugate (Pritchett 

et al., 2002). 

As noted in Table 7, one human study reported B2­

fold higher blood bisphenol A concentrations in Japanese 

men than women (Takeuchi and Tsutsumi, 2002). Based 

on positive correlation between serum bisphenol A and 

testosterone concentrations, authors speculated that sexrelated differences in bisphenol A concentrations might 

be due to androgen-related metabolism (Takeuchi and 

Tsutsumi, 2002). There are no known human studies 

showing inter-individual or sex-related variations in 

metabolism that could lead to higher bisphenol A 

concentrations in blood. Experimental animal studies 

have not consistently demonstrated higher concentrations of bisphenol A or radioactive dose in one sex 

(Pottenger et al., 2000; Kurebayashi et al., 2005). In Wistar 

rats orally administered 1 mg bisphenol A every 2 days 

Table 60 

Human Toxicokinetic Values for Total Bisphenol A Dose 

Endpoint Value Reference 

Oral absorption, % Z84% Vo ̈lkel et al.(2002, 2005) 

Dermal absorption, in vitro, % B10% European-Union (2003) 

Tmax, min 80 Vo ̈lkel et al. (2002) 

Elimination half-life, hr 4–5.4 Vo ̈lkel et al. (2002, 2005) 

for 2 or 4 weeks, liver microsomal UDPGT activity 

toward 17b-estradiol and testosterone and expression of 

UGT2B1 protein and mRNA were reduced in males but 

not females (Shibata et al., 2002). One study reported an 

B3-fold higher concentration of blood bisphenol A in 

male than in female Wistar–Imamichi rats that were 

apparently not treated, but there were was no sex-related 

difference in percent glucuronidated bisphenol A in 

serum (Takeuchi et al., 2004b). However, in an in vitro 

study conducted with hepatic microsomes, glucuronidation of bisphenol A and expression of UGT2B1 mRNA 

were higher in microsomes from female than male rats. 

As described in more detail in Section 2.1.2.3, one study 

showed reduced capacity to glucuronidate bisphenol A 

in livers from pregnant than in non-pregnant rats (Inoue 

et al., 2004). 

2.6 Summary of General Toxicology and Biologic 

Effects 

Analytical considerations. Free concentrations of 

BPA measured in various matrices can be affected by 

analytic techniques and methodology. Free bisphenol A 

contamination from reagents and plastic ware may 

contribute to the measured free concentration of bisphenol A (Tsukioka et al., 2004; Vo ̈lkel et al., 2005). 

Analytical techniques employed may incorrectly overestimate the free concentration of measured bisphenol A. 

HPLC with ultraviolet, fluorescence, or electrochemical 

detection is unable to make definitive identification of 

bisphenol A or bisphenol A glucuronides, because 

similar retention times may occur for the metabolites of 

other endogenous and exogenous compounds (Vo ̈lkel 

et al., 2005). Bisphenol A glucuronide can also be 

hydrolyzed and in some cases degraded to unknown 

components either in acidic or basic pH solutions of 

diluted urine, adding another potential source of error in 

the measurement of sample levels of bisphenol A and its 

Table 61
 

Concentrations of Bisphenol A in Maternal and Fetal Samplesa
 

Bisphenol A concentrations, mg/L, median (range) or mean7SD 

Serum or plasma 

Study description; analytical 

method Maternal Fetal 

Other fetal 

compartments Reference 

21 samples collected in women in 0.5 (Non-detectable 

the U.S. before 20 weeks o0.5–1.96) 10% of 

gestation; LC with amniotic fluid 

electrochemical detection samples detectable 

37 German women, 32–41 weeks 3.1 (0.3–18.9); 4.473.9 2.3 (0.2–9.2); 2.972.5 12.7 (ng/g) (1.0–104.9) 

gestation; GC/MS 11.279.1) Placental 

tissue 

9 sets of maternal and umbilical 0.43 (0.21–0.79) 0.64 (0.45–0.76) 

cord blood samples obtained at 0.4670.2 0.6270.13 

birth in Japanese patients; 

HPLC 

180 Malaysian newborns; GC/MS Non-detectable 

(o0.05) to 4.05 88% 

of samples 

detectable 

Engel et al. (2006) 

Scho ̈nfelder et al. 

(2002b) 

Kuroda et al. (2003) 

Tan and Mohd (2003) 

a

As discussed in Section 1.1.5, ELISA may overestimate bisphenol A concentrations so only results from studies based on HPLC, GC/ 

MS, and LC/MS are presented. 

Birth Defects Research (Part B) 83:157–395, 2008
230 CHAPIN ET AL. 

Table 62 

Toxicokinetic Values for Bisphenol A in Non-Pregnant Animals 

Model Endpoint Value Reference 

Rats orally exposed to r100 mg/kg bw 

Ovariectomized, adult rats gavaged 

with bisphenol A at 10 and 100 mg/ 

kg bw 

Immature rats orally dosed with 

r10 mg/kg bw 

Monkeys orally dosed with r100 mg/ 

kg bw 

Chimpanzees orally dosed with 10 mg/ 

kg bw 

Rats s.c. dosed with r100 mg/kg bw 

Monkeys s.c. dosed with r100 mg/kg 

bw 

Chimpanzees s.c. dosed with 10 mg/kg 

bw 

Ovariectomized, adult rats orally 

dosed with bisphenol A at 10 and 

100 mg/kg bw 

Rats orally dosed with 10 mg/kg bw 

Rat 

Rats orally dosed with 10 mg/kg bw 

Rats orally dosed with 100 mg/kg bw 

Ovariectomized, adult rats orally 

dosed with (mg/kg bw): 

10 

100 

Oral doses (mg/kg bw) in immature 

rats at each age: 

1 (PND 4) 

10 (PND 4) 

1 (PND 7) 

10 (PND 7) 

1 (PND 21) 

10 (PND 21) 

Monkeys orally dosed with 10 and 

100 mg/kg bw 

Monkeys orally dosed with 10 mg/kg 

bw 

Rats s.c. dosed with 10 and 100 mg/kg 

bw 

Monkeys s.c. dosed with 10 and 

100 mg/kg bw 

Chimpanzees s.c. dosed with 10 mg/kg 

bw 

Oral doses (mg/kg bw) in immature 

rats at each age: 

1 (PND 4) 

10 (PND 4) 

1 (PND 7) 

10 (PND 7) 

10 (PND 21) 

Rats orally dosed with 10 mg/kg bw 

Rats orally dosed with 100 mg/kg bw 

Monkeys orally dosed with 10 and 

100 mg/kg bw 

Chimpanzees orally dosed with 10 mg/ 

kg bw 

Rats s.c. dosed with 10 and 100 mg/kg 

bw 

Tmax, hr 

Tmax1 /Tmax2, hr 

Tmax hr 

Tmax, hr 

Tmax, hr 

Tmax, hr 

Tmax, hr 

Tmax, hr 

Bioavailability, % 

Bioavailability, % 

Plasma protein 

binding, % 

Cmax, mg/L 

Cmax, mg/L 

Cmax1/Cmax2, mg/L 

Cmax (mg/L) 

Cmax, mg/L 

Cmax, mg/L 

Cmax, mg/L 

Cmax, mg/L 

Cmax, mg/L 

AUC, mg-hr/L 

AUC, mg-hr/L 

AUC0–24 h, mg-hr/L 

AUC0–24 h, mg-hr/L 

AUC0–24 h, mg-hr/L 

AUC0–24 h, mg-hr/L 

0.083–0.75 

0.5–1.5 / 3–6 

0.25–3 

0.7 

0.5 

1 

2 

2 

16.4 and 5.6a 

5.3 

90–95%. 

14.7–63 

580 

Range of values for 

males and females: 

30–60 

10,200–48,300 

40–80 

1100–1400 

5–6 

200 

2793 and 5732a 

96–325 

872 and 3439a 

57,934 and 10,851a 

1026–2058 

Range of values for 

males and females: 

85.6 

1353 

3247 and 52,595a 

813–1167 

3377 and 23,001a 

Domoradzk et al. (2004); Pottenger 

et al. (2000); Negishi et al. (2004b); 

Takahashi and Oishi (2000); Yoo et al. 

(2001) 

Upmeier et al. (2000) 

Domoradzk et al. (2004) 

Negeshi et al. (2004b) 

Negeshi et al. (2004b) 

Negeshi et al. (2004b) 

Negeshi et al. (2004b) 

Negeshi et al. (2004b) 

Upmeier et al. (2000) 

Yoo et al. (2001) 

Kurebayashi et al. (2003); Teeguarden 

et al. (2005) 

Domoradzk et al., (2004); Yoo et al. 

(2001) 

Negeshi et al. (2004b). 

Upmeier et al. (2000) 

30/40 

150/134 

Domoradzki et al. (2004) 

Negeshi et al. (2004b) 

Negeshi et al. (2004b) 

Negeshi et al. (2004b) 

Negeshi et al. (2004b) 

Negeshi et al. (2004b) 

Domoradzki et al. (2004) 

100–200 

7200–23,100 

100 

1700–1900 

1000–1100 

Yoo et al. (2001) 

Negeshi et al. (2004b). 

Negeshi et al. (2004b). 

Negeshi et al. (2004b). 

Negeshi et al. (2004b). 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 231 

Table 62
 

Continued
 

Model	 Endpoint Value Reference 

Monkeys s.c. dosed with 10 and AUC0–24 h, mg-hr/L 3247 and 39,040a Negeshi et al. (2004b). 

100 mg/kg bw 

Chimpanzees s.c. dosed with 10 mg/kg AUC0–24 h, mg-hr/L 12,492–21,141 Negeshi et al. (2004b). 

bw 

Rats orally dosed with 10 mg/kg bw Apparent volume of 4273 Yoo et al. (2001) 

distribution, L/kg 

Immature rats orally dosed with Half-life, hr 4.3–21.8 Domoradzki et al. (2004) 

r10 mg/kg bw 

Rats orally dosed with 10 mg/kg bw Terminal elimination 21.3 Yoo et al. (2001) 

half-life, hr 

Rats orally dosed with 10 mg/kg bw Oral clearance, mL/ 2352.1 Yoo et al. (2001) 

min/kg 

a

Results presented for low and high dose. 

Table 63 

Age and Route Factors Affecting Free Bisphenol A Concentrations in Blood 

Model and regimen	 Findings for free bisphenol A in blood Reference 

Age effects of rat oral dosing at 1 or 10 mg/kg: Domoradzki et al. (2004) 

4 days of age 1.5–56.8 mg/L 

7 days of age 1.1–12.2 mg/L 

21 days of age 0.8–8 mg/L 

Adulthood 0.07–0.6 mg/L 

S.C. or gavage dosing of 18–21-day-old rats with	 [93% lower] with oral than s.c. dosing 2.9 mg/L Yamasaki et al. (2000) 

160 mg/kg bw/day s.c. (plasma) 0.2 mg/L oral (plasma) 

Route effects in rats administered 10 or Pottenger et al. (2000) 

100 mg/kg bw: 

Oral [o2–8%] BLQ (males); 0.04 mg/L (females) 

(at 10 mg/kg) 

s.c.	 [65–76%] 0.69 (males); 0.87 mg/L (females)
 

(at 10 mg/kg)
 

i.p.	 [27–51%] 0.39 (males); 0.34 mg/L (females) 

(at 10 mg/kg) 

Route effects in monkeys: Percent of dose: Kurebayashi et al. (2002) 

i.v. 5–29
 

Oral 0–1
 

conjugates (Waechter et al., 2007). These considerations 

taken together, suggest that it is possible that free 

bisphenol A concentrations reported in biological samples may be overestimated. 

2.6.1 Toxicokinetics and metabolism. Human 

toxicokinetic data for bisphenol A are summarized in 

Table 60. In humans ingested bisphenol A is rapidly 

glucuronidated and circulated as bisphenol A glucuronide (Vo ̈lkel et al., 2002). There is no evidence of 

enterohepatic circulation (Vo ̈lkel et al., 2002). Most of the 

bisphenol A dose is excreted by humans through urine; 

bisphenol A recoveries in urine were reported at Z84% 

within 5 hr of dosing (Vo ̈lkel et al., 2005) and 100% within 

42 hr of dosing (Vo ̈lkel et al., 2002). Human urinary 

profiles were reported at B33–70% bisphenol A glucuronide, B10–33% parent compound, and B5–34% bisphenol A sulfate conjugate (Kim et al., 2003b; Ye et al., 

2005). The presence of bisphenol A in human fetal tissues 

or fluids demonstrates that bisphenol A is distributed to 

the human conceptus (Ikezuki et al., 2002; Scho ̈nfelder 

et al., 2002b; Yamada et al., 2002; Kuroda et al., 2003; Tan 

and Mohd, 2003; Engel et al., 2006) (Table 61). Results 

from a limited number of studies indicated that fetal 

bisphenol A concentrations are within the same order of 

magnitude as maternal blood concentrations (Scho ̈nfelder et al., 2002b; Kuroda et al., 2003) and amniotic fluid 

bisphenol A concentrations are B1 order of magnitude 

lower than maternal blood concentrations (Yamada et al., 

2002). Significantly higher mean bisphenol A concentrations were reported in the blood of male than female 

fetuses (3.572.7 vs. 1.771.5 ng/mL, P 5 0.016). Bisphenol A concentrations were measured in placenta samples 

at 1.0–104.9, median 12.7 mg/kg (Scho ̈nfelder et al., 

2002b). There were no differences between pregnant 

and non-pregnant blood levels (median in mg/L 0.44, 

range 5 0.22–0.87; mean1SD 5 0.4610.20) (Kuroda et al., 

2003). Median bisphenol A concentrations in human milk 

were reported to be r1.4 mg/L (Calafat et al., 2006; Ye 

et al., 2006). One of the studies reported a milk/serum 

ratio of 1.3 (Sun et al., 2004). 

Animal toxicokinetic data for bisphenol A are summarized in Table 62. Following oral intake of bisphenol A 

by rats, most of the dose (Z77%) is glucuronidated and 

circulated as bisphenol A glucuronide (Miyakoda et al., 

2000; Domoradzki et al., 2003; Kurebayashi et al., 2005). 

Most bisphenol A (90–95%) circulates bound to plasma 

proteins (Kurebayashi et al., 2003) [reviewed in (Teeguarden et al., 2005)]. In a single study in mice injected 

Birth Defects Research (Part B) 83:157–395, 2008
232 CHAPIN ET AL. 

Table 64 

Tissue Radioactivity in Pregnant and Fetal Rats After Oral Administration of 500 mg/kg 14C-Bisphenol A to Dams 

Radioactivity concentration (ng bisphenol A eq. g -1 or mL -1) 

2 days of gestation 15 days of gestation 18 days of gestation 

Dam and fetal tissues 30 mina 124 hr 30 mina 24 hr 30 mina 24 hr 

Dams 

Amniotic fluid 

Blood 

Ovary 

Placenta 

Uterus 

Fetus 

Fetal membrane 

Yolk sac 

ND 

43.32 

21.94 

15.43 

22.68 

NQ 

NQ 

NQ 

ND 

4.33 

3.96 

NQ 

ND 

NQ 

NQ 

ND 

NQ 

37.51 

13.91 

18.12 

NQ 

NQ 

NQ 

ND 

NQ 

3.83 

NQ 

NQ 

NQ 

NQ 

NQ 

ND 

NQ 

30.99 

15.67 

9.91 

15.31 

NQ 

NQ 

NQ 

NQ 

10.79 

3.49 

3.86 

NQ 

3.28 

10.87 

54.14 

a

Each time shows the sacrifice time after oral administration of 14C-bisphenol A to each pregnant rat. 

ND, not determined (indistinguishable); NQ, nonquantifiable (below LOQ). 

Table 65 

Summary of Elimination Information for Bisphenol A 

Elimination Dose eliminated Compound and metabolite 

Model route (%) profile Reference 

Pregnant or non-pregnant Feces 50–83 Bisphenol A (83–93%); Domoradzki et al. (2003); 

rats orally, i.p., or s.c. dosed Bisphenol A glucuronide Snyder et al. (2000); 

with o100 mg/kg bw (2–3%) Pottenger et al. (2000) 

Urine 13–42 Bisphenol A (3–23%); 

Bisphenol A glucuronide 

(57–87%); Bisphenol A 

sulfate (2–7%) 

Rats orally or i.v. dosed with Feces 64–88 Not reported Kurebayashi et al. (2003) 

0.1 mg/kg bw 

Urine 10–34 

Rats orally or i.v. dosed with Bile 45–66 within 5 hr Bisphenol A glucuronide (84– Kurebayashi et al. (2003) 

0.1 mg/kg bw 88%) 

Rats orally dosed with Feces Not reported Bisphenol A (61% of dose) Kurebayashi et al. (2003) 

100 mg/kg bw/day 

Urine Bisphenol A and bisphenol A 

sulfate (r1.1% of dose); 

Bisphenol A glucuronide 

(6.5% of the dose) 

Bile Bisphenol A glucuronide 

(41% of dose) 

Pregnant mice injected with Feces Not reported Bisphenol A (495%) Zalko et al. (2003) 

0.025 mg/kg bw bisphenol 

A 

Urine Major metabolites: bisphenol 

A glucuronide and 

hydroxylated bisphenol A 

glucuronide 

Bile Bisphenol A glucuronide 

(490%) 

Monkeys orally or i.v. dosed Feces 2–3 Not reported Kurebayashi et al. (2002) 

with 0.1 mg/kg bw 

Urine 79–86 

with a low dose (0.025 mg/kg bw), the most abundant 

compound in most tissues was bisphenol A glucuronide 

(Zalko et al., 2003). Most of a bisphenol A dose is 

circulated as the glucuronide in monkeys (Kurebayashi 

et al., 2002). As noted in Table 63, free bisphenol A in 

blood represents r8% of the dose in rats and r1% of the 

dose in monkeys following oral dosing; higher concentrations of free bisphenol A in blood were observed 

following parenteral dosing. The presence of 2 or more 

Cmax values for radioactivity or bisphenol A, an indication of enterohepatic circulation, was noted in some rat 

studies (Upmeier et al., 2000; Domoradzki et al., 2003; 

Kurebayashi et al., 2005). In rats, glucuronidation of 

bisphenol A was shown to occur in intestine (Sakamoto 

et al., 2002; Inoue et al., 2003a) and liver (Inoue, 2004). 

UGT2B1 was identified as a liver enzyme capable of 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 233 

glucuronidating bisphenol A, and possible involvement 

of other liver isoforms was noted (Yokota et al., 1999). 

There are some data indicating that glucuronidation 

capacity is very limited in fetuses and lower in immature 

than adult animals. Little-to-no UGT2B activity toward 

bisphenol A was detected in microsomes of rat fetuses; 

activity of the enzyme increased linearly following birth 

(Matsumoto et al., 2002). In an in vitro study comparing 

clearance of bisphenol A by hepatic microsomes from 

rats of different ages, activity was lower in microsomes 

from fetuses than in those from immature animals and 

adults [reviewed in (European-Union, 2003)]. As noted in 

Table 63, immature rats are capable of glucuronidating 

bisphenol A, but activity appears to increase with age. 

One study demonstrated that neonatal rats were able to 

glucuronidate a larger fraction of a lower (1 mg/kg bw) 

than higher (10 mg/kg bw) bisphenol A dose (Domoradzki et al., 2004). 

Kurbayashi et al. (2005) evaluated fetal and maternal 

rat bisphenol A during different stages of pregnancy. 

Bisphenol A labeled with carbon-14 was administered 

p.o. to male and female Fischer (F344) rats at relatively 

low doses (20, 100, and 500 mg/kg), and i.v. injected at 100 

and 500 mg/kg). 14C-bisphenol A (500 mg/kg) was also 

administered orally to pregnant and lactating rats to 

examine the transfer of radioactivity to fetuses, neonatal 

rats, and milk (Table 64). Radioluminographic determination using phosphor imaging plates was employed to 

achieve highly sensitive determination of radioactivity. 

Absorption ratios of radioactivity after three oral doses 

were high (35–82%); parent 14C- bisphenol A in the 

circulating blood was quite low, however, suggesting 

considerable first-pass effect. After an oral dose of 

100 mg/kg 14C- bisphenol A, the radioactivity was 

distributed and eliminated rapidly, but remained in the 

intestinal contents, liver, and kidney for 72 hr. The major 

metabolite in the plasma and urine was bisphenol A 

glucuronide, whereas most of the bisphenol A was 

excreted with the feces as free bisphenol A. A second 

peak in the time-course of plasma radioactivity suggested enterohepatic recirculation of bisphenol A glucuronide. There was limited distribution of 14C- bisphenol A 

to the fetus and neonate after oral administration to the 

dam. Significant radioactivity was not detected in fetuses 

on GD 12 and 15. On GD 18, however, radioactivity was 

detected in the fetal intestine and urinary bladder 24 hr 

after oral dosing of 14C- bisphenol A to the dam. The 

distribution pattern of radioactivity in pregnant rats was 

essentially the same as that in non-pregnant female rats. 

The distribution levels were dose-dependent in most of 

the tissues. There was limited distribution of 14Cbisphenol A to the fetus. Radioactivity in fetal tissues 

was undetectable except on GD 18 in the fetal urinary 

bladder and intestine. On GD 18, the amount of radioactivity in fetal tissues at 24 hr was about 30% that in 

maternal blood, and the yolk sac contained a much 

higher level of radioactivity than the maternal blood. The 

Expert Panel thought these differences were a consequence of the routes of administration, i.v. or p.o., 

because only trace amounts of parent bisphenol A dosed 

orally appeared in the plasma. 

The major metabolite of bisphenol A is the glucuronide 

conjugate. Another metabolite that has been commonly 

detected in urine is bisphenol A sulfate. Excretion 

patterns for bisphenol A are summarized in Table 65. 

As noted in Table 65, the major elimination routes 

for bisphenol A in rodents are feces and bile; a 

lower percentage of the dose is eliminated through 

urine. The major compound detected in feces is 

bisphenol A and the major compound detected in bile 

and urine is bisphenol A glucuronide. Excretion 

patterns and metabolic profiles observed in rodents 

dosed orally or parenterally with low (o1 mg/kg bw/ 

Table 67
 

Toxicokinetic Values for Radioactive Dose in Pregnant
 

Rats (Total Bisphenol A)a
 

Endpoint Value 

Cmax1/ Cmax2, mg eq/L 370–1006/211–336 

Tmax1/ Tmax2, hr 0.25/12–24 

Time to non-quantifiable concentration, hr 72–96 

AUC 14C, mg –eq � hr/L 7100–12,400 

AUC Bisphenol A glucuronide, 6800–12,300 

mg –eq � hr/L 

a

Dormoradzki et al. (2003).
 

Dams were gavaged with 10 mg/kg bw/day on GD 6–10, 14–18,
 

or 17–21.
 

GD, gestation day.
 

Table 66
 

Toxicokinetic Values for Free Bisphenol A in Pregnant Rats and Fetuses
 

Dose Endpoint Maternal Fetal Reference 

1000 mg/kg bw orally on GD 18 Cmax, mg/L 14,700 9220 Takahashi and Oishi (2000) 

10 mg/kg bw orally on GD 19 Concentration 1-hr post-dosing, mg/L 34 11 Miyakoda et al. (1999) 

2 mg/kg bw i.v. on 1 day between GD 17–19 Cmax, mg/L 927.3 794 Shin et al. (2002) 

1000 mg/kg bw orally on GD 18 Tmax, min 20 20 Takahashi and Oishi (2000) 

2 mg/kg bw i.v. on 1 day between GD 17–19 Tmax, hr No data 0.670.3 Shin et al. (2002) 

1000 mg/kg bw orally on GD 18 AUC, mg � hr/L 13,100 22,600 Takahashi and Oishi (2000) 

2 mg/kg bw i.v. on 1 day between GD 17–19 AUC, mg � hr/L 905.5 1964.7 Shin et al. (2002) 

1000 mg/kg bw orally on GD 18 Mean retention time, hr 10.6 20.0 Takahashi and Oishi (2000) 

1000 mg/kg bw orally on GD 18 Variance in retention time, hr2 203 419 Takahashi and Oishi (2000) 

2 mg/kg bw i.v. on 1 day between GD 17–19 Mean residence time, hr 3.0 3.0 Shin et al. (2002) 

1000 mg/kg bw orally on GD 18 Half-life, hr: Takahashi and Oishi (2000) 

From 20–40 min 0.0952 0.55 

From 40 min–6 hr 2.58 1.60 

From 6–48 hr 4.65 173 

2 mg/kg bw i.v. on 1 day between GD 17–19 Elimination half-life, hr 2.5 2.2 Shin et al. (2002) 

Birth Defects Research (Part B) 83:157–395, 2008
234 CHAPIN ET AL. 

Table 68 

Toxicokinetic Values for Free Bisphenol A in Lactating 

Ratsa 

Endpoint Blood value Milk value 

Systemic clearance, 119.2/142.4/154.1b 

mL/min/kg 

Steady state 66.1/120.0/217.1b 173.1/317.4/493.9b 

bisphenol 

A concentration, 

ng/mL 

Milk/serum ratio 2.7/2.6/2.4b 

Rats were i.v. injected 0.47, 0.94, or 1.88 mg/kg bw and then
 

infused over a 4-hr time period with 0.13, 0.27, 0.54 mg/hr.
 

a

Yoo et al. (2001).
 

b

Effect at each dose, from low to high dose.
 

Table 69
 

Toxicokinetic Values for Radioactive Dose in Lactating
 

Rats (Total Bisphenol A)a
 

Endpoint Blood value Milk value 

Cmax, mg – eq/L 27.2 4.46 

Tmax, hr 4 8 

Elimination half-life, hr 31 26 

AUC (0–48 hr), mg – eq � hr/L) 689 156 

a

Kurebayashi et al. (2005).
 

Rats were orally dosed with 0.5 mg/kg bw on PND 11.
 

day) or high-doses (10–100 mg/kg bw/day) were similar. 

In contrast to rodents and similar to humans, most of the 

dose in orally- or i.v.-exposed monkeys was eliminated 

through urine. 

Toxicokinetics of bisphenol A were examined in 

pregnant rats and are summarized in Table 66 for free 

bisphenol A and Table 67 for total dose. One study 

demonstrated similar disposition, metabolism, and elimination of bisphenol A in pregnant and non-pregnant rats 

(Domoradzki et al., 2003). A number of rodent studies 

demonstrated distribution of bisphenol A or radioactive 

dose to fetuses following oral dosing of the dam 

(Miyakoda et al., 1999; Takahashi and Oishi, 2000; 

Domoradzki et al., 2003; Kim and Hwang, 2003; Kabuto 

et al., 2004; Kurebayashi et al., 2005). Bisphenol A 

distribution to fetus was also demonstrated with i.v. 

dosing of rats (Shin et al., 2002) and s.c. dosing of mice or 

monkeys (Uchida et al., 2002; Zalko et al., 2003). In a 

study in which bisphenol A was orally administered to 

rats on GD 19, bisphenol A glucuronide was not detected 

in fetuses (Miyakoda et al., 2000); study authors noted 

the possibilities that bisphenol A glucuronide was not 

likely transferred from dams to fetuses and that fetuses 

do not likely possess glucuronidation ability. Some of the 

studies demonstrated slower elimination of bisphenol A 

from fetuses than maternal blood following oral dosing 

(Miyakoda et al., 1999; Takahashi and Oishi, 2000). 

Toxicokinetics data in lactating rats are summarized in 

Table 68 for free bisphenol A and Table 69 for total dose. 

Distribution of bisphenol A to milk and/or nursing pups 

was demonstrated in rodent studies with oral or i.v. 

exposures (Snyder et al., 2000; Yoo et al., 2001; 

Kurebayashi et al., 2005). One study reported that most of 

the bisphenol A dose is present as bisphenol A glucuronide 

in milk of lactating rats (Snyder et al., 2000). In a study that 

compared bisphenol A concentrations in maternal serum, 

milk, and offspring after rat dams were administered low 

oral doses (0.006 or 6 mg/kg bw/day), a significant 

increase in bisphenol A concentration was only observed 

in the serum of dams from the high-dose group on PND 21; 

no increase was observed in milk or pups (Yoshida et al., 

2004). Another study demonstrated higher concentrations 

of bisphenol A in milk compared to maternal serum after 

i.v. dosing of rat dams (Yoo et al., 2001). 

A number of in vitro studies compared bisphenol A 

metabolic velocity rates in microsomes or hepatocytes 

from rodents and humans. Generally, faster rates were 

demonstrated by rodent than human hepatocytes and 

microsomes (Elsby et al., 2001; Pritchett et al., 2002) 

[reviewed in (European-Union, 2003)]. One of the studies 

noted that adjustment for total hepatocyte number in 

vivo resulted in higher predicted rates for humans than 

rodents (Pritchett et al., 2002). The European Union 

(2003) noted that the interpretation of such studies 

should included knowledge about in vivo conditions 

such as varying metabolic capacity of hepatic cells, 

relationship of hepatic size to body size, and possibly 

important physiological endpoints such as blood flow. 

2.6.2 General toxicity. Gross signs of toxicity 

observed in rats acutely exposed to bisphenol A included 

pale livers and gastrointestinal hemorrhage [reviewed by 

the (European-Union, 2003)]. Acute effects of inhalation 

exposure in rats included transient and slight inflammation of nasal epithelium and ulceration of the oronasal 

duct. Based on LD50 observed in animals, the European 

Union (2003) concluded that bisphenol A is of low acute 

toxicity through all exposure routes relevant to humans. 

According to the European Union (2003), there is evidence 

that bisphenol A is irritating and damaging to the eye and 

is irritating to the respiratory tract and possibly the skin. 

Findings regarding sensitization potential were not clear. 

Possible target organs or systems of toxicity identified 

in repeat-dose animal studies with oral dosing included 

intestine, liver, kidney, and male, and female reproductive 

systems [reviewed in (NTP, 1982; Yamasaki et al., 2002a; 

European-Union, 2003)]. Intestinal findings (effect levels) 

in rats included cecal enlargement (Z25 mg/kg bw/day) 

and cecal mucosal hyperplasia (Z200 mg/kg bw/day). 

Hepatic effects included prominent hepatocyte nuclei or 

inflammation in rats (Z500 mg/kg bw/day), multinucleated giant hepatocytes in mice (Z120 mg/kg bw/day), 

and increased weight with no evidence of histopathology 

in dogs (Z270 mg/kg bw/day). Renal tubule degeneration or necrosis was observed in rats dosed with 

Z500 mg/kg bw/day. Reproductive findings are discussed in Section 4.0. Effects in subchronic inhalation 

studies in rats included cecal enlargement resulting from 

distention by food and transient, slight hyperplasia and 

inflammation of epithelium in the anterior nasal cavity; 

both effects occurred at (Z50 mg/m3

). 

2.6.3 Estrogenicity. Estrogenicity of bisphenol A 

has been evaluated using in vitro (Table 52) and in vivo 

(Table 53) assays. In those studies estrogenic potency was 

compared to 17b-estradiol, ethinyl estradiol, diethylstilbestrol, and, in one study, estrone. There is considerable 

variability in the results of these studies with the estrogenic 

potency of bisphenol A ranging over about 8 orders of 

magnitude (Fig. 2). On the other hand, the average potency 

only differs by 1 order of magnitude and there is very little 

difference between rat and mouse means. 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 235 

Most in vivo estrogenicity studies examined effects on 

uterine weights of intact weanling or ovariectomized 

adult rats or mice. The potency of bisphenol A in 

increasing uterine weight varied over B4 orders of 

magnitude. Uterine weight findings can be affected by 

the time period between dosing and measurement. Most, 

but not all studies, showed a greater effect on uterine 

weight with s.c. than with oral dosing. The greater 

activity of s.c. than oral bisphenol A is presumably due to 

glucuronidation of the orally administered compound 

with consequent loss of estrogenicity (Matthews et al., 

2001). Inter-strain variability in rats has been evaluated 

as a source of variability in estrogenicity assays. (see 

Section 4.0 for additional discussion) Inter-laboratory 

variability has been noted for uterotropic effects in intact 

weanling mice exposed to bisphenol A (Tinwell and 

Joiner, 2000); one factor that can result in variability is 

body weight of the animal. Use of mice with lower body 

weights results in lower and less variable control uterine 

weights and greater likelihood of bisphenol A effect 

(Tinwell and Joiner, 2000; Ashby et al., 2004). In in vivo 

studies examining gene expression profiles, some but not 

all gene expression changes were consistent between 

bisphenol A and reference estrogens (Tinwell and Joiner, 

2000; Naciff et al., 2002; Singleton et al., 2004; Terasaka 

et al., 2006); ER-independent activity was suggested by 

1investigator (Singleton et al., 2004). [Based on one 

comprehensive study of the effects of bisphenol A 

orally delivered from 60–1000 mg/kg for 3–7 days, the 

Expert Panel concludes that the uterotrophic responses 

were only found at higher does (Ashby, 2002; Kanno 

et al., 2003a) whereas s.c. dosing produced consistent 

uterine weight increases at lower doses.] 

2.6.4 Androgenic activity. In the majority of in 

vitro tests conducted, bisphenol A was not demonstrated 

to have androgenic activity (Sohoni and Sumpter, 1998; 

Gaido et al., 2000; Kitamura et al., 2005; Xu et al., 2005). 

Anti-androgenic activity was demonstrated in systems 

using cells transfected with three different androgen 

receptor reporting systems (ARE-luc, MMTV-lacZ, and 

C3-luc) (Table 56). No consistent effects were observed 

on male accessory reproductive organ weights in 3 in 

vivo studies in which rats were dosed with bisphenol A 

at r600 mg/kg bw/day; the study authors concluded 

that bisphenol A does not have anti-androgenic or 

androgenic activity (Kim et al., 2002a; Yamasaki et al., 

2003; Nishino et al., 2006). 

2.6.5 Genetic toxicity. In in vitro genetic toxicity 

studies reviewed by the European Union (2003) and/or 

Haighton et al. (2002), evidence of aneugenic potential, 

chromosomal aberration, micronuclei formation, and 

DNA adducts was observed (Table 57). Because of the 

lack of chromosomal effects in in vivo studies (Table 58) 

and unknown relevance of DNA adduct formation, 

which only occurred at high-doses, both groups concluded that bisphenol A is not likely to have genotoxic 

activity in vivo. 

2.6.6 Carcinogenicity. Carcinogenic potential of 

bisphenol A was evaluated in rats and mice by the NTP 

(1982) and Huff (2001). NTP concluded that under the 

conditions of the study, there was no convincing 

evidence that bisphenol A was carcinogenic in F344 rats 

or B6C3F1 mice. However, NTP stated that there was 

suggestive evidence of increased cancer in the hematopoietic system based on marginally significant increases 

in leukemia in male rats, non-statistically significant 

increases in leukemia in female rats, and a marginally 

significant increase in combined incidence of lymphoma 

and leukemia in male mice. A statistically significant 

increase in testicular interstitial cell tumors in aging F344 

rats was also considered suggestive evidence of carcinogenesis. The effect was not considered conclusive 

evidence because of the high incidence of the testicular 

neoplasm in aging F344 rats (88% incidence in historical 

controls). Both the European Union (2003) and Haighton 

et al. (2002) stated that the evidence does not suggest 

carcinogenic activity of bisphenol A in rats or mice. 

Conclusions by the European Union (2003) and Haighton 

et al. (2002) were based on factors such as lack of 

statistical significance for leukemia, mammary gland 

fibroadenoma, and Leydig cell tumors, lack of activity at 

noncytotoxic concentrations in both in vitro genetic 

toxicity tests and an in vivo mouse micronucleus test, 

and unlikely formation of reactive intermediates at doses 

that do not saturate detoxification pathways. 

2.6.7 Potentially sensitive subpopulations. Studies in humans and laboratory animals demonstrated 

developmental changes in UDPGT gene expression or 

enzyme activity that could potentially affect the concentration of free bisphenol A reaching target organs because 

of a differential capacity for bisphenol A glucuronidation. 

In humans, activities for some UDPGT isozymes were 

reported to be very low at birth but increased with age 

(Coughtrie et al., 1988). No transcripts for UDPGT were 

detected in samples from 20-week-old human fetuses and 

activity for some UDPGT enzymes was lower in children 

than adults (Strassburg et al., 2002). Compared to adults, 

human fetal uridine 50

-diphosphoglucuronic acid concentrations were 5-fold lower in liver and 1.5-fold lower in 

kidney (Cappiello et al., 2000). It is not clear if any of the 

isozymes examined are involved in bisphenol A glucuronidation by humans. Human findings were consistent 

with rodent studies that demonstrated no or limited 

glucuronidation capacity by fetuses (Miyakoda et al., 

2000; Matsumoto et al., 2002; Domoradzki et al., 2003) 

and lower glucuronidation capacity in immature than 

adult rats (Matsumoto et al., 2002; European-Union, 2003; 

Matsumoto et al., 2004). 

Some studies suggested possible gender-related differences in sulfation capacity in humans (Pritchett et al., 

2002; Kim et al., 2003b) and laboratory animals (Pritchett 

et al., 2002). One study in humans demonstrated no 

differences in urinary bisphenol A concentrations in 

individuals carrying a sulfotransferase genotype associated with greater activity (Yang et al., 2003). 

3.0 DEVELOPMENTAL TOXICITY DATA 

The Panel attended to multiple design and analysis 

characteristics in judging the acceptability of individual 

studies. It was our consensus that for a study to be 

acceptable for this review process, several conditions had 

to be met. First, effects related to litter of origin needed to 

be accounted for in design and statistical procedures. 

Second, animals needed to be dosed via the dam or 

directly under individual housing conditions. Concern 

that multiple exposures within a cage to different 

animals could cause cross-animal contamination across 

cage-mates led to the determination that this design was 

not acceptable. Third, a minimum of 6 animals per 

Birth Defects Research (Part B) 83:157–395, 2008
236 CHAPIN ET AL. 

treatment condition needed to be used to provide 

minimal confidence in results. Fourth, if similar tests 

were conducted at multiple ages, the statistical analyses 

needed to account for repeated measurement in order not 

to inflate degrees of freedom. The Panel carefullyconsidered the merits of each study according to these 

primary criteria, and the related design characteristics 

represent the most common reasons for judging a study 

to be unacceptable for our review process. Our intent was 

to have our review depend most heavily on studies that 

would have reduced risks for false negative or false 

positive findings. 

In addition, the Panel carefully considered the value of 

studies where bisphenol A was administered anywhere 

other than to the mouth or stomach of the experimental 

animal. Human exposure is overwhelmingly oral, and 

oral exposure produces an internal metabolite profile 

which is overwhelmingly dominated by the (inactive) 

glucuronide in both rats and humans. Subcutaneous or 

parenteral injections result in blood levels of active 

parent compound which are much higher than those 

seen after oral exposure. In light of these pharmacokinetic differences, the Panel concluded that injection 

studies, unless they proved otherwise, would produce 

irrelevantly high internal doses of the active parent 

compound, and would tend to produce ‘‘false positive’’ 

effects from the point of view of the human oral 

situation. Thus, the Panel viewed those otherwise 

adequate studies that injected bisphenol A as providing 

‘‘supplemental’’ information (i.e., of limited utility), 

unless they also analyzed the levels of parent compound 

and metabolites after the injection. The intent of this 

approach is limit the impact of those studies which 

produced an unrealistic and irrelevant internal metabolite profile (i.e., one which is significantly different from 

that experienced by humans). Thus, the closer any given 

study came to replicating the human situation, the more 

weight it had in the final analysis. 

The report below mentions ‘‘dosing procedures’’ as 

reasons for limiting the adequacy or utility of various 

studies. This has been used to mean non-gastric administration (s.c. injection, intramuscular [i.m.] injection, i.p. 

injection, or intracerebroventricular injection). 

The Panel also had extensive discussion about dosing 

vehicles. Dimethyl sulfoxide (DMSO) has significant 

biological activities of its own (Santos et al., 2003), and 

the experience of the Panel is that DMSO can help move 

solutes into cells. Increasing the DMSO concentration can 

produce a greater solute effect, even when holding that 

solute concentration stable. The real impact of this for in 

vivo injections is uncertain, and this effect is likely to be 

small at the dosing volumes administered in the studies 

considered here. The use of 100% DMSO as a vehicle for 

ALZET mini-pump studies is a clear contravention of the 

directions for mini-pump use3

, as it accelerates the 

3

Manufacturer instructions specify use of up to 50% DMSO (http://

www.alzet.com/products/checklist.php). One hundred percent DMSO is

completely incompatible with the pump reservoir material and will

dissolve reservoirs within 24–36 hr. Eighty percent DMSO causes pinholes

to appear in the reservoirs within 4–7 days. Thus, if a high concentration

of DMSO is used, one most likely will infuse both degraded reservoir

material as well as the salt compound which makes up the osmotic layer.

These two things, combined with DMSO itself (a strong tissue irritant) will

most likely cause tissue inflammation and edema (Kurt Kemling ALZET

Associate Product Manager, personal communication, September 14,

2007).

breakdown of the mini-pumps and produces blood 

levels that are not predictable and therefore not useful 

for the Evaluative Process. Various oils each can bring 

their own potential issues, such as oxidative damage, but 

these were considered and discussed by a sub-team of 

the Panel and not considered to be consequential for this 

analysis 

The Panel also examined the issue of data that would 

be expected to result when positive controls were 

employed. While we did not feel that positive controls 

were required for studies, when they were used, 

expected effects needed to be demonstrated to validate 

that the experimental model was capable of responding 

to a certain stimulus. This is of even more value when 

there is no response to the main exposure under study. 

When looking for estrogenic responses, investigators 

often use 17b estradiol or diethylstilbestrol. These must 

be used at adequate doses to produce the desired 

response. Inadequate challenge by the positive control, 

resulting in no response, leaves the reader uncertain 

whether the lack of response is due to the selection of too 

low a dose, or whether the experimental model is 

incapable of responding to a sufficient challenge. Even 

though the Panel, based on its own scientific experience, 

might conclude that inappropriately low doses had been 

selected and thus a lack of response is not surprising, the 

Panel was left with little choice in such situations but to 

give much less weight to studies where non-effective 

doses of a positive control compound were used. 

The Panel is confident in our assessment of those 

studies judged adequate and useful, and are focusing our 

limited time on the consistency and utilization of these 

data. 

3.1 Human 

No studies were located on possible human developmental effects of bisphenol A. 

3.2 Experimental Animal 

Studies are presented by species (rat, mouse, other), 

route (oral, parenteral), and by whether exposure was 

during pregnancy or the postnatal period. Studies in 

which exposures were started during pregnancy and 

continued after pregnancy are discussed with studies in 

which exposures occurred postnatally. 

3.2.1 Rat—oral exposure only during pregnancy. 

3.2.1.1 Evaluation of pre- or perinatal growth and 

development: Morrissey et al. (1987), supported by 

NTP/NCTR, examined the effects of prenatal bisphenol 

A exposure in rats and mice in a study conducted 

according to GLP. Studies are also available as NTP 

publications for rats (NTP, 1985c) and mice (NTP, 1985b). 

The study was conducted in two sets of rats and mice, 

and data were pooled for each species. [The data for 

mice are discussed in Section 3.2.5.1.] Pregnant CD rats 

were randomly assigned to groups of Z10 animals in 

each set of the study, for a total of Z20 animals/dose. On 

GD 6–15 (GD 0 5 sperm or plug), rats were gavaged with 

bisphenol A at 0 (corn oil vehicle), 160, 320, 640, or 

1280 mg/kg bw/day. Doses were based on results of 

preliminary studies and were expected to result in 10% 

maternal mortality at the high-dose and no toxicity at the 

low dose. Purity of bisphenol A was 495% and 2,40

­

bisphenol A was reported as an impurity. Dosing 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 237 

Table 70 

Maternal and Developmental Effects in Rats Exposed to Bisphenol Aa 

Dose, mg/kg bw/day 

Endpoint 100 300 1000 BMD10 BMDL10 BMD1SD BMDL1SD 

Dams 

No. pregnant 

Body weight gain 

Corrected body weight 

Food intake on GD 4 

No. fetal deaths 

No. early resorptions 

Post-implantation losses 

Fetuses 

No. live /litter 

Male body weight 

Female body weight 

Ossification 

2

2

2

2

2

2

2

2

2

2

2

2

k35% 

k14% 

k24% 

2

2

2

2

k14% 

2

2

k30% 

k52% 

k15% 

k57% 

m6.5-fold 

m6-fold 

m11-fold 

k36% 

k20% 

k21% 

k

178 

631 

168 

827 

821 

1278 

929 

456 

439 

152 

490 

147 

13 

14 

394 

348 

339 

328 

379 

566 

313 

978 

980 

982 

694 

682 

304 

424 

257 

585 

584 

713 

497 

490 

a

Kim et al. (2001b).
 

m,k Statistically significant increase, decrease compared to controls; 2 No statistically significant effect compared to controls.
 

solution concentrations were verified. Pregnant animals 

were weighed during the study. Rats were killed on GD 

20. Liver and uterus were weighed, and corpora lutea 

and implantation sites were examined. Fetuses were 

sexed, weighed, and examined for viability and external, 

visceral, and skeletal malformations. Data were analyzed 

by Bartlett test for homogeneity of variance, ANOVA 

and/or William multiple comparison, Dunnett, or Fisher 

exact probability tests. [Data were presented and 

analyzed on a per litter basis.] 

An unexpectedly high number of dams (7 of 27) died 

in the 1280 mg/kg bw/day group, with most deaths 

occurring in the second set of animals. Because of the 

high death rate, the study authors decided not to 

evaluate data in the 1280 mg/kg bw/day group. Clinical 

signs that occurred most frequently in dams from the 

640 mg/kg bw/day group included lethargy, piloerection, pica, rough coat, wet urogenital area, weight loss, 

and alopecia. Significant and dose-related decreases in 

maternal body weights were observed during the entire 

gestation period and thus were not confined to the GD 6– 

15 treatment period in rats from the 160, 320, and 

640 mg/kg bw/day groups. Body weight corrected for 

gravid uterine weight was also decreased in all three 

dose groups. Effects on maternal body weight were most 

pronounced during the treatment period. [During the 

treatment period, dam body weights were 35, 53, and 

54% lower in the 160, 320, and 640 mg/kg bw/day 

groups than in control groups; estimated benchmark 

doses4 in mg/kg bw/day were BMD10 113, BMDL10 94, 

4

Benchmark doses are used commonly in a regulatory setting; however,

they are used in this study when the underlying data permit their

calculation, and are only supplied to provide one kind of description of

the dose–response relationship in the underlying study. Calculation of a

benchmark dose in this report does not mean that regulation based on the

underlying data is recommended, or even that the underlying data are

suitable for regulatory decision-making. The BMD10 is the benchmark

dose associated with a 10% effect, estimated from a curve fit to the

experimental data. The BMDL10 represents the dose associated with the

lower 95% confidence interval around this estimate. Unless otherwise

indicated, BMD values in this report were calculated using a power model

for continuous data and a probit model for dichotomous data using

Environmental Protection Agency (EPA) Benchmark Dose Software

version 1.3.2.

BMD1SD 416, BMDL1SD 321.] Despite this large effect on 

maternal body weight, there were no effects on numbers 

of implantation sites or resorptions, gravid uterine 

weight, or liver weight. The numbers of litters available 

for evaluation in the control and 160, 320, and 640 mg/kg 

bw/day dose group were 23, 26, 24, or 29. There were no 

significant effects on fetal body weight or viability, 

percentage males/litter, or malformed fetuses/litter. 

Study authors concluded that bisphenol A was not 

teratogenic in rats at doses that cause maternal toxicity. 

Strengths/Weaknesses: This study used adequate 

sample sizes to evaluate the effects of GD 6–15 exposure 

on maternal body weight during gestation and on 

implantation and resorption sites/dam, fetal body 

weight, and fetal viability to GD 20. Strengths are the 

verification of dosing solutions, use of GLP, adequate n, 

sensitive evaluation of soft and hard-tissue structures. 

Weaknesses include no postnatal examination, as well as 

the absence of data from the 1280 mg/kg bw/day group, 

the absence of a no-effect dose. The absence of effects on 

fetal endpoints despite marked reductions in maternal 

body weight corrected for gravid uterine weight warrants the appropriate conclusion that bisphenol was not 

teratogenic when based on GD 20 data. Further, a gross 

visceral exam is likely insensitive to certain abnormalities 

of the reproductive tract and brain, as noted above. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

Kim et al. (2001b), support not indicated, examined 

the effects of prenatal bisphenol A exposure on developmental toxicity in rats. Sprague–Dawley rats were fed 

commercial rodent chow (Jeil Feed Co., Daejon, Korea) 

and housed in polycarbonate cages; no information was 

provided about bedding. Twenty dams/group were 

gavaged with 0 (corn oil vehicle), 100, 300, or 1000 mg/ 

kg bw/day bisphenol A [purity not provided] on GD 1– 

20 (GD 0 5 first 24 hr after detection of vaginal sperm or 

plug). Dose selection was based on the results of a 

preliminary study that demonstrated maternal and 

developmental toxicity at doses Z400 mg/kg bw/day 

and a lack of effect at doses r200 mg/kg bw/day. 

Endpoints examined in dams during the study were 

Birth Defects Research (Part B) 83:157–395, 2008
238 CHAPIN ET AL. 

clinical signs, body weight gain, and food intake. Dams 

were killed on GD 21 and examined for corpora lutea and 

implantation sites. Fetuses were sexed, weighed, and 

examined for viability and external abnormalities. 

Anogenital distance was measured and alternate fetuses 

were examined for visceral and skeletal malformations. 

The dam or litter was considered the statistical unit. Data 

were analyzed by ANOVA, Scheffe ́ multiple comparison 

test, Kruskal–Wallis nonparametric ANOVA, Mann– 

Whitney U-test, and Fisher exact probability test. 

Statistically significant effects are summarized in 

Table 70. Dose-dependent clinical signs observed in 

dams at the 2 highest doses included piloerection, dull 

fur, reduced locomotor activity, emaciation, sedation, 

red-colored tears, soft stool, diarrhea, urination, and 

perineal soiling. Pregnancy failure, as observed by lack of 

implantation sites, was increased in females from the 

high-dose group. Maternal body weight, body weight 

gain, and body weight corrected for gravid uterus weight 

were reduced at the mid- and high-dose. GD 4 was the 

only time period when food intake was significantly 

reduced at the mid- and high-dose. Expansion and 

congestion of stomach and/or intestines were observed 

in dams from the high-dose group. Body weights of male 

fetuses were decreased at the mid- and high-dose, and 

body weights of female fetuses were reduced at the highdose. Increases in fetal death, early resorption, and postimplantation loss, accompanied by reduced number of 

live fetuses, were observed at the high-dose. Anogenital 

distance was significantly reduced in males from the 

mid- and high-dose groups, but there were no differences in anogenital distance of males or females when 

the values were normalized by the cube root of body 

weight. Significantly reduced ossification was observed 

in the high-dose group. There were no treatment-related 

differences in fetal sex ratio or external, visceral, or 

skeletal malformations. Study authors concluded that 

exposure of rats to a maternally toxic dose of bisphenol A 

during the entire gestation period resulted in pregnancy 

failure, post-implantation loss, reduced fetal body 

weight, and retarded fetal ossification but not 

dysmorphogenesis. 

Strengths/Weaknesses: This report presents a fairly 

standard embryo–fetal developmental toxicity study. 

One strength is that the doses utilized incorporated both 

a no-effect dose and a high maternally toxic dose, 

revealing fetal effects only at the high-dose that showed 

marked maternal toxicity. Measurement of anogenital 

distance is another strength. Weaknesses include the 

absence in all groups of information about postnatal 

viability, and postnatal function. Further, a gross visceral 

exam is likely insensitive to certain abnormalities of the 

reproductive tract and brain. However, this type of study 

does report on the ability of the exposure to cause 

structural malformations, which are notably absent. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

Kim et al. (2003), support not indicated, examined the 

effects of prenatal bisphenol A exposure on postnatal 

body and organ weights of Sprague–Dawley rats. Rats 

were housed in polycarbonate cages. [No information 

was provided on feed or bedding material.] Rats were 

grouped according to body weight and randomly 

assigned to dose groups. On GD 7–17 (GD 0 5 day of 

vaginal sperm or plug), at least 10 rats/dose group were 

gavaged with bisphenol A (499.7% purity) at doses of 0 

(corn oil vehicle), 0.002, 0.020, 0.200, 2, or 20 mg/kg bw/ 

day. Dosing solution concentrations were verified. Dams 

were weighed and observed for clinical signs of toxicity 

during the study. Dams were killed on Day 21 of the 

postpartum period. Corpora lutea, implantation sites, 

resorptions, and fetal viability were assessed. Maternal 

liver, kidney, spleen, ovary, and gravid uterus were 

weighed. Live fetuses were weighed and examined for 

external and visceral abnormalities. Fetal liver, kidneys, 

spleen, and reproductive organs were weighed in half 

the fetuses. [These methods are produced here as 

written in the original; although dams were clearly 

stated to have been killed on PND 21, the ‘‘fetal’’ 

examinations described appear more consistent with 

killing of the dams on GD 21.] Data were analyzed by 

ANOVA and Student t-test. [It was not clear if the litter 

or fetus was considered the statistical unit in the 

evaluation of developmental toxicity data.] 

A significant but non-dose-related increase in dam 

body weight occurred in the 0.2 mg/kg bw/day group 

on GD 0–15. Dam body weight was significantly 

increased on GD 21 in the 2 (by 53%) and 20 (by 43%) 

mg/kg bw/day groups. No significant differences in 

dam body weight were noted during the lactation period. 

Significant changes in dam relative organ weights (dose 

at which effects were observed) were: increased liver 

(0.002, 0.020, and 20 mg/kg bw/day); decreased right 

kidney (0.2 mg/kg bw/day); increased right kidney 

(2 mg/kg bw/day), and increased uterine (0.2 mg/kg 

bw/day). There was no effect on ovary weight of 

dams. The majority of dams were in diestrus when 

killed. One of 7 dams in the 0.2 mg/kg bw/day 

group was in proestrus. One of 7 dams in the 0.2 mg/ 

kg bw/day, 1 of 6 dams in the 2 mg/kg bw/day 

group, and 2 of 8 dams in the 20 mg/kg bw/day 

group were in diestrus. Body weight effects in male 

and female offspring were reported in most treatment 

groups when evaluated at various time points between 

birth and PND 22. In general, when body weights effects 

were detected it was an increase in weight of B12–65%. 

[Changes occurred at most dose levels but were not 

consistent over time and there was little evidence of 

dose–response relationships. In general, effects appeared to be most pronounced in the lowest dose 

group.] Relative weights for several tissues attained 

statistical significance at 1 or more doses in offspring of 

both sexes: liver, spleen and right kidney. In addition, 

relative organ weights for were altered in males for the 

left kidney, both testes, right epididymis, left seminal 

vesicle, and prostate gland. There were no effects on 

ovary or uterus weights. [In most cases, there was little 

evidence of a dose–response relationship for organ 

weights, including male reproductive organs, in offspring.] Study authors concluded that bisphenol A had 

estrogenic effects on rat dams and offspring exposed 

during the gestation period. 

Strengths/Weaknesses: While the verification of the 

dosing solution is a strength, this study is of unclear 

quality, to the point that there is real confusion about 

what was actually done. It is indicated that 10 dams were 

assigned to each dose group but numbers at sacrifice 

were 7, 7, 6, and 8 across the 4 doses. It is unclear 

whether fetal data were appropriately analyzed with 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 239 

litter as the unit. It is unclear when the dams were killed 

and analyzed. The absence of understandable doserelated effects complicates interpretation at these low 

doses; although the possibility of unusual low dose 

effects cannot be discounted. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for inclusion into the evaluation 

process, due to small sample size and poor documentation and communication about what was done. 

3.2.1.2 Evaluation of reproductive organ development: Talsness et al. (2000), supported by the German 

Federal Ministry for Environmental Protection and 

Radiation Security, examined the effect of prenatal 

bisphenol A exposure on the reproductive systems of 

male and female rats. [No information was provided 

about feed, caging, and bedding materials used.] On 

GD 6–21, Sprague–Dawley rats (n 5 18–20/group) were 

gavaged with 2% corn starch vehicle or bisphenol A 

[purity not indicated] at 0.1 or 50 mg/kg bw/day. A 

group of 11 dams was gavaged with 0.2 mg/kg bw/day 

ethinyl estradiol. Litters were weighed during the 

lactation period. Pups were weaned on PND 22 (according to Table 1 of the study, PND 1 was apparently the day 

of birth) and males and females were separated around 

PND 30. Vaginal opening was examined in 42–91 female 

offspring/group, and estrous cyclicity was monitored 

over a 3-week period in 42–53 females/group. At 4 

months of age, 5–10 females/group were killed during 

diestrus and 20 females/group were killed while in 

estrus. A histopathological evaluation of vaginal tissue 

was conducted in 5 animals [assumed 5/group]. In 44– 

112 male offspring/group, anogenital distance was 

measured on PND 3, 15, and 21 and days of testicular 

descent and preputial separation were recorded. Males 

were killed on PND 70 (n 5 20/group) or 170 (n 5 17–20/ 

group). Blood LH and testosterone concentrations were 

measured in 14–20 animals/group/time period. Sperm 

and spermatid numbers and sperm production and 

transit rates were determined in all offspring. Histopathological evaluation of the testis was conducted in 2 

animals [assumed/group]. Body, reproductive organ, and 

liver weights were measured in all male and female 

offspring killed. Data from female rats were analyzed by 

ANOVA with post-hoc Dunnett test or Fisher test. Data 

from male rats were analyzed by ANOVA and Dunnett 

test. [It appears that offspring were considered the 

statistical unit.] 

Pup body weights at birth were unaffected in the 

bisphenol A group, but on PND 22, pup body weights 

were lower [by 28%] in the low-dose group than in the 

control group. Study authors noted that the mean litter 

size in the low-dose group was larger by 2.6 pups than in 

the control group. Vaginal opening was delayed in the 

low-dose group and accelerated in the high-dose group. 

When estrous cyclicity data were evaluated according to 

total number of cycles, there was an increase in estrous 

phases lasting more than 1 day and prolongation of the 

cycle length in the high-dose group. Evaluation of 

estrous cycles by individual rat indicated a decrease in 

the percentage of low-dose females with 3 consecutive 1­

day estrus phases. The only terminal body and organ 

weight effects occurred in the low-dose group and 

included decreased absolute liver weight in females 

killed in estrus and decreased body and uterus weights 

in females killed in diestrus or in estrus. There were no 

effects on relative organ weights. Histological observations in vaginal tissue of bisphenol A-exposed rats 

included less pronounced cornification during estrus 

and more pronounced mucification during diestrus, with 

magnitude of effect greater in the low- than the highdose group. Observations in the animals exposed to 

ethinyl estradiol included decreased pup birth weight, 

delayed vaginal opening, near-persistent estrus, decreased absolute and relative uterus weights, and 

changes in vaginal histology similar to those described 

for the low-dose bisphenol A group. 

Decreased anogenital distances was observed in the 

bisphenol A groups during all three time periods for 

male offspring, but the effect remained statistically 

significant only in the high-dose group when normalized 

for body weight. Testicular descent and preputial 

separation were delayed in the low-dose group. Organ 

weight effects that remained significant following 

adjustment for body weight included increased prostate 

weight in the high-dose group on PND 70 and increased 

testicular and epididymal weights in the low-dose group 

on PND 170. There was no effect on sperm morphology. 

Blood testosterone concentration was decreased in the 

high-dose group on PND 70, and blood LH concentration 

was increased in the high-dose group on PND 170. 

Testicular histopathology observations in the low-dose 

group on PND 70 included cellular debris in lumens, 

pyknotic nuclei in spermatids, and apoptotic debris in 

the region of the spermatogonia and primary spermatocyte. In testes of 70-day-old animals of the high-dose 

group, there were central necrotic masses, low numbers 

of meiotic figures in spermatocytes, and low spermatozoa numbers. On PND 170, observations in testes from 

the low-dose group included low spermatozoa numbers, 

a thin layer of spermatocyte meiotic figures, and 

apoptotic debris in region of spermatids. Low spermatocyte meiotic figures were the only testicular observation in the high-group on PND 170. Effects observed in 

the ethinyl estradiol group included increased anogenital 

distance, delayed testicular descent, accelerated preputial 

separation, decreased testis and prostate weights, decreased sperm counts and production, increased LH 

concentrations, increased testosterone concentrations on 

PND 170, apoptotic debris, and/or low sperm numbers 

in testes. 

Study authors concluded that prenatal exposure to 

bisphenol A disrupts the reproductive systems of both 

male and female rats and that the effects do not occur 

according to a classic dose–response curve, which is 

generally observed in toxicology studies. 

Strengths/Weaknesses: Strengths are the postnatal 

evaluation of various endpoints to ‘‘pup’’ adulthood 

and that the concentration of the dosing solutions was 

verified. Based on the description of numbers of pups 

contributing to various endpoints, however, the authors 

do not appear to have used the litter as the unit of 

analysis. These inflated numbers subjected to analysis 

complicate the interpretation of findings, especially for 

PND 1–21 measures. A weakness also is that only 2 dose 

levels were examined. The vaginal opening data for the 

controls were outside the normal range for Sprague– 

Dawley rats. It is unclear how the estrous cycle data were 

analyzed. The F1 data were not analyzed correctly. Data 

may be suggestive of developmental disruptions at both 

doses, but the magnitudes are likely unreliable, and the 

Birth Defects Research (Part B) 83:157–395, 2008
240 CHAPIN ET AL. 

Table 71 

Benchmark Doses for Rat Reproductive Organ Endpoints 

Affected by Prenatal Bisphenol Aa 

Benchmark dose, mg/kg bw/day 

Endpoint BMD10 BMDL10 BMD1SD BMDL1SD 

Delayed vaginal opening 68 51 35 16 

Sperm count/testis 55 30 57 31 

Sperm count/g testis 81 41 68 34 

Daily sperm count/testis 56 31 59 31 

Daily sperm count/g testis 83 42 70 34 

a

Calculated from data in Tinwell et al. (2002). 

authors’ statements about dose–response peculiarities 

must be viewed with caution until more complete dose– 

response assessments are published. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process. 

Tinwell et al. (2002), support not indicated, examined 

the effects of in utero exposure to bisphenol A on sexual 

development of male rats. The study attempted to 

duplicate findings that were reported in several abstracts 

and as a full report (Talsness et al., 2000). Sprague– 

Dawley and Wistar-derived Alderley Park rats were 

housed in plastic-bottomed cages containing sawdust 

and shredded paper bedding. Rats were assigned to 

groups based on body weights and 6–7/group/strain 

were gavaged on GD 6–21 with bisphenol A (99% purity) 

at 0 (arachis oil vehicle), 0.020, 0.100, or 50 mg/kg bw/ 

day. A positive control group initially received 200 mg/kg 

bw/day ethinyl estradiol, but the dose was reduced to 

100 mg/kg bw/day between GD 11 and 14 due to 

maternal toxicity. Dosing solution concentrations and 

stability were verified. Dams were fed RM3 breeding diet 

(18.5% soybean protein; Special Diet Services, Ltd.) 

during gestation and lactation. At birth, pups were 

counted, sexed, and weighed. Anogenital distance was 

measured 24 hr following birth (PND 1). On PND 5, pups 

were culled to 8/litter, with equal numbers of males and 

females when possible. On PND 23, rats were weighed 

and housed according to sex. Following weaning, pups 

were fed RM1 feed (6.5% soybean protein). Pups were 

weighed throughout the post-lactation period. Ages at 

preputial separation, vaginal opening, and first estrus 

were assessed. Males were killed on PND 90–91 and 

females on PND 98. Liver and reproductive organs were 

weighed. Daily sperm production was determined. Data 

were analyzed using the litter and grouped individuals 

as the statistical unit. [Litter values are discussed 

below.] Data were analyzed by ANOVA, ANCOVA, 

and Dunnett test. 

The only significant effect observed in female rats 

exposed to bisphenol A was a 1.6-day delay in vaginal 

opening in Alderley-Park rats of the high-dose group. 

The study authors stated that effect on vaginal opening 

was correlated with body weight. [Data were not shown 

by study authors.] In Alderley Park males of the highdose group, significant reductions were observed for 

total sperm count/testis [12% lower than controls], 

sperm count/g testis [10% reduction], daily sperm 

count/testis [12% reduction], and daily sperm count/g 

testis [10% reduction]. Benchmark doses for the endpoints with statistically significant changes are shown in 

Table 71. In both strains, bisphenol A treatment had no 

effect on litter size, sex ratio, birth weight, anogenital 

distance, first day of estrus, or age of preputial 

separation. There were no significant effects on weights 

of liver, ovary, cervix, uterus, vagina, testis, epididymis, 

seminal vesicle, or prostate. Rats treated with ethinyl 

estradiol also experienced decreased sperm counts, in 

addition to decreased weights of male reproductive 

organs and advanced age of vaginal opening. Several 

findings (Talsness et al., 2000) were not duplicated in this 

study including: reduced anogenital distance; altered age 

of sexual maturation in males and females; variable 

changes in male reproductive organ weight, including 

prostate weight; and reduced sperm production at low 

doses. Study authors concluded that this study failed to 

confirm low-dose endocrine effects. 

Strengths/Weaknesses: Strengths of this study are the 

range and appropriateness of selected measures, the use 

of 2 strains of rat, the verification of dosing solutions, 

and the use of ethinyl estradiol, which produced 

expected responses. An unfortunate weakness is the 

small sample size of 6–7 dams/strain/group. Nevertheless, data were analyzed appropriately with the litter 

as the experimental unit, and significance judgments 

were apparently based on 7/group. Modest effects were 

noted in male and female offspring in the 50 mg/kg 

exposure group. While effects on the lowest doses in this 

study were not seen, it is important to recognize the 

effects seen at 50 mg/kg bw/day (the high-dose in this 

study) dosing on GD 6–21. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

Scho ̈nfelder et al. (2002a), supported by the German 

Federal Ministry for Education and Research, examined 

the effects of prenatal bisphenol A exposure on the rat 

vagina. Sprague–Dawley rats were gavaged on GD 6–21 

with bisphenol A at 0 [2% corn starch vehicle (Mondamin)], 0.1, or 50 mg/kg bw/day. A positive control 

group was treated with 0.2 mg/kg bw/day 17a-ethinyl 

estradiol in a peanut oil vehicle. [No information was 

provided on the number of dams treated, the day of 

vaginal plug, purity of bisphenol A, or the type of 

chow, bedding, and caging materials used.] [According 

to the author the number of litters treated were: 

Mondamin 5 20, 0.1 mg/kg bw/day bisphenol A 5 20, 

50 mg/kg bw/day bisphenol A 5 18, and 0.2 mg/kg bw/ 

day 17a ethinyl estradiol 5 11; day of sperm positive 

smear was considered to be GD 0 and was used instead 

of day of vaginal plug; purity of bisphenol A was 

Z98%; Altromin 1324 rodent chow was used (obtained 

from Altromin GmbH); bedding was wood shavings 

obtained from Altromin GmbH; caging was Type III 

macrolon cages (G. Scho ̈nfelder, personal communication, July 20, 2007).] At 3 months of age, estrous cyclicity 

was evaluated for 3 weeks in 42 female offspring of the 

control group, 21 offspring of the 0.1 mg/kg bw/day 

group, 18 offspring of the 50 mg/kg bw/day group, and 

24 offspring of the 17b-estradiol group. [The number of 

litters represented was not stated.] At 4 months of age, 

female offspring were killed in either estrus or diestrus. 

[Authored clarified that each estrus group contained 22 

offspring from 20 dams in the cornstarch group, 13 

offspring from 13 dams in the 0.1 mg/kg/d and 12 

offspring from 12 dams in the 50 mg/kg/d bisphenol A 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 241 

group, as well as 19 offspring from 11 dams in the 

0.2 mg/kg/d 17a-ethinyl estradiol group (G. Scho ̈nfelder, personal communication, July 20, 2007)]. [Exact 

litter representation for animals collected during 

diestrus was not provided.]Vaginas were fixed in Bouin 

solution and a histopathological evaluation was conducted. Western blot analyses were conducted to 

measure expression of ERa and ERb. [It does not appear 

that statistical evaluations were conducted.] 

Qualitative descriptions of vaginal histopathology 

changes and ER expression were provided by the study 

authors. Low-dose animals killed during the estrous 

stage lacked keratinization of the surface epithelium and 

demonstrated reduced thickness of the total epithelium. 

Similar but less pronounced effects were observed in rats 

of the high-dose bisphenol A group. Vaginal findings 

were similar in the positive control group, and slight 

desquamation of the superficial layers was also observed. There were no differences in vaginal histopathology findings in rats killed during the diestrous stage. No 

ERb was observed in vaginas of rats from any treatment 

group. Full-length ERa expression was not observed in 

either bisphenol A group during estrus, but ERa in the 

bisphenol A-exposed groups did not differ from the 

control group during the diestrous stage. ERa in vaginas 

obtained from the positive control group was either 

reduced or was not detected. The study authors 

concluded that altered vaginal morphology following 

bisphenol A treatment appears to be due to downregulation of ERa. 

Strengths/Weaknesses: Vaginal histopathology of female offspring is of interest but the quality of the study 

cannot be judged due to unclear methodology. Uncertainty about the numbers of animals, the number of 

offspring examined and the lack of statistical accounting 

for litter effects are significant weaknesses. 

Utility (Adequacy) of the CERHR Evaluation Process: 

This study is inadequate for the evaluation process for 

the reasons detailed above. 

Scho ̈nfelder et al. (2004), supported by the German 

Federal Ministry for Environmental Protection and 

Radiation Security, examined the effects of prenatal 

bisphenol A exposure on the rat uterus. [No information 

was provided about composition of feed, caging, or 

bedding.] Sprague–Dawley rats [number treated not 

specified] were gavaged with bisphenol A [purity not 

reported] at 0 (2% corn starch vehicle), 0.1, or 50 mg/kg 

bw/day on GD 6–21. [Author clarified that the purity of 

Table 72
 

Uterine Effects in Rats Exposed to Bisphenol A During
 

Prenatal Development
 

Dose, mg/kg bw/day 

Endpoint 0.1 50 

Thickness of luminal epithelium 

Epithelial nucleia 

Epithelial nuclei with 

condensed chromatin 

2

m69% 

m2.7-fold 

k38% 

m89% 

m3.1-fold 

Epithelial cells with cavities 

ERa positive cells in epithelium 

ERb-positive cells in uterine tissue 

m2.1-fold 

2

k88% 

m 1.9-fold 

m67% 

k88% 

a

It is unclear if authors were referring to numbers of nuclei. 

bisphenol A was Z98%; Altromin 1324 rodent chow 

was used (obtained from Altromin GmbH); bedding 

was wood shavings obtained from Altromin GmbH; 

caging was Type III macrolon cages (G. Scho ̈nfelder, 

personal communication, July 20, 2007).] The high 

bisphenol A dose was selected because it was reported 

to be the no observed effect level (NOEL) recommended 

by the Society of the Plastics Industry. A positive control 

group was gavaged with 0.2 mg/kg bw/day ethinyl 

estradiol on GD 6–21. Estrous cyclicity was examined for 

3 weeks in 6 female offspring/group beginning at 3 

months of age. Six female offspring/group were killed at 

4 months of age on the day of estrus. Body and 

reproductive organ weights were measured. Uteri were 

fixed in methacarn solution and sectioned. Examinations 

of uterine morphology were conducted. Immunohistochemistry techniques were used to detect ERa and ERb

in the uterus, and results were verified by Western blot. 

Data were analyzed by Mann–Whitney test. [It was not 

clear if data were analyzed on a per litter or per 

offspring basis.] [Author states that each female came 

from a different litter so the data were analyzed on a 

per litter basis (G. Scho ̈nfelder, personal communication, July 20, 2007).] Statistically significant findings are 

summarized in Table 72. Effects observed at both dose 

levels were increased epithelial cell nuclei, epithelial 

nuclei with condensed chromatin, and epithelial cells 

with cavities and reduced ERb-positive cells in uterine 

tissue. Additional effects observed only at the high-dose 

included decreased thickness of luminal epithelium and 

increased ERa-positive cells in the epithelium. Similar 

findings were observed following treatment with ethinyl 

estradiol. The study authors concluded that prenatal 

bisphenol A exposure causes uterine effects in rat 

offspring. 

Strengths/Weaknesses: A strength is the examination 

of effects on uterine indices in female offspring. A slight 

weakness is the use of only 6 females per group; 

however, the panel noted that the results appeared to 

be consistent across animals and across endpoints, 

especially in the 50 mg/kg bw/day treatment group. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

Wistuba et al. (2003), supported by the German 

Federal Ministry of Education and Science, examined 

the effects of prenatal exposure on testicular histology 

and sperm endpoints in rats. [No information was 

provided about chow, bedding, or caging.] Sprague– 

Dawley rats were gavaged with 0 (2% corn starch 

suspension vehicle), 0.1, or 50 mg/kg bw/day bisphenol 

A [purity not reported] on GD 6–21 (GD 0 5 day of 

sperm detection). A third group was treated with 

0.02 mg/kg bw/day ethinyl estradiol. The high-dose 

was said to correspond to the current accepted no 

observed adverse effect level (NOAEL) and the lower 

dose was selected to determine if effects occurred at 

lower doses. It appears that the number of dams treated 

was 2 in the control group, 4 in the low-dose group, 1 in 

the high-dose group, and 4 in the ethinyl estradiol group. 

Litters were weighed during the lactation period. Pups 

were weaned on PND 22 [day of birth not defined]. 

Male offspring were killed between the ages of B9–12 

months. The number of males killed was 5 from 2 litters 

in the control group, 15 from 4 litters in the low-dose 

Birth Defects Research (Part B) 83:157–395, 2008
242 CHAPIN ET AL. 

group, 5 from 1 litter in the high-dose group, and 10 from 

4litters in the ethinyl estradiol group. Testes were fixed in 

Bouin solution, and Sertoli cells were counted. Spermatogenesis was evaluated by examining germinal epithelia 

for cell death and distribution of various cell populations. Data were analyzed by ANOVA. [It appears that at 

least some data were analyzed on a per litter basis. In 

addition, analyses were done to determine intralitter 

variability and thus the numbers of animals per group 

that needed to be analyzed.] 

Examination of tubule cross sections revealed qualitatively normal spermatogenesis in the bisphenol A 

groups. A comparison of Sertoli cell numbers in 

littermates revealed high variability (20–27%) in the 

0.1 mg/kg bw/day group. A comparison of Sertoli cell 

numbers in the 4 litters from the 0.1 mg/kg bw/day 

group revealed almost identical results between litters. 

Sertoli cell numbers/organ were significantly increased 

by 19.4% in the low-dose group and 19% in the high-dose 

group. Bisphenol A had no significant effect on Sertoli 

cell numbers/g testis weight. The opposite situation 

occurred in the ethinyl estradiol group, with no 

significant effects on Sertoli cell numbers/organ but a 

significant increase in Sertoli cell numbers/g testis 

weight. Testis weight was not affected by bisphenol A 

treatment but was decreased in the ethinyl estradiol 

group. The study authors concluded that the study does 

not support the hypothesis of disruption of the male 

reproductive system by bisphenol A exposure. 

Strengths/Weaknesses: The conceptual strength is the 

focus on the male reproductive tract/function. However, 

a weakness is that there were too few animals to provide 

reliable data. 

Utility (adequacy) for the CERHR Evaluation Process: This study is inadequate based on insufficient 

sample size (n 5 2–4). 

Thuillier et al. (2003), supported by National Institute 

of Environmental Health Sciences (NIEHS), examined a 

possible role for the platelet-derived growth factor 

system in estrogenic effects induced by bisphenol A in 

rats exposed during gestation. The effects of other 

compounds such as genistein and coumestrol were also 

examined but will not be discussed here. Pregnant 

Sprague–Dawley rats were gavaged with bisphenol A 

at 0 (corn oil vehicle) or 0.1, 1, 10, or 200 mg/kg bw/day 

from GD 14 through birth (PND 0). Additional rats were 

s.c. injected with diethylstilbestrol at 0.01–2 mg/kg bw/ 

day during the same period. [No information was 

provided about number of rats treated, purity of 

bisphenol A, feed, or materials used in bedding and 

caging.] Male offspring were killed on GD 21 or PND 3 

and testes were collected. Expression of mRNA or 

protein for platelet-derived growth factor receptor-a

and platelet-derived growth factor receptor-b were 

determined in testes using RT-PCR, in situ hybridization, 

or immunohistochemistry. Statistical analyses included 

unpaired t-test with Welch correction. [It was not clear if 

the litter or offspring were considered the statistical 

unit.] 

Expression of mRNA for platelet-derived growth 

factor receptor-a and -b was significantly increased at 

bisphenol A doses Z1 mg/kg bw/day in testes from 3­

day-old rats. All other experiments with bisphenol A 

were conducted with a single dose of 200 mg/kg bw/ 

day. In situ hybridization examination of testes from 3­

day-old rats from the bisphenol A group revealed an 

increase in expression of platelet-derived growth factor 

receptor-a mRNA in testicular interstitium and plateletderived growth factor receptor-b mRNA in interstitium 

and seminiferous cords. Exposure to bisphenol A 

resulted in slightly increased platelet-derived growth 

factor receptor-a protein expression and strong expression of platelet-derived growth factor receptor-b in 

gonocytes from 3-day old rat testes. Immunolocalization 

studies in testes from 21-day-old fetuses revealed that 

exposure to 200 mg/kg bw/day bisphenol A did not 

affect expression of platelet-derived growth factor 

receptor-a protein in gonocytes, but platelet-derived 

growth factor receptor-b protein appeared to be increased in gonocytes and Sertoli cells. Diethylstilbestrol 

tended to have a biphasic effect with increased expression of platelet-derived growth factor receptor-a and -b

mRNA in 3-day-old rat testis at low doses and decreased 

expression at the high-dose. Treatment with 1 mg/kg bw/ 

day diethylstilbestrol decreased mRNA expression of 

platelet-derived growth factor receptor-a in interstitium 

and increased platelet-derived growth factor receptor-b

mRNA expression in seminiferous cords. Immunoreactivity for platelet-derived growth factor receptor-a

protein was maintained but there was a minimal level 

of platelet-derived growth factor receptor-b protein 

expression in 3-day-old rat testes following exposure to 

1 mg/kg bw/day diethylstilbestrol. In testes obtained 

from 21-day-old fetuses, expression of platelet-derived 

growth factor receptor-a protein was decreased in Sertoli 

and interstitial cells and expression of platelet-derived 

growth factor receptor-b protein was apparently increased following exposure to diethylstilbestrol. The 

study authors concluded that the platelet-derived growth 

factor receptor pathway may be a target for estrogens in 

the testis, but the findings do not exclude the possibility 

that effects may have occurred through an ER-independent mechanism. 

Strengths/Weaknesses: Endpoints are a strength, but 

inadequate methodological detail (i.e., sample size or 

adequate control for litter effects) precludes any informed judgment of study quality. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process based 

on insufficient methodological details. 

Wang et al. (2004), supported by NIEHS, examined the 

effects of prenatal bisphenol A exposure on expression of 

ER-associated proteins in rat testis. The effects of 

genistein and coumestrol were also examined but will 

not be discussed here. Pregnant Sprague–Dawley 

rats [apparently 3/group] were gavaged with corn oil 

vehicle or bisphenol A at 0.1–200 mg/kg bw/day from 

GD 14 (14 days post-coitum) through birth. Additional 

rats were s.c. injected with 0.01–2 mg/kg bw/day 

diethylstilbestrol during the same time period. [No 

information was provided about feed, caging 

and bedding material, or compound purity.] Male 

offspring from three independent litters were killed on 

GD 21, PND 3, or PND 21. Western blot, RT-PCR, and 

immunohistochemistry techniques were used to measure 

expression of protein or mRNA for Hsp90, Hsp90a, p23, 

CYP40, Hsp70, and/or ERb. Spermatogonia were 

quantified in PND 21 rat testis. Data were analyzed 

by unpaired t-test. The dam was considered the 

statistical unit. 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 243 

In testes from 3-day-old rats, RT-PCR revealed significant increases in mRNA for hsp90 at bisphenol A dose 

levels of 10 and 200 mg/kg bw/day, and significant 

decreases in expression of CYP40 at 200 mg/kg bw/day 

and p23 at 1 mg/kg bw/day. In situ hybridization 

analyses in 3-day-old rat testes revealed that bisphenol 

A tended to increase expression of hsp90 throughout the 

testis, with patterns indicating increased expression in 

gonocytes and interstitial Leydig cells. Examination of 

protein in testes from 3-day old rats exposed to 200 mg/ 

kg bw/day bisphenol A revealed significantly increased 

levels of hsp90 and hsp70, but no effect on levels of 

CYP40, p23, or ERb. Immunohistochemistry revealed 

that hsp90 protein in testes from 3-day-old rats was most 

increased in gonocytes and less so in interstitium 

following exposure to 200 mg/kg bw/day bisphenol A. 

Use of a probe specific for hsp90a protein revealed that 

increased protein expression of hsp90 was due in a large 

part to the hsp90a isoform. Examination of testes from 

GD 21 fetuses and PND 21 pups revealed that the 

amount of hsp90 protein in the bisphenol A treatment 

group was similar to that observed on PND 3 but that the 

amount of protein did not differ from controls on PND 

21. In 21-day-old rats from the bisphenol A group, the 

number of spermatogonia/tubule was significantly higher by B2-fold compared to the control group. [It is not 

clear which bisphenol A dose induced an increase in 

spermatogonia, but it was most likely 200 mg/kg bw/ 

day, because that dose appeared to be used in all 

studies not examining dose–response relationships.] 

Effects following diethylstilbestrol exposure included 

increased expression of hsp90 mRNA at 1.0 mg/kg bw/ 

day and decreased CYP40 mRNA expression at 0.01 and 

1 mg/kg bw/day, but no effect on protein levels of those 

compounds was reported in testes from 3-day-old rats. 

The number of spermatogonia/tubule was also increased 

after prenatal exposure to diethylstilbestrol. The study 

authors concluded that prenatal exposure to bisphenol A 

affects hsp90 expression in gonocytes of rats, and because 

hsp90 interacts with several signaling molecules, changes 

in its expression could affect gonocyte development. 

Strengths/Weaknesses: This study was generally well 

conceived, but the small sample size suggests it presents 

pilot data only. A full study is needed to provide reliable 

data. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate based on insufficient sample 

size (n 5 3). 

3.2.1.3 Neurodevelopmental endpoints: Funabashi 

et al. (2004a), supported in part by Yokohama City 

University, examined the effects of bisphenol A on the 

numbers of corticotropin-releasing hormone neurons in 

the preoptic area and bed nucleus of the stria terminalis 

of rats exposed during development. [No information 

was provided about chow or composition of bedding 

and caging.] Pregnant Wistar rats (n 5 8–11/treatment 

group) were given drinking water containing the 0.1% 

ethanol vehicle or 10 mg/L bisphenol A [purity not 

reported] until their offspring were weaned at 3 weeks of 

age. [It is implied but not stated that exposure occurred 

during the entire gestation period.] Bisphenol A intake 

was estimated by study authors at 2.5 mg/kg bw/day. 

Male and female offspring (n 5 8–11/group) were killed 

at 4–7 months of age, and immunocytochemistry 

techniques were used to determine the number of 

corticotropin-releasing hormone neurons in brain. Female rats were killed during proestrus. [Although the 

number of litters represented in each group was not 

specified, the number of rats examined suggests that 1 

rat/sex/litter was examined.] Histological slides of brain 

were evaluated by an investigator blinded to treatment 

conditions. Two series of experiments were conducted, 

and data from both experiments were combined. Data 

were analyzed by ANOVA followed by Fisher protected 

least significant difference post-hoc test. [It was not 

stated if data were analyzed on a per litter or per 

offspring basis, but as stated earlier, it appears that 1 

rat/sex/litter was examined.] In the control group, 

females had more corticotropin-releasing hormone neurons in the preoptic area and anterior and posterior bed 

nucleus of the stria terminalis than males. Bisphenol A 

treatment did not change the number of corticotropinreleasing hormone neurons in the preoptic areas of males. 

A loss in sex difference occurred in the anterior and 

posterior bed nuclei of the stria terminalis following 

bisphenol A treatment because differences in numbers of 

corticotropin-releasing hormone neurons between males 

and females were no longer evident. It appears that 

bisphenol A treatment increased the number of corticotropin-releasing hormone neurons in males and decreased 

the number in females. The study authors concluded that 

exposure to bisphenol A during gestation and lactation 

results in a loss of sex difference in corticotropin-releasing 

hormone neurons in the bed nucleus of the stria terminalis 

but not in the preoptic area. 

Strengths/Weaknesses: This study was appropriately 

designed to examine effects on the development of brain 

areas known to be influenced by hormonal levels. 

Strengths include the relevance and subtleties of the 

endpoints measured; weaknesses include uncertainties 

about the numbers of animals examined and the duration 

of the dosing period. The results suggest a disruption of 

the normal pattern of sexually dimorphic neurons, a result 

of critical importance to concerns about disruptions 

relevant to reproductive function and sexually dimorphic 

behaviors. While the sample size was 8–11/group, the 

design and statistics appear to be appropriate. It is a 

weakness that the control for litter effects was not clear. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate for inclusion in the evaluation 

process, although of limited utility due to uncertainties 

about the sample size, duration of dosing, and control for 

litter effects. 

Fujimoto et al. (2006), supported by the Japanese 

Ministry of Education, Culture, Sports, Science, and 

Technology, examined the effect of prenatal bisphenol A 

exposure on sexual differentiation of neurobehavioral 

development in rats. Wistar rats were fed CE-2 feed 

(CLEA, Japan). [Caging and bedding materials were not 

described.] From GD 13 (day of vaginal sperm not 

defined) to the day of birth (PND 0), 6 rats/group were 

given tap water containing bisphenol A [purity not 

reported] at 0 or 0.1 ppm. The study authors estimated 

the bisphenol A dose at 0.015 mg/kg bw/day. On PND 1, 

pups were weighed and litters were culled to 4 pups/ 

sex. Pups were weaned on PND 21. Neurobehavioral 

evaluations conducted in 20–24 offspring/sex/group at 

6–9 weeks of age included open-field, elevated plus 

maze, passive avoidance, and forced swimming tests. 

Statistical analyses included ANOVA, Fisher protected 

Birth Defects Research (Part B) 83:157–395, 2008
244 CHAPIN ET AL. 

least significant difference test, and Mann–Whitney Utest. [It appears that offspring were considered the 

statistical unit.] 

In the control group, rearing frequency and duration 

were significantly higher in females than males, but there 

were no sex-related differences in rearing frequency or 

duration in the bisphenol A group. Bisphenol A exposure 

caused an increase in rearing duration in males when 

compared to males from the control group. In the forced 

swim test, females in the control group struggled more 

than males but no sex-related differences in struggling 

were observed in the bisphenol A group. The duration of 

immobility in the swimming test was longer in males 

from the bisphenol A compared to males from the control 

group. Immobility was described as non-significantly 

increased in females exposed to bisphenol A compared to 

control females. Bisphenol A exposure had no effect on 

performance in passive avoidance and elevated plus 

maze test. The study authors concluded that exposure of 

male offspring to bisphenol A during the final week of 

gestation resulted in impaired sexual differentiation in 

rearing and struggling behaviors and facilitated depression-like behavior. 

Strengths/Weaknesses: This study used a good choice 

of methods to examine functional disruptions in sexually 

dimorphic behaviors. Weaknesses include a lack of clarity 

about the nature of disruption of sexually dimorphic 

behavior patterns that was indicated in the authors’ 

conclusions, the somewhat small sample size, the use of 

a single dose level, which was not confirmed, and the lack 

of clarity of the statistical methods regarding litter. 

Utility (Adequacy) for CERHR Process: This study is 

inadequate for the evaluation process due to statistical 

methodology. 

3.2.2 Rat—parenteral exposure only during 

pregnancy. Ramos et al. (2001), supported by the 

Argentine National Council for Science and Technology, 

the Argentine National Agency for the Promotion of 

Science and Technology, and the Ministry of Health, 

examined the effects of bisphenol A exposure on the rat 

prostate. Wistar rats were housed in stainless steel cages. 

[No information was provided about chow or bedding 

material.] Four dams/group were exposed to bisphenol 

A [purity not reported] at 0 (DMSO vehicle), 0.025, or 

0.250 mg/kg bw/day by s.c. pump on GD 8–23 (GD 

1 5 day of vaginal sperm). Pups were weighed and sexed 

at birth. Litters were culled to 8 pups, with 4/sex when 

possible. Pups were weaned on PND 22 [day of birth not 

defined]. On PND 30, pups were injected with bromodeoxyuridine and killed 2 hr later. Ventral prostates were 

dissected and fixed in 10% neutral buffered formalin. 

Immunohistochemical techniques were used to measure 

proteins associated with cell proliferation and cell 

phenotypes. Morphometric measurements were taken. 

[It was not clear how many rats/treatment group were 

examined for each endpoint. Although a statement was 

made that males from a single dam were evaluated, it 

was later stated that siblings were excluded from the 

same experimental group. Therefore it appears that 

different litters were represented.] Data were analyzed 

by Kruskal–Wallis ANOVA and Mann–Whitney U-test. 

[It was not clear if the dam or offspring were 

considered the statistical unit.] 

In the periductal stroma, the fibroblastic layer was 

increased, the smooth muscle layer was reduced, and 

androgen receptor-positive cells were decreased. Prostatic acid phosphatase-positive cells were reduced in 

epithelial cells. There were no effects on cell proliferation 

and ERa was not detected. No changes were observed in 

interductal stromal cells. 

Strengths/Weaknesses: This study has an interesting 

design with respect to choice of endpoints. Certain 

design aspects are unclear and statistical approaches 

are inadequate. The sample size was small (4 dams/ 

group) and there was considerable uncertainty about 

numbers of offspring examined and accounting for litter 

effects. The use of DMSO (% not specified) is of concern, 

as this can modify the effects of the solute. Of additional 

concern is the route of administration (s.c. pump). 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is considered inadequate. 

Ramos et al. (2003), supported by the Argentine 

Ministry of Health, Argentine National Agency for the 

Promotion of Science and Technology, and the National 

University of Litoral, examined the effects of bisphenol A 

exposure on the prostate and the hypothalamic-pituitarygonadal axis in Wistar rats. Rats were housed in stainless 

steel cages and 7–9/group were administered DMSO 

vehicle or bisphenol A at 0.025 or 0.250 mg/kg bw/day 

by s.c. pump on GD 8–23 (GD 1 5 day of vaginal sperm). 

[No information was provided on purity of bisphenol 

A, the type of feed used, or composition of bedding.] 

After birth, pups were weighed and sexed. Litters were 

culled to eight pups with equal numbers of male and 

female pups when possible. Pups were weaned on PND 

22 [day of birth not defined]. During prepuberty (PND 

15), peripuberty (PND 30), and adulthood (PND 120), 6–8 

males/group were injected with bromodeoxyuridine and 

killed 2 hr later. Serum was collected for measurement of 

LH and prolactin by RIA. Immunohistochemistry techniques were used to evaluate markers of cell proliferation, estrogen/androgen receptors, and prostatic cells. 

Expression of mRNA for ERa and ERb in the preoptic 

area and medial basal hypothalamus was determined by 

RT-PCR. Data were analyzed by Kruskal–Wallis 1-way 

ANOVA using Dunn post-test. 

No significant effects were observed for ventral 

prostate weight. Numerous transient effects were observed in both bisphenol A dose groups. On PND 15, 

cellular proliferation was increased in the periductal 

stroma of the prostate, and serum testosterone 

levels were elevated. On PND 30, the fibroblasts 

(vimentin-positive cells) in the prostatic periductal 

stroma was increased and the area of smooth muscle 

cells a-smooth muscle actin) was decreased. Also 

observed on PND 30 was a reduction in androgenreceptor positive stromal cells, a decrease in epithelial 

cells positive for prostatic acid phosphatase, and an 

increase in serum prolactin levels. Expression of ERb

mRNA was increased in the preoptic areas on PND 30 

and 120, and the study authors considered the effect to 

be permanent because it occurred on both days. The 

study authors concluded that prenatal exposure to 

environmental concentrations of bisphenol A during 

gestation results in transient and permanent changes in 

the male reproductive axis. 

Strengths/Weaknesses: The design appears reasonable 

as a means to address the study questions. Like many of 

these studies, altered values are given without addressing the normal range of variation or the likely functional 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 245 

significance of the changes. Weaknesses include use of 

the s.c. pump as a route of administration and use of 

DMSO as a vehicle. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for inclusion due to the use of 

99.9% DMSO as a vehicle to administer BPA via s.c. 

pump. As discussed in earlier, the use of 450% DMSO as 

a vehicle for ALZET mini-pump studies is a clear 

contravention of the directions for mini-pump use, as it 

accelerates the breakdown of the mini-pumps 

Naciff et al. (2002), from the Procter and Gamble 

Company, examined the effects of prenatal bisphenol A 

exposure on gene expression and, to a limited extent, 

development in female rat reproductive organs. Pregnant 

Sprague–Dawley rats were fed Purina 5K96, a caseinbased soy- and alfalfa-free diet. [Composition of caging 

and bedding materials was not reported.] The rats were 

assigned to groups (Z7 rats/group) s.c. injected with 

bisphenol A (B99% purity) in DMSO vehicle at 0, 5, 50, 

or 400 mg/kg bw/day on GD 11–20 (day of sperm 

detection 5 GD 0). Dams were killed on GD 20, and 

ovaries and uteri were removed from fetuses. In 4 litters/ 

group, 1 female fetus/litter was examined for ovarian 

and uterine histopathology. In 5 litters/group, ovaries 

and uteri from at least 5 littermates were pooled for a 

microarray analysis of gene expression. Changes in gene 

expression were further quantified using RT-PCR. Data 

were analyzed by t-test, ANOVA, and Jonkheere-Terpstra 

test. Comparisons of gene expression among estrogenic 

compounds were made by Wilcoxon-Mann–Whitney and 

Jonkheere-Terpstra tests. Results of gene expression assays 

are discussed in Section 2. Vaginal bleeding and early 

parturition occurred in 1 of 8 dams in the high-dose group. 

Bisphenol A treatment had no effect on maternal body 

weight or number of live fetuses/litter, and there were no 

gross or histopathological effects on ovary or uterus. 

Prominent nipples and areolas were observed in males 

and females in the high-dose bisphenol A group [number 

of fetuses and litters affected were not reported]. 

Strengths/Weaknesses: Strengths are that these endpoints appear appropriate; weaknesses are the limited 

nature of the endpoints and the use of neat DMSO as 

vehicle. The sample sizes are 4–5/endpoint/group and 

judged to be inadequate. Of additional concern is the 

route of administration. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process. 

Naciff et al. (2005), from The Procter and Gamble 

Company, examined the effect of prenatal bisphenol A 

exposure on male rat reproductive organ histology and 

gene expression. Pregnant Sprague–Dawley rats were fed 

Purina 5K96, a casein-based soy- and alfalfa-free diet. 

Rats were housed in stainless steel cages before mating. 

Rats were randomly assigned to groups (Z8 rats/group) 

and s.c. injected with bisphenol A (B99% purity) in 

DMSO at 0, 0.002, 0.02, 0.5, 50, or 400 mg/kg bw/day on 

GD 11–20 (day of sperm detection 5 GD 0). Dams were 

killed on GD 20, and testes and epididymides were 

removed from fetuses. In 4 litters/dose group, 1 male 

fetus/litter was examined for testicular histopathology. 

In 5 litters/group, testes and epididymides from 5 

littermates were pooled for a microarray analysis of 

gene expression. Changes in gene expression were 

further quantified using RT-PCR. Data were analyzed 

by t-test, ANOVA, and Jonkheere-Terpstra test. 

Comparisons of gene expression among estrogenic 

compounds were analyzed by Wilcoxon-Mann–Whitney 

and Jonkheere-Terpstra tests. 

Bisphenol A treatment had no effect on maternal body 

weight or number of live fetuses/litter, and there were no 

gross or histopathological effects on the testis or 

epididymis. Prominent nipples/areolas were observed 

in male and female fetuses from the high-dose group 

[numbers of fetuses and litters affected were not 

reported]. In pooled testis and epididymis samples from 

the high-dose bisphenol A group, expression of 15 genes 

was significantly altered in a dose-related manner. When 

bisphenol A data were pooled with data obtained from 

ethinyl estradiol and genistein and globally analyzed, 

there were 50 genes that were significantly altered in the 

same direction by all three compounds. The study 

authors concluded that transplacental exposure to highdoses of bisphenol A alters the expression of certain 

genes in the testis and epididymis of fetal rats without 

causing malformations in those organs. The study 

authors noted that the dose response to bisphenol A 

was monotonic with no evidence of robust quantifiable 

responses at low doses. 

Strengths/Weaknesses: Strengths are that these endpoints appear appropriate; weaknesses are the limited 

nature of the endpoints and the use of neat DMSO as 

vehicle. The sample sizes are 4–5/endpoint/group and 

judged to be inadequate. Of additional concern is the 

route of administration. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process. 

Saito et al. (2003b), support not indicated, examined 

the effect of prenatal bisphenol A exposure on testosterone production during adulthood in rats. On GD 12–19 

(day of vaginal plug not reported), 2 Wistar rats were s.c. 

injected with the corn oil vehicle, 4 rats were s.c. injected 

with 0.005 mg/day bisphenol A [purity not indicated], 

and 2 rats were injected with 5 mg/day 17b-estradiol. 

[Assuming a pregnant Wistar rat weights B0.33 kg, 

0.005 mg/day would be equivalent to 0.015 mg/kg bw/ 

day bisphenol A.] Other materials found in dental 

composites were also evaluated but will not be discussed. During the lactation period, rats were housed in 

polypropylene cages with synthetic bedding. [No information was provided on feed.] Offspring were 

housed separately at 3 weeks of age and killed at 13 

weeks of age. Body and testis weights were measured in 

all male offspring (22 in the bisphenol A group, 11 in the 

vehicle control group, and 5 in the 17b-estradiol group). 

Plasma testosterone level was measured by RIA, and 

plasma cholesterol level was measured using a kit. 

Activities of testicular enzymes involved in the production of testosterone from progesterone were also examined in an in vitro assay in which testicular microsomes 

were incubated with 14C-progesterone and 14C-d4

-androstenedione for 20 min. Data were analyzed using 

unspecified post-hoc tests. [Although not clear, it 

appears that offspring were considered the statistical 

unit for some analyses.] 

Bisphenol A exposure had no effect on pup sex ratio. 

No effects on body weight or absolute testicular weight 

were observed in the bisphenol A group at 13 weeks of 

age. However, relative (to body weight) testicular weight 

was lower [by 6%] in rats of the bisphenol A compared to 

the control group. Also observed in the bisphenol A 

Birth Defects Research (Part B) 83:157–395, 2008
246 CHAPIN ET AL. 

group was a reduction in plasma testosterone level [by 

B28%]. No effect was observed on cholesterol level. In 

the ex vivo study, prenatal bisphenol A exposure 

increased activities of 17a -hydroxysteroid dehydrogenase [by B140%] and 17b-hydroxysteroid dehydrogenase 

[by B70%]. Observations in the 17b-estradiol compared 

to the control group included decreased numbers of 

offspring delivered, higher body weight of male offspring at 13 weeks of age, reduced plasma testosterone 

level, and increased testicular 17a-hydroxysteroid dehydrogenase activity. The study authors concluded that 

bisphenol A had an estrogenic effect on the testis but did 

not decrease activities of enzymes involved in testosterone production. 

Strengths/Weaknesses: A strength of this study is the 

examination of testosterone levels at 13 weeks of age. 

This strength is negated by the sample size (n 5 2–4), 

which is too small to draw any firm conclusions. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate based on insufficient sample size. 

Murray et al. (2007), supported by NIH, examined the 

effect of prenatal bisphenol A exposure on in situ 

induction of mammary tumors. Wistar-Furth rats were 

fed Harlan Teklad 2018, which was reported to contain 20 

fmol/g estrogen equivalents. Water was supplied in glass 

bottles. Caging and bedding materials were not reported, 

but they were stated that to test negative in the ESCREEN. From GD 9 (GD 1 5 day of vaginal sperm) 

through PND 1 [The day of birth was PND 0 (A. Soto, 

personal communication, March 2, 2007)], rats received 

the 50% DMSO vehicle or bisphenol A [purity not 

reported] at 0.0025, 0.025, 0.250, or 1 mg/kg bw/day. 

Dosing solutions were delivered by implanted [assumed 

s.c.] osmotic pumps. [Number of dams treated was not 

reported. Based on a limited amount of information 

provided on the number of offspring examined, it 

appears that r6 dams/group were treated.] Pup 

viability was assessed on PND 1. On PND 2 pups were 

sexed and litters were culled to 8 pups. Anogenital 

distance was measured on PND 4. Litters were weighed 

during the lactation period. Female offspring were 

monitored for body weight and vaginal opening in the 

post weaning period. Female offspring were killed on 

PND 50 or 95. Mammary glands were collected and 

whole-mounted or sectioned for histopathological examination. Morphometric analyses were conducted to 

examine possible presence of preneoplastic lesions. 

Mammary glands were examined for ERa and Ki-67 

protein by an immunohistochemistry technique. Maximal numbers of ‘‘maternal units’’ were represented in 

each dose group. One female/litter was included in 

histological examinations. [Apparently r6 offspring/ 

group were examined in histopathological examinations. Number of offspring examined for other endpoints was not reported in the manuscript. According 

to an author, n 5 7–21 for the other endpoints (A. Soto, 

personal communication, March 2, 2007).] Statistical 

analyses included ANOVA followed by post-hoc tests 

(Bonferroni or t-test) when significant effects were 

observed by ANOVA. [It was not clear if dams or 

offspring were considered the statistical unit.] 

Bisphenol A exposure did not affect offspring viability, 

sex ratio, age at vaginal opening, or female anogenital 

distance. Anogenital distance was reduced on PND 4 in 

males from the 0.250 mg/kg bw/day group. Percent 

hyperplastic ducts was increased in all dose groups on 

PND 50 and in the 0.0025 mg/kg bw/day group on PND 

95; the study authors noted that the effect on PND 50 was 

quantitatively similar in all dose groups (i.e. 3–4-fold 

increase). Cribriform structures were observed in the 0.25 

and 1 mg/kg bw/day groups. [Incidence was not 

reported for the control and lower dose groups.] The 

structures were classified as carcinomas-in-situ and were 

characterized by increased ductal size resulting from 

luminal epithelium proliferation, enlarged luminal 

epithelial cells, presence of a nucleolus, variable chromatin pattern, and rounded luminal spaces consisting of 

trabecular rods of cells perpendicularly aligned to the 

longer duct axis. Numbers of Ki-67- and ER-a positive 

cells were increased in aberrant compared to normal 

tissues, regardless of dose. [Results in treated compared 

to control groups were not reported.] The study authors 

concluded that fetal bisphenol A exposure is rats is 

sufficient to induce development of preneoplastic and 

neoplastic mammary lesions. 

Strengths/Weaknesses: Relevance of endpoints is a 

strength, as is the use of multiple dose levels. Weaknesses 

include an unstated number of dams (and by inference, a 

small number of these, and thus, because of dam-related 

effects, a small overall n), the uncertainty of the response 

rate of histopathology in the controls, and the use of 50% 

DMSO as vehicle. 

Utility/Adequacy for CERHR Evaluation: This study 

was inadequate due to small sample size, route of 

administration, and lack of clarity on statistical analysis. 

Durando et al. (2007), supported by Universidad 

National del Litoral, Argentine National Agency for the 

Promotion of Science and technology, and NIH, examined the effects of prenatal bisphenol A exposure on 

susceptibility to mammary tumors in rats. Wistar rats 

were fed Cooperacio ́n (Buenos Aires, Argentina) and 

housed in stainless steel cages. [It was not clear if 

bedding was used.] On GD 8–23 (GD 1 5 day of vaginal 

sperm), 11–14 dams/group were s.c. dosed by osmotic 

pump with the DMSO vehicle or 0.025 mg/kg bw/day 

bisphenol A [purity not indicated]. Pups were delivered 

on GD 23 and weaned on PND 21. It was not indicated if 

day of birth was considered PND 0 or 1. During the 

study, body weights and day of vaginal opening were 

monitored. Offspring were killed before puberty (PND 

30), after puberty (PND 50), or in adulthood (PND 110 

and 180). In mammary gland stroma and epithelium, 

proliferation was assessed by BrdU incorporation and 

apoptotic cells were identified by TUNEL method. 

Morphometric analyses were conducted in sectioned 

mammary glands. Mast cells were identified by immunostaining for proteinase. At least 6 offspring/group/ 

time point were evaluated. [No littermates were used in 

the evaluation at any given time point (A. Soto, 

personal communication, March 2, 2007).] Additional 

offspring were examined for responsiveness to chemically-induced mammary preneoplastic or neoplastic 

lesions. On PND 50, N-nitroso-N-methylurea was administered to 10–16 offspring from the vehicle control 

group at 25 or 50 mg/kg bw and 21 offspring from the 

bisphenol A group at 25 mg/kg bw. Based on findings 

from a pilot study, 25 mg/kg bw was considered a 

subcarcinogenic dose and 50 mg/kg bw was considered 

a positive control. During the study, rats were palpated 

for tumors. Rats that received 50 mg/kg bw N-nitrosoBirth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 247 

N-methylurea were killed on PND 180 and rats that 

received 25 mg/kg bw N-nitroso-N-methylurea were 

killed on PND 110 or 180. Whole-mounted mammary 

glands were examined for tumors. Immunostaining was 

conducted to identify cytokeratin 8 (an epithelial marker) 

and p63 (a myoepithelial marker). Data were statistically 

analyzed using the Mann–Whitney U-test. 

Bisphenol A exposure did not affect successful 

pregnancies, dam weight gain, pregnancy duration, 

number of pups/litter, or percent females/litter. Anogenital distance on PND 1 or 5 and postnatal body 

weights were unaffected in pups exposed to bisphenol 

A. Vaginal opening was accelerated in pups from the 

bisphenol A group (mean 34 days of age compared to 39 

days of age in controls). On PND 50, the BrdU/apoptosis 

ratio was significantly increased and apoptosis was 

significantly decreased in mammary parenchyma and 

stroma of bisphenol A-exposed animals; the effects 

were not observed on PND 30 or 110. Significantly 

increased percentages of hyperplastic ducts, density of 

stromal nuclei, and numbers of mast cells were 

observed in the bisphenol A group on PND 110 and 

180. Exposure to bisphenol A resulted in formation 

of a dense stroma layer around mammary epithelial 

structures and replacement of normal adipose tissue with 

a fibroblastic stroma. In rats exposed to 25 mg/kg bw 

N-nitroso-N-methylurea on PND 50, incidence of 

hyperplastic lesions on PND 180 was significantly higher 

in the group with prenatal bisphenol A compared to 

DMSO exposure (mean incidence of 35.5% compared to 

15.7% in controls). Although statistical significance was 

not achieved, exposure to 25 mg/kg bw N-nitroso-Nmethylurea resulted in tumors in 2 of 15 rats in the 

prenatal bisphenol A group and 0 of 10 rats in the 

prenatal vehicle control group on PND 180. Cytokeratin 8 

immunostaining revealed no invasion by stromal epithelial cells. The study authors concluded that rats prenatally exposed to environmentally relevant doses of 

bisphenol A may have an increased risk of developing 

mammary tumors. 

Strengths/Weaknesses: Weaknesses include route of 

administration and the high single dose is a weakness as 

is the use of pure DMSO. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for inclusion due to the use of 

99.9% DMSO as a vehicle to administer bisphenol A via 

s.c. osmotic pump. 

Hong et al. (2005), sponsored by the Korea Research 

Foundation, investigated the effects of acute exposures to 

bisphenol A during late pregnancy on expression and 

protein level of calbindin-D9k, a putative biomarker of 

estrogen activity, in the uteri of offspring and lactating 

rats on PND 5. Pregnant Sprague–Dawley rats were 

given free access to water and a diet of soy-free pellets in 

polycarbonate caging. [Housing conditions (individual 

or group) and bedding material were not indicated.] On 

GD 17–19, pregnant rats were s.c. injected daily with 200, 

400, or 600 mg/kg bw/day bisphenol A [purity not 

provided] in corn oil (n 5 5/group). Negative and 

positive control groups (n 5 10/group) were administered corn oil or 17b-estradiol 40 mg/kg bw/day. On PND 

5, lactating dams and female pups were killed and their 

uteri harvested. Dose response changes in calbindin-D9k, 

expression levels in uteri of lactating dams and female 

offspring (3/group) were analyzed by Northern blot and 

RT-PCR, with appropriate housekeeping gene controls. 

Protein levels and localization of calbindin-D9k were 

performed by Western blot and immunohistochemistry 

for lactating dams only. Statistical analyses were performed using the Kruskall–Wallis and Dunnett tests. [It 

was not clear if dams or offspring were considered the 

statistical unit.] 

Northern blot analysis revealed a significant increase 

[B6.4-fold] in the level of calbindin-D9k expression in the 

uteri of lactating dams exposed to 600 mg/kg bw/day 

bisphenol A compared to oil controls. 17b-Estradiol 

treatment produced a significant [B3.9-fold] increase 

in calbindin-D9k mRNA expression in the dam uterus 

that was not statistically distinct from the effect of the 

high bisphenol A dose. Uteri of offspring exposed to the 

highest dose level of bisphenol A also showed a 

significant upregulation [B4.4-fold] in calbindin-D9k 

expression. Expression levels of ERa were unaffected in 

maternal uteri exposed to bisphenol A. However, ERa

expression was increased significantly in uteri of pups 

exposed to 400 and 600 mg/kg bw bisphenol A [m33% 

and 66%, estimated from a graph]. Protein levels of 

calbindin-D9k in lactating dam uteri were elevated 

significantly at all dose points [50, 40, and 50%, for 

200, 400, and 600 mg/kg bw/day, respectively]. 17bEstradiol-treatment was not associated with a significant 

increase in calbindin-D9k protein. The density of calbindin-D9k-immunopositive cells was increased in uterine 

sections from lactating dams exposed to all doses of 

bisphenol A relative to oil controls, correlating with 

Western blot results. Authors note insufficient material 

or low detectability of calbindin-D9k protein in offspring 

tissue, and protein analyses were not performed. 

The authors suggest that calbindin-D9k can serve as a 

reliable biomarker of acute estrogenic exposure, particularly for insight into maternal-fetal metabolic exchange, 

given that calbindin-D9k is tightly regulated and rapidly 

induced by 17b-estradiol, diethylstilbestrol, alkylphenols, and now, bisphenol A. They further point out that 

calbindin-D9k expression is absent in immature rat and 

ovariectomized rat uteri. 

Strengths/Weaknesses: This study supports the use of 

calbindin-D9k as a uterine biomarker of estrogenic effect 

in the perinatal period in the rat, and provides some 

dose–response information for bisphenol A induction of 

an estrogenic response. Limitations are the subcutaneous 

route of exposure, small sample size, high-doses and 

uncertain statistical analyses of the F1 data. 

Utility (Adequacy) for CERHR Evaluation Process: 

While providing some dose–response information regarding bisphenol A-induced estrogenic effects following exposure of rats in the perinatal period, the lack of 

clarity regarding whether the dam or offspring was 

considered the statistical unit, route of exposure, and use 

of high doses render this study inadequate for consideration in the evaluation process. 

3.2.3 Rat—oral exposure postnatally with or 

without prenatal exposure. 

3.2.3.1 Reproductive studies: The International Research and Development Corporation (General Electric, 

1976), sponsored by General Electric, examined the 

effects of bisphenol A exposure on CD rats and their 

offspring. Male and female F0 rats were housed in wire 

mesh cages and fed Purina Laboratory Chow. Ten rats/ 

sex/group (body weights of 110–170 g for males and 

Birth Defects Research (Part B) 83:157–395, 2008
248 CHAPIN ET AL. 

100–151 g for females) were given feed containing bisphenol A [purity not specified] at 0, 1000, 3000, or 

9000 ppm for 17 weeks. [It was not clear how long before 

mating that the dosing was started or if dosing was 

continued through the gestation and lactation periods.] 

The European Union (2003) estimated bisphenol A intake 

at 0, 70, 200, or 650 mg/kg bw/day in males and 0, 100, 

300, or 950 mg/kg bw/day in females. F0 rats were mated 

at B100 days of age and assessed for fertility. F1 pups 

were counted and weighed at birth and on PND 21 (day of 

birth not defined). Fifteen male and female F1 rats/group/ 

sex that were exposed in utero were selected for a 13-week 

feeding study and were fed diets containing the same 

concentration of bisphenol A as their parents. F1 rats were 

weighed and observed for clinical signs. Hematological, 

clinical chemistry, and urinalysis parameters were examined in 5 rats/sex/group in the control and 2 highest dose 

groups at 1, 2, and 3 months of F1 exposure. Ophthalmoscopic examinations were conducted at 3 months of F1 

exposure. After 13 weeks of dosing, the F1 rats were killed 

and necropsied. Organs were weighed and fixed in 10% 

neutral buffered formalin. Included among organs 

weighed were testis and ovary. Histopathological examinations were conducted in tissues from 10 rats/sex/ 

group in the control and high-dose group. Included 

among organs histologically examined were prostate, 

uterus, testis, and ovary. Statistical analyses included w2 

test with Yates correction, Fisher exact probability test, 

Mann–Whitney U-test, ANOVA, t-test, and Dunnett 

multiple comparison test. 

Fertility was unaffected in F0 rats. Body weight gain 

was lower in F0 rats from the 3000 and 9000 ppm groups. 

Body weight at Week 17 followed the same patterns as 

body weight gain [6–7% decrease in the 3000 ppm group 

and 12–18% decrease in the 9000 ppm group compared 

to controls]. There were no differences in food intake. 

[Statistical significance for body weight effects was not 

reported. It was not clear if statistical analyses were not 

conducted or if the effects did not attain statistical 

significance.] 

There were no effects on number of F1 pups/litter or 

survival of pups. Pup birth weights in the 9000 ppm 

group were slighter decreased but were said to be within 

normal range. Body weight gains on PND 21 were 

slightly decreased in pups from the 3000 and 9000 ppm 

dose groups. Body weights on PND 21 were significantly 

lower in pups from the 3000 and 9000 ppm groups [7 and 

12% lower compared to controls; benchmark dose 

analysis not conducted because variances not reported]. 

One male F1 rat in the control group and 2 female F1 rats 

in each of the 3000 and 9000 ppm group died during the 

study. Post-weaning body weight gain was lower in 

females from all dose group and in males from the 3000 

and 9000 ppm dose groups. Body weight at week 13 

followed the same patterns as body weight gain [13% 

decrease in the 1000 ppm group, 11–17% in the 

3000 ppm group, and 22–24% decrease in the 9000 ppm 

group compared to controls]. Food intake was decreased 

in females from all dose groups and in males from the 

9000 ppm group. Examination by ophthalmoscopy revealed no treatment-related effects. No treatment-related 

effects were observed for hematology, biochemistry, or 

urinalysis. No changes in organ weights or gross or 

histopathological lesions were considered treatmentrelated. The study authors noted increases in mean 

weights of spleen, brain, thyroid, and adrenals in the 

treated groups but concluded that the effects resulted 

from decreased body weight. [With the exception of 

PND 21 pup weights, there was no discussion of 

statistical significance for effects observed in F1 rats. It 

was not clear if statistical analyses were not conducted 

or if statistical significance was not attained.] 

Strengths/Weaknesses: This study is a conventional, 

state-of-the-art-at-the-time two-generation toxicity study. 

The inclusion of a breeding period and a second 

generation are strengths. Weaknesses are magnified in 

hindsight: these include the limited number of animals 

examined, the lack of close examination of the reproductive processes in the F1 animals, and uncertainty about 

the statistical significances. The study has not been peerreviewed. 

Utility (Adequacy) for CERHR Evaluation Process: 

Although this study was not designed to find non-linear 

dose–responses, it represents a conventional-for-the-time 

2-generation toxicity study, and is adequate for the 

evaluation process but of limited utility because the high 

doses preclude evaluation of low dose effects and limit 

its utility in showing a lack of marked organ toxicity or 

gross reproductive toxicity in a limited number of 

animals at very high-doses. 

The International Research and Development Corporation (General Electric, 1978), sponsored by General 

Electric, examined the effects of bisphenol A exposure on 

male and female CD rats and their offspring. In the first 

part of the experiment, male and female rats were 

housed in wire mesh cages and were fed Purina 

Laboratory Chow containing bisphenol A [purity not 

specified] for 18 weeks. Ten rats/group (body weights of 

135–179 g for males and 114–158 g for females) were 

assigned to each treatment group based on even 

distribution of body weight and litter mates. [Based on 

information provided in study tables, it appears that 

the rats were B30 days old at the start of dosing.] 

Bisphenol A was added to feed at concentrations of 0, 

100, 250, 500, 750, or 1000 ppm. The European Union 

(2003) estimated bisphenol A intake at 0, 5, 15, 30, 50, and 

60 mg/kg bw/day in males and 0, 10, 25, 50, 75, and 

100 mg/kg bw/day in females. Rats were examined for 

clinical signs, body weight gain, and food intake 

throughout the study. Estrous cyclicity was examined 

in females for 3 weeks before breeding and during 

breeding. At 100 days of age (Week 10 of the study), rats 

were moved to plastic cages with corncob bedding and 

mated for 3 weeks. GD 0 was defined as the day that 

vaginal sperm or plug was observed. Rats were assessed 

for fertility and gestation length. Day of delivery was 

designated lactation day 0 (PND 0). Pups were counted, 

sexed, and weighed, assessed for viability at birth and 

through the lactation period. After weaning, 15 male and 

female F1 rats/group that were exposed in utero were 

selected for a 90-day feeding study. Parental rats and 

unselected F1 rats were killed and discarded. 

During a 90-day period, F1 rats were fed diets 

containing the same concentration of bisphenol A as 

their parents. [Ages at the start of dosing were not 

reported, but based on body weight ranges reported 

(64–138 g for males and 57–118 g for females) it appears 

that rats were different ages at the start of dosing.] F1 

rats were weighed and observed for clinical signs. 

Hematological, clinical chemistry, and urinalysis 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 249 

parameters were examined at Day 30, 60, and 90 of the 

study. Ophthalmoscopic examinations were conducted 

before initiation of and following 90 days of dosing. The 

rats were killed and organs weighed. Adrenals, pituitary, 

ovaries, and thyroid were weighed following fixation in 

10% neutral buffered formalin. Histopathological examinations were conducted in tissues from 10 rats/sex/group 

in the control and high-dose groups. Organs histologically 

examined included prostate, uterus, testis, and ovary. 

Statistical analyses included w2 test with Yates correction, 

Fisher exact probability test, Mann–Whitney U-test, ANOVA, t-test, and Dunnett multiple comparison test. 

In parental rats, bisphenol A exposure did not affect 

general behavior, appearance, or survival. Mean body 

weight of males in the 1000 ppm group was 6% lower 

than control males. Food intake was increased [by B7– 

11%, no dose–response] in females of all dose groups. 

Bisphenol A exposure had no effect on estrous cyclicity 

or gestation length [data were not shown], male or 

female fertility, number of pups/litter, or pup survival 

Body weights of pups in the 750 ppm group were 

significantly higher [by B10%] compared to controls 

on PND 21, but the study authors did not consider the 

effect to be treatment-related. 

In the F1 offspring, a slight decrease in body weight 

gain was observed for males in the 750 ppm group. [At 

the end of the study, body weights of males in the 

750 ppm group were B7% less than controls]. Food 

intake was similar in treated and control groups. 

Ophthalmoscope examinations did not reveal any treatment-related effects. Although mean blood urea nitrogen 

levels were slightly lower and mean serum glutamicoxaloacetic transaminase values were sporadically increased in treated rats, the study authors noted that the 

values were within physiological ranges. There were no 

effects on hematological or urinalysis parameters. Some 

significant organ weight changes were noted by the 

study authors, but they stated that the biological 

significance of the effects was not known. [There did 

not appear to be dose–response relationships for any 

organ weight change.] The study authors stated that no 

compound-related lesions were observed in organs, 

including reproductive organs. 

Strengths/Weaknesses: The use of multiple dose levels 

(going down to fairly low exposure levels) is a plus, as is a 

breeding phase. Weaknesses include the limited number of 

animals per group, discarding of the parental animals 

without examination, the fact that not all F1 animals were 

examined at least for structural effects, the lack of close 

examination of F1 animals for reproductive effects (cyclicity 

and sperm measures), and the use of the conventional ‘‘topdown’’ pathology evaluation, wherein the lower dose 

groups were examined only if effects were noted in the 

high-dose. The study has not been peer-reviewed. 

Utility (Adequacy) for CERHR Evaluation Process: 

For what it is, this study is adequate and of limited 

utility for the evaluative process, showing no gross 

changes in the structure of a limited number of tissues in 

a limited number of F1 animals, exposed from preconception. This study was not designed to find unusual 

effects or non-linear dose–response relationships or to 

address the issue of low-dose functional responses or 

non-linear responses. 

Ema et al. (2001), supported by the Japanese Ministry 

of Health and Welfare, examined developmental toxicity 

endpoints, in a 2 generation rats study described in detail 

in Section 4.2.3.1. Two generations of rats were gavaged 

with 0, 0.0002, 0.002, 0.020, or 0.200 mg/kg bw/day 

bisphenol A (99.9% purity) before and during mating 

and throughout the gestation and lactation period. These 

doses were based on previous studies that found effects 

at 0.002 and 0.020 mg/kg bw/day. There were some nondose-related and sporadic effects, but the study authors 

concluded that none of the effects were related to 

bisphenol A treatment. Bisphenol A exposure did not 

adversely affect prenatal or postnatal growth or survival, 

developmental landmarks, anogenital distance, or age of 

puberty. In adult animals exposed to bisphenol A during 

development, there was no evidence of adverse effects 

on reproductive endpoints such as fertility, estrous 

cyclicity, or sperm counts. Prostate and other male 

reproductive organ weights were unaffected. 

Strengths/Weaknesses: Strengths of this study were 

the thoroughness of the evaluation, the size of the dose 

range, the large number of animals, the litter-based 

analysis, and the verification of the dosing solution. A 

minor weakness is the lack of a positive control group, 

which leaves a question about the ability of this group of 

rats to respond. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

Tyl et al. (2002b), supported by The Society of the 

Plastics Industry, Inc., reported some developmental 

toxicity effects in a multigeneration bisphenol A study 

in Sprague–Dawley rats that is reported in detail in 

Section 4.2.3.1. In that study, F1, F2, and F3 rats were 

exposed to bisphenol A [99.70%-99.76% pure] indirectly 

during gestation and lactation and directly through feed 

after weaning. Dietary doses were 0, 0.015, 0.3, 4.5, 75, 

750, or 7500 ppm, and target intakes were B0.001, 0.02, 

0.30, 5, 50, and 500 mg/kg bw/day. At the 7500 ppm dose 

there were fewer pups and live pups/litter and body 

weight gain of pups was lower during the lactation 

period. Delayed puberty in both males and females of the 

7500 ppm group was most likely related to reduced body 

weights according to the study authors. Bisphenol A 

exposure during development did not increase the 

weight of the prostate in adult rats. Although some 

decreases in epididymal sperm concentration and 

daily sperm endpoints were each observed in 1 generation of males from the high-dose group, the study authors 

concluded there were no treatment-related effects on 

sperm endpoints or reproductive function. The study 

authors identified an offspring and reproductive NOAEL 

of 750 ppm (B50 mg/kg bw/day). A systemic NOAEL 

for adult rats was identified at 75 ppm (B5 mg/kg bw/ 

day) by the study authors; therefore, bisphenol A was not 

considered a selective developmental toxicant. 

Strengths/Weaknesses: This study has numerous 

strengths, including the quality and number of the 

endpoints evaluated, the number of dose groups and 

generations examined, and the confirmation of dosing 

solutions. This study incorporated screening-level endpoints within the context of a multigeneration study. As 

such, it addresses gross issues but does provide helpful 

data regarding the NOAEL. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

Birth Defects Research (Part B) 83:157–395, 2008
250 CHAPIN ET AL. 

3.2.3.2 Development of the reproductive or endocrine 

systems: Cagen et al. (1999b), support not indicated 

(but all authors affiliated with industry), conducted a 

study to examine the effects of prenatal and lactational 

bisphenol A exposure on reproductive development of 

rats. The study attempted to replicate findings by Sharpe 

et al. that appeared in an unpublished meeting abstract. 

The protocol used by Cagen et al. (1999b) was the same 

as that used by Sharpe et al. with the exception that more 

dose levels were included, group sizes were larger, and a 

greater number of reproductive endpoints were examined. Animals were fed Certified Rodent Chow 5002. 

Music was played at a low volume to provide background noise. Female Han-Wistar rats were randomly 

assigned to groups. For 2 weeks before mating, during a 

2-week mating period, and during the gestation and 

lactation periods, 28 rats/group were given drinking 

water containing bisphenol A (499% purity) at 0.01, 0.1, 

1.0, or 10 ppm (0.001–0.004, 0.008–0.038, 0.100–0.391, or 

0.775–4.022 mg/kg bw/day). Two negative control 

groups of 28 rats each were given undosed drinking 

water. Because the two control groups were determined 

to be statistically equivalent, data from the two groups 

were pooled. A positive control group of 28 rats was 

given drinking water with diethylstilbestrol at 0.1 ppm 

(0.006–0.036 mg/kg bw/day). Dosing solutions were 

prepared weekly, and concentrations were verified. 

Dams were evaluated for food and water intake, weight 

gain, and fertility endpoints. Pups were sexed, weighed, 

and counted at birth. During the postnatal period, pups 

were evaluated for growth and survival. On PND 4, 

litters were culled to 8 pups with as many male pups 

retained as possible. At weaning on PND 22, up to 4 

males/litter (86–109 pups/group) were randomly selected to continue in the study until 90 days of age and 

were individually housed. At necropsy, brain, liver, 

kidneys, and reproductive organs were weighed, daily 

sperm production was determined, and testes were 

examined histologically. Technicians were blinded to 

treatment group. The litter was considered the experimental unit in statistical analyses. Data were analyzed by 

Levene test, ANOVA, Dunnett test, rank transformation, 

and Wilcoxon rank sum test with Bonferroni correction. 

In the bisphenol A groups, there were no significant 

effects on dam body weight gain or food or water intake. 

[Food and water intake data were not shown.] There 

were also no effects on dam fertility, mating, gestation 

index and duration, live litter size, or pup survival and 

body weight gain during the postnatal period. Male sex 

ratio was increased in the 0.1 ppm bisphenol A group 

(56.7% males vs. 48.4% in control), but the study authors 

did not consider the effect to be treatment-related due to 

the lack of a dose response relationship. Dams in the 

diethylstilbestrol group experienced decreased body 

weight gain and food intake, increased duration of 

gestation, smaller litter size at birth, and decreased pup 

survival in the postnatal period. 

In adult offspring from the bisphenol A groups, there 

were no significant effects on terminal body weight or 

organ weights including prostate, epididymis, preputial 

gland, seminal vesicle, or testis. There were also no 

significant effects on epididymal sperm concentration, 

efficiency of sperm production, or daily sperm production. No histopathological alterations were observed in 

the testis. Reproductive development in male offspring 

was also unaffected by prenatal exposure to diethylstilbestrol. The study authors noted that the reduced 

testis weight and sperm production reported by Sharpe 

et al. was not confirmed in this study and that bisphenol 

A should not be considered a selective reproductive or 

developmental toxicant. 

[The NTP Statistics Subpanel (NTP, 2001) concluded 

that the statistical methods used by Cagen et al. (1999b) 

were appropriate. Although the Subpanel agreed with 

the study author conclusions, 2 matters were noted. The 

first was that a significant ANOVA is not a requirement for Dunnett test. The second was that a 

Bonferroni correction of Wilcoxon-rank sum test was 

not needed because the authors already required 

significance by ANOVA, which was sufficient.] 

Strengths/Weaknesses: Significant strengths of this 

study include the large number of dose levels and 

animals per dose level and the technical care with which 

the study was performed, as well as the inclusion of a 

positive control group and two negative controls. The 

lack of much effect with diethylstilbestrol treatment is a 

weakness. 

Utility (Adequacy) for CERHR Evaluation Process: 

Although only weak effects were observed for the 

diethylstilbestrol positive control the panel considered 

this study adequate and of high utility. 

Elswick et al. (2000), from the Chemical Industry 

Institute of Toxicology [CIIT], examined the effects of 

sampling design on conclusions made about bisphenol A 

effects on prostate weight. Two of the 3 studies discussed 

in the study relate to bisphenol A research in Sprague– 

Dawley rats performed at CIIT between 1997–1999. One 

study is Kwon et al. (2000) which is discussed in detail in 

Section 3.2.3.3. The other study was unreferenced at the 

time and remains so. This section discussed the analysis 

of the unpublished study. In that study, the litter was 

considered the experimental unit in statistical analyses. 

Organ weights were analyzed using a nested ANOVA 

with litter within dose as the random effect. Post-hoc 

tests were conducted when appropriate. 

Dams were given drinking water containing 0, 0.005, 

0.05, 0.5, 5, or 50 mg/L bisphenol A [purity not 

indicated] from GD 2 to PND 21. The study authors 

estimated bisphenol A intakes at B0.001–10 mg/kg bw/ 

day. The lowest doses were reported to be similar to 

human exposure levels. The study was conducted in 2 

blocks separated by 4 months. A total of 16 dams/group 

were exposed, and the overall sample size was ultimately 

13–16/group. In the first block, 2 males/litter were most 

often retained and in the second block, 1 male/litter was 

retained until 6 months of age. Fresh ventral prostate 

weights were recorded. Analysis of data from the first 

study block revealed no treatment-related effects on 

ventral prostate weight. Within litters, ventral prostate 

weights were observed to be very variable, with weights 

sometimes differing by values of 2-fold or more. In the 

second study block, mean weights in the 0.05, 5, and 

50 mg/kg bw/day groups were significantly higher than 

those of the control group. It was noted that mean 

prostate weight in the control group from Block 2 

(0.387 g) was much lower than the mean weight observed 

in Block 1 (0.517 g) and that the standard error in Block 2 

(0.174 g) was almost two times higher than the standard 

error in Block 1 (0.092 g). When data from the 2 blocks 

were combined, statistical significance remained. The 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 251 

study authors noted that no historical control database 

was available at CIIT at the time of the analysis. 

[The NTP Statistics Subpanel (NTP, 2001) reanalyzed 

these data agreed with its results and conclusions 

showed a consistent increase in ventral prostate weight 

in the 2 replicates. Note that the NTP Statistics 

Subpanel rejected the conclusions in Elswick et al. 

(2000) that use of multiple pups per litter can decrease 

false positive rates in these studies.] 

Strengths/Weaknesses: This study demonstrated an 

increase in ventral prostate weight. These data argue for 

multiple pup/litter sampling, a characteristics that has 

been uncommon in this literature. The fact that significant effects were noted in only in 1 block raise the 

question of a lack of experience or training among the 

technicians. The study referred to in Elswick et al. (2000) 

is unpublished and not peer-reviewed. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate because it is primarily a 

discussion of results published previously and the new 

data presented have inconsistencies in block replicates. 

Rubin et al. (2001), supported by the Tufts Institute of 

the Environment and NIH, examined the effects of 

perinatal bisphenol A exposure on estrous cyclicity and 

LH levels in rats. Uterotropic responses were examined 

in a second group of rats, and those results are listed in 

Table 53. Sprague–Dawley rats were fed Purina Rodent 

Chow and provided drinking water in glass bottles. The 

rats were housed in plastic cages; estrogenicity testing of 

ethanol extracts indicated that estrogenic compounds did 

not leach from cages at detectable levels. [No information was provided about bedding.] Dams were weighed 

and randomly assigned to treatment groups of 6 animals 

given drinking water containing bisphenol A [purity not 

reported] at 0 (1% ethanol vehicle), 1, or 10 mg/L from 

GD 6 (plug day not indicated) through the lactation 

period. Mean bisphenol A doses were estimated by study 

authors at 0.1 and 1.2 mg/kg bw/day. At weaning, pups 

were given untreated water. Dams were examined and 

weighed during the studies. Offspring were sexed on 

PND 2 and weighed beginning in the postnatal period 

and continuing through adulthood (n 5 40–53/group 

during the neonatal period and 19–27/sex/group during 

adulthood). Anogenital distance was examined during 

the neonatal period. [It was not clear how many time 

points and animals were examined. According to 1 

study author, anogenital distance was measured on 

PND 2 (A. Soto, personal communication, March 2, 

2007).] Genital tracts were examined for gross abnormalities in males killed during the neonatal period, at 3 

months, and at 5 months of age and in females killed 

during the neonatal period, at 8 months, and at 12–16 

months of age. [The total number of animals examined 

at each time period was reported as 12–34, but it is not 

known how many/dose group were examined.] Animals 

were selected from as many different litters as possible at 

each time point. Day of vaginal opening was monitored. 

Estrous cyclicity was evaluated daily for 18 days at 4 and 

6 months of age in 18–28 rats/group. Eight female 

offspring/group were killed 3 months later following 

ovariectomy to measure serum LH levels using an LH 

assay kit; a total of 6–8 values/group were obtained. 

Body and uterine weights and LH levels were analyzed 

by ANOVA followed by t-test, Tukey test, or least 

significant difference test. Mammary tumors were 

analyzed by w2 test, and estrous cyclicity data were 

analyzed by Kruskall–Wallis test and Mann–Whitney Utest. [It appears that offspring were considered the 

statistical unit.] 

On PND 4, 7, and 11, body weights were significantly 

higher in pups from the bisphenol A groups than in the 

control group; body weights were higher in animals of 

the low compared to the high-dose group. Body weights 

of low-dose females were higher than body weights of 

control and high-dose females at PND 28 and beyond. 

Although the percentage of control females with regular 

estrous cycles was 83% at 4 months of age and 60% at 6 

months of age, the values were reduced significantly in 

the high-dose group to 21% at 4 months of age and 23% 

at 6 months of age. There were no clear patterns of 

estrous cycle changes. Periods of diestrus were extended 

in some animals and other animals had extended 

periods of proestrus and/or estrus. The mean number 

of 4–5-day estrous cycles was reduced significantly in 

rats of the high-dose group at 6 months of age. Serum 

LH levels in the high-dose group were reduced significantly by B19% compared to the control group 

[BMD10 5 0.94, BMDL10 5 0.48, BMD1 SD 5 1.6, and 

BMDL1 SD 5 0.78 mg/kg bw/day]. The treatment group 

incidences of females with mammary tumors (10% in 

controls, 20% in the low-dose group, and 28% in the 

high-dose group) were not statistically different. The 

study authors noted that the study was not designed to 

detect mammary tumors and that the tumors were 

detected during routine handling. No effects were 

reported for mean number of pups/litter, sex ratio, day 

of vaginal opening, or anogenital distance in the neonatal 

period. [Data were not shown for anogenital distance.] 

In comparing the effects on estrous cycles and LH levels 

in animals exposed in the perinatal period to the lack of 

uterotropic effects in animals exposed in the postpubertal period, the study authors concluded that there 

was evidence of increased sensitivity to bisphenol A 

during the perinatal period. 

Strengths/Weaknesses: This study incorporates a 

range of basic developmental and gross functional 

reproductive endpoints, but the sample sizes are small 

(6 dams/group) and the statistical approach does not 

appear to use litter as the unit. Actual exposures are 

poorly defined, particularly postnatally. The plausibility 

of the estrous cycle changes is a strength. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process, 

based on a lack of adequate control for litter effects 

Takashima et al. (2001), supported by a Grant-in-Aid 

for Health Sciences Research [sponsor not indicated], 

examined the effect of bisphenol A exposure during 

development on carcinogenicity induced by N-nitrosobis 

(2-hydroxypropyl)amine. [No information was provided 

about caging and bedding materials used in this study.] 

Female Wistar rats were fed either MF diet or soybeandevoid powder diet (Oriental Yeast Co.). In each dietary 

group, 10–11 rats/group received bisphenol A [purity 

not indicated] at 0 or 1.0% diet. Bisphenol A exposure 

commenced 10 weeks before mating and was continued 

through the mating, gestation, and lactation periods. 

Total intakes of bisphenol A were reported at 21–22 g/rat. 

[Assuming an exposure period of B16 weeks, mean 

bisphenol A intake over the course of the study was 

estimated at B200 mg/day. Based on reported body 

Birth Defects Research (Part B) 83:157–395, 2008
252 CHAPIN ET AL. 

weights, bisphenol A intake was B1600 mg/kg bw/day 

during the prebreeding stage and 1000 mg/kg bw/day 

during gestation and at weaning.] The rats were mated 

to males fed CE-2 basal pellet diet (Clea, Inc.), and GD 0 

was defined as the day of the vaginal plug. Endpoints 

associated with pregnancy, delivery, and nursing were 

evaluated. Dam body weight and food intake were 

measured. Offspring were not culled and were weaned 

at 3 weeks of age. Dams were killed following weaning of 

offspring. Serum levels of thyroid hormones were 

measured in 2–4 dams/group. Implantation sites were 

evaluated. Weights of several organs, including ovary, 

were measured. The organs were fixed in 10% buffered 

formalin and processed for histopathological evaluation. 

Offspring (n 5 32–50/group) were evaluated for body 

weight gain, preputial separation, and vaginal opening. 

Beginning at 5 weeks of age and continuing for 12 weeks, 

offspring in each group were subdivided into 2 groups 

(n 5 17–21/group/sex) that received either undosed tap 

water or tap water containing 2000 ppm N-nitrosobis (2­

hydroxypropyl)amine. Offspring were killed at 25 weeks 

of age. Serum thyroid hormone levels were measured. 

Organs, including testis, ovary, and uterus were 

weighed. In 5–19 offspring/sex/group, histopathological 

examinations were conducted in organs targeted by Nnitrosobis (2-hydroxypropyl)amine (lungs, thyroid, esophagus, liver, and thymus). Data were analyzed by 

Dunnett and w2 tests. [Data for pre-and postnatal 

survival were presented and apparently analyzed on a 

litter basis. The offspring were apparently used as the 

statistical unit in body weight analyses. It was not clear 

if the dam or offspring were considered the statistical 

unit in other analyses.] 

Dam body weight was lower in the 1.0% bisphenol A 

group fed MF diet compared to the MF diet control 

during the gestation period and at weaning. Food intake 

and maternal serum levels of triiodothyronine, thyroxine, 

and thyroid-stimulating hormone were unaffected by 

bisphenol A exposure. Changes in weights or histopathological alterations of maternal organs, including 

uterus and ovary, were not observed in the bisphenol A 

groups. [Data were not shown by the study authors.] 

Bisphenol A had no significant effect on mating, fertility, 

duration of gestation, live-born pups, implantation loss, 

or offspring viability through PND 21. In pups from 

dams exposed to 1.0% bisphenol A fed MF diet 

compared to pups from MF controls, body weights were 

higher [by 11%] in females at 3 days of age and lower 

in males and females at 10 days and 2 weeks of age [16– 

22% decreases in males and 12–19% decreases in 

females]. In pups from dams exposed to 1.0% bisphenol 

A and fed soybean-free diet compared to pups from the 

soybean-free controls, body weights of pups were 

increased in males at 3 weeks of age [13% increase] 

and in females at 10 days and 3 weeks of age [13–19% 

increase]. Prenatal exposure to bisphenol A did not 

affect preputial separation or vaginal opening. In 25­

week-old rats that were not exposed to N-nitrosobis (2­

hydroxypropyl)amine, prenatal bisphenol A exposure 

was associated with some thyroid-stimulating hormone 

elevations in males and females from the MF and 

soybean-free diet groups. According to a statement in 

the study abstract, the study authors did not consider the 

effect on thyroid-stimulating hormone to be related to 

bisphenol A exposure. There were no effects of Nnitrosobis (2-hydroxypropyl)amine exposure on serum 

thyroid-stimulating hormone, triiodothyronine, or thyroxin levels or on thyroid histopathology. No effects were 

observed on offspring organ weights. [With the exception of uterus and ovary, no organ weight data were 

shown.] Prenatal bisphenol A exposure was not associated with significant differences in the development of 

N-nitrosobis (2-hydroxypropyl)amine-induced neoplasms in the offspring. The study authors concluded 

that bisphenol A exposure did not induce tissue injury in 

rat dams or their offspring or affect the development of 

tumors in offspring exposed to N-nitrosobis (2­

hydroxypropyl)amine. 

Strengths/Weaknesses: Weaknesses include highdoses and inadequate sample sizes. This study appears 

to discount the importance of certain effects on body 

weight and thyroid-stimulating hormone levels that 

might have received more attention in a study with a 

non-tumor focus. Sample size is inadequate to address 

neoplasm endpoints. Information is insufficient to judge 

the appropriateness of the statistical analyses and hence 

the reliability of findings. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process due 

to small sample size, high-dose levels, and inappropriate 

statistics. 

Kobayashi et al. (2002), supported by the Japanese 

Ministry of the Environment, examined the effect of 

prenatal and lactational bisphenol A exposure on somatic 

growth and anogenital distance in Sprague–Dawley rats. 

The same rats were used to measure plasma hormone 

levels and testicular testosterone content in a study by 

Watanabe et al. (2003) and apparently thyroid function in 

a study by Koybayashi et al. (2005). Rats were fed 

standard laboratory feed (CE-2, CLEA Japan, Inc.). [No 

information was provided about caging or bedding 

materials.] Rats were randomly assigned to groups and 6 

rats/group were gavaged with bisphenol A (99.8% 

purity) at 0 (corn oil vehicle), 4, 40, or 400 mg/kg bw/ 

day from GD 6 through PND 20. GD 0 was defined as the 

day a vaginal plug was observed, but the day of birth 

was not defined. Doses were based on the study by 

Kwon et al. (2000) [discussed in Section 3.2.3.3]. On 

PND 7, litters were culled to 10 pups, with equal 

numbers of males and females when possible. Offspring 

were weaned on PND 21. Dams were weighed during 

the study. Body weight and anogenital distance were 

measured in offspring at 1, 3, and 9 weeks of age. Plasma 

and testicular testosterone levels were measured at 9 and 

36 weeks of age, and plasma LH and FSH concentration 

were measured at 9 weeks of age Weights of liver, kidney, 

and testis were examined in offspring at 3 and 9 weeks of 

age. One to 10 (most often 6–10) offspring/group/sex 

were examined for body weight and anogenital distance 

at 1 week of age and 4–6/sex/group at 3 and 9 weeks of 

age. A pair of male and female offspring/litter [assuming authors meant 1/sex/litter] was examined for organ 

weights, and 4–6 males/group were used in hormone 

analyses at 3 and 9 weeks of age. Statistical analyses 

included ANOVA followed by Dunnett test. [It was 

not clear if the dam or litter was considered the 

statistical unit.] 

In the 40 mg/kg bw/day group, all pups from 1 dam 

were found dead on PND 2. Four of 6 dams of the 

400 mg/kg bw/day group died on GD 21, and all pups 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 253 

born to 1 dam in that group died by PND 2. Maternal 

body weight gain during pregnancy was reduced in the 

400 mg/kg bw/day group. A transient decrease in body 

weight gain was observed early in the lactation period in 

dams of the 40 mg/kg bw/day group. In offspring from 

the 4 and 40 mg/kg bw/day group, no statistically 

significant effects were observed for body or organ 

weights, anogenital distance, anogenital distance/g body 

weight, or anogenital distance/body weight cubed at any 

time point in the study. At 9 weeks of age, plasma 

testosterone levels were significantly increased by 88% in 

the 4 mg/kg bw/day group and by 123% in the 40 mg/ 

kg bw/day group. [Benchmark dose was not calculated 

because the SD was provided only graphically.] The 

study authors stated that there was a tendency for 

plasma testosterone to increase with dose at 36 weeks, 

but neither of the values were significantly increased 

compared to control. Testis testosterone was not statistically different from control at either dose at 9 or 36 

weeks of age. There were no significant effects on plasma 

LH and FSH levels at 9 weeks of age. Plasma levels of 

17b-estradiol were also unaffected by bisphenol A 

exposure. [Data were not shown.] The study authors 

concluded that gestational and lactational exposure to 

bisphenol A did not affect somatic growth or anogenital 

distance but did have a significant effect on testosterone 

homeostasis in rat offspring. 

Strengths/Weaknesses: The study appears better able 

to address maternal toxicity than offspring outcomes, for 

which it appears to be best considered a screening study. 

Sample sizes are too small to reliably judge postnatal 

endpoints. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation of bisphenol 

A effects on postnatal outcome. 

Yoshino et al. (2002), supported by the Japanese 

Ministry of Health, Labor, and Welfare, examined the 

effects of prenatal and lactational bisphenol A exposure 

in the prostate and testis of rats. In this study, pregnant 

and lactating dams were fed nMF feed and offspring 

were fed MF feed (Oriental Yeast Co., Tokyo). The 

animals were housed in an unspecified type of cage 

containing wood chip bedding. F344 rat dams (n 5 19– 

22/group) were gavaged with bisphenol A (99.9% 

purity) at 0 (0.5% sodium carboxymethylcellulose vehicle), 7.5, or 120 mg/kg bw/day during mating, gestation, 

and lactation periods. Doses were based on the result of 

an NTP study (NTP, 1982). Clinical signs, food intake, 

and body weight were monitored in dams during the 

study. After birth, pups were counted and weighed. Pups 

were randomly culled to 8/litter on PND 4 (day of birth 

not defined). On PND 21, weaning occurred and female 

pups were killed and discarded. Dams were killed at 

weaning for examination of implantation sites. Male 

pups were weighed during the post-weaning period. On 

PND 23, 28, and 91, five male offspring/group were 

killed. Ventral prostate weights were measured during 

each evaluation period, and anterior and dorsolateral 

prostate, testis, and epididymis weight were also 

measured on PND 91. Reproductive organs were 

preserved in 10% buffered formalin and examined 

histologically. Sperm count, motility, and morphology 

were examined on PND 91. The study was repeated with 

evaluation of 10 male offspring/group. [The number of 

dams treated/group in the repeat study was not 

reported. Based on body weights reported for rats in 

Experiment 2, it appears they were evaluated at 

adulthood, but it was not specified if they were 

evaluated on PND 91.] Data were analyzed by Student 

t-test. [It appears that offspring were considered the 

statistical unit.] 

In the first experiment, bisphenol A exposure had no 

effect on dam body weights during gestation or lactation, 

duration of the gestation period, or number of implantation sites. There were no effects on litter size, pup 

viability, or sex ratio. On PND 21, relative dorsolateral 

prostate weight was significantly higher [by 23%] in the 

low-dose group than in controls. [It was not stated if 

organ weights were relative to body weight.] There 

were no effects on final body weight or weights of 

anterior and ventral prostate, testis, or epididymis. There 

were no increases in malformations of reproductive 

organs. [Data were not shown by study authors.] 

Testicular sperm counts were significantly lower [by 

22%] in males of the high-dose group, but there were no 

effects on epididymal sperm counts. There were also no 

effects on sperm motility or abnormalities. [Data were 

not shown by authors.] In the second experiment 

examining 10 males/group, exposure to bisphenol A 

had no effects on final body weights or relative weights 

of testis, epididymis, or ventral, anterior, or dorsolateral 

prostate. There were no adverse effects on testicular or 

epididymal sperm count, motility, or morphology. 

Morphologically abnormal sperm were reduced in rats 

of the low-dose group. Study authors concluded that 

under the conditions of their study, exposure of dams to 

bisphenol A during the gestation and lactation periods 

did not result in adverse effect on the reproductive 

system of male offspring. 

Strengths/Weaknesses: The number of dams used in 

Experiment 1 appears adequate and 10 males/group 

were used to examine various organ endpoints at 

multiple time points. It is unfortunate that these data 

were then analyzed by many t-tests rather than multivariate analyses. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is considered inadequate due to statistical 

insufficiencies. 

Ichihara et al. (2003), supported by the Japanese 

Ministry of Health, Labor, and Welfare, examined the 

effects of prenatal and lactational exposure to bisphenol 

A on the development of prostate cancer in rats. F344 rat 

dams were fed nMF feed during pregnancy and lactation 

and their offspring were fed MF (Oriental Yeast Co.) 

following weaning. Rats were housed in cages containing 

wood chip bedding. [No information was provided 

about caging materials.] During pregnancy and lactation, B8–15 dams/group were gavaged with bisphenol 

A (99.9% purity) at 0 (0.5% carboxymethyl cellulose 

sodium salt vehicle), 0.05, 7.5, 30, or 120 mg/kg bw/day. 

Doses were based on findings from an NTP study 

[citation not provided]. Dam body weight and food 

intake were monitored during the study. Gestation 

period duration and implantation sites were evaluated. 

Pups were counted and sexed at birth. Litters were culled 

randomly to 8 pups on PND 4, and pups were weaned 

on PND 21 [day of birth not defined]. At 5 weeks of age, 

21 male rats/group were injected s.c. with 50 mg/kg bw 

3,2-dimethyl-4-aminobiphenyl 10 times at 2-week intervals. An additional 12 rats/group in the 0, 0.05, 7.5, and 

Birth Defects Research (Part B) 83:157–395, 2008
254 CHAPIN ET AL. 

120 mg/kg bw/day bisphenol A groups were injected 

with corn oil during the same time period. Surviving 

male offspring were killed and necropsied at 65 weeks of 

age. Blood was collected for analysis of serum testosterone levels in 5 rats/group. Reproductive organs were 

examined for gross abnormalities, weighed, and fixed in 

10% buffered formalin. A histopathological examination 

of the prostate was conducted. Body and organ weight 

data were analyzed by Student t-test. The incidence of 

histopathological lesions was evaluated by Fisher exact 

probability test. [It appears that the litter was considered the statistical unit in analyses for numbers and 

survival of pups at birth. Offspring were apparently 

considered the statistical unit for other analyses.] 

Body weights of dams in the 120 mg/kg bw/day 

group were significantly lower than control values from 

GD 14–20. There were no consistent or dose-related 

effects on dam body weights during lactation, although a 

significant increase in body weight was observed in 

dams of the 0.05 mg/kg bw/day group on PND 14. 

Exposure to bisphenol A had no effect on gestation 

period duration or number of implantation sites. In pups 

exposed to bisphenol A, there were no differences in 

number of live births, sex ratio, external anomalies, or 

body weights during the lactation period. [Data for pup 

body weights were not shown by study authors.] 

Terminal body weight of pups exposed to 0.05 mg/kg 

bw/day bisphenol A before treatment with 3,2-dimethyl4-aminobiphenyl were significantly higher than controls 

[by 12%]. Exposure to bisphenol A had no effect on 

weights of prostate, testis, or epididymis. Incidences of 

prostatic intraepithelial neoplasia, carcinoma, and atypical hyperplasia were not increased by bisphenol A 

treatment, and there were no increases in tumors found 

in non-reproductive organs. No effect was observed on 

serum testosterone levels. The study authors concluded 

that exposure of rat dams to bisphenol A during the 

gestation and lactation periods does not predispose their 

offspring to prostate cancer development. 

Strengths/Weaknesses: Strengths are the range of low 

dose levels, the use of an additional strain (Fischer 344 

rat), and the endpoints evaluated. The design is reasonable for some of the endpoints measured, but sample 

sizes are inadequate for the prostate cancer endpoint and 

hormonal endpoints in particular. Statistical accounting 

for litter effects is unclear for neonatal measures, body 

weight, and fertility endpoints. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate based on insufficient sample 

size given the endpoints (i.e., tumors response) and lack 

of consistently accounting for litter effects. 

Yoshida et al. (2004), supported by the Japanese 

Ministry of Health, Labor, and Welfare, examined the 

effects of bisphenol A exposure on development of the 

rat female reproductive tract. Donryu rats (12–19/group) 

were gavaged with bisphenol A [purity not reported] at 

0 (carboxymethylcellulose solution), 0.006, or 6 mg/kg 

bw/day from GD 2 to the day before weaning of pups at 

21 days post delivery. The low dose was said to represent 

average daily intake from canned foods and the highdose was reported to represent the maximum dose level 

detected in plastic plates for children. [It is assumed the 

authors meant estimated exposure levels for children 

eating off plastic plates.] Bisphenol A levels were 

measured in maternal and pup tissues, and those values 

are reported in Section 2.1.2.2.1. After delivery, dams and 

litters were housed in plastic cages with wood chip 

bedding. Tap water was stored in plastic containers. The 

only information provided about feed was that it was a 

commercial pellet diet. Samples of tap water, drinking 

water from plastic containers, and feed were measured 

for bisphenol A content by HPLC. Offspring were sexed, 

weighed, and examined for external abnormalities on 

PND 1 and then weighed weekly through PND 21. 

Litters were adjusted to 8–10 pups at PND 4 or 6 (day of 

birth 5 PND 0). Dams were weighed, and observed 

during the study and killed following weaning of litters 

on PND 21. Implantation sites were examined and 

organs including uterus, vagina, and ovaries were fixed 

in 10% neutral buffered formalin and examined histologically. [It does not appear that results of histopathological testing in dams were reported.] All female 

offspring were examined for vaginal opening, and 

following vaginal opening, vaginal smears were taken 

for the remainder of the study. Three to 5 offspring/ 

group from different litters were killed on PND 10, 14, 

21, or 28 and at 8 weeks of age. At most time 

periods, uteri were weighed, preserved in 10% neutral 

buffered formalin, and examined histopathologically to 

determine development of uterine glands. Ovaries and 

vagina were also examined histologically. ERa was 

determined using an immunohistochemical method. 

Serum was collected for measurement of FSH and LH 

by RIA. Four offspring/group from different litters were 

killed at 8 weeks of age on the morning of estrus to 

examine ovulation by counting ova in oviducts. Initiation 

of carcinogenesis following injection of the uterine 

horn with N-ethyl-N0

-nitro-nitrosoguanidine was examined at 11 weeks of age in 35 or 36 animals/group. 

At B24 weeks following cancer initiation, the 24–30 

surviving animals/group were killed and uteri were 

examined histologically to determine the presence of 

tumors and other lesions. Statistical analyses included 

ANOVA and Dunnett test. [Most of the data for 

endpoints evaluated at birth appeared to be presented 

and apparently analyzed on a litter basis. For other 

data, it appears that offspring were considered the 

statistical unit.] 

Bisphenol A was not detected in fresh tap water but 

was detected at B3 ng/mL following storage in plastic 

containers. The bisphenol A concentration in feed was 

B40 ng/g. In dams exposed to bisphenol A, there 

were no clinical signs of toxicity or effects on body 

weight, implantation sites, or gestation length. Bisphenol 

A exposure had no effect on litter size, pup body 

weight at birth and through PND 21, external abnormalities in pups or age of vaginal opening. In uteri of 

bisphenol A-exposed offspring, there were no effects on 

weight, gland development, ERa, or cell proliferation. 

No increase in lesions was reported in organs of the 

alimentary, urinary, respiratory, or nervous system. 

[Data were not shown by study authors.] Bisphenol A 

exposure had no effect on ovulation, estrous cyclicity, 

or serum FSH or LH levels. There were no effects on 

uterine preneoplastic or neoplastic lesions or ovarian 

histopathology following bisphenol A treatment. 

The study authors concluded that perinatal exposure 

to bisphenol A at levels comparable to human 

exposure did not affect the reproductive system of 

female rats. 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 255 

Strengths/Weaknesses: Strengths of this study were 

the bisphenol A determinations that were made and the 

anchoring of animal exposure levels to human exposures. The design appears sound with a good range of 

endpoints measured. Small numbers of animals were 

sacrificed at several time points and cellular analyses 

were performed; these numbers were too small for a 

definitive cancer evaluation and were, in fact, too small 

for definitive conclusions to be reached for most of the 

adult reproductive endpoints. Statistics are not described 

in enough detail to determine how data from multiple 

sampling points were evaluated. This experiment represents a thorough screening study. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate based on insufficient sample 

size (3–5/group). 

Takagi et al. (2004), supported by the Japanese 

Ministry of Health, Labor, and Welfare, examined the 

effect of perinatal bisphenol A exposure on the reproductive and endocrine systems of rats. Nonylphenol was 

also examined but will not be discussed. Sprague– 

Dawley rat dams were fed a soy-free diet (Oriental Yeast 

Co.) prepared according to the formula for NIH-07. At 

weaning, the offspring were fed CRF-1 diet (Oriental 

Yeast Co.), which contains soybean and alfalfa-derived 

proteins. Rats were housed in polycarbonate cages 

containing wood chip bedding. Dams were randomly 

assigned to groups, and 5–6 dams/group were fed diets 

containing bisphenol A (96.5% purity) at 0, 60, 600, or 

3000 ppm from GD 15 (GD 0 5 day of vaginal plug) to 

PND10 (PND 1 5 day of birth). The study authors 

estimated bisphenol A intake at B5, 49, and 232 mg/kg 

bw/day during the gestation period and B9, 80, and 

384 mg/kg bw/day during the lactation period. Dose 

levels were based on results of preliminary studies, and 

selected with a goal of achieving weak to moderate 

toxicity in dams at the highest dose. In a separate study, 

rats were fed diets containing ethinyl estradiol at 0 or 

0.5 ppm from GD 15 to PND 10. On PND 2, offspring 

were counted, sexed, and weighed and anogenital 

distance was measured. Litters were culled to 6 pups 

on PND 10, and pups were weaned on PND 21. Five 

pups/sex/group (1/sex/litter) were selected for necropsy on PND 21 and brain, adrenals, testis, ovary, 

and uterus were weighed. Eight offspring/sex/group (at 

least 1/sex/litter) were selected for evaluation in adulthood, and these rats were observed for age and body 

weight at puberty. Estrous cyclicity was observed from 8– 

11 weeks of age. Offspring were killed at 11 weeks of age, 

on the day of diestrus for cycling female rats. Brain, 

pituitary, thyroid, adrenal mammary gland, epididymis, 

prostate, seminal vesicles, ovary, uterus, and vagina were 

weighed and examined histologically. The testis was 

fixed in Bouin solution, and other organs were fixed in 

10% neutral buffered formalin. The volume of the 

sexually dimorphic nucleus of the preoptic area (SDNPOA) was measured. It appears that endpoints were 

assessed in 8 adult rats/sex/group, with the exception of 

histopathological evaluations, which were conducted in 5 

rats/sex/group. The litter was considered the experimental unit in statistical analyses of data from PND 21 

offspring, and the individual animal was considered the 

statistical unit for data obtained from adult offspring. 

Homogenous numerical data were analyzed by ANOVA 

and Dunnett test, and heterogeneous numerical data 

were analyzed by Kruskall–Wallis H-test and Dunnetttype rank sum test. Data for histopathological lesions and 

vaginal cyclicity were analyzed by Fisher exact probability test or Mann–Whitney U-test. 

Maternal body weight gain was significantly decreased the high-dose bisphenol A group during gestation, but there were no effects on body weight gain 

during lactation or food intake. In offspring evaluated on 

PND 2, there were significant decreases in body weight 

in low- and high-dose males [13 and 22%] and in highdose females [20%], but there were no effects on number 

of live offspring or anogenital distance. Body weight gain 

was lower in high-dose males [21%] and females [29%] 

from PND 2–10. Increased relative brain weight as a 

result of growth retardation was reported in high-dose 

offspring evaluated on PND 21. [Data were not shown 

by study authors.] Exposure to bisphenol A did not 

affect onset of vaginal opening, preputial separation, or 

estrous cyclicity. Body weight of males was significantly 

lower [by 9.3%] at adult necropsy. Weights and histopathology of brain, pituitary, thyroid, adrenal mammary 

gland, epididymis, prostate, seminal vesicles, ovary 

uterus, and vagina in adulthood were unaffected in rats 

from the bisphenol A group. [Organ weight data were 

not shown by study authors.] Bisphenol A did not affect 

SDN-POA volume. Effects observed in offspring from 

the ethinyl estradiol study included reduced numbers of 

live offspring, increased male:female ratio, decreased 

body weight and body weight gain, accelerated vaginal 

opening, delayed preputial separation, increased estrous 

cycle irregularities, and histopathological alterations in 

pituitary, ovary, uterus, vagina, and mammary gland. 

The study authors concluded that bisphenol A did not 

affect endocrine or reproductive system development of 

rats at doses that induced maternal toxicity. 

Strengths/Weaknesses: Strengths include the range of 

doses and endpoints measured and the use of the ethinyl 

estradiol comparator group. The study used small 

sample sizes of dams (n 5 5–6/group) and inadequate 

statistical procedures to control for litter effects. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is considered inadequate for the evaluative 

process, based on sample size and statistical procedures. 

Akingbemi et al. (2004), supported by NIEHS, USEPA, 

NICHHD, and NIH, conducted a series of studies in 

Long–Evans rats to determine the effects of postweaning 

and perinatal exposure to bisphenol A on testicular 

steroidogenesis. In vitro studies were also conducted and 

are described in Section 4 because cells used in the 

studies were obtained from adult animals. Rats were fed 

Purina chow, which contains soybean meal, and given 

drinking water in polycarbonate bottles. Pregnant and 

nursing dams were housed in polycarbonate cages lined 

with wood bedding, but no information was provided on 

caging used at the other life stages. To reduce leaching of 

bisphenol A, the cages were washed, rinsed, and dried at 

least twice/week and were discarded once they began 

getting cloudy; water bottles were cleaned daily. Corn oil 

vehicle was used for bisphenol A and was administered 

to control animals. Rats were stratified according to body 

weight and randomly assigned to treatment groups. RIA 

methods were used to measure steroid hormone concentrations in serum or testicular fluid. RT/PCR methods 

were used to examine changes in mRNA expression. 

Statistical analyses included ANOVA with multiple 

Birth Defects Research (Part B) 83:157–395, 2008
256 CHAPIN ET AL. 

comparisons conducted by the Duncan multiple range 

test. [In the part of the study in which dams were 

dosed, it appears that offspring were considered the 

statistical unit.] 

In the first study, rats were gavaged with bisphenol A 

[purity not given] at 0, 0.0024, 0.010, 100, or 200 mg/kg 

bw/day from PND 21–35. The two lowest doses were 

selected to represent environmental exposures, and the 

higher doses were selected to compare the effects 

between low and high-doses. Rats were killed at the 

end of treatment and blood was collected for measurement of serum LH, testosterone, and 17b-estradiol levels. 

Leydig cell cultures were prepared for measurement of 

ex vivo testosterone production with and without the 

addition of LH, testosterone precursors, or metabolizing 

enzymes. Additional weanling rats were exposed to 

bisphenol A at 0 or 0.0024 mg/kg bw/day on PND 21–35. 

At the end of treatment, mRNA for LHb, ERb, and ERa

was measured in pituitary using an RT-PCR technique. 

All endpoints were reported for 7–12 rats/group. 

Compared to rats in the control group, rats exposed to 

bisphenol A at 0.0024 mg/kg bw/day had significantly 

lower levels of serum LH [by 62%] and testosterone [by 

40%]. Serum 17b-estradiol levels were decreased in rats 

exposed to 0.0024, 0.010, and 100 mg/kg bw/day bisphenol A [by B30, 40, and 25% in each respective dose 

group]. There were no effects on basal ex vivo 

testosterone production by Leydig cells. In Leydig cells 

obtained from rats exposed to 0.0024 mg/kg bw/day 

bisphenol A, testosterone production was significantly 

reduced when cells were exposed to LH or CYP450 17ahydroxylase/17–20 lyase. In Leydig cells obtained from 

rats exposed to 0.0024 or 0.010 mg/kg bw/day bisphenol 

A, testosterone production was significantly reduced 

following exposure of the cells to pregnenolone or 

progesterone. No effects on blood hormone levels or ex 

vivo testosterone production were observed at higher 

doses. Significant effects observed in pituitaries obtained 

from rats exposed to 0.0024 mg/kg bw/day bisphenol A 

were decreased LHb mRNA and increased ERb mRNA. 

The study authors concluded that the decreased serum 

LH level resulted from bisphenol A effects on the 

pituitary and that decreased LH stimulation of Leydig 

cells was the cause of reduced serum testosterone levels. 

In the second experiment, 7 dams/group were 

gavaged with bisphenol A at 0 or 0.0024 mg/kg bw/ 

day from GD 12–PND 21. Male offspring received no 

further treatment following weaning. Males were randomly selected from each dam and killed on PND 90. 

Endpoints evaluated in 10–12 male offspring/group 

included serum LH and testosterone levels, ex vivo 

testosterone production by Leydig cells, testosterone 

levels in testicular interstitial fluid, and seminal vesicle 

and prostate weight. Significant (Po0.01 or 0.05) effects 

observed in 90-day-old males that had been perinatally 

exposed to bisphenol A compared to the control group 

included increased body weight [10%], decreased relative weight (to body weight) of paired testes [17%] and 

seminal vesicles [17%], reduced testicular testosterone 

level [B39%], and reduced basal and LH-induced ex 

vivo testosterone production. 

In the third experiment, 10–12 rats/group were 

gavaged with bisphenol A at 0 or 0.0024 mg/kg bw/ 

day from PND 21–90. Within 24 hr following treatment, 

rats were killed and examined for the same endpoints 

described for the second experiment. Significant (Po0.01 

or 0.05) effects compared to the control group included 

increased serum LH level [117%], decreased seminal 

vesicles weight [absolute: 15%, relative: 16%], reduced 

testicular testosterone level [B24%], and decreased basal 

and LH-induced ex vivo testosterone production. For the 

second and third experiments, the study authors concluded that bisphenol A exposure inhibits androgen 

production by Leydig cells. 

Strengths/Weaknesses: Significant strengths of this 

report were the sequential nature of the work, in which 

later studies built on the previous data, and the clear 

expertise that the authors brought to this endeavor. 

Experiment 1 provided a helpful examination of postnatal effects following adolescent exposure and examined hormonal levels under stimulated and unstimulated 

conditions, thus separating pituitary from target organ 

contributions to serum levels. In Experiment 2, the 

sample size of 7 dams/prenatal treatment group and 

the examination of 10–12 offspring/group raise questions about the adequacy of the sample size with respect 

to the number of litters represented and the number of 

offspring used to represent each litter. In Experiment 3, 

10–12 rats/group were treated from PND 21–90 (through 

adolescence and into early adulthood) and then examined according to endpoints common to Experiments 1 

and 2. Weaknesses include an inadequate number of 

animals to obtain confidence about the hormonal 

changes (indeed, LH was decreased in the first experiment and increased in the third), the lack of histopathology evaluation, and lack of an estrogenic positive control. 

Utility (Adequacy) for CERHR Evaluation Process: 

Experiments 1 and 3 are adequate but of limited utility 

because of the mechanistic nature of the endpoints 

examined. Experiment 2 is inadequate for consideration 

due to inappropriate statistics that failed to account for 

litter effects. 

Masutomi et al. (2004), supported by the Japanese 

Ministry of Health, Labour, and Welfare, examined the 

potential effects in rats of neonatal bisphenol A exposure 

through maternal dietary intake on the number of 

offspring pituitary cells positive for LH, FSH, and 

prolactin. The authors exposed 5–8 pregnant CD(SG)IGS 

dams from GD 15–PND 10 to soy-free diet containing: (1) 

genistein 20, 200, or 1000 ppm; (2) diisononyl phthalate 

400, 4000, or 20,000 ppm; (3) methoxyclor 24, 240, or 

1200 ppm; (4) 4-nonylphenol 60, 600, or 3000 ppm; or (5) 

bisphenol A [96.5% purity] 60, 600, or 3000 ppm. Ethinyl 

estradiol at 0.5 ppm was also administered to a positive 

control group and the regular soy-free diet to a control 

group. [Only the bisphenol A-treated group will be 

considered here. Feed consumption and dam body 

weight were not reported, but would be expected to 

have changed dramatically over the treatment period, 

making it difficult to estimate the bisphenol A doses 

received by the rats.] After weaning, offspring were 

placed on CRF-1 rodent chow. Animals were housed in 

polycarbonate cages with wood-chip bedding. 

During postnatal week 3 or 11, offspring were killed 

and anterior pituitary glands from 5 male and 5 female 

offspring/group were harvested. Immunohistochemistry 

using paraffin-embedded sections for LH, FSH, and 

prolactin was conducted and the percentage of cells 

positive for LH, FSH, and prolactin was determined in 2 

sections/gland. Statistical analyses were performed by 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 257 

Student or Welch’s t-test using values from the highest 

bisphenol A dose group and the control. There was no 

effect of bisphenol A treatment on relative pituitary 

weight or on cell counts for LH, FSH, or prolactin. There 

was an increase in cells staining for prolactin in female 

offspring from the ethinyl estradiol-treated dams at 3 

weeks. 

Strengths/Weaknesses: This hypothesis-driven study 

was carefully designed with respect to exposure during 

established periods relevant to the sexual differentiation 

of the brain and with respect to assessment of appropriate parameters related to reproductive function. A 

large number of dose levels were examined across 5 

compounds, one being bisphenol A with evaluation of 4 

dose levels, including controls. Five to eight animals/ 

sex/dose were used in evaluations and animals were 

selected as 1 male and 1 female/litter. Findings were 

judged against an incorporated positive control that 

resulted in predicted findings. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate but of limited utility for the 

evaluation because of the secondary nature of the 

endpoints for a human health evaluation. 

Tan et al. (2003), supported by the University of 

Malaya and the Ministry of Science, Technology, and 

Environment, examined the effects of bisphenol A 

exposure on pubertal development of male rats. Sprague–Dawley rats were fed soy-free feed (Gold Coin 

Feedmills) and housed in aluminum cages containing 

shredded recycled paper as bedding. On PND 23–53, 12 

rats/group were gavaged with 100 mg/kg bw/day 

bisphenol A [purity not reported] in a Tween-80/water 

solution (1:9 v/v), 100 mg/kg bw/day nonylphenol in 

corn oil, or a mixture of 100 mg/kg bw/day bisphenol A 

and nonylphenol. A control group of 12 rats was gavaged 

with Tween 80 in corn oil. Dosage selection was based on 

published studies reporting NOAELs of 50 mg/kg bw/ 

day for both compounds. Rats were examined for 

preputial separation during the study. Six rats/group 

were killed on PND 52, and the other 6/group were 

killed on PND 53. Testes, epididymides, liver, kidney, 

adrenal, seminal vesicles plus coagulation gland, and 

thyroid were weighed. [The Expert Panel assumes that 

by coagulation gland, the authors mean the anterior 

prostate or coagulating gland.] Thyroid, testis, kidney, 

and liver were fixed in 10% formalin and examined 

histologically. Statistical analyses included ANOVA and 

Fisher protected least significant difference test. 

There was no significant effect on weight gain in rats 

treated with bisphenol A. In the bisphenol A group, 

preputial separation occurred by PND 53 in 66.7% of rats 

compared to 100% of rats in the control group. In the 

bisphenol A group, significant increases were observed in 

absolute and relative (to body weight) kidney and thyroid 

weights and significant decreases were observed for 

absolute and relative liver weight. Cortical thickness of 

the kidney was significantly decreased [by B13% compared to controls according to CERHR calculations and 

B30% according to study authors]. There was no effect on 

testicular weight or tubule diameter. Normal patterns of 

spermatogenesis were observed in rats from the control 

group. Multinucleated giant cells were observed in 

seminiferous tubules and there was no indication of 

spermatogenesis in 4 of 12 rats of the bisphenol A group. 

Giant cells were observed and spermatogenesis was found 

to occur in only some seminiferous tubules of the 

remaining rats treated with bisphenol A. Moderate-tosevere hydronephrosis was observed in 50% of rats and 

mild hydronephrosis was observed in the other 50% of rats 

from the bisphenol A group. 

Preputial separation occurred by PND 53 in 33.3% of 

rats in the nonylphenol group and 58.3% of rats exposed 

to the bisphenol A/nonylphenol mixture. In animals 

treated with nonylphenol, relative liver weight was 

increased, absolute and relative seminal vesicle weights 

were decreased, and the diameter of testicular tubules 

was reduced. A decrease in relative seminal vesicle 

weight was the only significant organ weight effect 

observed in the group treated with both bisphenol A and 

nonylphenol. Moderate hydronephrosis was observed in 

25% of rats exposed to the bisphenol A/nonylphenol 

mixture and mild hydronephrosis was observed in the 

other rats from that exposure group. No spermatogenesis 

was observed in 3–5 of 12 rats/group treated with 

nonylphenol or the mixture of bisphenol A/nonylphenol. The study authors concluded that both bisphenol A 

and nonylphenol affected the reproductive system of 

rats, while only bisphenol A affected the kidneys. They 

also noted a less-than-additive effect with administration 

of the bisphenol A/nonylphenol mixture. 

Strengths/Weaknesses: This study was apparently 

well performed and documents the endpoints tested. A 

weakness is the use of a single high-dose level of 

bisphenol A. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

Kobayashi et al. (2005), supported by the Japanese 

Ministry of Health, Labor, and Welfare, examined the 

effects of developmental exposure to bisphenol A on 

thyroid status in rats. Rats used in this study were fed 

standard laboratory chow (CE-2, Clea Japan). [No 

information was provided about caging or bedding 

materials.] From GD 6 (day of copulatory plug 5 GD 0) 

through PND 20 (day of birth not defined), 6 maternal CD 

rats/group were gavaged with bisphenol A (499.8% 

purity) at 0 (corn oil vehicle), 4, 40, or 400 mg/kg bw/day. 

The 400 mg/kg bw/day maternal group was excluded 

from analysis because of excessive maternal toxicity. 

Details about maternal toxicity and additional aspects of 

this study are available in the summary for the study by 

Kobayashi et al. (2002). On PND 7, litters were culled to 5 

pups/sex when possible. It appears that culled pups may 

have been used in analyses conducted at 1 week of age. 

Pups were weaned on PND 21. Approximately 1 male and 

1 female pup/litter were killed at 3 and 9 weeks of age. 

Plasma thyroxin levels were measured by chemiluminescence immunoassay in 1–9 offspring/group/sex at 1 week 

of age and 3–6 offspring/sex/group at 3 and 9 weeks of 

age. At 9 weeks of age, thyroid stimulating hormone test 

was conducted in 2–7 rats/sex/group by measuring 

thyroxin levels after injection with bovine thyroid stimulating hormone. Statistical analyses included ANOVA 

followed by Dunnet test or Student or Welch’s t-test. [It 

was not clear if the litter or offspring were considered 

the statistical unit.] 

In the 4 and 40 mg/kg bw/day groups, there were no 

significant differences in thyroxine levels at 1, 3, or 9 

weeks of age or in thyroid stimulating hormone-induced 

increases in thyroxine levels at 9 weeks of age. Based on 

Birth Defects Research (Part B) 83:157–395, 2008
258 CHAPIN ET AL. 

the findings of this study, the study authors concluded 

that prenatal and lactational exposure of rats to bisphenol 

A does not appear to affect thyroid function. 

Strengths/Weaknesses: Strengths of this study include 

the use of a range of dose levels of bisphenol A. 

Weaknesses include the limited endpoints addressed 

(thyroid function), concern that the number of animals 

used (6 dams per treatment group) may not provide 

adequate statistical power to assess changes in hormone 

levels and response given the variability inherent in these 

measures, and failure to account for litter in the analyses. 

Utility (Adequacy) for CERHR Evaluation Process: 

As presented, this study is inadequate because it is 

unclear whether there were adequate controls for litter 

effects. 

Zoeller et al. (2005), supported in part by NIH, 

examined the effect of bisphenol A exposure on the 

thyroid of developing rats. Sprague–Dawley rats were 

housed in plastic cages. [No information was provided 

about composition of feed or bedding materials.] 

Before initiation of dosing, rats were trained to eat an 

untreated wafer each day. On GD 6 (day of vaginal plug 

not defined) through the remainder of the experiment 

(the remainder of the gestation and lactation periods), 9 

rats/group were given a wafer dosed with bisphenol A 

[purity not reported] at levels resulting in exposure to 0 

(methanol vehicle), 1, 10, or 50 mg/kg bw/day. Doses 

were based on those used in the study by Tyl et al. 

(2002b). Pups (n 5 7–9/group/sex/time period) were 

weighed and killed on PND 4, 8, 15, or 35 (day of birth 

not defined). During each of those time periods, serum 

thyroxin was measured by RIA. On PND 15, brains of 

male pups were sectioned and examined for presence of 

RC3/neurogranin mRNA, a thyroid hormone-responsive 

gene, using an in situ hybridization and autoradiography 

technique. Serum thyroid-stimulating hormone was 

measured using an unspecified method in 6–8 male 

pups/group (1/litter) on PND 15. Statistical analyses 

included ANOVA and Bonferroni t-test. 

The text of the study indicated a significant reduction 

in maternal body weight gain during pregnancy in the 

high-dose group, while Figure 1 of the study indicated a 

significant reduction in maternal body weight gain 

during pregnancy at all dose levels. Maternal 

body weight gain during the lactation period was 

unaffected by bisphenol A treatment. Bisphenol A 

exposure had no effect on litter size at birth. [Data were 

not shown by study authors.] Bisphenol A had no effect 

on pup body weights on PND 4, 8, or 15. On PND 15, but 

at no other time period, there was a significant increase 

in serum thyroxin levels in all dose groups of male and 

female pups [percent increases compared to controls 

were B11, 35, and 37% in each respective dose group.] 

Significant increases in expression of RC3/neurogranin 

mRNA were observed in the upper and lower dentate 

gyrus in males from each treatment group [with no 

apparent dose–response relationship]. Expression of 

RC3/neurogranin mRNA in cortex was unaffected by 

bisphenol A treatment. No significant effects were 

observed for thyroid-stimulating hormone levels in 

males on PND 15. The study authors concluded that 

bisphenol A acts as a thyroid antagonist at these 

concentrations. 

Strengths/Weaknesses: Strengths of the study include 

use of a range of doses and examination of a role of 

bisphenol A as a thyroid hormone antagonist. Weaknesses include the lack of litter-based analysis. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate based on inappropriate statistics 

(i.e., not accounting for repeated measures over time or 

the use of more than one pup per litter for a given 

endpoint). 

3.2.3.3 Studies with neurobehavioral endpoints: 

Kwon et al. (2000), from CIIT, examined the effects of 

bisphenol A exposure during pre- and postnatal development on reproductive endpoints and the SDN-POA of 

rats. Sprague–Dawley rats were fed NIH-07 feed and 

housed in polycarbonate cages with cellulose fiber-chip 

bedding. Water was provided in glass bottles with 

Teflon-lined caps. Pregnant rats were randomly assigned 

to groups according to body weight. Rats (n 5 8/group) 

were gavaged with bisphenol A (B99% purity) at 0 (corn 

oil vehicle), 3.2, 32, or 320 mg/kg bw/day from GD 11 

(GD 0 5 day of sperm detection) through PND 20, 

excluding the day of parturition. It was not stated if the 

day of parturition was considered PND 0 or 1. A positive 

control group was treated with 15 mg/kg bw/day 

diethylstilbestrol. Rats were examined for clinical signs 

of toxicity and weighed during the study. Pups were 

weighed on PND 1 and 7. Pups were weaned on PND 21. 

After pups were weaned, dams were killed for assessment of body and organ weights. On PND 10, brains 

were collected from 1–3 female offspring/litter from 6–8 

litters/group for measurement of SDN-POA volume. All 

remaining female pups were examined for age of vaginal 

opening and day of first estrus, and estrous cyclicity was 

monitored for 22 days, beginning at B4 months of age. 

Lordosis behavior was examined at 6 months of age in 1– 

2 female offspring/litter from 7–9 litters/group that had 

been ovariectomized 2 weeks before reproductive 

behavior testing and primed with estradiol benzoate 

and progesterone. Male offspring were killed on PND 

180 for measurement of body and reproductive 

organ weights and histopathological evaluation of 

ventral prostates fixed in 10% neutral buffered formalin. 

[Based on information presented in the Results 

section, it also appears that ovaries and uteri were 

examined in an unspecified number of female offspring at 6 months of age.] Statistical analyses included 

ANOVA, Dunnett test, ANCOVA, and pair-wise comparison of least square means. The litter was considered the 

experimental unit. 

Bisphenol A treatment had no significant effect on 

maternal body weight during pregnancy or lactation or 

on maternal liver, kidney, adrenal, ovary, or uterus 

weights. There was no effect on number of live pups/ 

litter at birth or pup weight on PND 1 or 7. In female 

offspring, bisphenol A exposure had no significant effect 

on volume of SDN-POA, age or weight at vaginal 

opening or first estrus, estrous cyclicity, or mean lordosis 

intensity. In male offspring, there were no significant 

effects on body weight or weights of testis, epididymis, 

seminal vesicle, or prostate. The study authors noted that 

a 23% increase in ventral prostate weight in the highdose group did not reach statistical significance. No 

treatment-related histopathological alterations were reported for ventral prostate, ovary, or uterus. Effects 

observed in the diethylstilbestrol group included increased maternal liver weight, increased SDN-POA 

volume in female offspring, and disrupted estrous 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 259 

cycles. The study authors concluded that indirect 

exposure of offspring to bisphenol A at these levels 

during gestation and lactation did not affect estrogenmediated reproductive endpoints. A similar study with 

comparable findings in females was reported in abstract 

form (Kwon et al, 1999). 

Strengths/Weaknesses: This study was well performed and presented. The wide coverage of the dose 

range (across a three-log range) is a major strength. The 

use of diethylstilbestrol as a positive control is a strength, 

as is the number of reproductive organs and endpoints 

evaluated. A weakness was the limited analysis of those 

reproductive organs (wet weight only; histopathology 

was only performed on the prostate) and a lack of 

determination of pup exposure during lactation. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

Kubo et al. (2001), supported by the Japanese Ministry 

of Education, Science, and Culture, examined the effect 

of prenatal bisphenol A exposure on sexually dimorphic 

behavior and brain development in rats. [No information was provided about the type of chow, bedding, or 

caging materials used.] Throughout the gestation (from 

the day that sperm were detected in the vagina) and 

lactation periods, 5 Wistar rats/group were administered 

bisphenol A through drinking water at 0 or 5 mg/L. [No 

information was provided on bisphenol A purity or use 

of a vehicle.] The study authors estimated the bisphenol 

A dose at 1.5 mg/kg bw/day. [It is not clear whether 

this is an estimate based on water consumption or 

dosing by a separate, unspecified route. If based on 

drinking, this estimate is suspect because it implies a 

daily consumption of approximately 70 mL water 

(because the weight of the rats is not supplied this 

must of necessity be a guess), which is well in excess of 

published intakes for post-partum rats (generally noted 

as around 20 mL/day). It is also noted that water 

consumption varies widely in non-lactating rats and 

throughout the period of lactation in rats, reflecting 

milk production, so any such estimate would of 

necessity be suspect, and doses will vary with time 

post-partum.] Litters were adjusted to 5 pups/sex on the 

day following birth. Pups were weaned on PND 21 [day 

of birth not defined] and housed according to sex and 

litter. Behavior was tested for 10 min in an open-field 

apparatus at 6 weeks of age (n 5 11–14) [It was not clear 

if the number of animals examined included total 

animals, total/group, or total/sex/group. Litter distribution was not indicated.] A passive avoidance test was 

conducted at 7 weeks of age (n 5 11–14); the test included 

a habituation period and testing of retention 24 hr later. 

An unspecified number of rats were killed and necropsied at 12 weeks of age, with females killed in diestrus. 

Reproductive organs were weighed (n 5 12–14) and 

sperm endpoints were evaluated in an unspecified 

number of rats. Serum hormone levels were measured 

by RIA (n 5 5–10/group). At 20 weeks of age, 6 rats/sex 

from the control group and 7 rats/sex from the treated 

group were killed to measure the volume of the SDNPOA and the locus ceruleus. Behavioral data were 

analyzed by Student t-test or Mann–Whitney U-test, 

and brain morphology data were analyzed by Student 

t-test. [It was not clear if the litter or offspring was 

considered the statistical unit.] No information was 

provided on data analyses for reproductive organ 

weight, serum hormone levels, or sperm endpoints.] 

In open-field testing of controls, females moved 

significantly greater distances, reared more times, and 

stayed in the center of the apparatus for a longer period 

of time than males. In passive avoidance testing of 

controls, latency to enter the dark chamber following 

electric shock was significantly longer in male than 

female rats. In rats exposed to bisphenol A, there were no 

significant differences in the behaviors of males compared to females. Study authors attributed the loss of 

sexually dimorphic behaviors to demasculinization of 

male behavior and defeminization of female behavior. 

Bisphenol A treatment did not affect brain weight, which 

was higher in male than female controls. The larger size 

of SDN-POA in male compared to female controls was 

retained following bisphenol A treatment. The volume of 

the locus ceruleus was significantly larger in females 

than males of the control group. In the bisphenol A 

group, the volume of the locus ceruleus was described as 

larger in males than females, but the stated increase was 

not statistically significant (P 5 0.12). [Graphically, there 

is an estimated 14% difference between male and 

female locus ceruleus volume in controls and in 

bisphenol A-exposed animals, with the direction of 

the difference apparently reversed by treatment.] Bisphenol A treatment had no effect on absolute weight of 

the testis or epididymis or relative weights of the ventral 

prostate, ovaries, or uterus. There were no significant 

effects on serum levels of LH, FSH, testosterone, or 17bestradiol. Sperm count and motility were also unaffected 

by bisphenol A exposure. The study authors concluded 

that current methods for establishing NOAELs may not 

be sufficient to detect disrupted sexual dimorphism in 

the brain. 

Strengths/Weaknesses: A strength is the variety of 

biological and behavioral endpoints assessed. The major 

weakness of the study is the lack of experimental detail, 

which makes it difficult to determine whether litter 

effects were adequately controlled for and how much 

bisphenol A was received by the animals. 

Utility (Adequacy) for CERHR Evaluation Process: 

Given the lack of methodological data provided in the 

study, this communication is inadequate for the evaluation process. 

Kubo et al. (2003) examined the effect of prenatal 

bisphenol A exposure on sexually dimorphic behavior 

and brain structure of rats. [No information was provided 

on the type of feed or materials used in bedding or 

caging.] Wistar rats were dosed with the 0.1% ethanol in 

distilled water vehicle (n 5 5 dams/group) or bisphenol A 

[purity not reported] at 0.1 or 1 mg/L (n 5 6 dams/ 

group). The study authors estimated bisphenol A intake at 

0.030 and 0.3 mg/kg bw/day and noted that the levels 

were below the tolerated daily intake. [Although not 

clearly stated, it appears that as in the previous study by 

Kubo et al. (2001), exposures occurred through drinking 

water during the entire gestation and lactation period.] 

Five dams/group were exposed to trans-resveratrol, an 

estrogenic compound found in grapes, at 5 mg/L or 

diethylstilbestrol at 50 mg/L. Body weight and anogenital 

distance were measured in pups on PND 1 (the day 

following birth). [All litters were examined and although 

the number of pups examined in each litter was not 

clearly stated, it was implied that all pups were 

Birth Defects Research (Part B) 83:157–395, 2008
260 CHAPIN ET AL. 

analyzed.] Anogenital distance was adjusted by the cube 

root of body weight. Following the evaluations on PND 1, 

litters were standardized to 5 pups/sex. Pups were 

weaned on PND 21 and housed according to sex and 

litter. Day of testicular descent or vaginal opening was 

monitored in all remaining offspring (n 5 25/sex in the 

control group and 30–31/sex in the treated group). Openfield testing was conducted in 20–24 animals/group at 6 

weeks of age. [It is not clear if the authors meant 20–24 

animals/group or animals/group/sex]. Sexual behavior of 

7–13 male and female rats/sex/group was tested at 11–12 

weeks of age. Males and females (n 5 11–15/sex/group) 

were killed at 12 weeks of age, females during proestrus. 

Reproductive organs were weighed. Serum hormone 

levels were measured by RIA. Sperm from one testis 

and cauda epididymis were counted. Histological examinations were conducted on testis fixed in Bouin solution 

and ovary fixed in 10% neutral buffered formalin. Rats 

were killed at 14 weeks of age for measurement of SDNPOA and locus ceruleus volume in 7–8 males and 

females/group. 

Because of the large number of animals used, the 

experiment was conducted in 3 blocks representing 

identical experiments. All data were collected and 

analyzed following completion of the third block of the 

study. The litter was considered the statistical unit in 

analyses of data collected before weaning of animals. 

Individual animals were considered the statistical unit in 

analyses of data collected subsequent to weaning. 

Behavior and brain structure data were analyzed by 

ANOVA and differences between sexes were analyzed 

by Student t-test. Reproductive data were analyzed by 

ANOVA followed by Fisher protected least significant 

difference test for each sex. 

Bisphenol A exposure had no significant effect on 

body weight on PND 1, anogenital distance in males and 

females, day of testicular descent, or day of vaginal 

opening. Body weight at vaginal opening was significantly higher [by 7%] in the high-dose bisphenol A 

group. In sexual behavior testing of males, a non-doserelated decrease in the intromission rate observed in the 

low-dose group was the only significant effect reported 

following bisphenol A exposure. There were no effects 

on mounting or ejaculation. Bisphenol A exposure had 

no significant effects on female sexual behavior as 

measured by ear wiggle, lordosis quotient, and rejection 

of males. The study authors concluded that bisphenol A 

exposure had no remarkable effects on male or female 

sexual behavior. The only significant effect on organ 

weights was an [9%] increase in testis weight in the highdose bisphenol group. There were no significant effects 

on absolute weight or relative (to body weight) weights 

of ventral prostate, seminal vesicle, uterus, or ovary. 

Bisphenol A treatment did not affect sperm count or 

motility or estrous cycles. Serum levels of LH, FSH, 

prolactin, testosterone, and 17b-estradiol were also 

unaffected by treatment. No histopathological findings 

were observed in testis or ovary. [Data were not shown.] 

In open-field testing of control rats, females moved 

greater distances, reared more often, and spent more 

time in the center of the testing apparatus. Following 

treatment with the low or high-dose of bisphenol A, there 

were no longer significant differences between males 

and females in frequency of rearing and or duration of 

time spent in the center of the apparatus. Differences in 

distances moved by males versus females were no longer 

significant following exposure to the high bisphenol A 

dose. Males in the low bisphenol A group reared 

significantly more times than males in the control group. 

Bisphenol A treatment had no significant effect on the 

sex-related difference in size of the SDN-POA, which 

was significantly larger in males than females in the 

control and treatment groups. Although the volume of 

the locus ceruleus was significantly greater in females 

than males of the control group, locus ceruleus volume 

was significantly larger in males than females of both 

bisphenol A groups. The change was due to a significant 

increase in volume in males at the low dose and 

significant decrease in volume in females at both dose 

levels of bisphenol A. [Magnitude of locus ceruleus 

volume changes in males and females was B12–17% 

compared to controls, as estimated from a graph.] The 

numbers of neurons in the locus ceruleus was affected in 

the same manner as volume by bisphenol A treatment, 

except that increases in neuron numbers following 

bisphenol A treatment were also significant in males of 

the high-dose group. 

Diethylstilbestrol mainly affected open-field behavior, 

locus ceruleus volume, and the reproductive system. 

Trans-resveratrol mainly affected locus ceruleus volume 

and the reproductive system. The study authors concluded that the brain is highly sensitive to bisphenol A at 

levels below the tolerable daily intake and disruptions in 

sexual differentiation may differ from effects observed 

with diethylstilbestrol and trans-resveratrol. 

Strengths/Weaknesses: As with the previous study by 

this group (Kubo et al., 2001) the main weakness of the 

study lies in the failure to accurately describe the 

methods to allow a reader to determine how much 

bisphenol A the dams received during the experiment. 

Despite well-selected endpoints, the sample size of 5 

dams/group and lack of clarity on the number of pups 

analyzed per litter are weaknesses. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process due 

to insufficient sample size and lack of experimental 

detail. 

Facciolo et al. (2002), supported in part by the Italian 

Ministry of University Education and Research, examined the effects of developmental exposure to bisphenol 

A on the somatostatin receptor subtype sst2 in the limbic 

circuit of rats. Sprague–Dawley dams were exposed 

orally to bisphenol A at 0 (arachis oil vehicle), 0.040, or 

0.400 mg/kg bw/day. [No information was provided on 

the specific method of oral dosing, the purity of 

bisphenol A, or the number of dams treated/group. 

There was no information on the type of chow used or 

composition of cage and bedding materials.] [Author 

states that 32 dams were subdivided into three 

treatment subgroups: controls (n 5 8;), low bisphenol 

A and high bisphenol A (n 5 12/group) (R. Facciolo, 

personal communication, July 17, 2007).] The authors 

stated that the doses selected were relevant to human 

exposures from can linings and dental sealants and had 

been reported to induce morphometric changes in 

offspring. The rats were mated for 5 days during the 

treatment period, and treatment was continued through 

gestation and lactation. Litters (minimum 8/group) were 

culled to 8 pups at birth and 1 pup/litter was randomly 

assigned to a dam in the same treatment group for 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 261 

postnatal rearing. Pups were weaned on PND 23 (day of 

birth not defined). On PND 10 and 23, 4–7 rats/group 

[10–11/group according to figures in the study] were 

killed and their brains were removed to examine effects 

on sst2 receptors in the limbic region. Receptor binding 

was assessed using 125I-Tyr0

-somatostatin-14 as a ligand. 

At the same ages, interactions of sst2 with a-containing gaminobutyric acid (GABA) receptors, using the agonists 

zolpidem and Ro 15-4513, were examined in 12–13 rats/ 

group. Results were reported for only the high-dose of 

bisphenol A (0.400 mg/kg bw/day) because higher 

affinity was obtained for receptor ligand binding. 

Statistical analyses included Student t-test, ANOVA, 

and Newman–Keuls multiple range test. Analyses did 

not account for litter of origin. 

Strengths/Weaknesses: Strengths of this study are the 

fact that it appears to have been carefully performed and 

used biologically-relevant concentrations delivered orally. A weakness is the purposeful confounding of litter 

of origin through cross-fostering 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate due to experimental design that 

did not sufficiently account for litter effects. 

Facciollo et al. (2005), supported by the Italian 

Ministry of University Education and Research, examined the effects of bisphenol A on expression of 

somatostatin subtype 3 (sst3) receptor mRNA in brains 

of female rats exposed during development and investigated whether the aGABAA receptor is also involved in 

this effect. Sprague–Dawley rats were housed in stainless 

steel cages. [No information was provided about the 

type of feed or bedding used.] Beginning 8 days before 

mating and continuing through the mating period (5 or 8 

days) and during pregnancy and lactation (42 days), 8 

rats received the arachis oil vehicle and 12 rats/group 

received bisphenol A [purity not reported] at 0.040 or 

0.400 mg/kg bw/day. Vehicle or bisphenol A were orally 

administered by pipette. To minimize litter effects, 1 

female pup from each litter was fostered to a dam from 

the same treatment group (8 pups/dam). Pups were 

weaned on PND 23. On PND 7 and at 55 days of age, 4 

rats/group/time period were killed. Brains were sectioned and a 32S-labeled probe was used in an in situ 

hybridization method to measure sst3 mRNA expression. 

The effects of aGABAA receptor subunits on expression 

of sst3 mRNA was examined by incubating the brain 

sections in 1 nM–100 mM of aGABAA receptor agonists 

(zolpidem, flunitrazepam, RY 080, and RO 15-4513). 

Additional brain sections from high-dose rats were used 

to determine interactions between sst3 with a1 and a5 

subunits with or without addition of 5–500 nM zolpidem 

or RY 080. Statistical analyses included ANOVA followed 

by Dunnett t-test or Neuman–Keuls multiple range posthoc test, when analysis by ANOVA indicated statistical 

significance. 

Changes in sst3 expression varied with dose and age. 

Expression patterns were changed in the presence of 

aGABAA receptor agonists. Based on their findings, the 

study authors concluded that bisphenol A exposure can 

affect cross-talking mechanisms involved in the plasticity 

of neural circuits with resulting influences on neuroendocrine/sociosexual behaviors. 

Strengths/Weaknesses: Strengths of this study are the 

fact that it appears to have been carefully performed and 

used biologically-relevant concentrations. A weakness is 

the purposeful confounding of litter of origin through 

cross-fostering. 

Utility (Adequacy) for CERHR Evaluation 

Process: This study is inadequate due to experimental 

design. 

Aloisi et al. (2002), supported in part by the Italian 

Ministry for Universities and Scientific and Technological Research (MURST), examined the effects of prenatal 

or postnatal bisphenol A exposure on the pain response 

of rats. [No information was provided in the manuscript on chow or composition of caging and bedding. 

The Expert Panel has been informed that Harlan Teklad 

2018 feed, Lignocel bedding, and polysulfone cages 

were used (F. Farabollini et al., personal communication, March 1, 2007).] Sprague–Dawley rats were fed 

peanut oil vehicle (n 5 13) or 0.040 mg/kg bw/day 

bisphenol A [purity not given in the manuscript; 

Z95% according to the authors (F. Farabollini et al., 

personal communication, March 1, 2007)] (n 5 7/group) 

by pipette during pregnancy and lactation. Within 48 hr 

after birth, the offspring were sexed and cross-fostered to 

form the following groups: 

*	 Prenatal exposure group—born to dams receiving 

bisphenol A and nursed by dams receiving the peanut 

oil vehicle (n 5 11 males; 9 females); 

*	 Postnatal exposure group—born to vehicle control 

dams but fostered to bisphenol A treated dams (n 5 11 

males; 9 females); and 

*	 Vehicle control group—born to and nursed by dams 

exposed to the vehicle control (n 5 16 males and 11 

females). 

At 22 weeks of age, the rats were randomly assigned to 

sham or formalin treatment groups, but the sham group 

was not analyzed. The formalin group was s.c. injected 

with 10% formalin on the dorsal surface of the right hind 

paw. Pain behaviors, such as licking, flexing, and jerking 

of the paw were recorded for 60 min. Following testing, 

the phase of the estrous cycle was determined and blood 

was drawn to measure plasma levels of testosterone in 

males and corticosterone and 17b-estradiol in both sexes 

by RIA. Data were analyzed by ANOVA followed by 

post-hoc least significant difference test. 

The frequency of paw jerking was decreased at 30– 

60 min following formalin injection in postnatally exposed rats. [The study abstract and results section 

indicate that the effect occurred in males and females, 

but according to data presented in figures of the study, 

the effect only appeared to have occurred in males.] 

Duration of flexion was increased 0–30 min following 

formalin injection in both sexes exposed prenatally to 

bisphenol A. Although statistical significance was not 

attained, the study authors noted an increase in licking 

duration at 0–30 min following formalin injection in 

females exposed to bisphenol A during prenatal development. No effects were observed on open-field behaviors or plasma levels of testosterone, 17b-estradiol, or 

corticosterone. The study authors concluded that their 

findings indicated sex- and exposure-related modifications of neural pathway activity or nociception centers 

following exposure to bisphenol A. 

Strengths/Weaknesses: A strength of this study is the 

added dimension being investigated (pain response). A 

weaknesses, however, are use of a single dose and the 

Birth Defects Research (Part B) 83:157–395, 2008
262 CHAPIN ET AL. 

purposeful confounding of litter of origin during the crossfostering process. In addition, the sample size of 7 dams in 

the 0.040 mg/kg bw/day bisphenol A group and the 

examination of n 5 11 male and n 5 9 female offspring in 

the prenatal exposure group raise questions about experimental or statistical accounting for litter effects. 

Utility (Adequacy) for CERHR Evaluation Process: 

The data presented are inadequate due to the methodological design and lack of clarity on accounting for litter 

effects. 

Negishi et al. (2003), support not indicated, examined 

the effect of perinatal bisphenol A exposure on behavior 

of rats. F344/N rats (n 5 8–9/group) were orally exposed 

to bisphenol A at 0 (olive oil vehicle), 4, 40, or 400 mg/kg 

bw/day from GD 10–PND 20. GD 0 was defined as the 

day that vaginal sperm were detected and PND 0 was 

defined as the day of parturition. [No information was 

provided on purity of bisphenol A, the specific method 

of oral dosing, type of chow used, or composition of 

bedding or caging materials.] Dams were observed and 

weighed throughout the study. On PND 0, pups were 

counted, weighed, and culled to 8/litter with equal 

numbers/sex when possible. Pups were weighed periodically from PND 7–84. Pups were housed as same-sex 

littermates following weaning on PND 21. On weaning 

of pups, dams were killed and body and organ 

weights were recorded. Behavioral testing of offspring 

consisted of spontaneous motor activity measured at 28– 

34 days of age (n 5 12–27/group), active avoidance 

testing conducted at 28–34 and 56–62 days of age 

(n 5 8–9/group), and open-field behavior evaluations at 

56–62 days of age (n 5 9–18/group). Litter was not 

accounted for in the analyses. On PND 62, offspring 

were randomly selected (8/sex/group) and killed for 

evaluation of body and organ weights. Statistical 

analyses included ANOVA, nested ANCOVA, and posthoc Fisher protected least significant difference test. 

[Data analyzed at birth were presented and analyzed on 

a per litter basis. Postnatal data were apparently 

analyzed on a pup basis.] 

Maternal body weight gain was reduced during the 

gestation and lactation period in dams exposed to the 

mid- or high-dose. The only organ weight effects in dams 

were reduced absolute and relative (to body weight) 

thymus weight. There were no effects on weights of liver, 

kidney, or spleen in dams. Bisphenol A treatment did not 

affect the number of pups/litter or sex ratio. In male 

offspring, body weights were lower than control values 

on PND 7 and 28 at the mid-dose, and PND 7, 21, 28, and 

56 at the high-dose. Body weights of female offspring 

were lower than controls at PND 7 and 28 at the low- and 

mid-dose and PND 7, 21, and 28 at the high-dose. On 

PND 62, there were no effects on body weight or liver, 

kidney, spleen, thymus, brain, or testis weights. There 

were no effects on spontaneous activity, but total 

immobile time was increased in females of the mid-dose 

group. Performance of males in avoidance testing 

improved in the mid- and high-dose group at 4 weeks 

of age but decreased in the low-dose group at 8 weeks of 

age. Increased grooming by males of the low-dose group 

was observed in open-field testing. The study authors 

concluded that perinatal bisphenol A exposure caused 

behavioral alterations that differed by sex. 

Strengths/Weaknesses: Doses were sufficiently high 

to produce gross body weight changes, and 3 different 

measures of behavior were collected, as well as organ 

weights at necropsy from the same animals. Weaknesses 

include a lack of statistical accounting for possible litter 

effects in the postnatal analysis, the lack of an evaluation 

of hormone-dependent behaviors, and the lack of 

assessment of more hormone-dependent tissues (prostate, levator ani muscle, etc.) or processes (age at 

balanopreputial separation, postnatal anogenital 

distance). 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process due 

to a failure to account for litter effects. 

Negishi et al. (2004a), support not indicated, examined 

the effect of perinatal bisphenol A [purity not indicated] 

exposure on the behavior of rats. The effects of nonylphenol were also examined but will not be discussed. 

F344/N rats (10 or 11/group) were gavaged with bisphenol A at 0 (corn oil vehicle) or 0.1 mg/kg bw/day 

from GD 3–PND 20. GD 0 was defined as the day that 

vaginal sperm was detected, and PND 0 was the day of 

parturition. At birth, pups were counted and weighed. 

Litters were culled to 6 pups, with equal numbers of each 

sex when possible. Pups were weighed throughout the 

postnatal period. At weaning, dams were killed and organ 

weights were measured. One male pup/litter (n 5 8–10/ 

group) was subjected to a series of behavioral tests. The 

remaining male pups were killed for measurement of 

organ weights at 21 days or 8 weeks of age. Neurobehavioral endpoints evaluated included open-field behavior 

at 8 weeks of age, spontaneous motor activity at 12 weeks 

of age, passive avoidance at 13 weeks of age, performance 

in the elevated-plus maze at 14 weeks of age, and active 

avoidance at 15 weeks of age. At 22–24 weeks of age, a 

monoamine reduction test was performed: rats were 

injected with the monoamine oxidase inhibitor trans-2­

phenylcyclopropylamine hydrochloride or with saline, 

and behavior was then evaluated. Data were analyzed by 

ANOVA, and if statistical significance was obtained, 

Fisher protected least significant difference test was 

conducted. Behavioral endpoints were measured on 1 

male pup/litter, thus accounting for litter issues. 

Bisphenol A exposure did not affect dam body weights 

during gestation or lactation, gestation duration, litter size, 

number of male and female pups, or final dam body and 

organ weights. [Data were not shown.] Body and organ 

weights of male offspring at 21 days and 8 weeks of age, 

behavior in open-field testing, spontaneous motor activity, 

and performance in the elevated-plus maze were also 

unaffected by bisphenol A exposure. [Data were not 

shown by study authors.] Bisphenol A had no significant 

effect on performance in the passive avoidance test, 

although tendencies for increased latency were observed. 

In active avoidance testing, rats from the bisphenol A 

group had significantly (Po0.01) fewer correct avoidance 

responses during the first, second, and third of five 

sessions, and failure of avoidance was significantly 

increased [B2.5% in the bisphenol A group compared 

to 0.2% in controls]. In contrast to control rats, bisphenol 

A-treated rats did not show an increase in locomotion 

following a challenge with trans-2-phenylcyclopropylamine hydrochloride. The number of rearings following 2­

phenylcyclopropylamine hydrochloride exposure did not 

differ significantly between rats from the bisphenol A and 

control groups. The study authors concluded that perinatal exposure of rat dams to bisphenol A at concentrations 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 263 

slightly higher than environmental exposures irreversibly 

affected perception of fear-provoking stimuli and monoaminergic neural pathways in male offspring. 

Strengths/Weaknesses: The use of a single dose level is a 

weakness. Strengths include the variety of endpoints used 

to provide data, which point to effects that are not gross 

structural changes but relatively subtle behavioral effects. 

Utility (Adequacy) for CERHR Evaluation Process: 

These data are adequate and of high utility for the 

evaluation process. 

Farabollini et al. (1999), supported by the University of 

Siena, University of Firenze, MURST, and the Italian 

National Research Council, examined the effects of 

perinatal bisphenol A exposure on behavior in male and 

female rats. [No information was provided in the 

manuscript about chow or composition of cage and 

bedding materials. The Expert Panel has been informed 

that Morini MIL chow, lignocel bedding, and polysulfone cages were used (F. Farabolli et al., personal 

communication, March 1, 2007).] Three groups of 

Sprague–Dawley rats were orally dosed with the arachis 

oil vehicle or bisphenol A [purity not reported in the 

manuscript; Z95% according to the authors (F. Farabolli 

et al., personal communication, March 1, 2007)] by 

micropipette. One group of 11 rats was administered 

0.040 mg/kg bw/day bisphenol A from 10 days before 

conception until weaning of pups at 21 days of age. A 

second group of 11 rats was given arachis oil from 10 days 

before conception through GD 13, 0.400 mg/kg bw/day 

bisphenol A from GD 14 [day of vaginal plug not stated] 

through 6 days following delivery of pups, and arachis oil 

until weaning of pups. A control group of 9 rats was given 

arachis oil from 10 days before conception until weaning 

of pups. Beginning at 85 days of age and continuing for 3 

days, behavioral testing was conducted using a hole board 

and elevated-plus maze in 15 offspring/sex from the lowdose group, 11–12 offspring/sex from the high-dose 

group, and 14 pups/sex from the control group. [Litter 

distribution was not reported.] Separate sessions were 

conducted for each sex and treatment group. Data were 

analyzed by ANOVA and Fisher least significant difference test. A factor analysis was conducted using the 

principal components method with an orthogonal rotation 

of the factor matrix. [It appears that offspring were 

considered the statistical unit.] 

In general, head dipping (extending head over edge of 

apparatus) and arm entries were reduced and selfgrooming was increased in exposed females. Head 

dipping and stretched-attend posture (moving body 

forward without moving paws and then returning to 

original position) were inhibited and arm entries were 

increased in exposed males. A factor analysis indicated 

reduced anxiety and motivation to explore in treated 

males and reduced activity and motivation to explore in 

treated females. The study authors concluded that 

although sex-related differences in behavior were noted 

following bisphenol A treatment, there was no clear 

masculinization of behavior in females. The authors also 

noted the lack of substantial differences in results 

between the two exposure protocols. 

Strengths/Weaknesses: The unusual exposure scenario in this study is both a strength and a weakness; 

however, the use of 11–15 pups from 9–11 litters raises 

concern for possible litter effects that were unaccounted 

for in the statistical analysis. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate due to insufficient control for 

possible litter effects. 

Farabollini et al., (2002), supported by the University 

of Siena, University of Firenze, and MURST, examined 

the effects of perinatal bisphenol A exposure on sociosexual behavior in rats. Sprague–Dawley rats were 

housed in polysulfone cages. [No information was 

provided in the manuscript on type of feed or 

composition of bedding materials. The Expert Panel 

has been informed that Harlan Teklad 2018 chow and 

Lignocel bedding were used (F. Farabolli et al., personal 

communication, March 1, 2007).] Dams received arachis 

oil vehicle (n 5 13) or 0.040 mg/kg bw/day bisphenol A 

[purity not indicated] (n 5 7) through a micropipette 

from mating through weaning of pups. On Day 2 

following delivery, litters were culled to 4 pups/sex 

and cross-fostered to obtain the following exposure 

groups of 12 animals/sex: 

*	 Prenatal exposure group: born to bisphenol A-treated 

dams and nursed by vehicle-treated dams; 

*	 Postnatal group: born to vehicle-treated dams and 

nursed by bisphenol A-treated dams; and 

*	 Control group: born to and nursed by vehicle-treated 

dams. 

Litters were weaned on PND 21 (day of birth not 

defined). On day 45 [assumed to be PND 45], animals of 

the same sex were randomly chosen and housed 4/cage, 

with no siblings in any cage. At 100 days of age, behavior 

in the presence of an intruder rat was observed. In female 

rats, vaginal smears were taken at the end of intruder 

testing and only females in diestrus were considered 

(n 5 8–9/group). One week later, sexual orientation was 

tested in 12 rats/sex/group by placing a rat between 

cages containing a sexually receptive female and sexually 

mature male and recording the number of visits to each 

rat. Sexual performance was tested next in males; 

evaluation was restricted to only males that ejaculated 

(n 5 10–12 group). One week later, sexual behavior was 

tested in females during the diestrous or proestrous 

phase. [It is not clear how many females were evaluated 

for sexual behavior.] Behavior testing sessions were 

video recorded and later evaluated by a blinded 

observer. Data were analyzed by ANOVA followed by 

post-hoc Fisher least significant difference test. Litter 

effects were purposely confounded through crossfostering. 

In intruder testing, statistically significant effects 

observed in males exposed prenatally to bisphenol A 

included an increased number showing defensive behavior (9 of 10 vs. 4 of 10 in the control group), a decreased 

number showing ambivalent behavior (3/10 vs. 8/10 in 

the control group), and increased ratio of defensive/ 

agonistic behaviors [by 280% compared to controls]. No 

significant effects were observed in intruder testing of 

female rats. There was no effect on sexual preference of 

males or females. For sexual behavior testing of females, 

data from the pre- and postnatal exposure groups were 

pooled because there were no significant differences 

between groups. Bisphenol A exposure significantly 

decreased exit latency in females in diestrus [by B66%] 

and proestrus [by B83%] and significantly (Po0.05) 

Birth Defects Research (Part B) 83:157–395, 2008
264 CHAPIN ET AL. 

increased lordosis frequency in females in proestrus 

[B11.75 vs. 3.75 times in controls]. Statistically significant effects on sexual performance of treated males 

included an increased number of intromissions [B15 

compared to 11 in controls] in the postnatal exposure 

group and increased duration of intromission latency 

[B115 vs. 40 sec in controls] and genital sniffing [B40 

vs. 16 sec in controls] in the prenatal exposure group. 

The study authors stated that the results suggested a 

slight intensification of sexual behavior in females, 

slightly reduced performance in a limited number of 

endpoints in males, but no effect on other important 

sexual endpoints in males (e.g., latency of ejaculation and 

refractory period). It was concluded that pre- or postnatal 

exposure to bisphenol A potentiated female behavior and 

depotentiated male behavior. 

Strengths/Weaknesses: The work was carefully performed. The use of a single dose level of bisphenol A is a 

weakness; however, this dosing paradigm is consistent 

with many other studies by this group making comparisons between the studies relevant. Addressing aggressive/defensive behavior as well as sexual performance 

and interest in both male and female offspring is a 

strength. The failure to address underlying biological 

mechanisms is a weakness. Further weaknesses include 

the inability to account for litter effects as the use of 

multiple pups from some litters without appropriate 

statistical control raises concern for possible litter effects 

due to unequal litter representation 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for evaluation purposes due to 

the inability to fully account for possible litter effects. 

Dessi-Fulgheri et al. (2002), supported by the University of Firenze, University of Siena, and MURST, 

examined the effect of perinatal bisphenol A exposure on 

play behavior in rats. Sprague–Dawley rats were housed 

in polysulfone cages. [No information was provided in 

the manuscript on chow or bedding material. The 

Expert Panel has been informed that Morini MIL chow 

and Lignocel bedding were used (F. Farabolli et al., 

personal communication, March 1, 2007).] Using a 

pipette, rats were fed solutions containing the arachis 

oil vehicle and/or bisphenol A according to 1 of 3 

exposure scenarios. A control group of 9 rats was given 

arachis oil from 10 days before mating until weaning of 

pups on PND 21 [day of birth not defined]. Eleven rats 

in the low-dose group were given 0.040 mg/kg bw/day 

bisphenol A [purity not provided] from 10 days before 

mating until weaning of pups. Eleven rats in the highdose group received arachis oil vehicle from 10 days 

before mating until GD 13 [day of vaginal plug not 

defined], 0.400 mg/kg bw/day bisphenol A from GD 

14–PND 6, and arachis oil from PND 7 until weaning. 

Both doses were considered to be within the range of 

human exposure. The low dose was said to represent 

exposures through food occurring over a long period of 

time. The high-dose was said to represent exposures 

occurring through dental procedures occurring over a 

short period of time. Litters were culled to 8 pups at 

birth. [No information was provided in the study on the 

sex distribution of the retained pups; the Expert Panel 

was advised that there were 4 males and 4 females/litter 

(F. Farabolli et al., personal communication, March 1, 

2007).] After pups were weaned, 3 male and 3 female 

pups were randomly caged together, with no siblings 

co-housed in any cage. Behavioral testing was conducted 

on PND 35, 45, and 55. For the behavioral testing, rats 

from the same cage were individually identified by 

marking them with dye. On each day of testing, the 6 

cage mates were transferred to a neutral arena that was 

covered in clean sawdust and video recorded for 6 min. 

Behaviors recorded during the second and third minute 

of each testing session were evaluated. There were 12– 

15rats/sex/group. [The methods section indicates that 

15 rats/sex were tested at the high-dose, 12 rats/sex at 

the low dose, and 15 rats/sex in the control group. 

According to Table 4 of the study, which gives the 

pooled number of rats tested for 3 age periods, it 

appears that 12/sex were tested in the high-dose group, 

15/sex in the low-dose group, and 15/sex in the control 

group. The Expert Panel has been informed that Table 4 

is correct (F. Farabolli et al., personal communication, 

March 1, 2007).] For statistical analyses, individual 

factor scores were used as independent variables in a 3­

way ANOVA that considered treatment, sex, and age. 

Fisher least significant difference test was used when 

appropriate. At weaning, housing conditions confounded litter of origin that was not then accounted 

for in statistical analyses. 

Behavioral elements were categorized under 8 general 

factors. The authors first presented results that were 

pooled for the 3 different age groups. In females of the 

low-dose group, bisphenol A treatment was found to 

significantly increase factors addressing play directed 

toward females. Factors affecting low-intensity mating 

elements (e.g., crawling-under behavior) were reduced 

significantly in high-dose males and females. Factors of 

sociosexual exploration (e.g., genital and body sniffing) 

were reduced significantly in high-dose females and in 

males from both dose groups. Factors of social interest 

(e.g., approaching) were reduced significantly in both 

sexes at the high-dose but increased in low-dose males. 

The authors next discussed results for PND 35, because it 

is the approximate time period of vaginal opening in 

females. Factors that were affected significantly at PND 

35 included increased social interest by males and 

females of the low-dose group, decreased low-intensity 

mating elements by females of both dose groups, and 

decreased sociosexual exploration by males of both dose 

groups. The study authors concluded that 2 factors of 

female behavior were masculinized by treatment: play 

with females and sociosexual exploration. 

Strengths/Weaknesses: A strength of this work is that 

it evaluated the socio-sexual consequences of exposure, 

and specifically at a young age. Weaknesses include 

absence of accounting for litter influences and inadequate statistical procedures (i.e., failure to consider the 

repeated measures design). In addition, the hypothesis is 

not biologically plausible (i.e., consistent with expected 

effects of a chemical with an estrogenic mode of action) 

and the factor analysis does not necessarily cluster the 

play behaviors that are known to be sexually dimorphic. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for evaluation process due to 

faulty statistical procedures. 

Porrini et al. (2005), supported by MURST, the 

University of Firenze, and the University of Siena, 

examined the effects of perinatal bisphenol A exposure 

on play behavior of female rats. [No information was 

provided in the manuscript about the type of feed or 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 265 

bedding and caging materials. The Expert Panel has 

been informed that Harlan Teklad 2018 chow, polysulfone cages, and Lignocel bedding were used (F. 

Farabolli et al., personal communication, March 1, 

2007).] Female Sprague–Dawley rats were co-housed 

with males for 36 hr and then fed the peanut oil vehicle 

(n 5 10) or 0.040 mg/kg bw/day bisphenol A [purity not 

stated] (n 5 12) by micropipette during the gestation and 

lactation period. Two days following delivery, litters 

were adjusted to 4 pups/sex and pups were fostered by a 

dam from the same treatment group. Pups were weaned 

on Day 21 [assumed to be PND 21; day of birth not 

defined]. Offspring were housed in cages containing 3 

pairs of male–female siblings, with no siblings of the 

same sex in the same cage. Each group contained 18 

female pups. Before examination of behavior in rats from 

the same cages at 35, 45, and 55 days of age, animals were 

individually identified with dye. Behavior was observed 

in a neutral arena in which the floor was covered with 

clean sawdust. Animals were allowed to familiarize 

themselves to the new environment for 1 min and then 

behavior was video recorded for 6 min. Video recordings 

were analyzed by an investigator blinded to treatment 

conditions. Only behavior of female rats was considered. 

Data were analyzed by ANOVA for repeated measures. 

The cross fostering design precluded the ability to 

examine litter effects. 

Factors were defined by study authors based on 

groups of behaviors. Significant effects were reported 

for 3 factors. Social and non-social exploration was 

increased [by B34%] at 35 days of age and [by B25%] 

at 45 days of age. Frequency of play behavior with males 

was decreased [by B100%] at 45 days of age. Grooming 

behavior was also decreased [by B63%] at 45 days of 

age. The study authors concluded that bisphenol A does 

not clearly induce masculinization of female behavior, 

but some aspects of female behavior were defeminized. 

Strengths/Weakness: This study reports a well-performed study with a poorly researched endpoint 

(juvenile play behavior) that has implications for 

reproductive behavior later in life. The use of only a 

single dose level of bisphenol A is a weakness. The 

fostering of pups within treatment group prevents the 

evaluation of litter effects and the use of multiple pups 

from some litters without appropriate statistical correction raises concern for possible litter effects. 

Utility (Adequacy) for CERHR Evaluation Process: 

This work is inadequate for the evaluation process due to 

insufficient control for possible litter effects. 

Adriani et al. (2003), supported by the Nervous and 

Mental Disorders Research Area, Istituto Superiore di 

Sanita`, Italy, and by MURST, examined the effects of 

perinatal exposure to bisphenol A on behavior in rats. 

Sprague–Dawley rats were housed in Plexiglas cages 

with sawdust bedding. [No information was 

provided in the manuscript about feed. The Expert 

Panel has been informed that Morini MIL feed was 

used (F. Farabolli et al., personal communication, 

March 1, 2007).] Nine dams/group were dosed 

with bisphenol A [purity not reported] orally by 

micropipette at doses of 0 (arachis oil vehicle) or 

0.040 mg/kg bw/day from the day of mating to the 

day pups were weaned. Pups were weaned on PND 25 

(PND 0 5 day of birth) and housed in groups of 3 

according to sex. One male and 1 female/litter were 

observed in testing that included novelty-seeking behavior during adolescence (PND 30–45), impulsivity during 

adulthood (PND 70), and open-field behavior following 

injection with 1 mg/kg bw d-amphetamine during 

adulthood. It appears that the same animals were tested 

at each time period. Data were analyzed by Tukey HSD 

test and ANOVA. 

In novelty testing, the time spent in a new area of the 

testing apparatus was lower in females exposed to 

bisphenol A [B45–55% compared to vehicle control, 

Po0.05]. Males and females of the bisphenol A group 

exhibited increased activity in the novel area [increases 

of B75% in males and 35–55% in females, Po0.05]. The 

study authors interpreted the effects of novelty testing as 

suggesting a less pronounced habituation profile and 

increased stress in a novel situation. In the impulsivity 

testing, food-restricted animals were placed in an 

apparatus that involved nose poking in a small hole to 

immediately deliver 1 pellet of feed or a larger hole to 

deliver 5 pellets of feed following a delay that was 

increased over the time of the study. Lights were turned 

on during the delay periods following nose poking and 

for 25 sec after delivery of feed, time periods in which no 

feed could be delivered. Both groups of rats preferred the 

larger hole with delayed delivery, but treatment with 

bisphenol A resulted in a more marked preference for the 

larger hole (Po0.05), thus indicating reduced impulsivity. When the length of the delay was increased for the 

large hole, the frequency of inadequate responding (i.e., 

nose poking during the delay) was decreased in males 

from the bisphenol A group; the study authors interpreted the effect as indicating a demasculinization of the 

restlessness profile. [The study report originally mislabeled the control and bisphenol A-treated groups in 

Figure 3a. A corrected version of the figure was 

included in an erratum statement released by the study 

authors (Adriani et al., 2005).] In open-field testing, 

vehicle control males displayed significantly more 

rearing and crossing behaviors following injection 

with d-amphetamine, but an increase in rearing and 

crossing behavior following d-amphetamine injection 

did not occur in males exposed perinatally to bisphenol 

A. The study authors concluded that perinatal exposure 

of rats to bisphenol A resulted in altered behavior in 

rats. 

Strengths/Weakness: This study used protocols that 

are well established by this group. The use of only a 

single exposure level of bisphenol A is a weakness, with 

the proviso that the dose used is directly comparable to 

other studies. The degrees of freedom reported for 

behavioral measures suggest inflation of sample size 

due to failure to account for multiple time sampling. 

Utility (Adequacy) for CERHR Evaluation Process: 

The study is inadequate for evaluation due to inappropriate statistical procedures. 

Carr et al. (2003), supported by the National Science 

Foundation, the Mississippi Agricultural and Forestry 

Experiment Station, and the College of Veterinary 

Medicine at Mississippi State University, examined the 

effects of bisphenol A exposure on performance of rats in 

the Morris water maze. In this study, F344 rat dams and 

pups were fed Purina Test Diet 8117, a casein-based 

rodent chow. [No information was provided about 

caging or bedding materials.] Treatment groups were 

assembled by including pups from different litters such 

Birth Defects Research (Part B) 83:157–395, 2008
266 CHAPIN ET AL. 

that there was a member of each treatment group from 

each sex from each litter: a control animal was always 

present in each litter. Ten pups/sex/group were gavage 

dosed from PND 1 (day of birth 5 PND 14 with 

bisphenol A (499% purity) at 0 (safflower oil vehicle), 

0.1, and 0.25 mg/kg bw/day. An additional group of rats 

was gavaged with 17b-estradiol 72 mg/kg bw/day during the same time period. Straight channel swimming 

was tested on PND 33. Spatial learning and memory 

were tested by Morris water maze for 4 days beginning 

on PND 34. In the test, acquisition of maze solution 

occurred when the rat found a platform. A probe trial 

measuring the amount of time spent in an escape 

quadrant from which the platform had been removed 

was conducted on PND 40. Data were analyzed by 

ANOVA followed by means separation by least squared 

means or Greenhouse-Geisser adjusted F ratios. 

There were no significant effects of bisphenol A 

treatment on straight channel swimming or time to 

acquisition of maze solution in the Morris maze test. 

Time spent in the escape quadrant was significantly 

lower in females of the high-dose group [by B38%] than 

in controls. The study authors noted that acquisition of 

maze performance was significantly better in control 

males than control females. However, no sex-related 

difference was observed following treatment with the 

low bisphenol A dose. Increased time to acquisition in 

males on the third day of testing, and no sex-related 

differences in performance were reported for the 17bestradiol group. The study authors concluded ‘‘These 

data indicate that [17b-estradiol] and low dosages of 

[bisphenol A] can alter the normal sex-dependent pattern 

of acquisition, while higher dosages of [bisphenol A] 

alter the retention of spatial information without significantly affecting acquisition.’’ 

Strengths/Weaknesses: Strengths are the additional 

behavioral dimensions captured by this paper and the 

use of a positive control. The analyses appeared appropriate. The within litter dosing design raises concerns 

about cross-contamination that would decrease differences between groups and challenge interpretation of 

results of non-standard dose–response curves. Analyses 

did not account for the repeated measures design, thus 

inflating degrees of freedom. A weakness is the limited 

number of endpoints investigated. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is considered inadequate because of the 

limitations noted. 

Della Seta et al. (2006), supported by MURST and the 

University of Siena, examined the effects of pubertal 

bisphenol A exposure on behavior of male rats. [No 

information was provided in the manuscript about 

feed, caging, or bedding. The Expert Panel has been 

informed that Harlan Teklad 2018 chow, Lignocel 

bedding, and polysulfone cages were used (F. Farabolli 

et al., personal communication, March 1, 2007).] 

Seventy-eight Sprague–Dawley males were obtained 

from 16 dams and housed in groups of 4 with each from 

a different litter. On PND 23–30 (day of birth not 

defined), the rats were fed (by micropipette) peanut oil 

vehicle, 0.040 mg/kg bw/day bisphenol A [purity not 

reported in the manuscript; Z95% according to the 

authors (F. Farabollini et al., personal communication, 

March 1, 2007)], or 0.4 mg/kg bw/day ethinyl estradiol. 

[The number of rats treated in each group was not 

specifically indicated, but can be inferred to be 24–26/ 

group.] On PND 45, 12 males/group were tested for 

social and non-social behavior in response to a black PVC 

tube introduced into the cage. Behaviors were examined 

according to factor clusters of play and social interaction, 

environmental exploration and social investigation, and 

elements directed to the object. Twelve adults/group 

(490 days of age) were tested for sexual behavior with a 

sexually receptive female. Males that were not used in 

behavioral testing were killed on PND 37 (n 5 7 or 8/ 

group) and 105 (n 5 5 or 6/group) to measure plasma 

17b-estradiol and testosterone levels by RIA. Data were 

assessed by ANOVA and Fisher least significant difference test. 

Around the time of treatment, bisphenol A effects on 

juvenile behavior were not found on factors associated 

with environmental exploration and social investigation 

or with play and social interaction. However, juvenile 

behaviors directed to the object (biting, sniffing, climbing) occurred at a significantly lower frequency in the 

bisphenol A than control group. Compared to the vehicle 

controls, the ethinyl estradiol group exhibited lower 

frequencies of behaviors associated with environmental 

exploration or social investigation and with behaviors 

directed to the object. With respect to adult sexual 

behavior, data from the 9 or 10 of 12 animals/group that 

were sexually active were analyzed. Decreased intromission latency was significantly affected in males from the 

bisphenol A group. Significant effects in the ethinyl 

estradiol compared to the control group included 

decreased intromission latency as well as decreased 

latency to mount, increased frequency of intromission, 

increased ratio of intromissions/mount, and decreased 

duration of genital sniffing. On PND 37, the plasma 

testosterone level was significantly lower in the bisphenol A and ethinyl estradiol group than in controls. 

The plasma testosterone level was also significantly 

lower in the bisphenol A than control group on PND 105. 

No effects were observed on plasma 17b-estradiol levels. 

The study authors concluded that the behavioral effects 

observed in the bisphenol A-exposed rats occurred in the 

same direction as those observed in the ethinyl estradiol 

group and could be interpreted as consistent with 

estrogenic mediation. 

Strengths/Weaknesses: This study was well-conceived 

and executed. Appropriate dosing periods, design, and 

testing methods and timeframes were used to capture 

developmental effects of pubertal bisphenol A exposure 

of a short-term (juvenile period) and long-term (into 

adulthood) nature. Sample sizes were adequate. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for use in the 

evaluation process. 

Ceccarelli et al. (2007), supported by the University of 

Siena and MIUR, investigated the effects of orally 

administered bisphenol A and ethinyl estradiol during 

puberty in Sprague–Dawley rats. Sixteen pregnant 

Sprague–Dawley rats gave birth to offspring that were 

cross-fostered on PND 1, weaned on PND 21, and 

housed in groups of 4 males and 4 females. [No details 

of housing conditions during gestation were provided, 

including individual or group residency, bedding or 

cage material, or diet.] On PND 31, male and female 

offspring were separately housed in groups of 4 in 

Plexiglas cages with free access to water and food and 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 267 

Table 73 

Effects of Pubertal Exposure to Bisphenol A on ERa Levels in Sexually Dimorphic Hypothalamic Regions in the Rata 

Comparison, % change 

To oil control 

Bisphenol A Ethinyl estradiol Males to females 

Region PND Males Females Males Females Control Bisphenol A Ethinyl estradiol 

Arcuate nucleus 

Ventromedial nucleus 

Medial preoptic area 

37 

90 

37 

90 

37 

90 

2

2

2

2

2

2

2

2

[m50] 

2

2

2

2

2

[m112] 

2

2

2

2

2

2

2

2

[m85] 

2

2

2

2

2

2

2

2

2

2

2

[m50 in females] 

2

2

[m70 in males] 

2

2

[m118 in females] 

a

Ceccarelli et al. (2007).
 

Comparisons estimated from a graph.
 

m,k,2 Statistically significant increase, decrease, or no change compared to vehicle-treated, orchiectomized control.
 

maintained under a reversed light cycle. On PND 23–30, 

rats (n 5 14/group) were given bisphenol A 40 mg/kg 

bw/day, ethinyl estradiol 0.4 mg/kg bw/day, or peanut 

oil vehicle. Half the offspring (n 5 7/group) were killed 

on PND 37 and half on PND 90. Females killed on PND 

90 were killed in estrus. Blood samples were taken and 

animals were formalin perfused. Brains were harvested, 

post-fixed, and cryopreserved. Immuno-histochemistry 

was performed on frozen sections for comparative ERa

level analysis, with a focus on sexually dimorphic 

regions of the hypothalamus: the arcuate nucleus, 

ventromedial nucleus, and medial preoptic area. Two 

or three sections/rat were stained, equivalent field areas 

outlined, and ERa-positively stained nuclei counted 

under light microscopy by an evaluator blinded to all 

experimental parameters. Serum testosterone and 17bestradiol were determined by RIA. Statistical analyses 

were performed using ANOVA and post-hoc least 

significant difference test. 

The results for ERa are shown in Table 73. There were 

few statistically significant difference between controls 

and treated rats. Effects identified for ethinyl estradiol 

were not seen with bisphenol A with the exception of an 

increase in bisphenol A-treated females compared to 

males in ERa at 90 days in the medial preoptic area. On 

PND 37, testosterone was significantly reduced [B40%] 

in bisphenol A treated males compared to control males. 

There were no significant effects of bisphenol A 

treatment on 17b-estradiol or on testosterone/17b-estradiol ratio. 

The authors conclude that exposures to bisphenol A at 

40 mg/kg bw/day during early puberty can induce both 

short-term and long-term changes in sexually dimorphic 

regions of the brain and circulating testosterone/17bestradiol ratio. 

Strengths/Weaknesses: This interesting and novel 

manuscript examined the potential for the ethinyl 

estradiol positive control and bisphenol A administered 

before puberty, but after the most sensitive period (i.e., 

PND 3–10), to modulate ER and steroid hormones during 

puberty and sexual maturity. It appears that the authors 

tried to remove the potential for bias by blinded 

quantification of ER-positive neurons. The oral route of 

exposure was relevant. These data must be linked 

functionally to the results of Della-Seta et al. (2006). A 

weakness is that hormonal measurements were taken at 

single time points. 

Utility (Adequacy) for CERHR Evaluation Process: 

These data are adequate and of high utility for the 

evaluation process. 

3.2.4 Rat—parenteral exposure postnatally. 

3.2.4.1 Reproductive endpoints: Fisher et al. (1999), 

supported by the European Centre for Ecotoxicology of 

Chemicals and Zeneca, examined the effect of neonatal 

bisphenol A exposure on excurrent ducts of the rat testis. 

On PND 2–12 (PND 1 5 day of birth), Wistar rat pups were 

s.c. injected with the corn oil vehicle or 37 mg/kg bw/day 

bisphenol A [purity not given]. The dose was based on the 

solubility limit in oil. [The number of rats treated was not 

indicated nor was relationship to litter, but based on the 

number of rats examined in each time period (B3–7 in 

treated group and 5–20 in control group), it appears that 

there were B25/group in the bisphenol A group and B48 

in the vehicle control group. No information was 

provided about caging or bedding materials.] Seven other 

compounds were also examined but will not be discussed, 

with the exception of a brief explanation of results obtained 

with 0.0037–0.37 mg/kg bw/day diethylstilbestrol. Rats 

were killed at 10, 18, 25, 35, and 75 days of age. Testes 

and epididymides were removed and fixed in Bouin 

solution. Immunohistochemistry techniques were used to 

examine water channel aquaporin-1 levels. Morphology of 

rete testis and efferent duct were examined. Data were 

analyzed by ANOVA. 

In the bisphenol A group, the only effect on testis 

weight was a significant decrease [B40%] at 35 days of 

age. Epithelial cell height in the efferent ducts was 

significantly reduced [by B15%] at 18 and 25 days of 

age, but not at later time periods. There was no effect on 

expression of water channel aquaporin-1 protein or 

morphology of the rete testis. Treatment with most 

diethylstilbestrol doses resulted in reduced testicular 

weights at all ages, decreased expression of water 

channel aquaporin-1 protein, and decreased epithelial 

cell height in efferent ducts at 25 days of age and 

younger, and fluid retention and enlargement of rete 

testis, which was most severe at PND 18 and 25. The 

study authors concluded that the magnitude and 

Birth Defects Research (Part B) 83:157–395, 2008
268 CHAPIN ET AL. 

duration of adverse effects induced by estrogenic 

compounds were broadly similar to the estrogenic 

potencies of the compounds. 

Strengths/Weaknesses: This is a carefully performed 

study, although the inclusion of many methodological 

details (vide supra) would have improved it. Strengths 

include the use of a wide range of estrogenic compounds 

to alter testicular development. A limitation for the 

present purpose is that only a single dose level of 

bisphenol A was administered subcutaneously. A weakness is that tissues other than the testis were not 

examined. Other weaknesses include sample sizes ranging from 3–20 examined pups across groups and s.c. 

administration. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for evaluation due to lack of 

clarity about experimental or statistical control for litter 

effects. 

Nagao et al. (1999), supported by the Japanese 

Ministry of Health and Welfare, examined the effects of 

neonatal bisphenol A exposure on reproductive function 

of male and female Sprague–Dawley given CE-2 feed 

(Clea Japan). [No information was provided about 

caging or bedding materials.] From PND 1–5 (birth by 

16:00 considered PND 0), 28–31 pups/sex/group were 

s.c. injected with corn oil vehicle, 300 mg/kg bw/day 

bisphenol A [purity not reported], or 2 mg/kg bw/day 

estradiol benzoate. Pups within litters were treated with 

the same dose. Doses were based on results of preliminary studies that demonstrated no effect on growth 

or viability at bisphenol A doses up to 300 mg/kg bw/ 

day administered by s.c. injection in the neonatal period. 

Pups were examined for viability from PND 6–21. On 

PND 21, 5 pups/sex/group were randomly selected and 

killed. Pups were transcardially perfused, and reproductive organs were collected for histopathological evaluation. At 12 weeks of age, 22–25 rats/sex were mated with 

untreated rats. Females were killed on GD 13 for an 

evaluation of implant number and viability of embryos. 

After fertility evaluation, sexual behavior with a sexually 

receptive female was assessed in 10 males/group. 

Following evaluation of sexual behavior, 15 male rats/ 

group were killed for measurement of reproductive 

organ and brain weight. Histopathology of reproductive 

organs and SDN-POA volume were measured in 5 

males/group. Copulation and fertility indices were 

analyzed by w2 and Fisher exact one-tailed test. Data 

for other endpoints were analyzed by Student t-test. 

In rats treated with bisphenol A, there were no clinical 

signs of toxicity or effects on pup viability or body 

weight gain during or following the lactation period 

[data for pup viability not shown by study authors]. 

There were no effects on age of vaginal opening or 

preputial separation. Copulation and fertility indices and 

numbers of live embryos/litter were not affected in male 

or female rats treated with bisphenol A. Bisphenol A 

treatment did not affect sexual behaviors of males, as 

determined by number of mounts, intromissions, and 

ejaculations. No histopathological alterations were observed in the ovaries of treated females at 21 days of age 

or in the epididymis, prostate, or seminal vesicles of 

treated male rats at 21 days or 14 weeks of age. [The 

prostatic lobe not specified; based on the figure 

provided, the lobe appears to have been ventral 

prostate. The Expert Panel notes that the number of 

apically located nuclei may be elevated by 14 weeks of 

age over what would normally be expected; however, 

this observation cannot be determined definitively 

based on a single high power field and in the absence 

of a matched control.] No effect of treatment was 

observed on the SDN-POA of males. In contrast to the 

bisphenol A groups, rats treated with estradiol benzoate 

experienced decreased body weight gain, compromised 

male sexual behavior, infertility, lesions in reproductive 

organs, and reduced volume of the SDN-POA. The study 

authors concluded that neonatal exposure to a relatively 

high-dose of bisphenol A had no effect on morphological 

development or function of the reproductive system. 

Strengths/Weaknesses: Strengths include a well performed and documented study that compared effects of 

bisphenol A and estradiol benzoate. Additional strengths 

include documentation of both behavioral (mating 

behavior) and biological (genital tract development) 

endpoints in both male and female rats. Weaknesses 

include the use of only a single dose level of bisphenol A 

via s.c. injection, and no accounting for litter effects 

within the context of individual animal treatments 

within litters. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate for evaluation, however, utility is 

limited by subcutaneous administration. 

Stoker et al. (1999), support not indicated, examined 

the effects of prepubertal bisphenol A exposure on 

prolactin secretion and prostate size in rats. [No 

information was provided about feed, bedding, or 

caging materials.] On PND 22–32 (day of birth 5 PND 

0), 15–17 male Wistar rats from different litters/group 

were s.c. injected with bisphenol A [purity not reported] 

at 0 (sesame oil vehicle) or 50 mg/kg bw [assumed to be 

50 mg/kg bw/day]. Another group of rats was administered 17b-estradiol through a s.c. Silastic tube implant 

[dose administered not clear]. On PND 29, 6 animals/ 

dose were killed and blood was collected for measurement of serum prolactin concentration. The remaining 

rats (n 5 9–11/group) were killed at 120 days of age. 

Prolactin levels were measured in serum and anterior 

pituitary by RIA. Inflammation was visually examined in 

the ventral and lateral prostate. Left lateral and ventral 

prostates were weighed and lateral prostate was analyzed for myeloperoxidase (an indicator of neutrophil 

numbers) and DNA. The right lateral prostate was 

subjected to histological examination. Statistical analyses 

included ANOVA, Dunnett t-test for multiple comparison, and Fisher exact probability test. 

On PND 29, serum prolactin levels were significantly 

increased by B210% in rats of the bisphenol A group 

compared to the control group. On PND 120, there was 

no effect on prolactin levels in serum or pituitary in the 

bisphenol A group. Ventral prostate weight was unaffected but lateral prostate weight was increased [by 

B25%] in the bisphenol A group. Exposure to bisphenol 

A had no effect on body or testis weight. [Data were not 

shown by study authors.] The myeloperoxidase assay 

was reported to show a ‘‘trend’’ for lateral prostate 

inflammation in the bisphenol A group. [Trend was not 

defined; there was no statistical difference between the 

bisphenol A group and the control in the myeloperoxidase assay.] No histological evidence of inflammation 

was observed in prostates from the control group. In the 

bisphenol A group, histopathological analyses revealed 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 269 

that 44.4% of prostates contained increased a focal 

luminal polymorphonuclear cellular infiltrate that was 

milder in severity compared to prostates from the 17bestradiol group. The study authors noted the discrepancy 

between the results obtained by myeloperoxidase assay 

and histological observation in the bisphenol A group 

and stated that the discrepancy may have been due to 

evaluation of the whole tissue by myeloperoxidase assay 

versus only one section of the tissue by histological 

evaluation. Bisphenol A had no effect on prostate DNA 

content. In addition to prostate inflammation, effects 

observed in the 17b-estradiol group were increased 

serum prolactin levels on PND 29 and elevated myeloperoxidase and DNA content in lateral prostate on PND 

120. Based on these findings, the study authors concluded that chemically induced, transient increases in 

prolactin secretion in the prepubertal period can lead to 

increased incidence of lateral prostate inflammation in 

120-day-old rats. 

Strengths/Weaknesses: Comparison with other agents 

is a strength. Weaknesses include low to moderate 

sample sizes and the use of a single high-dose level of 

bisphenol A through subcutaneous administration. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate for the evaluation process but has 

limited utility due to concerns about sample sizes and 

route of administration of treatment. 

Atanassova et al. (2000), supported by the European 

Center for the Ecotoxicology of Chemicals and AstraZeneca, examined the effects of neonatal bisphenol A 

exposure on the reproductive system of male rats. Wistar 

rats were fed rat and mouse breeding diet No. 3, which 

contains 15.5% soy meal flour. [No information was 

provided about caging and bedding materials.] Litters 

of 8–12 male rats from randomized litter origin were 

assembled by cross-fostering pups on PND 1 (day of 

birth). On PND 2–12, rats were s.c. injected with corn oil 

vehicle or bisphenol A [purity not given] 0.5 mg/day. 

[Assuming a 5–25 g body weight during this interval, 

this dose would be B100 mg/kg bw/day at the beginning of the interval and B20 mg/kg bw/day at the end 

of the interval.] Other groups of rats were s.c. injected 

with 0.01–10 mg diethylstilbestrol every other day between PND 2–12 or 2 mg 4-tert-octylphenol/day during 

PND 2–12. Rats were killed on PND 18, 25, and 90–100. 

At PND 18 and 25, testes were weighed and fixed in 

Bouin solution. Testicular cell numbers and seminiferous 

tubule lumen formation were determined by standard 

point counting of cell nuclei. Apoptosis was assessed by 

DNA fragmentation detected by in situ DNA 30

-end 

labeling. Spermatocyte nuclear volume as a fraction of 

Sertoli cell nuclear volume was calculated as ‘‘an index of 

spermatogenic efficiency.’’ Plasma FSH and inhibin B 

were measured by RIA and ELISA methods, respectively. 

Fertility was assessed at 80–90 days of age; rats were 

mated for 7 days and number of pups was counted at 

birth. The number of rats/group examined was 7–14 at 

18 days of age, 4–12 at 25 days of age, and 6 in fertility 

testing. Data were analyzed by ANOVA. 

Significant effects observed on PND 18 were advanced 

testicular lumen formation and increases in testis weight, 

Sertoli cell volume/testis, and spermatocyte nuclear 

volume/unit Sertoli cell. A decrease in germ cell 

apoptosis was also described on PND 18 but was not 

statistically significant. Plasma FSH levels were 

increased significantly on PND 18, but there was no 

effect on plasma inhibin B concentration. The only 

significant effect observed on PND 25 was increased 

plasma FSH levels. Testis weight was increased in 

adulthood, but there were no effects on fertility or litter 

size. Effects observed with octylphenol were similar to 

those observed with bisphenol A. In contrast, exposure to 

one or more doses of diethylstilbestrol resulted in 

increased apoptosis, decreased plasma inhibin levels, 

decreased Sertoli cell nuclear volume, and changes in 

spermatocyte/Sertoli cell ratios. The study authors 

concluded that the effect of bisphenol A on spermatogenic processes is benign. 

Strengths/Weaknesses: Comparison with other agents 

is a strength. Weaknesses include low to moderate 

sample sizes, the use of a single high-dose level of 

bisphenol A through subcutaneous administration, and 

no accounting for litter effects within the context of 

individual animal treatments within litters. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate for the evaluation process but has 

limited utility due to concerns about sample sizes and 

route of administration of treatment. 

Williams et al. (2001), supported by the European 

Centre for Ecotoxicology, examined the effect of neonatal 

bisphenol A exposure on seminal vesicle structure and 

expression of sex steroid receptors in rats. On PND 2 

(day of birth 5 PND 1), litters consisting of 8–14 male 

Wistar rat pups were derived through cross-fostering. 

Rats were s.c. injected with corn oil vehicle or 0.5 mg/ 

day bisphenol A on PND 2–12. [Assuming a 5–25 g body 

weight during this interval, the dose would be 

B100 mg/kg/day at the beginning of the interval and 

B20 mg/kg bw/day at the end of the interval.] The dose 

was based on the highest amount that could remain in 

solution. A positive control group was injected with 

diethylstilbestrol at 0.1, 1, or 10 mg/day on PND 2, 4, 6, 8, 

10, and 12. Ethinyl estradiol was administered at 10 mg/ 

day, according to the protocol for diethylstilbestrol. 

Control animals for each compound were dosed with 

vehicle on the appropriate days, and because no 

differences were noted for controls, data were pooled. 

The effects of 4-tert-octylphenol, genistein, Antarelix, 

flutamide, and tamoxifen were also examined but will 

not be discussed. [No information was provided about 

feed, caging or bedding materials, or purity of 

compounds.] Animals were killed on PND 18, and 

seminal vesicles from 11–15 animals/group were collected and stored in Bouin solution. Seminal vesicles 

were examined for gross abnormalities in stroma and 

epithelium. Immunolocalization studies were conducted 

to assess ERb, ERa androgen receptor, and progesterone 

receptor proteins in the seminal vesicle. Studies were 

replicated 3–5 times using samples from at least 6 

animals/group. Results were scored subjectively. 

The gross structure of the seminal vesicles from 

bisphenol A-treated rats appeared normal, and there 

were no changes in ERb, ERa, androgen receptor, or 

progesterone receptor proteins in the seminal vesicle. In 

contrast, diethylstilbestrol induced changes in seminal 

vesicle morphology, increased ERa and progesterone 

receptor, and decreased androgen receptor. Effects of 

ethinyl estradiol were similar to those observed with 

diethylstilbestrol. The study authors concluded that the 

lack of bisphenol A effects suggested that only highBirth Defects Research (Part B) 83:157–395, 2008
270 CHAPIN ET AL. 

doses of potent estrogens induce gross abnormalities in 

the male reproductive system; and that only agents that 

suppress androgen receptor while increasing ERa and 

progesterone receptor are likely to cause gross developmental abnormalities in the male reproductive system. 

Strengths/Weaknesses: Strengths include expertise of 

the group coupled to well-performed experiments, data 

recording, and interpretation. Bisphenol A was not a 

primary target in this study but was one of a series of 

estrogenic compounds, allowing comparison with other 

similar compounds. However, a significant weakness are 

the s.c. route of administration, only a single varying 

dose level of bisphenol A was used, and there was no 

accounting for litter effects within the context of 

individual animal treatments within litters. 

Utility (Adequacy) for CERHR Evaluation Process: 

This work is inadequate for the evaluation process, based 

on lack of clarity for experimental or statistical control for 

litter effects. 

Rivas et al. (2002), supported by the European Union 

and the Spanish Ministry of Education, examined the 

effects of bisphenol A exposure on reproductive tract 

development of male rats. The main focus of the 

study was determining the effects of decreased androgen 

production in combination with a low dose of 

diethylstilbestrol. Effects of flutamide were also examined but will not be discussed. Wistar rats were fed a soyfree diet (rat and mouse soya-free breeding diet; SDS, 

Dundee, Scotland). [No information was provided about 

caging and bedding materials.] Litters of 8–12 male pups 

were assembled by cross-fostering on PND 1 (day of 

birth). Male rats were s.c. injected with the corn oil vehicle 

or 0.1 mg bisphenol A [purity not indicated] on PND 2, 4, 

6, 8, 10, and 12 with and without co-administration of 

10 mg/kg GnRH antagonist (a suppressor of androgen 

production). [Assuming a 5–25 g body weight during 

this interval, the bisphenol A dose would be B20 mg/kg 

bw/day at the beginning of the interval and B4 mg/kg 

bw/day at the end of the interval.] Additional rats were 

s.c. injected with diethylstilbestrol at doses of 0.1 or 10 mg 

on PND 2, 4, 6, 8, 10, and 12 with and without 

administration of GnRH antagonist. Rats were killed on 

PND 15. The testis was fixed in Bouin solution and 

testicular structures were measured. Plasma testosterone 

levels were measured using an ELISA technique. From 3– 

10 animals/group were examined for each endpoint. 

Data were analyzed by ANOVA. 

Treatment with bisphenol A alone did not affect 

plasma testosterone levels but treatment with GnRH 

antagonist alone and in combination with bisphenol A 

significantly lowered plasma testosterone levels. Treatment of rats with bisphenol A alone or in combination 

with GnRH antagonist had no significant effect on rete 

testis luminal area, efferent duct luminal area, efferent 

duct epithelial cell height, or vas deferens epithelial cell 

height. Exposure to the high diethylstilbestrol dose 

increased rete area, and both doses of diethylstilbestrol 

decreased plasma testosterone levels, increased efferent 

duct luminal area, and decreased epithelial cell height in 

efferent duct and vas deferens. The study authors 

concluded that the estrogenicity of bisphenol A when 

injected at a moderately high-dose was insufficient for 

disrupting the estrogen-androgen balance in rats. 

Strengths/Weaknesses: This study was performed 

carefully and well-documented. Weaknesses include: 

the dose of bisphenol A was high and only a single dose 

level administered subcutaneously was examined; and 

litter effects were not addressed in the context within 

litter dosing of cross-fostered litters. 

Utility (Adequacy) for CERHR Evaluation Process: 

This work is inadequate for the evaluation process, based 

on lack of clarity on control for litter effects. 

Sharpe et al. (2003), supported in part by the European 

Union and the Spanish Ministry of Education, examined 

the effects of neonatal exposure of rats to bisphenol A on 

Leydig cell development and function. Wistar rat dams 

were fed a standard soy-containing feed (rat and mouse 

breeding diet; SDS). [No information was provided on 

feed given to male offspring following weaning or 

bedding and caging materials.] Litters of 9–12 male 

pups were created by cross fostering pups on PND 1 (day 

of birth). Male pups were s.c. injected with the corn oil 

vehicle or 0.5 mg/day bisphenol A [purity not reported] 

on PND 2–12. [Assuming 5–25 g body weight during 

this interval, the dose would be B100 mg/kg bw/day at 

the beginning of the interval and B20 mg/kg bw/day at 

the end of the interval.] Other groups of rats received 

diethylstilbestrol at 0.1–10 mg/day on PND 2, 4, 6, 8, 10, 

and 12. Additional rats were treated with GnRH 

antagonist Antarelix or 4-tert-octylphenol, but those 

results will not be discussed. Rats were killed on PND 

18, 25, 35, or 90. Testes were weighed and fixed in Bouin 

solution. Sections of testes were immunostained with the 

Leydig cell marker 3b-hydroxysteroid dehydrogenase to 

evaluate Leydig cell development in 5–7 animals/group. 

Plasma testosterone levels were measured by ELISA. 

Group sizes for evaluation of testes weight and plasma 

testosterone were 2–23, with most groups containing at 

least 8 animals. Data were analyzed by ANOVA. 

The only significant effect on plasma testosterone level 

following exposure to bisphenol A was an increase on 

PND 18 (n 5 4). In rats of the bisphenol A group 

examined at each time period, there were no significant 

effects on testis weight, percent Leydig cell nuclear 

volume/testis, Leydig cell nuclear volume/testis, or total 

Leydig cell volume (nuclear1cytoplasmic volume/testis). Significant results in rats exposed to diethylstilbestrol included decreased Leydig nuclear cell volume at the 

mid- or high-dose on or before PND 35 and reduced 

plasma testosterone level and testis weight at all doses 

and most time points of evaluation. The study authors 

concluded that there were no consistent changes in 

Leydig cell development following exposure to bisphenol A. 

Strengths/Weaknesses: A strength is that bisphenol A 

was one of a number of compounds examined enabling 

internal comparison with other similar molecules. Limitations include use of a single high but variable dose of 

bisphenol A and small sample sizes for critical 

endpoints. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate due to small or uncertain 

sample sizes for key endpoints. 

Khurana et al. (2000), supported by NIH, March of 

Dimes, and Pardee Foundation, examined the effects of 

neonatal bisphenol A exposure on prolactin levels in rats. 

[The type of chow used and composition of bedding 

and caging materials were not reported.] On PND 1–5 

(day of birth 5 PND 0), 8–10 Fischer 344 rat pups/sex/ 

group (litter relationships are unclear) were s.c. 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 271 

injected with the tocopherol-stripped corn oil vehicle, 

bisphenol A [purity not indicated] at 0.1 or 0.5 mg/day, 

diethylstilbestrol at 5 mg/day, or octylphenol at 0.1 or 

0.5 mg/day. [Assuming a pup body weight of 5 g, 

bisphenol A intakes were estimated at 20 and 100 mg/ 

kg bw/day.] On PND 15, 20, and 25, blood was collected 

for measurement of serum prolactin level by RIA. A final 

sample for prolactin analysis was obtained when animals 

were killed on PND 30. Medial basal hypothalamus, 

anterior pituitary, uterus, and prostate were collected for 

measurement of ERa and ERb mRNA expression by RTPCR in animals of the low-dose group. Statistical 

analyses included ANOVA followed by Student-Newman–Keuls test. 

In male and female rats, hyperprolactemia was 

observed on PND 25 and 30. [On PND 30, prolactin 

levels in the low- and high-dose groups compared to 

the control group were B150 and 95% higher in females 

and 120 and 80% higher in males]. In females exposed to 

the low dose, ERa mRNA in the medial basal hypothalamus was higher [by 25%] than control levels. In 

anterior pituitary of low-dose males, ERa mRNA was 

higher [by B80%] and ERb mRNA was higher by 

35–40% compared to control levels. There were no 

effects on ERb mRNA in female tissues. Most effects 

observed with octylphenol exposure were similar to 

those observed with bisphenol A exposure. Diethylstilbestrol induced transient increases in prolactin levels, 

decreased expression of ERa in medial basal hypothalamus of males, upregulated ERa and ERb expression in 

the pituitary of males, decreased expression of ERa

in the uterus, and upregulated ERb expression in 

prostate. The study authors concluded that exposure of 

neonatal rats to bisphenol A resulted in delayed and 

sustained hyperprolactemia and changes in ER mRNA 

expression. 

Strengths/Weaknesses: A strength is that both male 

and female animals were assessed following administration of two dose levels. Weaknesses include small 

treatment groups consisting of unclear numbers of litters 

and composition and limited experimental details regarding design. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process due 

to lack of design clarity. 

Fukumori et al. (2003), support not indicated, examined the effect of postnatal bisphenol A exposure on 

ultrastructure of the prostate in rats. [The study was 

published in Japanese; a translation was provided by 

the American Plastics Council.] On Day 1–21 following 

birth, F344 rats were s.c. injected with bisphenol A 5 

days/week at doses of 0 (DMSO vehicle), 0.0008, 0.004, 

0.020, and 0.500 mg/kg bw/day. A positive control group 

received 100 mg/kg bw 17b-estradiol by s.c. injection 

during the same time period. Rats were killed at 22 days 

of age. Ventral prostates were fixed in glutaraldehyde, 

sectioned, and examined by electron microscopy. [The 

number of rats treated and examined/group and the 

number of litters represented were not reported. No 

information was provided on purity of bisphenol A, 

type of feed, or composition of bedding and caging. 

The translated version of the report did not include 

figures from the original report.] 

In ventral prostates obtained from rats exposed to 17bestradiol, there was an increase in secretory granules 

accompanied by reductions in microvilli on the surface of 

the glandular epithelium. Proliferation of fibroblasts was 

observed in the fibromuscular layer of the stroma in rats 

from the 17b-estradiol group. In the 0.020 and 0.500 mg/kg 

bw/day bisphenol A groups, a slight increase in secretory 

granules and slight decrease in microvilli was observed in 

glandular epithelium. Effects in stroma were described as 

unremarkable for the bisphenol A groups. The study 

authors concluded that bisphenol A may have ultrastructural effects on the ventral prostates of suckling rats. 

Strengths/Weaknesses: This is a translation of an 

apparently carefully performed study to assess the 

effects of low doses of perinatal bisphenol A on prostatic 

structure. A major weakness is that the original figures 

were not provided in the translated version of the report, 

and the route is s.c. injection in DMSO. The young age at 

which the animals were sacrificed is also a concern 

because prostatic development is not complete at 22 days 

of age making comparisons with the bulk of established 

data problematic. The lack of data specifics raise the level 

of uncertainty about this study. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is considered inadequate for inclusion in the 

evaluative process because of the lack of detail on study 

design (i.e., litter representation, number of animals per 

group). 

Kato et al. (2003), supported by the Japanese Ministry 

of Education, Culture, Sports, Science, and Technology 

and the Ministry of Health, Labor, and Welfare, examined the effects of neonatal bisphenol A exposure on the 

reproductive organs of rats. Sprague–Dawley rats were 

fed CRF-1 diet. [No information was provided on caging 

or bedding materials.] Female offspring from 8 dams 

were grouped to achieve equal distribution of body 

weight. At least 8 female offspring/group were s.c. 

injected with 0 (ethanol/corn oil vehicle), 0.25, 1, or 

4 mg/day bisphenol A [purity not reported] from PND 0 

to 9 (day of delivery 5 PND 0). [Based on body weights 

reported on PND 0 and 9, CERHR calculated mean 

bisphenol A intakes of B26, 105, and 427 mg/kg bw/ 

day.] A positive control group was given 10 mg/day 17bestradiol [B3 mg/kg bw/day] during the same time 

period. Rats were weighed during and following the 

lactation period and examined for day of vaginal opening. External reproductive organs were examined on 

PND 60, and estrous cycles were assessed from PND 61– 

94. One group of rats was ovariectomized on PND 80; 

ovaries were weighed, and fixed in 10% neutral buffered 

formalin for evaluation of corpora lutea and polyovular 

follicles. Another group of bisphenol A-exposed and the 

vehicle-treated control females were given 1 mg/kg 17bestradiol from PND 94–96 and killed the day following 

final injection; uterus and vagina were weighed, and 

fixed in 10% formalin. For all endpoints, 5–8 rats/group 

were examined. Statistical analyses included Student 

t-test and Fisher exact probability test. 

Treatment–related results are summarized in Table 74. 

Two rats of the high-dose group died. Body weights of 

rats in the high-dose group were lower than controls on 

PND 9–30 but higher than controls on PND 61–97. Effects 

observed at the mid- and high-dose included accelerated 

vaginal opening, increased incidence of polycystic 

ovaries, decreased area of corpora lutea, and decreased 

uterine fluid weight. All rats of the mid-dose group had 

partial clefts in the clitoris, and all rats of the high-dose 

Birth Defects Research (Part B) 83:157–395, 2008
272 CHAPIN ET AL. 

Table 74 

Effects in Female Rats Exposed to Bisphenol A During the Neonatal Perioda 

Dose, mg/kg bw/day [CERHR estimate] 

Endpoint 26 105 427 BMD10 BMDL10 BMD1SD BMDL1SD 

Body weight gain 

PND 9 2 2 k16% 286 200 233 156 

PND 97 2 2 m10% 432 261 430 253 

Day of vaginal opening 2 k2.9 days k4.1 days 345 267 159 116 

No. with normal estrous cyclesb 2 (8/8) 2 (2/8) k (0/6) 81 28 

No. with cleft clitorisc 2 (0/8) m (0/8) m (6/6) 299 failed 

Relative organ weight 

Ovary 2 2 k59% 85 59 140 93 

Uterus, wet 2 2 k60% 66 55 128 96 

Uterus, blotted 2 2 k21% 273 128 318 168 

Uterine fluid weight 2 k42% k97% 42 34 139 104 

No. with polycystic ovariesc No data m (4/8) m (5/5) 81 24 

No. with corpora luteab No data 2 (8/8) k (0/5) 238 90 

No. of corpora lutea No data 2 k (none) 65 38 137 83 

Corpora lutea area No data k 30% k (none) 42 37 84 66 

a

Kato et al. (2003).
 

b

Control rate 8/8.
 

c

Control rate 0/8.
 

m,k Statistically significant increase or decrease compared to controls; 2 no statistically significant effect.
 

group had deep clefts in the clitoris. Additional effects 

observed in rats of the high-dose group included 

disrupted estrous cycles (e.g., irregular cycles or persistent estrous) and decreased relative (to body weight) 

ovary and wet or blotted uterus weights. Absolute 

weights of wet uterus and ovary were also reduced in 

the high-dose group. No corpora lutea were observed in 

rats of the high-dose group. Qualitatively similar effects 

were observed in the group treated with 17b-estradiol. 

The study authors concluded that exposure of rats to 

bisphenol A during the neonatal period resulted in 

changes in female reproductive organs. 

Strengths/Weaknesses: The strengths are the carefully 

performed and documented experiments. The major 

limitation is that the s.c. route of administration and 

the doses of bisphenol A were relatively high. The 

changes in the female reproductive organs seen are well 

documented, but given the extremely high-dose of agent 

used, broadly unsurprising. 

Utility (Adequacy) for CERHR Evaluation Process: 

The results of this study reflect a careful documentation 

of the experiments performed. The study is adequate for 

the evaluation process but has limited utility due to 

concerns about the route of administration. 

Toyama and Yuasa (2004), supported in part by the 

Japanese Ministry of Environment and Ministry of 

Education, Science, Sports and Culture, examined the 

effects of neonatal bisphenol A [purity not reported] 

exposure on spermatogenesis during puberty and adulthood in rats and mice. [No information was provided 

about chow or bedding and caging materials. The 

mouse data are reported in Section 3.2.8.] Wistar rats 

were s.c. injected on a mg/pup basis with bisphenol A in 

a DMSO and olive oil vehicle on PND 1, 3, 5, 7, 9, and 11 

(PND 0 5 day of birth). Bisphenol A doses were 1.0, 10.0, 

100.0, and 600.0 mg/pup. Additional animals were 

treated with 17b-estradiol and estradiol benzoate. Animals were killed weekly at 2–10 weeks of age, and other 

pups were killed at 24 and 31 days of age. There were 5 

animals/dose/time point in bisphenol A groups and 

apparently 5 vehicle control rats/time period. Testes 

were examined by light and electron microscopy. Males 

from each experimental group (a total of 11 rats) were 

mated with 2 females [number tested in each dose 

group not reported]. A total of 11 rat dams were allowed 

to complete pregnancy. [It does not appear that 

statistical analyses were conducted.] 

All rats given 0.600 mg/pup bisphenol A died before 20 

days of age and were excluded from analysis. In 

mature spermatids of 8-week-old rats in the vehicle 

control group, the incidences of deformed acrosomes, 

deformed nuclei, and abnormal ectoplasmic specialization were o0.3%. In 8-week-old rats treated with 

Z0.010 mg/pup bisphenol A, the incidence of deformed 

acrosomes was 450–60%, the incidence of deformed 

nuclei was 440%, and the incidence of abnormal 

ectoplasmic specialization was 460–70%. [Data were 

not shown for individual dose levels.] Similar effects 

were observed in the groups treated with 17b-estradiol 

and estradiol benzoate. No effects were reported at other 

ages. [Data were not shown by study authors.] The 

blood–testis barrier remained intact based on histologic 

observations. All tested males from the bisphenol A 

group were fertile, and sex ratio, litter sizes, and pup 

weights were reported to be normal. [No results were 

shown for individual dose levels. Fertility data presented in Table 4 and 5 of the study, were not clearly 

identified by dose level.] The study authors concluded 

that bisphenol A acts as an estrogen and induces 

transient changes in the male reproductive system of 

rodents that resolve in adulthood. 

Strengths/Weaknesses: The strengths include the use 

of multiple doses of bisphenol A and the use of both 

rats and mice, allowing interspecies comparisons. 

Weaknesses include selective and unclear data presentation, absence of statistical analyses, subcutaneous injection on a per pup basis, and failure to examine sperm 

morphology in the fertile 15-week-old animals to 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 273 

determine whether the changes in sperm maturation 

seen at earlier time points had resolved or whether the 

animals were fertile in the face of such abnormalities. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate and not useful for the evaluation 

process due to lack of clarity of design and analyses, route 

of administration, and dosing procedures. 

Kato et al. (2006), supported by the Japanese Ministry 

of Education, Culture, Sports, Science and Technology 

and Ministry of Health, Labor and Welfare, examined the 

effects of neonatal exposure to bisphenol A on reproductive function of male rats. Sprague–Dawley rats were 

fed CRF-1 diet, which was described as having relatively 

low estrogenic activity compared to other Japanese 

rodent feeds. [No information was provided on caging 

or bedding materials.] Male rats used in this study were 

born to 12 dams, assigned to 8 foster dams in groups of 

seven based on body weights, and distributed to dose 

groups. From PND 0–9 (PND 0 5 day of birth), 24 male 

pups/group were s.c. injected with bisphenol A [purity 

not indicated] at 0 (ethanol/corn oil vehicle), 0.000024, 

0.000120, 0.000600, 0.003, or 1 mg/pup/day bisphenol A. 

Study authors calculated average exposures of 0.002, 

0.011, 0.056, 0.277, or 97 mg/kg bw/day. An additional 

group was treated with 10 mg/day 17b-estradiol (0.9 mg/ 

kg bw/day) during the same time period. Eight rats/ 

group were killed and necropsied at PND 10, 35, and 150. 

At the PND 10 necropsy, serum testosterone levels were 

measured by RIA, the testis was weighed and examined 

histologically, and expression changes in genes for 

hormone receptors and steroidogenic enzymes were 

determined by RT-PCR. The same endpoints were 

examined at the PND 35 necropsy in addition to 

measuring seminal vesicle, ventral prostate, and epididymis weights. The remaining rats were assessed for day 

of preputial separation. From PND 105–130, they were 

mated for 1 day a maximum of 4 times with an untreated 

female in proestrus. Females were killed on GD 13 (day 

of sperm 5 GD 0) and examined for corpora lutea, 

embryonic mortality, and implantation sites. Male rats 

were killed on PND 150. In addition to endpoints 

examined at earlier time periods, sperm endpoints and 

histopathology of ventral prostate were assessed. Statistical analyses included Bartlett method for homogeneity 

of variance followed by Dunnett method for homogeneous variances or Dunnett-type method with rank order 

for heterogeneous variances. Reproductive data were 

analyzed by Fisher exact probability test. Data obtained 

from the 17b-estradiol group were analyzed by Student ttest. 

There were no deaths or decreases in body weight in 

animals of the bisphenol A group. There were no effects 

on age of preputial separation, copulation rate, or 

fertility. In dams impregnated by bisphenol A-treated 

males, there were no effects on numbers of implantation 

sites, implantation losses, or live fetuses. Bisphenol A 

treatment had no adverse effects on sperm count, 

motility, or morphology. There were no effects on serum 

testosterone levels, histopathology of testis or prostate, or 

weights of testis, epididymis, seminal vesicle, ventral 

prostate, or penis. No significant changes were observed 

in mRNA for estrogen, androgen, or progesterone 

receptor or steroidogenic enzymes. In contrast to the 

bisphenol A groups, rats treated with 17b-estradiol 

experienced decreases in reproductive organ weights, 

altered gene expression, delayed and incomplete preputial separation, decreased copulatory rate, and decreased sperm numbers. The study authors concluded 

that neonatal bisphenol A exposure caused no adverse 

effects on reproductive function or gene expression of 

steroidogenic enzymes in the rat testis. 

There were no deaths or decreases in body weight in 

animals of the bisphenol A group. There were no effects 

on age of preputial separation, copulation rate, or 

fertility. In dams impregnated by bisphenol A-treated 

males, there were no effects on numbers of implantation 

sites, implantation losses, or live fetuses. Bisphenol A 

treatment had no adverse effects on sperm count, 

motility, or morphology. There were no effects on serum 

testosterone levels, histopathology of testis or prostate, or 

weights of testis, epididymis, seminal vesicle, ventral 

prostate, or penis. No significant changes were observed 

in mRNA for estrogen, androgen, or progesterone 

receptor or steroidogenic enzymes. In contrast to the 

bisphenol A groups, rats treated with 17b-estradiol 

experienced decreases in reproductive organ weights, 

altered gene expression, delayed and incomplete preputial separation, decreased copulatory rate, and decreased sperm numbers. The study authors concluded 

that neonatal bisphenol A exposure caused no adverse 

effects on reproductive function or gene expression of 

steroidogenic enzymes in the rat testis. 

Strengths/Weaknesses: This study has a number of 

major strengths, notably the wide range of doses, 

appropriate use of statistics, inclusion of a positive 

control, and use of relevant endpoints. Weaknesses 

include route of administration and dosing on a per 

pup basis, thus not adjusting for body weight. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate but of limited utility due to route 

of administration and dosing on a per pup basis. 

Noda et al. (2005), support not indicated, examined the 

effect of neonatal bisphenol A exposure on reproductive 

organs of Sprague–Dawley rats. For 5 days beginning on 

PND 1 (day of birth 5 PND 0), 6–10 pups/sex/group 

(drawn from 2 litters) were s.c. injected with olive oil 

vehicle or bisphenol A [purity not reported] at 0.0001, 

0.001, or 0.010 mg/rat/day. According to the study 

authors, the doses were equivalent to B0.010, 0.100, or 

1 mg/kg bw/day. A positive control group received 

diethylstilbestrol at the same doses as bisphenol A. 

Nonylphenol and genistein were also examined but will 

not be discussed here. Dose selection was based on 

diethylstilbestrol doses reported to have an effect. 

Stability, homogeneity, and concentration of dosing 

solutions were verified. Pups in each group were 

obtained from 2 dams. On PND 7, litters were adjusted 

to 4 males and 4 females/dam when possible. Dams and 

pups were housed in polycarbonate cages until weaning 

at PND 21. At that time, pups were housed in wire mesh 

cages. Animals were fed MF feed (Oriental Yeast Co.). 

[No information was provided on bedding used in 

polycarbonate cages.] During the study, animals were 

examined for clinical signs, body weight, anogenital 

distance on PND 7, and day of vaginal opening or 

preputial separation. Estrous cycles were assessed from 

the time of vaginal opening until animals were killed on 

PND 47–50 (females in diestrus). Rats in persistent estrus 

were killed on PND 70. Reproductive organs were 

weighed. Testis was fixed in Bouin solution and all other 

Birth Defects Research (Part B) 83:157–395, 2008
274 CHAPIN ET AL. 

reproductive organs were fixed in 10% neutral buffered 

formalin for histopathological examination. [It was not 

indicated, but it is assumed that all pups were 

examined in each analysis.] Data were analyzed by 

Bartlett test for homogeneity of variance, ANOVA, 

Dunnett test, or Kruskal–Wallis test. 

In the bisphenol A groups, there were no abnormal 

clinical signs or effects on body weight. Absolute 

anogenital distance was not affected, but anogenital 

distance adjusted by the square root of body weight 

cubed was decreased in females treated with the midand high-bisphenol A dose. There were no effects on day 

of vaginal opening or preputial separation or on estrous 

cycles. [Data were not shown.] No gross or histopathological abnormalities were reported in male or female 

reproductive organs. The study authors only reported 

organ weight effects relative to body weight, because the 

rats were killed at different ages. The only dose-related 

effect on reproductive organ weight was increased 

relative ventral prostate weight in the high-dose group. 

Relative pituitary weight was increased in males of the 

low-dose group and females of the high-dose group. 

There were no effects on weights of testis, epididymis, 

seminal vesicle, uterus, or ovary in bisphenol A-treated 

animals. Effects observed in animals treated with 1 or 

more dose of diethylstilbestrol included delayed or 

incomplete preputial separation, estrous cycle disruption, underdeveloped reproductive organs (including 

ventral prostate), malformations in male and female 

reproductive organs, ovarian cysts, and uterine squamous metaplasia in glandular epithelium. The study 

authors noted that the shortened anogenital distance in 

females appeared to be biologically significant. However, 

it was stated that the effect is of unknown relevance in 

female rats and was not observed in the rats treated with 

diethylstilbestrol. The study authors concluded that 

findings observed with bisphenol A were not toxicologically relevant. 

Strengths/Weaknesses: Strengths of this report include 

the use of 3 dose levels, the use of a positive control 

(diethylstilbestrol), and the use of multiple endpoints to 

evaluate estrogenic effects. Weaknesses include the use 

of only 2 litters to constitute exposure groups, exposure 

by the subcutaneous route to bisphenol A (not the 

anticipated route of exposure in humans), and dosing on 

a per pup basis. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate due to the combination of 

small sample size (i.e., 2 litters) and s.c. route of 

administration. 

Ho et al. (2006b), supported by NIH and Department 

of Defense, examined the effect of developmental 

exposure to bisphenol A on susceptibility of Sprague– 

Dawley rats to prostate cancer. The dams and offspring 

used in this study were fed a soybean-free phytoestrogen-reduced diet (Zeigler Reduced Rodent Diet 2, Ziegler 

Brothers, Inc.), housed in polysulfone cages [with 

unspecified bedding], and provided drinking water in 

glass bottles. On PND 1, 3, and 5 (day of birth 5 PND 0), 

20–30 male pups/group were s.c. injected with tocopherol-stripped corn oil vehicle, bisphenol A [purity not 

indicated] at 0.1 mg/pup (0.010 mg/kg bw), or estradiol 

benzoate at 0.001 mg/pup (0.1 mg/kg bw) or 25 mg/pup 

(2500 mg/kg bw). Male rats from each litter were 

randomly assigned to treatment groups, but the total 

number of litters from which the pups were selected was 

not reported. Likewise, it is unclear, but assumed, that all 

doses were represented within litter rearing units. Pups 

were weaned on PND 21. At PND 90, half the rats from 

each treatment group were implanted with Silastic 

capsules containing 17b- estradiol and testosterone and 

the other half were implanted with empty capsules; the 

capsules were left in place for 16 weeks. The treatment 

was designed to result in a serum 17b-estradiol level of 

B75 ng/L and testosterone level of B3 mg/L, levels 

reported to induce prostatic intraepithelial neoplasia in 

33% of Sprague–Dawley rats. Rats were killed at 28 

weeks of age. Prostates were removed, and histopathological evaluations were conducted on each lobe. 

Immunohistochemistry techniques were used to measure 

proliferation. Apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling (TUNEL) technique. PCR techniques were 

used to study methylation pattern and expression 

changes in prostate cell signaling proteins on PND 10, 

90, and 200. Statistical analyses included w2 test, ANOVA, 

Fisher exact test, and Bonferroni test. The study authors 

stated that similar responses were observed in each of the 

3 prostate lobes; and thus results were presented only for 

dorsal prostate. In bisphenol A-exposed compared to 

vehicle controls rats that did not receive 17b-estradiol/ 

testosterone exposure in adulthood, there were no effects 

on dorsal prostate weight, histopathology alterations, 

proliferation index, or apoptotic index. In bisphenol Atreated compared to vehicle control rats that received 

17b-estradiol/testosterone exposure in adulthood, there 

was increased incidence and severity of prostatic 

intraepithelial neoplasia (100 vs. 40% incidence). In the 

bisphenol A/17b-estradiol/testosterone group, proliferation and apoptosis indices were increased in regions 

where prostatic intraepithelial neoplasia (PIN) was 

observed. In humans PIN is an accepted precursor lesion 

to prostate cancer. In rodents the significance of PIN is 

less clear. Some transgenic mouse models will form PIN 

lesions that progress to adenocarcinoma in a manner 

broadly similar to that seen in humans. However, there 

are many examples in which mice form PIN lesions that 

do not progress to invasive disease. In rats, testosterone 

plus estradiol classically induces PIN lesions that 

progress to adenocarcinoma. The increase in incidence 

of PIN lesions seen following testosterone and 

estradiol treatment in BPA exposed rats in this study 

are certainly a cause for concern. The data presented do 

not address whether these lesions progress to cancer in a 

manner similar to PIN lesions seen in the classic 

testosterone plus estradiol model, or whether such 

progression occurs at a higher or lower rate. Changes 

observed in rats exposed to the high estradiol benzoate 

dose in the neonatal period but not 17b-estradiol/ 

testosterone during adulthood included increased incidence and severity of prostatic intraepithelial neoplasia 

and elevated apoptosis and proliferation indices. The 

same effects, in addition to decreased prostate weight, 

were observed in rats receiving neonatal exposure to the 

high estradiol benzoate dose and adult exposure to 17bestradiol/testosterone. 

In the investigation of a molecular basis for increased 

susceptibility to PIN, exposure to estrogenic compounds 

altered methylation pattern in several cell signaling 

genes. Phosphodiesterase type 4 variant, an enzyme 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 275 

involved in cyclic AMP breakdown, was selected for 

further study. Neonatal bisphenol A exposure resulted in 

hypomethylation of the phosphodiesterase type 4 variant 

gene and increased expression of that gene at 90 and 200 

days of age, with or without 17b-estradiol/testosterone 

exposure in adulthood. Similar responses in phosphodiesterase type 4 variant gene methylation and expression were observed with exposure to the low and high 17 

estradiol benzoate doses. The study authors concluded 

that developmental exposures of rats to bisphenol A 

increased susceptibility to precancerous prostate lesions 

resulting from prostate epigenomic alteration. 

Strengths/Weaknesses: This is a carefully performed 

study by a group with significant expertise in this area of 

work. The study has many strengths, from the use of a 

relatively low dose level of bisphenol A to the search to 

identify molecular mechanisms, possibly including sitespecific promoter methylation, underlying the observations made. Weaknesses include the use of a single dose 

level with subcutaneous dosing. It could be suggested 

that carrying the study further in terms of animal age 

might have produced more dramatic phenotypes and 

clarified the relevance of PIN resulting from BPA 

exposure to prostate cancer (potentially enhancing cancer 

incidence) in this model. Failure to do this could be 

considered a weakness of the work. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of limited utility for the 

evaluation process due to use of subcutaneous route of 

administration. 

3.2.4.2 Neurobehavioral endpoints: Ishido et al. 

(2004), supported by the National Institute for Environmental Studies and the Ministry of Economy, Trade, and 

Industry, examined the effects of postnatal intracisternal 

bisphenol A exposure on behavior of rats. Dams in this 

study were fed standard laboratory chow (MF Diet; 

Oriental Yeast Corp.). [No information was provided 

about caging or bedding materials.] At 5 days of age, 5– 

7 male Wistar rat pups/group were injected intracisternally with a bisphenol A dose [purity not indicated] of 0 

(ethanol/olive oil vehicle), 0.00002, 0.0002, 0.002, or 

0.020 mg. Pups were weaned at 3 weeks of age. 

Spontaneous motor activity was measured over a 12– 

24-hr period at 4–5 weeks of age. Rats were killed at 4 

and 8 weeks of age, and brains were removed. RNA was 

isolated from midbrain and striatum for DNA microarray analysis. Expression of the gene for dopamine 

transporter in midbrain was studied by RT-PCR. Tyrosine hydroxylase expression in brain was measured at 8 

weeks of age using an immunostaining method. Statistical analyses included ANOVA and Student t-test. 

In 4–5-week-old rats from the 0.020 mg bisphenol A 

group, motor activity was significantly increased and 

was 1.6 times higher than in control rats during the 

nocturnal period. In a dose response experiment, it was 

noted that hyperactivity was increased significantly at 

doses Z0.0002 mg. Microarray analysis revealed that 

bisphenol A [at an unspecified dose] downregulated 

expression of dopamine D4 receptor gene 2-fold at 4 

weeks of age and dopamine transporter gene 2.8-fold at 8 

weeks of age. Numerous other gene expression changes 

were observed but not discussed in detail by study 

authors. Analysis by RT-PCR confirmed that expression 

of the dopamine transporter gene was downregulated 3­

fold in the midbrain of 8-week-old rats treated with 

bisphenol A in the neonatal period. In rats from the 

0.020 mg bisphenol A group, tyrosine hydroxylase 

immunoreactivity was reduced in the substantia nigra 

at 8 weeks of age. The study authors interpreted the 

decrease in tyrosine hydroxylase immunoreactivity as 

degeneration of dopaminergic neurons. They concluded 

that bisphenol A affected the central dopaminergic 

system, resulting in hyperactivity that most likely 

occurred as a result of decreased tyrosine hydroxylase 

activity in midbrain. 

Strengths/Weaknesses: A significant weakness is the 

inability to correlate the internal exposure to bisphenol A 

provided by the intracisternal route with that seen by the 

oral route. Strengths of this study include the use of a 

range of concentrations of bisphenol A. The correlation of 

changes in behavior patterns induced by bisphenol A 

with expression of specific dopamine receptor sets is also 

a strength. A significant weakness is the inability to 

correlate the doses of bisphenol A provided by this 

dosing mechanism with those seen by more common s.c. 

or oral routes, as well as uncertainty about the disposition of the bisphenol A that is injected into the 

cerebrospinal fluid. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process due 

to uncertainties around the intracisternal route of 

administration. 

Masuo et al. (2004a), of the Japanese National Institute 

of Advanced Industrial Science and Technology and 

National Institute for Environmental Studies, investigated the effects in rats of an acute neonatal exposure to 

6-hydroxydopamine, bisphenol A, nonylphenol, p-octylphenol, or diethylhexyl phthalate on spontaneous motor 

activity, as well as catecholamine levels, dopaminergic 

neuron integrity by immunohistochemistry, and gene 

expression profiles. In the 6-hydroxydopamine group, 5­

day-old male Wistar pups weighing about 10 g were first 

pretreated with 25 mg/kg desipramine i.p. on PND 5 in 

order to protect noradrenergic neurons from the effects 

of 6-hydroxydopamine. These pups were then injected 

intracisternally 30 min later with 6-hydroxydopamine 

[not discussed here]. Other groups of pups were treated 

intracisternally with 0 (olive oil vehicle) or 87 nmol 

bisphenol A [purity not provided], nonylphenol, poctylphenol, or diethylhexyl phthalate in olive oil (n 5 6 

or 7/group). In additional experiments, intracisternal 

bisphenol A treatments were used over a 0.087–87 nmol 

[19.8 ng to 19.8 lg] dose range. Following treatment, 

pups were randomly assigned to lactating dams and 

weaned at 3 weeks of age. Animals were housed in 

acrylic cages at 221C under 12-hr light/12-hr dark 

conditions and given free access to water and chow from 

Oriental Yeast Co. 

Spontaneous motor activity was assessed at 4–5 weeks 

of age using an automated activity-monitoring system 

over a 12-hr light/12-hr dark cycle, apparently for a 

single 24-hr period. [Total number of cycles not 

indicated.] Brain sections from 8–10-week-old rats were 

snap frozen in liquid nitrogen. The striatum and whole 

mid-brains were used for cDNA microarray analyses. 

The frontal cortex, striatum, limbic regions including 

nucleus accumbens, septum, and olfactory tubercles 

were used to measure catecholamine levels by HPLC. 

Immunohistochemistry from whole brain sections was 

used to evaluate dopamine neuron integrity using 

Birth Defects Research (Part B) 83:157–395, 2008
276 CHAPIN ET AL. 

tyrosine-hydroxlyase monoclonal antibody reactivity 

[number of rats not indicated]. Most statistical analyses 

were performed using ANOVA techniques. Activity data 

were analyzed using repeated measures ANOVA to 

examine activity in 2-hr intervals, as well as across the 

dark, light, or full 24-hr period. Student t-tests were used 

to compare catecholamine levels. 

Spontaneous motor activity in rats treated with bisphenol A increased in a dose-dependent manner over 

the 0.087–87 nmol range, with significance on pair-wise 

comparison with controls at dose levels Z0.87 nmol [198 

ng]. Activity was increased in both the dark and light 

periods. Tyrosine hydroxylase activity was reduced in 

bisphenol A-treated rats, compared to controls. [Quantification of immunohistochemical sections was not 

provided.] Gene expression patterns in the midbrain 

differed in bisphenol A and 6-hydroxydopamine-treated 

animals. 

The authors concluded that neonatal exposure to 

bisphenol A was associated with an increase in spontaneous motor activity and reduced tyrosine hydroxylase 

activity. They hypothesized that bisphenol A may cause a 

deficit in the development of mesostriatal dopaminergic 

neurons, and that this increase either is greater than that 

produced by 6-hydroxydopamine lesions or involves 

additional neurochemical systems. A follow-up study 

(Masuo et al., 2004b) addressed these issues. The authors 

also proposed that bisphenol A-exposed rats can serve as 

animal models of attention deficit-hyperactivity disorder. 

Strengths/Weaknesses: A significant weakness is the 

inability to correlate the internal exposure to bisphenol A 

provided by the intracisternal route with that seen by the 

oral route. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process due 

to uncertainties around the intracisternal route of 

administration. 

Masuo et al. (2004b), funded by the New Energy and 

Industrial Technology Development Organization, the 

Ministry of the Environment, and the Ministry of 

Economy, Trade, and Industry, Japan, followed up their 

previous study (Masuo et al., 2004a) with additional gene 

expression microarrays to elucidate potential molecular 

pathways associated with the effects of an acute, 

neonatal exposure to 6-hydroxydopamine, bisphenol A, 

nonylphenol, diethylhexyl phthalate, or dibutyl 

phthalate on spontaneous motor activity levels at 4–5 

weeks of age. Pregnant Wistar rats were housed in 

acrylic cages with free access to tap water and laboratory 

chow (Oriental Yeast) and maintained on a 12-hr light/ 

12-hr dark cycle. In the 6-hydroxydopamine group, 

5-day-old male pups, each about 10 g, were first 

pretreated with 25 mg/kg desipramine by i.p. injection 

(to protect noradrenergic neurons from the effects of 6­

hydroxydopamine) and then given 6-hydroxydopamine 

intracisternally 30 min later. PND 5 male pups in other 

groups were intracisternally injected with olive oil 

vehicle, 87 nM bisphenol A [19.8 lg] [purity not 

provided], nonylphenol, diethylhexyl phthalate, or 

dibutyl phthalate. [Only the bisphenol A experiments 

will be discussed here.] Following treatments, 

pups were randomly fostered to lactating dams (5–7 

pups/per dam) and weaned at 3 weeks of age. At 4–5 

weeks of age, the spontaneous motor activity of bisphenol A treated rats was compared to vehicle treated 

rats (n 5 6 or 7/group) using an automated activitymonitoring system over a 12-hr light/12-hr dark cycle. 

Bisphenol A and vehicle-treated rats were killed at 8–10 

weeks of age and the striatum and midbrain were 

harvested. RNA was extracted from 2 pooled striata/rat 

(n 5 3/group) or 1 midbrain/rat (n 5 3/group) for cDNA 

microarray analyses. Gene expression values were 

evaluated relative to those of control-treated rats. 

Repeated measures ANOVA was used for statistical 

analyses of spontaneous motor activity during 2-hr 

time intervals. Statistics were not described for microarray results. 

Neonatal exposure to bisphenol A in male rats 

increased spontaneous motor activity significantly at 4– 

5 weeks during both the dark and light periods of the 

cycle when compared to controls. Gene expression 

profiles examined at 8–10 weeks of age for select genes 

potentially impinging on dopamine function and/or 

other pathways were altered in the adult striatum and 

midbrain of bisphenol A treated mice. The authors 

concluded that neonatal exposure to bisphenol A 

resulted in elevated spontaneous motor activity during 

both the light and dark phases. 6-Hydroxydopamine 

lesions increased motor activity only during the dark 

period. Comparisons of genetic expression in 6-hydroxydopamine and bisphenol A-treated rats suggested that 

the effects of bisphenol A may be mediated by alterations 

in dopamine as well as other systems. This profile of 

adverse effects was suggested to potentially serve as a 

model for human hyperactivity disorders. 

Strengths/Weaknesses: A significant weakness is the 

inability to correlate the internal exposure to bisphenol A 

provided by the intracisternal route with that seen by the 

oral route. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process due 

to uncertainties around the intracisternal route of 

administration. 

Ishido et al. (2005), support not indicated, examined 

the effects of neonatal bisphenol A exposure of rats on 

motor activity and gene expression in brain. Wistar rat 

dams were fed MF diet (Oriental Yeast). Pups were born 

from 10 pregnant dams and 5–7 male pups were assigned 

to each dam. At 5 days of age, male pups were injected 

intracisternally with vehicle (50% ethanol in olive oil) or 

87 nmol [19.8 lg] bisphenol A. [No information was 

provided on number of pups treated, purity of bisphenol A, or caging and bedding materials.] Pups were 

also treated with 2 nonylphenol compounds and 3 

phthalate compounds, but results for those compounds 

will not be discussed. Pups were weaned at 3 weeks of 

age. Spontaneous motor activity was measured in pups 

at 4–5 weeks of age. Rats were killed at 8 weeks of age, 

and RNA was isolated from midbrain for microarray 

analyses of gene expression. [The number of rats 

examined was not reported for any endpoint.] Data 

for spontaneous motor activity were analyzed by 

ANOVA or Student t-test. [There were no statistical 

analyses for gene expression data.] 

Rats exposed to bisphenol A were significantly more 

active during the nocturnal phase than control rats (by 

B1.4–1.6-fold). In midbrains of 8-week-old rats, expression levels were altered for 46 G protein-coupled receptor 

genes, which are involved in dopaminergic neurotransduction and many peptidergic neurotransduction 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 277 

processes. The study authors noted altered dopamine 

transporter gene expression, which was impaired by all 

chemicals tested. Bisphenol A also lowered galanin 

receptor 2 expression. The study authors concluded that 

intracisternal exposure to bisphenol A induced hyperactivity in rats, possibly by regulating gene or protein 

expression of G protein-coupled receptor and dopaminergic neurotransduction systems. 

Strengths/Weaknesses: Despite certain strengths, a 

significant weakness is the inability to correlate the 

internal exposure to bisphenol A provided by the 

intracisternal route with that seen by the oral route. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process. 

Patisaul et al. (2006), supported by the American 

Chemistry Council, evaluated the effect of neonatal 

bisphenol A on the anteroventral periventricular nucleus 

of the Sprague–Dawley rat. Pregnant rats (n 5 5) were fed 

a phytoestrogen-free diet (Purina 5K96) during the last 

week of gestation. [No information was provided about 

caging or bedding.] Dams were permitted to litter. Pups 

were cross-fostered among all dams so that 4 dams 

reared 6 females and 6 males and 1 dam reared 5 males. 

Pups (n 5 5–8/group) were randomly assigned to receive 

s.c. injections of 17b-estradiol 50 mg/pup, genistein 

250 mg/pup, bisphenol A [purity not indicated] 250 mg/ 

pup, or sesame oil vehicle every 12 hr for 48 hr. The 

authors estimated that the twice daily dosing with 

250 mg/pup was approximately equivalent to 100 mg/ 

kg bw/day. Injections began the morning of PND 1 

(delivery 5 PND 0). On PND 19, the pups were transcardially perfused with ice-cold saline followed by paraformaldehyde. Brains were post-fixed in 20% sucrose in 

paraformaldehyde, sectioned coronally, and processed 

for immunohistochemistry for ERa and tyrosine hydroxylase. Sections were counterstained with Nissl stain. 

Cells of the anteroventral periventricular nucleus positive for ERa, tyrosine hydroxylase, or both were counted. 

Statistical analysis used 2-way ANOVA with sex and 

treatment as factors, followed by 1-way ANOVA and 

post-hoc Fisher least significant difference test. 

There was a significant, sex-related effect on tyrosine 

hydroxylase-positive cells in the anteroventral periventricular nucleus with the number in males about 29% that 

of females [estimated from a graph]. The authors 

concluded that neonatal treatment with bisphenol A 

interfered with the normal testosterone-associated masculinization of the anteroventral periventricular nucleus. 

Because 17b-estradiol is aromatized to testosterone in the 

brain, the authors interpreted this effect of bisphenol A 

as anti-estrogenic. Cells staining for both ERa and 

tyrosine hydroxylase are not present in rodents after 

puberty, and the authors stated that these cells may play 

a role in the organization of the LH-surge. They 

postulated that the decrease in these cells with neonatal 

exposure to bisphenol A may result in cycle disruption in 

adulthood. 

Strengths/Weaknesses: Strengths of this study are the 

use of 17b-estradiol as a positive control and the measurement of ERa receptors. Weaknesses are the relatively highdose level of bisphenol A and the use of the subcutaneous 

route of exposure on a per pup basis without adjustment 

for body weight. Critical weakness include small sample 

size (5 treated dams) and lack of adequate experimental 

and statistical control for litter effects. 

Utility (Adequacy) for CERHR Evaluation Process: 

Despite certain strengths, this study is inadequate for the 

evaluation process for the reasons cited above. 

Patisaul et al. (2006), supported by the American 

Chemistry Council, investigated the effects of an acute 

neonatal exposure to bisphenol A or genistein (not 

discussed here) on the SDN-POA and the anteroventral 

periventricular nucleus in the adult male rat. Five 

pregnant Sprague–Dawley rats were obtained and 

maintained on a 12-hr light/12-hr dark cycle, with free 

access to water and a soy-free, phytoestrogen-free diet 

that was maintained throughout the duration of the 

experiment. [Details on housing (individual or group), 

type of caging, and bedding material were not 

provided.] Most of the dams were cross-fostered with 6 

male and 6 female pups. Starting on PND 1, all male 

pups were given s.c. injections every 12 hr over 48 hr with 

250 mg bisphenol A [purity not provided] or oil vehicle. 

[Assuming a Sprague–Dawley pup weighs B7.5 g, this 

dose would be equivalent to B66 mg/kg bw/day.] On 

PND 85, males were gonadectomized. Six ovariectomized female rats served as controls. After a recovery 

period, the rats were given s.c. injections of 10 mg 

estradiol benzoate, and 48 hr later, a s.c. injection of 

500 mg progesterone. The authors note that this protocol 

has consistently induced fos expression in GnRH neurons, leading to LH release in females. About 8 hr later, 

the animals were killed, formalin-perfused, and brains 

were harvested. Regions containing the SDN-POA and 

anteroventral periventricular nucleus were cryopreserved. SDN-POA sections were serially stained with 

Nissl or labeled for calbindin-d28 K. The vascular organ 

of the lamina terminalis was double-immunostained for 

Fos and GnRH. An automated stereomicroscope was 

used to gauge the volume areas of the anteroventral 

periventricular nucleus, the SDN-POA, the calbindinimmunoreactive regions of the SDN-POA, and number 

of calbindin-positive nuclei. Calbindin-positive nuclei 

were also counted by independent evaluators blinded to 

the treatments. Quantification analyses of GnRH and Fos 

staining were evaluated visually. Statistical analysis was 

performed using ANOVA, and Fisher least significant 

difference test. 

Acute neonatal treatment of bisphenol A did not affect 

the volume of the SDN-POA. Similarly, the volumes of 

the calbindin-immunoreactive regions of the SDN-POA 

were roughly equivalent to SDN volumes [estimated 

from a graph] with no apparent bisphenol A treatment 

effect. Bisphenol A treatment induced a significant 

increase [B50–60% estimated from a graph] in calbindin-positive nuclei. Bisphenol A had no effect on the 

volume of the anteroventral periventricular nucleus or 

the total number of GNRH-positive nuclei, and no 

induction of Fos protein was identified. 

The authors noted that the long-term effect of neonatal 

exposure to bisphenol A on male brain development and 

reproductive behavior cannot be predicted solely on 

anatomical changes in sexually dimorphic brain regions. 

They concluded that the development of more precise 

and predictive biomarkers is needed. 

Strengths/Weaknesses: Strengths of this study are the 

use of 17b-estradiol as a positive control. Weaknesses are 

the relatively high-dose level of bisphenol A and the use 

of the subcutaneous route of exposure on a per pup basis 

without adjustment for body weight. Critical weakness 

Birth Defects Research (Part B) 83:157–395, 2008
278 CHAPIN ET AL. 

include small sample size (5 treated dams) and lack of 

adequate experimental and statistical control for litter 

effects. 

Utility (Adequacy) for CERHR Evaluation Process: 

Despite certain strengths, this study is inadequate for the 

evaluation process for the reasons cited above. 

Shikimi et al. (2004), supported by the Japan Society 

for the Promotion of Science for Young Scientists, 

examined the effects of bisphenol A exposure on 

Purkinje cell development in rats. [No information was 

provided about feed or composition of caging and 

bedding materials.] At 6–9 days of age, 4 male or female 

Fisher rats/group received bisphenol A [purity not 

provided] at 0 (sesame oil vehicle), 0.050, or 0.500 mg/ 

day by injection into the cerebrospinal fluid near the 

region of the cerebellum. During the same time period, 

additional groups of 4 rats received 0.500 mg/day 

tamoxifen, 0.500 mg/day bisphenol A10.500 mg/day 

tamoxifen, or 5 mg/day estradiol benzoate through the 

same exposure route. [Both male and female rats were 

treated, but it was not indicated if there were equal 

numbers in each group; both sexes were apparently 

evaluated together.] At 10 days of age, pups were killed 

and vermal cerebella were removed and sectioned. 

Purkinje cells were examined morphologically following 

identification by calbindin-D28 K immunostaining. Data 

were analyzed by ANOVA, followed by Duncan multiple 

range test. 

Treatment with the high-dose of bisphenol A increased 

Purkinje fiber length. There was no effect on crosssectional soma area or Purkinje cell number as a result of 

bisphenol A treatment. Co-treatment with tamoxifen 

inhibited the increase in dendritic length that was 

observed following treatment with bisphenol A alone. 

Estradiol benzoate also induced an increase in dendritic 

length of Purkinje fibers that was blocked by tamoxifen. 

Treatment with tamoxifen alone also reduced dendritic 

fiber length. The effects of octylphenol were also 

examined and an increase in dendrite length was 

observed. The study authors concluded that bisphenol 

A induced Purkinje dendritic growth, possibly through 

the ER. 

Strengths/Weaknesses: The use of estradiol benzoate 

as a positive control is a strength of this study. 

Weaknesses are the injection into cerebrospinal fluid. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process due 

to uncertainties surrounding the route of administration 

(i.e., difficulty of relating a cerebrospinal injection to 

human exposures). 

Zsarnovszky et al. (2005), supported by NIH, NIEHS, 

and the American Heart Association, evaluated the 

effect of intracerebellar injection of bisphenol A on the 

development of activated extracellular signal-regulated 

kinase (ERK)-positive cells in cerebellar sections in 

Sprague–Dawley rats. Neonatal rats on PND 4–19 

underwent a single direct injection under anesthesia of 

bisphenol A or 17b-estradiol under stereotactic 

guidance into cerebellar folia 6 and 7. [For bisphenol 

A, only PND 10 results were given. The number of 

animals at each age was not specified, but a figure 

legend indicated at least 6/dose group. The purity of the 

chemicals was not specified. The day of birth 

was not defined.] Concentrations of the chemicals were 

10 -12–10 -6 M [bisphenol A concentrations of 

0.23 ng/L to 0.23 mg/L]. Uninjected, mock-injected, and 

vehicle-injected controls were used. Brains were removed and fixed 6 min after the onset of the injection. 

Sections were processed for immunohistochemistry 

using an antibody that recognized activated ERK. 

Quantitative analysis was performed on images of 

folium 9. Statistical analysis was performed using 

ANOVA with post-hoc Tukey–Kramer multiple 

comparison test. Response to different chemicals and 

different concentrations on PND 10 were compared 

using 2-factor ANOVA with post-hoc Bonferroni test. 

Adult rats were also treated but were not included in the 

quantitative analysis. 

The qualitative appearance of the immunostained 

sections was similar after bisphenol A and 17b-estradiol. 

In the 10 -12–10 -9 M dose range, the quantitative 

responses to the two chemicals were similar. Activated 

ERK-positive cells increased with a median effect 

concentration of 7.46 pM for 17b-estradiol and 3.25 pM 

[0.74 ng/L] for bisphenol A. Both chemicals were described as having an inhibitory effect at higher doses. 

[The data graph shows drop-offs to control densities at 

10 -9 and 10 -10 M, with a second increase in density at 

10 -7 and 10 -5 M.] Co-administration of 10 -10 M 17bestradiol with bisphenol A 10 -12–10 -10 M [0.23–23 ng/L] 

resulted in a concentration-dependent decrease in activated ERK-positive cells compared to the administration 

of 17b-estradiol alone. The authors concluded that 17bestradiol regulates ERK signaling in the developing 

cerebellum and that bisphenol A can mimic and also 

inhibit this estrogenic effect, with potentially adverse 

affects on brain development and function. 

Strengths/Weaknesses: The use of 17b-estradiol as a 

positive control is a strength of this study. Weaknesses 

are the intracerebellar injection and the administration on 

a per pup basis. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process due 

uncertainties surrounding the route of administration 

(i.e., difficulty of relating a cerebrospinal injection to 

human exposures). 

3.2.5 Mouse—oral exposure only during 

pregnancy. 

3.2.5.1 Studies without neurobehavioral endpoints: Morrissey et al. (1987), supported by NTP/ 

NCTR, examined the effects of prenatal bisphenol A 

exposure in rats and mice in studies conducted according 

to GLP. The studies are also available as NTP publications for rats (NTP, 1985c) and mice (NTP, 1985b). The 

study was conducted in two sets of rats and mice and 

data were pooled for each species. [The data for rats 

were discussed in Section 3.2.1.] Animals were fed 

Purina 5002 diet, housed in polypropylene or polycarbonate cages with stainless steel wire lids with Ab-Sorb-Dri 

cage bedding. Pregnant CD-1 mice were randomly 

assigned to groups of Z10 animals in each set of the 

study, for a total of Z20 animals/dose. On GD 6–15 (GD 

0 5 sperm or plug), mice were gavaged with bisphenol A 

at 0 (food-grade corn oil), 500, 750, 1000, or 1250 mg/kg 

bw/day. Doses were based on results of preliminary 

studies and were expected to result in 10% maternal 

mortality at the high-dose and no toxicity at the low 

dose. The purity of bisphenol A was 495%, and 2,40

­

bisphenol A was reported as an impurity. Concentrations 

of dosing solutions were verified. Pregnant animals were 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 279 

Table 75 

Maternal and Developmental Toxicity in Mice Gavaged With Bisphenol Aa 

Dose in mg/kg bw/day 

Endpoint 500 750 1000 1250 BMD10 BMDL10 BMD1SD BMDL1SD 

Dam weight in treatment period 

Gravid uterine weight 

Relative dam liver weight 

Resorptions/litter 

Fetal body weight/litter 

2

2

m9% 

2

2

2

2

m13% 

2

2

2

2

m17% 

2

2

k43% 

k32% 

m26% 

m2.8-fold 

k15% 

881 

983 

618 

817 

1079 

661 

690 

411 

377 

785 

1159 

1243 

755 

1245 

1249 

1039 

1123 

541 

1162 

1024 

a

Morrissey et al. (1987).
 

m,k Statistically significant increase, decrease; 2 no statistically significant change.
 

Table 76 

Sperm Production and Male Reproductive Organ Weights in Mice Exposed to Bisphenol A During Gestationa 

Dose in mg/kg bw/dayb 

Endpoint 0.002 0.020 BMD10 BMDL10 BMD1SD BMDL1SD 

Sperm production efficiency 

Body weight 

Preputial weight 

Seminal vesicle weight 

Epididymal weight 

2

k 9% 

m 36% 

k 12% 

k 12% 

k 19% 

2

2

2

k 8% 

0.011 0.007 0.010 0.007 

a

vom Saal et al. (1998).
 

b

Benchmark doses were not estimated for values obtained from graphs and non-dose-related effects; errors were assumed to be SEM, as
 

reported earlier in the study.
 

weighed during the study. Mice were killed on GD 17. 

Liver and uteri were weighed, and corpora lutea and 

implantation sites were examined. Fetuses were sexed, 

weighed, and examined for viability and external, 

visceral, and skeletal malformations. Data were analyzed 

by Bartlett test for homogeneity of variance, ANOVA, 

and/or William multiple comparison, Dunnett, and/or 

Fisher exact probability tests. [Data were presented and 

analyzed on a per litter basis.] 

Clinical signs reported in mice treated with bisphenol 

A included arched back, lethargy, piloerection, 

rough coat, vaginal bleeding, vocalization, alopecia, 

weight loss, and wheezing. One or 2 of 29–34 dams 

died in each of the 3 lowest dose groups and 6 of 33 dams 

died in the 1250 mg/kg bw/day group. Statistically 

significant effects are summarized in Table 75. Absolute 

liver weight was increased in the 500, 750, and 1000 mg/ 

kg bw/day dose groups, and relative liver weights were 

increased in all bisphenol A dose groups. Decreased 

gravid uterine weight and dam body weight gain during 

the gestation and treatment periods attained statistical 

significance at the 1250 mg/kg bw/day dose. The 

number of litters available for evaluation in the control 

and each dose group was 26, 23, 21, 23, and 21. Increased 

resorptions/litter and decreased fetal body weights/ 

litter attained statistical significance in the high-dose 

group. There was no effect on the number of live 

fetuses/litter at birth or on fetal malformations/litter. 

The study authors concluded that bisphenol A is not 

teratogenic in mice at doses that result in maternal 

toxicity. 

Strengths/Weaknesses: Strengths include the oral 

route of exposure as well as the design and sample sizes 

used. The use of very high-doses is a weakness. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility in the 

evaluation in providing information on conventional 

teratogenic endpoints. 

vom Saal et al. (1998), supported by NIH, examined 

the effects of bisphenol A exposure on male reproductive 

organs and sperm production in mice. The CF-1 mice 

used in this study were purchased in 1979 and 

maintained as an outbred stock in a closed colony. Dams 

were fed Purina breeder chow (5008) during pregnancy 

and lactation, and male offspring were fed Purina 5001 

standard lab chow after weaning. Housing consisted of 

polypropylene cages with corn cob bedding. Bisphenol A 

[purity not reported] in tocopherol-stripped corn oil 

vehicle was fed to 7 mice/group by electronic micropipette at 0.002 or 0.020 mg/kg bw/day on GD 11–17 (day of 

vaginal plug 5 GD 0). One group of 6 mice was given the 

vehicle control, and a group of 5 mice was not handled. 

Based on results of in vitro assays conducted by the 

study authors, the 0.02 mg/kg bw/day bisphenol A dose 

was predicted to be bioactive in mice. Additional mice 

were treated with the same doses of octylphenol. Females 

delivered pups naturally on GD 19, and pups were 

weaned on PND 23 (day of birth not defined). Male 

siblings were housed 3/cage until 5 months of age. 

Randomly selected males were housed individually at 5 

months of age and killed 1 month later. Body, testes, 

epididymides, preputial glands, and seminal vesicles 

were weighed in 11 control mice and 7 treated mice/ 

group. Data from the two control groups did not differ 

significantly and were combined for analyses of organ 

and body weight. Data for prostate weight were reported 

by Nagel et al. (1997). Daily sperm production was 

determined in 8 control males/group and 5 treated 

Birth Defects Research (Part B) 83:157–395, 2008
280 CHAPIN ET AL. 

males/group. [It was not stated how data from the 2 

control groups were handled for sperm analyses.] 

Sperm data were analyzed by ANOVA. Organ weight 

data were analyzed by ANCOVA, Pearson’s correlation 

analysis, ANOVA, and least significant means test. [It 

was not clear if the offspring or litter were considered 

the statistical unit; only one randomly selected male 

per litter was used per F. vom Saal, personal communication, June 20, 2007.] 

Statistically significant findings are summarized in 

Table 76. Exposure to bisphenol A resulted in doserelated reductions in daily sperm production efficiency 

(i.e., per g testis) that attained statistical significance at 

the highest dose level. Some significant but non-doserelated effects were observed for body and organ 

weights. Epididymal weights were reduced at both 

doses. At the low dose, body and seminal vesicle weights 

were reduced and preputial weight was increased. In 

mice treated with octylphenol, daily sperm production 

was reduced at the low dose but there was no effect on 

reproductive organ weights. The study authors concluded that exposure of the fetus to low doses of 

endocrine-disrupting chemicals can affect the size and 

function of reproductive organs. 

[The NTP Statistics Subpanel (NTP, 2001) noted that 

vom Saal et al. (1998) did not apparently require overall 

differences by ANOVA to be significant before applying 

the least significant difference test, which is prone to 

false positive findings without the overall protection of 

ANOVA. The NTP Subpanel was not able to confirm any 

of the significant findings reported for bisphenol A. The 

NTP Subpanel noted that in theory, their reanalysis of 

organ weights was not necessarily in conflict with the 

findings of the study authors because of the use of 

different statistical methods (Dunnett test vs. Fisher least 

significant difference test).] 

Strengths/Weaknesses: Strengths are the use of oral 

delivery and low dose levels. Weakness are the inability 

to assume the genetic comparability and responsiveness 

of CF-1 mice maintained in a closed colony for almost 20 

years is comparable to other sources of CF-1 mice), 

failure to weight-adjust the maternal dose daily, the lack 

of information on testis weight (that is needed for 

consideration of daily sperm production), small sample 

size for sperm production measurement, and the questions about the statistical analysis. An additional weakness is the unusual/unexplained findings of low dose 

only effect on weights. 

Utility (Adequacy) for CERHR Evaluation Process: 

The body weight data contained in this study are 

adequate for the evaluation process, however overall 

utility is limited because of sample size and statistical 

concerns. Data on reproductive organ weights and sperm 

production are considered inadequate for the evaluation. 

Nagel et al. (1997), supported by NIH and the 

University of Missouri-Columbia, examined the effect 

of prenatal bisphenol A exposure on mouse prostate 

weight. The mice used in this study were the same ones 

used in the study by vom Saal et al. (1998), and 

experimental details are provided in the above summary 

of that study. CF-1 mice were fed Purina Laboratory 

Chow 5001 and housed in polypropylene cages with corn 

cob bedding. The mice (7/group) were dosed with 

bisphenol A [purity not reported] at 0.002 and 

0.020 mg/kg bw/day on GD 11–17. A control group of 

6 mice was given the tocopherol-stripped corn oil vehicle 

during the same time period. Vehicle and dosing 

solutions were fed to the mice using a micropipette. A 

second control group of 5 dams was unhandled. Because 

there were no significant differences between the 2 

control groups, data from the 2 groups were pooled. 

Females were allowed to litter. Pups were weaned at 23 

days of age and housed 3/cage. One male/litter was 

selected and housed individually for 1 month. Body 

weights of males were measured throughout the study. 

Selected males were killed at 6 months of age for 

measurement of prostate weight. Data for prostate 

weight were analyzed by ANCOVA using body weight 

as the covariate. If it was determined that body weight 

did not account for differences in prostate weight, data 

were reanalyzed by ANOVA without adjustment for 

body weight. Selection of 1 male/litter controlled for 

litter effects. Body weights were lower in males from the 

0.002 mg/kg bw/day group than in controls. Statistical 

analyses revealed that prostate weight was not related to 

body weight. Compared to control values, prostate 

weights were 30% higher in the 0.002 mg/kg bw/day 

group and 35% higher in the 0.020 mg/kg bw/day 

group. The study authors concluded that bisphenol A 

alters the reproductive system of mice at doses near 

reported ranges of human exposure. 

[The NTP Statistics Subpanel (NTP, 2001) concluded 

that Nagel et al. (1997) used appropriate statistical 

methods, and the Subpanel reached essentially the 

same conclusions as the study authors regarding 

elevated prostate weight.] 

Strengths/Weaknesses: Strengths are the use of the 

same methods as vom Saal et al. (1998) and the use of 

dose levels in the range of human exposure. The 

independent confirmation of the data analysis by the 

NTP Statistics Subpanel is another strength. The use of a 

small sample size, closed mouse colony, and the failure to 

present any histopathological analyses are weaknesses. 

The Purina 5001 chow has high and variable levels of soy 

phytoestrogens, and the corn cob bedding is known to be 

problematic due to antiestrogenic constituents. This 

study did not use a positive control, although there are 

earlier reports from this laboratory using 

diethylstilbestrol. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and useful for the evaluation 

process. 

Cagen et al. (1999a), support not indicated [authors 

noted to work in industry], examined the effects of 

prenatal bisphenol A exposure on the developing 

reproductive system of male mice. The study attempted 

to duplicate the findings by vom Saal et al. (1998) and 

Nagel et al. (1997) by repeating their procedures. 

Exceptions were: (1) use of larger group sizes to increase 

statistical power; (2) use of 4 dose levels instead of 2; (3) 

use of 2 methods to determine sperm counts; (4) killing of 

male offspring at 90 instead of 180 days; (5) conducting 

the study according to GLP; (6) obtaining mice from a 

commercial source instead of an in-house bred colony; 

and (7) housing males individually after weaning. In the 

study by Cagen et al. (1999a), CF-1 mice gaining more 

than 4.5 g weight from GD 0 to 10 were randomly 

assigned to groups of 28 animals and administered 

bisphenol A (499% pure) 0.0002, 0.002, 0.020, or 0.2 mg/ 

kg bw/day on GD 11–17. Two negative control groups 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 281 

with 28 dams each were given the tocopherol-stripped 

corn oil vehicle. Because results from the two vehicle 

control groups were statistically equivalent, data from 

the two groups were pooled. A positive control group of 

28 mice was given 0.2 mg/kg bw/day diethylstilbestrol. 

Dosing solutions were dripped into the animals’ mouths 

using a micropipette. Concentrations of dosing solutions 

were verified before dosing. Animals were fed certified 

rodent chow 5002. Water was provided in glass bottles 

with Teflon seals. Cages were made of polypropylene 

with steel lids. Corn cob bedding was used. Music was 

played at low volume to provide background noise. 

Dams were monitored for clinical signs, food intake, 

body weight gain, and fertility endpoints. Pups were 

counted and sexed at birth (PND 0) and monitored for 

survival and weight gain until weaning on PND 22. 

Litters were culled to 8 pups on PND 4, leaving as many 

males as possible. At weaning, no more than 4 males/ 

litter (65–95 males/group) were randomly selected to 

continue in the study and housed individually. The 

males were monitored for body weight gain and feed 

intake until they were killed on PND 90. Brain, liver, 

kidneys, and reproductive organs were weighed. Daily 

sperm production and epididymal sperm counts were 

determined and a histopathological examination of testes 

was conducted. The litter was considered the experimental unit in statistical analyses. Data were analyzed by 

Levene test, ANOVA, Dunnett test, rank transformation, 

Wilcoxon rank sum test with Bonferroni correction, 

Fisher exact probability test, and binomial distribution 

test. 

There were no clinical signs or significant differences 

in body weight gain or feed intake in dams. The numbers 

of dams that died of unknown causes during the study 

were: 2 receiving vehicle controls; 1 dosed with diethylstilbestrol; 3 dosed with 0.0002 mg/kg bw/day bisphenol 

A; and 1 each in the 0.002 and 0.020 mg/kg bw/day 

bisphenol A groups. The number of total pups/litter was 

significantly lower than controls in the 0.2 mg/kg bw/ 

day bisphenol group (mean7SD 5 9.6073.85 compared 

to 12.3773.02 in the control group). In communications 

with the animal vendor, it was determined that litter size 

in the control group exceeded typical litter sizes (9–10 

pups), and the study authors therefore concluded that 

the effect was not treatment-related. Bisphenol A had no 

significant effects on gestation index or duration, 

percentage of male pups at birth, or pup survival and 

body weight during the lactation period. The same 

endpoints were unaffected in the diethylstilbestrol 

group. 

Terminal body weights were increased [by 7%] in the 

0.020 mg/kg bw/day group and [by 5%] in the 2 mg/kg 

bw/day group. Bisphenol A did not affect absolute or 

relative (to body or brain) weights of reproductive organs 

including prostate, preputial gland, seminal vesicle, or 

epididymis. Non-dose-related effects were observed for 

brain and kidney weights, and the study authors 

concluded that the effects were not treatmentrelated. There were no significant effects on cauda 

epididymal sperm concentration, daily sperm production, or efficiency of sperm production. Testicular 

histopathology was not affected by bisphenol A treatment. [Data were not shown by authors.] Reproductive 

development of male offspring was also unaffected by 

diethylstilbestrol. The study authors noted that the 

diethylstilbestrol dose was considered the ‘‘maximum 

effect’’ oral dose by vom Saal but was lower than doses 

affecting male offspring in other studies. The study 

authors also noted that the effects of bisphenol A on 

prostate weight and sperm production reported by vom 

Saal et al. (1998) and Nagel et al. (1997) were not repeated 

in this study. They concluded that bisphenol A should 

not be considered a selective reproductive or developmental toxicant. 

[The NTP Statistics Subpanel (NTP, 2001) concluded 

that the statistical methods used by Cagen et al. (1999a) 

were appropriate. Although the Subpanel agreed with 

the study author conclusions, they noted that (1) a 

significant ANOVA is not a requirement for Dunnett 

test; and (2) a Bonferroni correction of the Wilcoxonrank sum test was not needed because the study 

authors already required significance by ANOVA, 

which was sufficient.] 

Strengths/Weaknesses: The attempt to replicate the 

studies of vom Saal et al. (1998) and Nagel et al. (1997), 

the use of litter analysis, the large sample sizes, and the 

agreement of the NTP Subpanel with the author 

conclusions are strengths. With respect to this study as 

a replication, weaknesses include design differences 

relating to strain, dietary differences, age at evaluation, 

and the use of solo housing rather than small group 

housing. The lack of response of the positive control DES 

group is problematic. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process due 

to absence of response of the positive control group. 

Ashby et al. (1999), support not indicated [2 authors 

from industry], examined the effects of prenatal bisphenol A exposure on the mouse reproductive system. 

The study attempted to duplicate the findings reported 

by vom Saal et al. (1998) and Nagel et al. (1997). Both 

generations of CF-1 mice were fed RM1 diet containing 

6.5% soy during periods when they were not pregnant or 

lactating, and dams were fed RM3 diet containing 18.5% 

soy during pregnancy and lactation. On post-conception 

days 11–17, 8 dams/group were dosed with bisphenol A 

(99% pure) at 0, 0.002, or 0.020 mg/kg bw/day. The 

negative control group was administered the tocopherol 

stripped corn oil vehicle. A positive control group of 7 

dams received diethylstilbestrol at 0.2 mg/kg bw/day. A 

naı ̈ve group of 7 dams was not weighed or dosed. The 

dosing solution was slowly expelled from a pipette 

placed in the animals’ mouths. Day of vaginal plug 

detection was designated post-conception day 1, however. females that had no vaginal plugs but gained 

43.5 g were arbitrarily considered to be 10 days 

pregnant. Females with vaginal plugs and those that 

gained 43.5 g were distributed evenly among treatment 

and control groups. Females that gained 41 but o3.5 g 

were considered to be pregnant, but because the day of 

pregnancy could not be determined, they were assigned 

to the naı ̈ve control group. Dams were allowed to litter. 

All female offspring were weighed and monitored for 

vaginal opening. Females were killed at B44 weeks of 

age, and liver, kidney, and reproductive organs were 

weighed. Male pups were housed as littermates until 

PND 112 (day of birth designated as PND 1). To 

determine the effects of housing, B3 males from 4–7 

litters/group (11–21 males/group) were randomly selected and housed separately from PND 112 until study 

Birth Defects Research (Part B) 83:157–395, 2008
282 CHAPIN ET AL. 

termination, which occurred B71 days later. The 

remaining male pups from 4–5 litters/group from each 

litter (11–17/group) were housed together. Singly housed 

males were weighed and killed on PND 183–185, and 

group-housed males were weighed and killed on PND 

186–187. Equal numbers of males from each group were 

killed each day. Liver, kidney, and reproductive organs 

were weighed, and testicular sperm count and efficiency 

were determined. Technicians were blinded to experimental conditions. Measures taken to reduce stress to 

animals included administering test agents by drip 

feeding, minimal handling of pups, and minimal 

environmental noise. Selection of 3 males from each 

litter increased statistical power compared to previous 

studies (Nagel et al., 1997; vom Saal et al., 1997). 

Statistical analyses were dually conducted using the 

individual offspring and the litter as the statistical unit. 

Data were evaluated by ANOVA and Dunnett test. 

Results from vehicle-treated and naı ̈ve controls were 

pooled when there was no evidence of a vehicle effect. 

Data from individually housed and group housed-males 

were pooled when they did not differ significantly. 

There were no significant differences in litter sizes or 

percentage of males/litter. In female offspring from the 

bisphenol A groups, there were no significant effects on 

body weight or organ weights, including cervix, uterus, 

vagina, and ovary. Age and weight at vaginal opening 

were also unaffected in groups exposed to bisphenol A. 

Vaginal opening was delayed in the diethylstilbestroltreated group and in the naı ̈ve control group. 

Significant effects included increased terminal body 

weights in the low-dose group, increased testis weight in 

both dose groups, and increased epididymis weight in 

the high-dose group. Because testis and epididymis 

weights relative to body weights were nearly identical 

to controls [data not shown by study authors], the 

authors considered the finding equivocal. Although 

prostate weights were slightly higher in the bisphenol 

A groups, there were no statistically significant effects on 

prostate weight when adjusted for body weight and litter 

effects. Daily sperm production was increased in both 

dose groups, but the study authors considered the 

finding equivocal due to low biological significance. 

The study authors noted that the study failed to confirm 

the increase in prostate weight and decrease in sperm 

production reported in the studies by vom Saal et al. 

(1997) and Nagel et al. (1997), but results were consistent 

with those reported by Cagen et al. (1999a). Possible 

reasons for variability between studies were stated as 

differences in background sound level, diet, and animal 

body weights. The study authors also mentioned the 

possibility of genetic drift occurring in mice bred inhouse in the vom Saal laboratory. 

[The NTP Statistics Subpanel (NTP, 2001) essentially 

reproduced the findings reported by Ashby et al. 

(1999).] 

Strengths/Weaknesses: Strengths are the rather close 

replication of the designs of the studies by vom Saal et al. 

(1998) and Nagel et al. (1997) with diet as the only major 

difference, the use of both solo and group housed mice, 

and the support of the conclusions by the NTP Statistics 

Subpanel. The use of small samples is an understandable 

weakness given that this study was designed to be a 

replicate study. The lack of response of the positive 

control DES group is problematic. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process due 

to absence of response of the positive control group and 

small sample sizes. 

Howdeshell et al. (1999), support not indicated, 

examined the effect of prenatal bisphenol A exposure 

on age of puberty in female mice. [No information was 

provided about chow or composition of bedding and 

cage materials.] CF-1 mice (n 5 21/group) were fed oil 

vehicle [type of oil not specified] or bisphenol A [purity 

not reported] at 0.0024 mg/kg bw/day on GD 11–17 [day 

of vaginal plug not defined]. On GD 19, pups were 

obtained by cesarean section. Intrauterine position of 

pups (i.e., located next to male or female pups) was noted 

at that time. Pups were fostered by untreated mothers 

and weaned on PND 22. Body weights were measured, 

and pups were monitored for vaginal opening and time 

to estrus. Results were analyzed according to all pups 

from each dose group or in relation to intrauterine 

position. The study authors stated that fetuses positioned 

between 2 male mice were exposed to the lowest levels of 

17b-estradiol, while exposures to 17b-estradiol were 

highest in fetuses positioned next to female fetuses. Data 

were analyzed on a litter basis to control for maternal 

effects. Age of vaginal opening was covaried with weight 

at weaning. Numbers of female offspring evaluated were 

75–111/group for body weight and 51–58/group for 

vaginal opening. The study authors attempted to 

evaluate females from each intrauterine position in each 

litter. [No additional information was provided for 

statistical analysis in this brief communication.] 

Body weight at weaning was significantly increased in 

females in the bisphenol A group. When analyzed 

according to intrauterine position, body weights were 

22% higher than controls in females who were not 

positioned next to a male fetus and 9% higher in females 

who had been positioned next to 1 male in utero. There 

were no significant effects on age of vaginal opening. [It 

was not clear if the data presented were covaried with 

body weight.] Bisphenol A treatment significantly 

reduced the period between vaginal opening and first 

estrus by B2.5 days. When evaluated according to 

intrauterine position, a significant decrease in time to 

first estrous was observed in females who were not 

positioned next to a male pup (accelerated by B5 days) 

and in females positioned next to 1 male [B2 days]. No 

statistically significant findings were observed in females 

who had been positioned next to 2 males in utero. The 

study authors concluded that prenatal exposure to 

bisphenol A at environmentally relevant levels altered 

postnatal growth and reproductive function in female 

mice but that natural variations in individual endogenous 17b-estradiol levels influenced the response to 

bisphenol A. 

The results of this study were also discussed in a 

publication by Howdeshell and vom Saal (2000), which 

indicated that the work was supported by NIH and 

reported additional findings. There was a bisphenol Aassociated reduction in pup survival between birth and 

weaning. Complete litter death occurred in 6 of 21 

litters in the bisphenol A group compared to 1 of 21 

litters in the control group. Significantly increased 

body weight of male pups at weaning was also reported 

for the bisphenol A group. Body weights were highest in 

males who were positioned next to 2 female pups in 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 283 

utero and were 10% higher than body weights of control 

males positioned next to 2 female fetuses in utero. No 

increase in body weight occurred in males that were 

positioned between two male fetuses in utero. Although 

the authors identified a litter-based analysis, it was not 

always clear that this applied to all analyses (in Study 

Figure 1, the n-values exceed the number of dams, 

suggesting that some of the data were analyzed on a per 

pup basis. 

[The NTP Statistics Subpanel (NTP, 2001) requested 

the Howdeshell et al. (1999) data set for reanalysis, but 

it was not provided by study authors.] 

Strengths/Weaknesses: Strengths are the oral route of 

exposure and the use of a low dose level of bisphenol A. 

The omission of a description of husbandry conditions 

and lack of clarity of statistical procedures are weaknesses. Use of only a single dose is a weakness. Further, 

the use of time from vaginal opening to first estrus is not 

a standard endpoint for assessing puberty in mice and is 

of questionable biological significance. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate for the evaluation process but 

utility is limited due to uncertainties in data analyses. 

Gupta (2000), supported by NIH, examined the effects 

of bisphenol A exposure on the reproductive system of 

male mice. CD-1 mice were received on GD 12 (GD 

0 5 day of breeding). The mice were fed Purina Chow-5 

L9 at the Charles Rivers Laboratory and Purina Chow 

5012 at the study author’s laboratory. [No information 

was provided on bedding or caging materials.] On GD 

16–18, 15 mice/group were fed the corn oil/12% ethanol 

vehicle or 0.050 mg/kg bw/day bisphenol A [purity not 

reported]. Additional groups of mice were administered 

diethylstilbestrol at 0.1 and 200 mg/kg bw/day and 

Aroclor at 0.050 mg/kg bw/day during the same time 

period. The bisphenol A dose level was based on a level 

reportedly considered safe by the FDA. Following 

delivery, litters were culled to 8 pups, with at least 3 

males. Body weight and anogenital distance were 

examined in 3 pups/litter (45 pups) on PND 3, 2 

pups/litter (30 pups) on PND 21, and 1 offspring/litter 

(15 offspring) on PND 60. [Although Table 1 of the 

study lists the n-value as 15–45/group, a statement in 

the methods section indicated that an equal number of 

pups (n 5 1–3) were pooled from each litter.] Prostate 

and epididymis were weighed in 15 offspring/group on 

PND 3, 21, and 60. Whole-tissue mounts of prostate 

were examined for growth in 15-day-old offspring 

(n 5 4/group). Androgen binding was measured in 

prostates isolated at 3, 21, and 60 days of age, with 2– 

6 prostates pooled, depending on age; an n of 5 was 

reported in Figure 2 of the study. Data were analyzed 

by ANOVA. [It was not clear if the offspring or litter 

was considered the statistical unit.] 

Body weights of male offspring were not affected by 

bisphenol A treatment. In male pups of the bisphenol A 

group compared to the control group, anogenital distance 

adjusted for body weight was significantly increased [by 

22%] on PND 3, [by 25%] on PND 21, and [by 33%] on 

PND 60. Prostate weights in males of the bisphenol A 

group were significantly increased [by 56%] on Day 3, [by 

39%] on Day 21, and [by 101%] on Day 60. Relative (to 

body weight) epididymis weight in the bisphenol A group 

was significantly reduced [by 35%] on PND 60. Prostate 

growth was reported to be qualitatively increased by 

bisphenol A exposure. Androgen receptor binding was 

increased on PND 21 and 60 [by B344% on PND 21 and 

358% on PND 60, estimated from a graph]. Similar effects 

were reported following treatment with the low dose of 

diethylstilbestrol and Aroclor. In contrast, the high-dose of 

diethylstilbestrol reduced body weights, anogenital distance, prostate weight, and androgen receptor binding. 

Presentation of pathology data are superficial, thus 

questioning interpretation. 

The report also included an in vitro study to examine 

the effects of bisphenol A on prostate growth. The 

urogenital sinus was dissected from GD 17 fetuses and 

cultured for 7 days in media containing 0, 5, or 50 ng/L 

bisphenol A with and without the addition of testosterone. The urogenital sinus was also incubated in 0.1 or 

0.5 ng/L diethylstilbestrol and 5 or 30 ng/L 

Aroclor. Prostates obtained from cultures were then 

fixed in Bouin solution and examined histologically. A 

similar protocol was used to examine androgen 

binding in cultured prostates, except that only the highdoses of each compound were examined, and cells were 

cultured for 6 days. Bisphenol A at 50 ng/L increased 

prostate size [by 140%] in the absence of testosterone and 

[by 150%] in the presence of testosterone. Androgen 

binding in prostate was increased [by 200%] following 

treatment with bisphenol A. Similar effects were reported with diethylstilbestrol and the high Aroclor dose. 

The study authors concluded that the effects of in vivo 

studies were reproduced in in vitro studies, which 

suggests a direct effect on reproductive organs of fetal 

mice. 

In a subsequent commentary, Elswick et al. (2000) 

noted several concerns and requested clarification of the 

data analysis performed by Gupta. It was noted that 

statistical analyses were insufficiently described to 

determine if analyses in addition to ANOVA were 

conducted. It was not indicated if post-hoc tests were 

used or if corrections were made for multiple comparisons. Table 1 of the study was noted to contain a footnote 

indicating Po0.05 (larger) or Po0.05 (smaller). It was 

stated that determining a mean and conducting a onetailed post-hoc test based on whether the mean is larger 

or smaller is a source of potential bias in the statistical 

analyses. Analyses conducted by Elswick et al. (2000) 

indicated that the assumption of homogeneity of 

variance, a requirement for ANOVA, was not met for 

some data such as anogenital distance on PND 3 (Table 1 

of the study) and prostate size (Table 3 of the study). 

Therefore, questions were raised about whether homogeneity testing was done or if data were transformed to 

account for lack of homogenous variances before 

ANOVA. Failure to consider the litter as the experimental 

unit was noted in cases where the sample size was listed 

as 30 and 45, while only 15 dams/group were treated. It 

was noted that if anogenital distance was measured in 

the same animal at different time points, a repeatedmeasures ANOVA would have been the appropriate 

statistical test. It was stated that correction of anogenital 

distance by the cube root of body weight instead of body 

weight would have been preferred to avoid overcorrection; ANCOVA with body weight as a covariate would 

have been a better method for correcting anogenital 

distance, and the best method would have been a nested 

ANCOVA (dam within treatment). Questions were 

raised about whether sampling 1 pup/litter on PND 60 

Birth Defects Research (Part B) 83:157–395, 2008
284 CHAPIN ET AL. 

provided a reliable estimate, especially for highly 

variable endpoints such as anogenital distance, which 

can be affected by sex of the adjacent fetuses in the 

uterus. Organ weights were also stated to be variable, 

and it was questioned whether sampling 1 offspring/ 

litter on PND 60 resulted in a reliable estimate. 

Gupta (2001) responded to the questions raised by 

Elswick et al. (2000). Regarding the question of post-hoc 

tests for data analyzed by ANOVA, Gupta stated that 

comparisons using the least significant difference test 

support the effect reported in the original study. Gupta 

stated that the use of 1-tailed tests was never mentioned 

and that the criticism was unfounded. The numbers of 

offspring examined at each age was reiterated [with no 

mention of considering the litter the statistical unit]. It 

was stated that individual animals were not identified 

because it would have required using a toe clip or tattoo, 

which is stressful to the animals. Therefore, it was not 

known if the same animals were examined for anogenital 

distance at the different time points and use of the 

repeated-measures ANOVA would not have been appropriate. Regarding use of 1 animal/litter, it was stated that 

it is the standard procedure accepted by NIEHS to 

control for litter effects. Correction of anogenital distance 

by body weight was stated to be appropriate because of a 

significant correlation between body weight and anogenital distance (r 5 0.47, Po0.001). Adjustment for litter 

effects was stated to occur because litter was nested 

within treatment in the ANOVA. Gupta noted a 

typographical error in Table 3 of the original study. 

Standard deviations for the 50 ng/L bisphenol A and 

Aroclor groups were mistakenly indicated to be 10-fold 

higher than the actual values (i.e., the actual values were 

0.024 for bisphenol A and 0.032 for Aroclor). The errors 

made it appear that there were differences in variances 

between groups, when actually there were not. Gupta 

stood by his original conclusion that low levels of 

bisphenol A alter the development of the male reproductive tract. 

Strengths/Weaknesses: Strengths are the oral 

route of administration, the use of a low dose level of 

bisphenol A, the use of diethylstilbestrol as a positive 

control, the prostate measurements at 3 postnatal time 

points, and the use of an in vitro study to support the in 

vivo results. The use of a single dose level, and 

questionable histopathological presentation and evaluation are weaknesses. An additional weakness is that 

more than one male per litter was used for some 

endpoints without adequate statistical control for litter 

effects. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for evaluation 

of prostate weight, biochemical endpoints, and body 

weight and AGD at PND 60 but not other endpoints 

where litter effects were not adequately controlled for 

(i.e., those where 30 or 45 pups were examined from 15 

litters). 

Iida et al. (2002), supported by the Japan Society for 

Promotion of Science, examined the effect of prenatal 

bisphenol A exposure on spermatogenesis in adult mice. 

[No information was provided about composition of 

feed, caging, or bedding.] On GD 10–17 [day of vaginal 

plug not defined], Z3 ddY mice/group were orally 

administered bisphenol A [purity not reported] at 0 

(corn oil vehicle), 1, 10, or 100 mg/kg bw/day. [The 

specific method of oral dosing was not stated.] At 60 

days of age, 4–5 male mice/dose group (obtained from 3 

litters/dose group) were weighed and killed. Testes were 

removed and fixed in paraformaldehyde for histopathological evaluation by light microscopy. At 120 days of 

age, testicular histopathology was examined by light and 

electron microscopy in 3 mice/group from the control 

and 10 mg/kg bw/day groups. Data were analyzed by 

ANOVA. [It was not clear if the litter or offspring were 

considered the statistical unit.] 

No effects on body weight were observed in 60-dayold mice. Significant and dose-related increases in the 

incidence of abnormal seminiferous tubules were observed in mice exposed to bisphenol A. The incidence of 

abnormal seminiferous tubules in the control and each 

respective treatment group was 3.7, 15.2, 17.7, and 31.5%. 

[Benchmark dose analysis using a probit model and 

n 5 3 litters gave a BMD10 5 44 and a BMDL10 5 17 mg/ 

kg bw/day.] Examples of seminiferous tubule lesions 

included luminal space loss in tubules, reduced numbers 

of maturing elongate spermatids, decreased tubular 

diameter, aberrant distribution of spermatogenic cells in 

epithelium, and accumulation of material within tubules. 

In the 120-day-old mice exposed to 10 mg/kg bw/day, 

the same types of lesions were observed at a higher 

incidence than controls (28.3 compared to 5.14%). 

Electron microscopic examinations of 2 abnormal seminiferous tubules from exposed 120-day-old mice revealed 

the presence of round but not elongated spermatids, 

leading study authors to suggest disrupted spermatogenesis. Disorganized arrangement of Sertoli cells was 

also observed in the 120-day-old mice of the 10 mg/kg 

bw/day group. The study authors noted that degeneration of Sertoli cells may be the cause of aberrant 

distribution of spermatogenic cells. 

Strengths/Weaknesses: The oral route of delivery is a 

strength of this study. The lack of information on details 

of husbandry, the small sample size (4–5 male mice from 

3 litters per dose group) and the lack of adjustment for 

litter effects, inadequate methods for histopathological 

preservation and evaluation (i.e., use of paraformaldehyde for paraffin embedding) are weaknesses. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process based 

on methodology. 

Timms et al. (2005), supported by NIEHS and U.S. 

EPA, examined the effects of bisphenol A exposure on 

development of the prostate in mice. CD-1 mice were fed 

soy-based Purina 5008 chow, provided drinking water in 

glass bottles, and housed in polypropylene cages. [The 

type of bedding material was not indicated.] On GD 14– 

18 (day of mating 5 GD 0), pregnant mice were fed by 

micropipette with 0.010 mg/kg bw/day bisphenol A 

[purity not indicated] (n 5 6), the tocopherol-stripped 

corn oil vehicle (n 5 5), 0.1 mg/kg bw/day ethinyl 

estradiol (n 5 5), or 0.1 mg/kg bw/day diethylstilbestrol 

(n 5 5), the positive control. The dose of bisphenol A was 

based on previous findings that suggested bisphenol A 

was 100-fold less potent than diethylstilbestrol in 

permanently increasing prostate size in mice. On GD 

19, fetuses were removed by cesarean section, and during 

the removal process, intrauterine position of male fetus 

relative to sex of adjacent fetuses was recorded. To 

reduce effects associated with sex hormone exposure 

from the adjacent fetus, 1 male/litter that developed 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 285 

Table 77 Table 78 

Effects on Prostate Development in Mice After Prenatal
 

Exposure to 0.010 mg/kg bw/day Bisphenol Aa
 

Prostate region 

Dorsolateral 

Endpointb Dorsolateral Ventral and ventral 

No. of prostate ducts m 41% 2 m 40% 

Prostate duct volume m 99% m 78% m 91% 

Proliferating cell m 44% 2 No data 

nuclear antigen staining 

a

Timms et al. (2005).
 

b

Percent changes calculated by CERHR differed slightly from
 

values presented by authors; it was not clear which part of the
 

prostate the authors’ values represented.
 

m,k Statistically significant increase, decrease; 2 no statistically
 

significant effect.
 

between a male and female fetus was examined. Prostate 

morphology was determined by a 3D computer reconstruction technique. Immunohistochemistry techniques 

were used to measure levels of proliferating cell nuclear 

antigen and mouse keratin 5. Statistical analyses included ANOVA, followed by Fisher least-squares mean 

test when statistical significance was obtained. In a 

separate study, prostate morphology was examined in 4 

pregnant mice/group that were dosed with vehicle or 

200 mg/kg bw/day diethylstilbestrol according to the 

procedures described above. 

Bisphenol A increased numbers of ducts, volume, and 

proliferation in one or more prostate regions, as outlined 

in Table 77. The pattern of proliferating cell nuclear 

antigen staining was similar to that observed with mouse 

keratin 5, a basal epithelial cell maker. The study authors 

also reported a 56% increase in the volume of the 

coagulating glands. [Data were not shown by study 

authors.] An abnormal narrowing was observed in the 

portion of the urethra near the neck of the bladder. [The 

volume of the cranial urethra was reduced by 35% 

compared to controls. Malformation of prostatic sulci 

was reported, but no information was provided on 

incidence or severity.] Similar effects on the prostate 

were reported in mice exposed to ethinyl estradiol and 

the low dose of diethylstilbestrol. Narrowing of the 

cranial urethra was observed in mice exposed to ethinyl 

estradiol. In contrast, exposure to the high diethylstilbestrol dose resulted in inhibited morphogenesis of the 

prostate. The study authors concluded that the differentiating urogenital system of male mice is very sensitive 

to a low dose of bisphenol A. 

Strengths/Weaknesses: Strengths are the oral route of 

administration, the low dose level of bisphenol A, the use 

of diethylstilbestrol and ethinyl estradiol as positive 

controls, and the sophisticated measures applied to the 

prostate. Weaknesses are the use of a single dose level 

and small sample size, although the Panel judged it to be 

adequate for the methodology. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation. 

Palanza et al. (2002), supported by NIEHS, NIH, 

MURST, the University of Parma, and the National 

Maternal Behavior Effects in Mice Exposed to Bisphenol
 

A During Gestation or Adulthooda
 

Bisphenol A exposure during gestation/adulthood 

Bisphenol Vehicle/ Bisphenol 

Percent timeb A/vehicle bisphenol A A/bisphenol A 

Nursing k 15% k 14% 2

Nest building m 73% m 146% 2

Resting alone m 67% m 29% m 46% 

Grooming m 25% m 18% 2

Active 2 m 18% 2

In nest k 12% k 10% 2

Out of nest m 17% m 12% 2

a

Palanza et al. (2002).
 

b

Data were presented graphically. Values were provided by the
 

study author (personal communication, P. Palanza, February 26,
 

2007).
 

m,k Statistically significant increase/decrease compared to
 

vehicle-vehicle group, 2 no statistically significant effect.
 

Council for Research, examined the effects of bisphenol 

A treatment on maternal behavior following exposure of 

mice during prenatal development and/or adulthood. 

The CD-1 mice used in this study were maintained as an 

outbred colony. Mice were housed in polypropylene 

cages with corn cob bedding. During pregnancy and 

lactation, mice were fed Purina 5008 (soy-based) chow. 

After weaning, mice were fed Purina 5001 (soy-based) 

chow. Water was provided in glass bottles. On GD 14–18 

(GD 0 5 day of vaginal plug), 14 mice were fed the 

tocopherol-stripped corn oil vehicle and 9 mice were fed 

0.010 mg/kg bw/day bisphenol A [purity not reported] 

using an electronic micropipette. Dams were housed 3/ 

cage after mating and individually housed on GD 17. 

Body weights of dams were measured during gestation. 

The day of birth was considered PND 1, and offspring 

were weaned on PND 20. At 2–2.5 months of age, F1 

female offspring from vehicle- and bisphenol A-treated 

dams were mated and exposed to vehicle or 0.010 mg/kg 

bw/day bisphenol A on GD 14–18. There were 4 groups 

of F1 females that were exposed during gestation– 

adulthood to vehicle–vehicle (n 5 20), vehicle–bisphenol 

A (n 5 15), bisphenol A–vehicle (n 5 15), and bisphenol 

A–bisphenol A (n 5 15). Maternal behavior was observed 

in F1 dams every 4 min during a 120-min period on PND 

2–15. On PND 1, F2 pups were weighed, sexed, and 

counted. Litters were then culled to 10 pups, with equal 

numbers of male and female pups when possible. Pups 

were weighed during the lactation period and cliff-drop 

aversion and righting reflex were evaluated in all pups of 

a subset of 8 litters/group on PND 3, 5, 7, and 9. For 

statistical analyses, all pup data were adjusted for litter. 

Data were analyzed by ANOVA, Holms t-test, and/or 

Fisher protected least-squared difference test. 

Bisphenol A treatment did not affect gestational body 

weight gain in F0 or F1 dams. Statistically significant 

effects for F1 maternal behavior collapsed across 14 

observation days are presented in Table 78. Exposure to 

bisphenol A either in gestation or in adulthood resulted 

in decreases in the percentage of time the dams spent 

nursing and in the nest and increases in the percentage of 

time the dams spent nest building, resting alone, 

Birth Defects Research (Part B) 83:157–395, 2008
286 CHAPIN ET AL. 

grooming, and out of the nest. Increased activity was also 

observed in the group exposed to bisphenol A in 

adulthood. The only significant effect observed in mice 

exposed to bisphenol A during gestation and adulthood 

was increased time resting. When data were presented 

for individual evaluation days, time resting was significantly increased on PND 9, 10, 11, 12, and 14 in the 

group exposed to bisphenol A during gestation. Time 

spent resting was significantly increased on PND 9 and 

14 in the group exposed to bisphenol A during gestation 

and adulthood. No other significant effects were 

observed on specific evaluation days. There were no 

significant differences in the number of live F2 pups/ 

litter, sex ratio, or body weight at birth or in weight gain 

during the lactation period. [Data were not shown]. No 

significant effects were observed for cliff aversion or 

righting reflexes. The study authors concluded that 

reduced levels of nursing behavior were observed in 

mice exposed to bisphenol A only as fetuses or only as 

adults. [Because this study involves effects of adult 

exposure on maternal behaviors, it is also discussed in 

Section 4.2.] 

Strengths/Weaknesses: Strengths are the oral route of 

administration, the low dose level of bisphenol A, and 

the exploration of effects on complex maternal behaviors. 

It is unusual that pre- and postnatal exposure had effects 

but not the combination of pre- and postnatal exposure, 

and failure to explain this finding is a weakness. The use 

of a diet high in soy isoflavones is an additional 

weakness. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

Nishizawa et al. (2003), supported by the Japanese 

Ministry of Education, Culture, Sports, Science, and 

Technology, examined the effects of prenatal bisphenol A 

exposure on expression of retinoic acid receptor a and 

retinoid X receptor a in mouse embryos. ICR mice were 

fed standard feed (CM; Oriental Yeast). [No information 

was provided about caging and bedding materials.] 

Mice were orally dosed with bisphenol A [purity not 

indicated] at 0 (olive oil vehicle) or 0.002 mg/kg bw/day 

on 6.5–11.5, 6.5–13.5, 6.5–15.5, and 6.5–17.5 days postcoitum. Day of vaginal plug was considered 0.5 days 

post-coitum. [No information was provided about the 

specific method of oral dosing.] Twelve dams/group 

were killed at 12.5, 14.5, 16.5, and 18.5 days post-coitum, 

24 hr after receiving the last dose. Expression of 

mRNA for retinoic acid receptor a and retinoid X 

receptor a was measured by RT-PCR in fetal cerebrum, 

cerebellum, and gonads. Data were analyzed by ANOVA. [It was not clear if the litter or offspring was 

considered the measurement unit.] Numerous changes 

in mRNA expression were observed following in utero 

exposure to bisphenol A, and they varied according to 

sex, tissue, and dosing period. The study authors 

concluded that these findings suggest a novel mechanism of bisphenol A toxicity mediation by disruption of 

the expression of retinoic acid receptor a and retinoid X 

receptor a. 

Strengths/Weaknesses: Strengths are the oral route of 

delivery, the use of a low dose level of bisphenol A, and 

the exposure at different time periods. The study has 

value for understanding mechanisms of action although 

these changes were not tied to any adverse findings that 

might be related to these changes. Weaknesses include 

the use of a single dose level and lack of clarity on 

number of embryos per litter sampled. This is not 

considered a critical weakness because it is known that 

standard procedures for these methods require pooling 

of embryos within litter. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate but of limited utility for the 

evaluation because of the mechanistic nature of the 

endpoints. 

Nishizawa et al. (2005b), supported by the Japanese 

Ministry of Education, Culture, Sports, Science, and 

Technology and by the Japan Society for the Promotion of 

Science, examined the effects of bisphenol A exposure on 

expression of mRNA for arylhydrocarbon and retinoid 

receptors in mouse embryos. ICR mice were fed standard 

diet (CM; Oriental Yeast). [No information was provided 

about caging or bedding materials.] Pregnant mice were 

orally dosed with bisphenol A [purity not indicated] at 0 

(olive oil vehicle), 0.00002, 0.002, 0.20, or 20 mg/kg bw/ 

day from 6.5 to 13.5 days post-coitum or 6.5 to 17.5 days 

post-coitum. Day of vaginal plug detection was considered 0.5 days post-coitum. [No information was provided about the specific method of oral dosing.] Twelve 

pregnant mice/group were killed on 14 and 18.5 days 

post-coitum, 24 hr after the last bisphenol A dose was 

administered. RT-PCR analyses were conducted to 

determine expression of mRNA for retinoic acid, retinoid 

X, and arylhydrocarbon receptors in fetal cerebrum, 

cerebellum, ovary, and testis. Data were analyzed by 

ANOVA. [It was not clear if the litter or offspring was 

considered the measurement unit.] Numerous changes 

in mRNA expression were observed following bisphenol 

A exposure and they varied according to dose, sex, 

tissue, and exposure period. The study authors concluded the this study shows a novel mechanism by 

which bisphenol can induce endocrine disruption 

through upregulation of arylhydrocarbon receptor (a 

key factor in the metabolism of some xenobiotics 

compounds) and retinoid receptors (key factors in 

nuclear receptor signal transduction). 

Strengths/Weaknesses: The wide dose range from 

0.00002 to 20 mg/kg bw/day and the oral route are 

strengths. The study has value for understanding 

mechanisms of action although these changes were not 

tied to any adverse findings that might be related to 

these changes. Weaknesses include the lack of specification of the method of oral dosing and lack of clarity on 

sample origins and sizes for each assay. Again, this is not 

considered a critical weakness because it is known that 

standard procedures for these methods require pooling 

of embryos within litter. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate but of limited utility for the 

evaluation because of the mechanistic nature of the 

endpoints. 

Nishizawa et al. (2005a), supported by the Japan 

Society for the Promotion of Science, examined the 

effects of bisphenol A exposure on expression of aryl 

hydrocarbon receptors, related factors, and metabolizing 

enzymes in mouse embryos. ICR mice were fed standard 

diet (CM; Oriental Yeast). [No information was provided 

about caging and bedding materials.] Mice were orally 

dosed with bisphenol A [purity not indicated] at 0 (olive 

oil vehicle), 0.00002, 0.002, 0.2, or 20 mg/kg bw/day from 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 287 

6.5–13.5 days post-coitum and 6.5 to 17.5 days postcoitum. Day of vaginal plug was considered 0.5 days 

post-partum. [No information was provided about the 

method of oral dosing.] Another group of mice was 

dosed with 5 mg/kg bw/day 17b-estradiol during the 

same time periods. Twelve mice/group were killed at 

14.5 and 18.5 days post-coitum, 24 hr after receiving the 

final dose. Embryos were dissected to obtain cerebrum, 

cerebellum, ovary, testis, and liver. RT-PCR analysis was 

used to measure mRNA levels of genes. Western 

immunoblotting was used to measure protein levels of 

CYP1A1 and glutathione-S-transferase in liver. Data were 

analyzed by ANOVA. [It was not clear if the litter or 

offspring was considered the measurement or statistical unit.] 

Numerous changes in mRNA expression were observed following bisphenol A exposure, and they varied 

according to dose, sex, tissue, and exposure period. In at 

least one sex and time period, exposure to 17b-estradiol 

increased expression of mRNA arylhydrocarbon receptor 

in all tissues, arylhydrocarbon receptor repressor in 

testes and ovaries, arylhydrocarbon receptor nuclear 

translocator in brain or testes, CYP1A1 in brain, and 

glutathione-S-transferase in brain. Changes in protein 

levels of CYP1A1 and glutathione-S-transferase in liver 

were also examined in embryos at 18.5 days post-coitum 

and levels of both proteins were increased with exposure 

to bisphenol A at doses Z0.2 mg/kg bw/day and with 

exposure to 17b-estradiol. The study authors proposed a 

novel mechanism of toxicity involving upregulation of 

mRNA for arylhydrocarbon receptor and other factors by 

bisphenol A. 

Strengths/Weaknesses: The wide dose range and the 

oral route are strengths. The study has value for 

understanding mechanisms of action although these 

changes were not tied to any adverse findings that might 

be related to these changes. Weaknesses include the lack 

of specification of the method of oral dosing and lack of 

clarity on sample origins and sizes for each assay. This is 

not considered a critical weakness because it is known 

that standard procedures for these methods require 

pooling of embryos within litter. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate but of limited utility for the 

evaluation because of the mechanistic nature of the 

endpoints. 

Imanishi et al. (2003), supported by the Ministry of 

Education, Culture, Sports, Science, and Technology of 

Japan, used DNA microarrays to investigate potential 

mode of action of bisphenol A on alterations in 

expression of 20 nuclear hormone receptors and a few 

other genes in the mouse placenta. ICR male and female 

mice were housed in polycarbonate cages, given ad lib 

access to tap water and CM rodent feed (Oriental Yeast), 

and maintained under standard 12-hr/12-hr light/dark 

cycle. Between 6.5 and 17 days post-coitum, pregnant 

dams were orally administered 0 or 0.002 mg/kg bw/ 

day bisphenol A [purity not provided] in olive oil 

[method of oral administration not given]. The dams 

were killed 18.5 days post-coitum, and placentas and 

fetuses were frozen at 801C. Placental RNA from male 

and female embryos was separately extracted, reverse 

transcribed, and hybridized to a microarray chip for 18 hr 

at 421C. Images were analyzed using Atlas navigator 

software, and statistical analyses were performed using 

the Pearson correlation coefficient, normalized to the 

Fisher z transformation. Differentially expressed genes 

were identified using paired t-test, and significant 

changes were noted in percent values increased or 

decreased relative to control mRNA expression values. 

[The number of dams used and arrays run was not 

given. It was not clear if the litter or offspring were 

considered the statistical unit.] 

Nuclear receptor genes that showed differential 

expression in male and/or female fetuses were: neuronderived orphan receptor 1, retinoic acid-related orphan 

receptor g, estrogen receptor b, liver X receptor a, 

progesterone receptor, chicken ovalbumin upstream 

promoter transcription factor a, germ cell nuclear factor, 

steroidogenic factor 1, and photoreceptor-specific nuclear receptor. Nuclear receptor genes that did not show 

differential expression included thyroid hormone receptor b, peroxisome proliferators activated receptor a and g, 

constitutive androstane receptor, farnesoid X receptor, 

chicken ovalbumin upstream promoter transcription 

factor b, testis receptor b, estrogen-related receptor g, 

aryl hydrocarbon receptor, small heterodimer partner, 

and dosage-sensitive sex reversal receptor. Other genes 

the expression of which was both significantly altered in 

pair-wise comparison with control treatment and exhibited opposing up- or downregulation in a sexdependent manner included fast skeletal troponin C, 

probasin, RNA-specific adenosine deaminase, and 

ADAM25/testase 2, a-fetoprotein, and kinesin light 

chain 1. These genes were downregulated in placentas 

of male fetuses and upregulated in placentas of female 

fetuses. Placentas of male and female fetuses exhibited 

downregulation if a-fetoprotein (k60%, male and k24%, 

female) and kinesin light chain 1 (k70%, male and k10%, 

female). 

The authors conclude that fetal sex-based differences 

in placental physiology resulting from bisphenol A 

exposure may lead to subsequent sex-specific developmental perturbance. They also indicated that important 

but largely unknown effects of bisphenol A may occur 

with respect to a cluster of orphan nuclear receptors, 

which exhibited significant changes in gene expression. 

Strengths/Weaknesses: Strengths: The evaluation of 

several molecular endpoints including gene activity for 

several receptors that are not commonly examined, oral 

dosing, and use of a low dose represent strengths. 

Weaknesses are the use of only one dose level of BPA and 

absence of many critical experimental details such as the 

number of litters used. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for inclusion due to lack of 

reporting key experimental details. 

Yoshino et al. (2004), supported by the Japanese 

Ministry of Education, Science, Sports, and Culture and 

the Japan Private School Promotion Foundation, examined the effect of prenatal bisphenol A exposure on 

immune response in mice. [No information was provided about feed or caging and bedding materials.] 

DBA/l J mice were fed bisphenol A [purity not 

indicated] at doses of 0 (ethanol/corn oil vehicle), 

0.003, 0.030, 0.300, or 3 mg/kg bw/day for 18 days 

[stated to be 17 days in the Methods section but 18 days 

in other parts of the report], beginning on the day of a 

24-hr mating period (Day 0). Twelve mice/group were 

treated and 7–9/group became pregnant. [The specific 

Birth Defects Research (Part B) 83:157–395, 2008
288 CHAPIN ET AL. 

method of oral dosing was not described.] At 8 weeks of 

age (Day 77) 5 mice/group/sex were randomly selected 

and immunized by i.p. injection with hen egg lysozyme. 

Representation of litter was not specified. Blood was 

collected and spleens were removed 3 weeks following 

immunization (Day 98). Serum levels of hen egg 

lysozyme-specific immunoglobulin G (IgG), IgG1, and 

IgG2A were measured by ELISA. Spleen cell suspensions 

were prepared, and proliferation was assessed by 

incorporation of 3

H-thymidine following a 72-hr incubation with hen egg lysozyme. Spleen cell suspensions 

were also prepared for measurement of interferon-g and 

interleukin-4 secretion by ELISA. An additional 6 mice/ 

group/sex were killed at 8 weeks of age (Day 77). 

Spleens were removed and expression of CD31CD81

and CD31CD41 molecules on splenic lymphocytes was 

examined using monoclonal antibodies and flow cytometry. Thymus and spleen were fixed in 4% formaldehyde and examined histologically. Data were analyzed 

by Mann–Whitney U-test. It was not clear if the litter of 

origin was accounted for in statistical analyses. 

Bisphenol A treatment had no significant effect on 

pregnancy rate, sex ratio, or body weight of offspring. 

There were several significant immune responses for 

male mice. [Results in female mice were said to be 

similar to those observed in male mice but the data 

were not show by study authors.] At bisphenol A doses 

Z0.03 mg/kg bw/day, production of anti-hen egg 

lysozyme IgG2a following immunization was increased. 

Effects observed at Z0.3 mg/kg bw/day included 

increases in production of anti-hen egg lysozyme IgG 

and secretion of interferon-g and interleukin-4. Additional findings at the high-dose (3 mg/kg bw/day) were 

increases in spleen cell proliferation and production of 

anti-hen egg lysozyme IgG1 following immunization. 

Augmentation of interferon-g and interleukin-4 secretion 

following incubation of spleen cells with hen egg 

lysozyme was examined in the high-dose group only 

and found to be increased. [Increases in CD31CD81 and 

CD31CD41 expression on lymphocytes were reported 

in males and females exposed to bisphenol A, but the 

doses at which the effects occurred were not specified.] 

No histopathological alterations were reported for the 

spleen or thymus. The study authors explained that 

effects on IgG2a and interferon-g were indicators of T 

helper 1 immune responses and effects on IgG1 and 

interleukin-4 were indicators of T helper 2 responses. 

They concluded that the findings suggest that prenatal 

exposure to bisphenol A may upregulate immune 

responses in mice. 

Strengths/Weaknesses: The oral route of administration and the wide range of doses are strengths. 

Weaknesses include small sample size (n 5 5), lack of 

clarity regarding statistical handling of factors such as 

litter and sex effects. 

Utility (Adequacy) of CERHR Evaluation Process: 

This study is inadequate for the evaluation process due 

to the reasons stated above. 

Berger et al. (2007), supported by The Natural Sciences 

and Engineering Research Council of Canada, examined 

the effect of bisphenol A exposure on ovum implantation 

and pup survival in mice. CF-1 mice were housed in 

polypropylene cages and were fed Harlan Teklad 22/5 

rodent feed, which was stated to contain soy. [No 

information was provided about bedding materials.] 

On GD 1–4 or 5 [described as GD 1–5 in Methods 

section and GD 1–4 in study figures and tables] (GD 

0 5 day of vaginal plug), 31 mice in the control group 

were s.c. injected with peanut oil vehicle and 5–15 mice/ 

group were s.c. injected with bisphenol A (97% purity) at 

0.0005, 0.0015, 0.0046, 0.0143, 0.0416, 0.125, 0.375, 1.125, 

3.375, or 10.125 mg/animal/day. In a second experimental group, BPA was administered through a diet containing 3% or 6% BPA added to peanut butter and chow. In a 

third experimental group maintained on chow, BPA was 

administered at 0.11, 1.0, 3.0, or 9.0% in separate 

offerings of peanut butter alone. Pregnancy disruptions 

in orally exposed mice are discussed in Section 3.2.5.1. 

[In the first experimental group, if it is assumed that 

the mice weighed 0.02 kg at the start of gestation 

(USEPA, 1988), CERHR estimated bisphenol A intakes 

of 0.025, 0.075, 0.23, 0.72, 2.1, 6.3, 19, 56, 170, and 500 mg/ 

kg bw/day.] Mice were allowed to litter. Pups were 

counted on the day of parturition and observed for 

survival for 5 days. Pups were weaned at 28 days after 

birth and at that time, body weight and sex ratio were 

determined. Data were analyzed by ANOVA, w2 test, and 

Newman–Keuls multiple comparisons. [It was not clear 

if all offspring data were analyzed on a pup or litter 

basis.] A study examining implantations in s.c.-treated 

females is discussed in Section 4.2.1.1 Percent of females 

giving birth was significantly decreased in the 

10.125 mg/day group (B28% vs. 97% in control group). 

Numbers of pups born were significantly decreased in 

the 3.375 and 10.125 mg/day group (B8 and 2 pups in 

each of the dose groups and 13 pups in the control 

group). There were no treatment-related effects on pup 

weight or sex ratio at weaning. [As discussed in Section 

3.2.5.1, it appears that with oral exposure, pregnancy 

disruption occurred at higher bisphenol A levels 

(68.8 mg/day, 3440 mg/kg bw/day) than with s.c. exposure (10.125 mg/day, B500 mg/kg bw/day)]. The study 

authors concluded that the amount of bisphenol A 

required for pregnancy disruption was higher than 

typical environmental levels but that it is not known if 

bisphenol A could have additive or synergistic effects 

with other environmental estrogens. 

Strengths/Weaknesses: A strength of the subcutaneous study is that it examined a wide range of 

bisphenol A dose levels. The comparison of the differential effects of s.c. and oral routes of bisphenol A 

administration is also a strength. Weaknesses include the 

limited/unequal number of mated mice in each dose 

group, absence of maternal data to ascertain the potential 

impact of maternal toxicity on pregnancy, methodological deficiencies regarding fertility assessment, and the 

use of a diet that contains phytoestrogens. 

Utility (Adequacy) for CERHR Evaluation Process: 

Due to the limited number of mated mice per dose level 

(n 5 5–15), methodological concerns, absence of key 

statistical information as well as maternal information, 

this study is inadequate for the CERHR evaluation 

process. 

3.2.5.2 Studies with neurobehavioral endpoints: 

Narita et al. (2006), supported by the Japanese Ministry 

of Health, Labor, and Welfare, and Ministry of Education, 

Culture, Sports, Science, and Technology, conducted a 

series of studies to examine the effects of bisphenol A on 

the dopaminergic system of mice exposed during 

development. Only brief details were provided about 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 289 

the studies. In each study, ddY mice received feed 

containing bisphenol A from mating to weaning of their 

offspring. [No information was provided on purity of 

bisphenol A, type of feed, caging and bedding 

materials, the number of dams treated, or the ages or 

sexes of offspring that were tested.] Statistical analyses 

included ANOVA with Bonferroni/Dunnett test. [It was 

not clear if the litter or offspring was considered the 

statistical unit.] In a place conditioning-study, testing 

was conducted in 6–14 mice/group born to dams 

exposed to bisphenol A at 0, 0.03, 0.3, 3, 500, or 

2000 mg/kg food. [Assuming a female mouse eats 

B0.2 kg feed/kg bw/day (USEPA, 1988), bisphenol A 

intake would have been 0.006, 0.06, 0.6, 100, or 400 mg/ 

kg bw/day.] During the preconditioning period, mice 

were placed in one section of a cage following injection 

with saline [specific route not reported] and in another 

section of the cage following s.c. injection with 1 mg/kg 

bw morphine. On the day of testing, the amount of time 

spent in each section of the cage was recorded. Mice from 

the lowest dose group (0.03 mg/kg food) and 2 highest 

dose groups (500 and 2000 mg/kg) food spent more time 

in the section of the cage associated with morphine 

injection. [Compared to controls, the time spent in the 

morphine-associated section of the cage was B9.5-, 7-, 

and 9-fold longer in each of the respective dose 

groups.] Total locomotor activity was measured for 3 hr 

in 5–15 mice/group born to dams exposed to 0, 0.03, 3, or 

2000 mg/kg food. Following s.c. injection with 10 mg/kg 

bw morphine, activity was increased in mice from the 

low- (0.03 mg/kg food) and high- (2000 mg/kg food) 

dose groups compared to the control group [increased 

by B9-fold in the low dose group and 12-fold in the 

high-dose group]. Binding of 35S-guanosine-50

[g-thio]­

triphosphate in the limbic system was measured in 3 

samples/group obtained from offspring of dams exposed to 0.03, 3, or 2000 mg/kg food. Dopamine-induced 

binding of 35S-guanosine-50

[g-thio]-triphosphate in the 

limbic system was increased at each dose level compared 

to controls [by B32, 18, and 56%]. Based on their 

findings, the study authors concluded that prenatal and 

neonatal exposures to low bisphenol A doses can 

potentiate central dopamine receptor-dependent neurotransmission in the mouse. 

Strengths/Weaknesses: This study is so poorly written 

that it is extremely difficult to understand many 

sentences (let alone paragraphs) and to determine 

precisely what was done, why, and what happened. 

The main weakness of the study is therefore its inability 

to pass its message to the reader. Given this limitation, it 

is difficult to determine whether the study has any 

strengths, and if so what they might be. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process 

because of the lack of methodological details and the 

poor communication of the study results. 

Kawai et al. (2003), supported by Core Research for 

Evolutional Science and Technology and Japan Science 

and Technology, examined the effects of prenatal bisphenol A exposure on aggressive behavior in male mice. 

[No information was provided about feed, bedding, or 

caging materials.] Pregnant CD-1 mice were randomly 

assigned to groups of 7 and orally dosed by micropipette 

with 0.002 or 0.020 mg/kg bw/day bisphenol A [purity 

not reported] on GD 11–17. A control group of 9 mice 

received the corn oil vehicle by micropipette during the 

same time period. Doses were said to be within the range 

of human exposures. Pups were weaned on PND 21 (day 

of birth 5 PND 0), and males randomly selected males 

from the same litter were housed in groups of 4 or 5. 

Aggression testing was conducted at 8, 12, and 16 weeks 

of age. For the testing, 15 control male mice from the 9 

litters were randomly selected to be opponents and 

housed 5/cage. Opponents were used only once/day for 

testing. During testing of mice from the control and 

treated groups, the subject was housed alone for 5 min 

before placing the opponent mouse into the cage. 

Behavior with the opponent mouse was observed for 

7 min. The numbers of mice evaluated were 26–32/group 

at 8 weeks of age, 18–24/group at 12 weeks of age, and 

10–16/group at 16 weeks of age. MiceRandomly selected 

were killed at 9, 13, and 17 weeks of age, 1 week 

following behavior testing, for measurement of testis 

weight and serum testosterone level. [The results section 

states that testis weights and serum testosterone levels 

were obtained at 8, 12, and 16 weeks of age.] Eight 

mice/group were killed after the first 2 test periods and 

10–16 mice/group were killed after the last test period. 

Mice that were not killed were tested at the next 

evaluation period, so that mice killed after 16 weeks of 

age were tested a total of 3 times. Statistical analyses 

included ANOVA and Spearman rank correlation test. [It 

does not appear that the litter was considered the 

statistical unit.] 

Aggression scores, as determined by contact time, 

were increased significantly compared to the control 

group at 8 weeks of age in both the low- (124% increase) 

and high- (146% increase) dose bisphenol A groups. No 

treatment-related effects on aggression score were 

observed at 12 and 16 weeks of age. In the low-dose 

group, relative (to body weight) testis weight was 10% 

lower than controls at 8 weeks of age and 18% lower than 

controls at 12 weeks of age. Relative testis weight was 

11% lower than control values in the high-dose group at 

12 weeks of age. No significant effects were observed for 

serum testosterone levels. There were no correlations 

between serum testosterone levels and contact time in 

aggression testing. The study authors concluded that 

prenatal bisphenol A exposure of mice resulted in 

behavioral changes and decreased relative testis weight 

that was more pronounced at the lower dose. 

Strengths/Weaknesses: Strengths are the use of 2 low 

dose levels and the oral route of administration. The lack 

of husbandry information, inappropriate presentation of 

testis weight data, variable degrees of repeated behavioral testing, and the apparent lack of consideration of 

possible litter effects are weaknesses. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process due 

to the reasons stated above. 

Kawai et al. (2007), supported by Japan Sciences 

Technology and Core Research for Evolutional Science 

and Technology, evaluated the brain expression of ERa

and ERb in male mice exposed in utero to bisphenol A. 

Pregnant ICR mice were fed bisphenol A in corn oil by 

micropipette on GD 11–17 at 0 or 0.002 mg/kg bw/Day 9, 

n 5 18/group). Mice were housed singly in polypropylene cages. [The first day of gestation was likely 

designated as GD 0, according to a figure. Type of 

feed and bedding material were not given.] Litters were 

Birth Defects Research (Part B) 83:157–395, 2008
290 CHAPIN ET AL. 

reared by their dams until weaning on PND 21 

[birth 5 PND 0]. Males from the same litters were 

housed 4 or 5/cage. Randomly selected males [8–12/ 

group, without mention of litter of origin] were killed at 

4–5, 8–9, or 12–13 weeks of age. Testosterone was 

measured by RIA in trunk blood serum. Brains were 

perfusion fixed and processed for immunostaining with 

antibody to ERa, ERb, serotonin, and serotonin transporter. Fields were selected within the dorsal raphe nucleus 

and ERa- or ERb-positive neurons were counted in every 

fourth section (n 5 8 or 9 animals/group). Staining for 

serotonin and serotonin transporter involved overlapping dendrites, making it difficult to count positive 

neurons, and densitometric methods were used to 

quantify staining for serotonin and serotonin transporter 

(n 5 8–12 animals/group). Data were analyzed using 2­

way ANOVA and post-hoc Student t-test. 

The number of neurons in the dorsal raphe nucleus 

expressing ERa and ERb was increased by bisphenol A at 

5 and 13 weeks but not at 9 weeks. There were no 

significant differences at any time point in serum 

testosterone concentrations. The authors identified a 

‘‘tendency’’ for serotonin and serotonin transporter 

immunoreactivity to be increased by bisphenol A in the 

dorsal raphe nucleus, but there were no statistical 

differences between bisphenol A-treated and control 

brains at any time point. The authors concluded that it is 

possible that alterations in ER in the brain may be 

responsible for emotional and behavioral alterations in 

mice. 

Strengths/Weaknesses: This was a reasonable attempt 

to detect effects and explore a connection between 

bisphenol A, brain receptors, and aggressive behavior. 

This study is weakened by the use of only one dose, lack 

of experimental details, and uncertain accounting for 

litter and repeated measures/sections effects in analyses. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is deemed inadequate for inclusion due to 

unclear statistical procedures regarding litter and nested 

factors associated with repeated measurements. 

Laviola et al. (2005), supported by Italian Ministry of 

Health, Ministry of Universities and Research, and the 

University of Parma, examined the effect of prenatal 

bisphenol A exposure on d-amphetamine-reinforcing 

effects in mice. [No information was provided about 

feed, housing, or bedding composition.] CD-1 mice 

were trained to drink the tocopherol-purified corn oil 

vehicle through a syringe. The mice were randomly 

assigned to groups, and 10–12/group were exposed to 

bisphenol A [purity not reported] at 0 (vehicle) or 

0.010 mg/kg bw by feeding from a syringe on GD 11–18 

[day of vaginal plug not defined]. Another group of 

mice was exposed to methoxychlor; those findings will 

not be discussed. Litters were culled to 10 pups (571 of 

each sex) within 12 hr of parturition. Offspring were 

weaned and group housed with littermates of the same 

sex on PND 25. At 60 days of age, 3 offspring/sex/litter 

(1 sex/litter at each d-amphetamine dose) were subjected 

to conditioned place-preference testing. For the test, 

animals were acclimated to the apparatus on the first day 

of testing. On alternate days over a 4-day period, animals 

were i.p. injected with 0, 1, or 2 mg/kg bw d-amphetamine and confined to one compartment of the apparatus 

for 20 min. On the other days of the 4-day period, 

animals were injected with saline and confined in 

another section of the apparatus for 20 min. On the fifth 

day of testing, animals were not treated and were given 

free access to the entire apparatus for 10 min. The amount 

of time spent in the compartment associated with damphetamine treatment was measured. Data were 

analyzed by a split-plot ANOVA, in which the litter 

was considered the block variable, and Tukey HSD test. 

Prenatal treatment was described as a between litters 

factor and all other variables were described as within 

litter factors. 

No differences were reported for birth weight and sex 

ratio at birth. [Data were not shown by authors.] There 

were no significant effects of bisphenol A treatment on 

locomotor activity. Conditioned place-preference occurred in control females following injection with either 

d-amphetamine dose, but was not observed in females 

treated with bisphenol A. In males, both the vehicle 

control and the bisphenol A group displayed a preference for the d-amphetamine-associated compartment 

following treatment with the high d-amphetamine dose. 

Therefore, there was no change in preference following 

bisphenol A treatment of males. The study authors 

concluded that prenatal bisphenol A exposure affected 

organization of the brain dopaminergic system in female 

mice leading to long-term alterations in neurobehavioral 

function. 

Strengths/Weaknesses: Strengths of this study include 

robust and appropriate design and analysis, adequate 

sample size, and oral dosing. The use of only one dose 

level is a weakness. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility in the 

evaluation. 

3.2.6 Mouse—parenteral exposure only during 

pregnancy. Markey et al. (2001a), supported by NIH, 

the Massachusetts Department of Health, the International Union Against Cancer, and the World Bank, 

examined the effect of prenatal bisphenol A exposure 

on mammary gland development in mice. CD-1 mice 

were fed RMH 3000 rodent diet, which showed negligible activity in estrogenicity testing. Caging and bedding were also reported to test negative in estrogenicity 

assays. Dams (6–10/group) were estimated to have 

received the DMSO vehicle or bisphenol A [purity not 

reported in the manuscript; 9772% per A. Soto, 

personal communication, March 2, 2007] at 0.000025 or 

0.000250 mg/kg bw/day through a s.c. pump from GD 

9–20 (GD 1 5 day of vaginal plug). [The original 

publication stated that bisphenol A doses were 25 

and 250 mg/kg bw/day, but units were corrected to ng/ 

kg bw/day in an addendum released for the study]. 

Doses were not adjusted for increasing body weight as 

dams gained weight during pregnancy. Dams were 

allowed to litter and offspring were weaned at 19 days 

of age. At 10 days, 1 month, and 6 months of age, 6–10 

female offspring/group were killed during each time 

period. [Number of litters represented was not stated 

but there may have been 1 offspring/litter based on the 

numbers examined.] Vaginal smears were assessed in 

mice following puberty, and post-pubertal mice were 

killed during proestrus. Before being killed, females 

were injected with bromodeoxyuridine, and incorporation of bromodeoxyuridine in mammary glands was 

determined by an immunohistochemistry method. Histological and morphometric analyses of mammary 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 291 

glands were also conducted. Data were analyzed by 

ANOVA, least significant difference test, and t-test. [The 

statistical analyses considered litter differences, method 

unstated.] 

At 1 month of age, the rate of ductal migration into the 

stroma was increased in the low-dose group and 

decreased in the high-dose group; values in the 2 

treatment groups were significantly different from one 

another but neither dose group was significantly 

different from the control group. Bisphenol A treatment 

increased percentages of ducts and buds at 6 months of 

age. Bromodeoxyuridine incorporation was decreased in 

epithelial cells at both doses at 10 days of age, decreased 

in stromal cells at the high-dose at 1 month of age, and 

increased in stromal cells at both dose levels at 6 months 

of age. At 1 month of age, the ratio of bromodeoxyuridine-positive epithelial to stromal cells was 4:1 in the 

control group, 2:1 in the 0.000025 mg/kg bw/day group, 

and 6:1 in the 0.000250 mg/kg/bw/day group. The 

percentage of alveoli containing secretory products was 

increased at the low dose at 6 months of age. The study 

authors concluded gestational exposure to low doses of 

bisphenol A alters timing of DNA synthesis in mammary 

epithelium and stroma, resulting in a histoarchitecture 

that is not typical for a virgin mouse. 

Strengths/Weaknesses: The examination of the mammary gland, a system not often studied, is a strength. A 

critical weakness is the uncertainty of the DMSO 

concentration as a vehicle and therefore pump performance. An additional weakness is that the proliferative 

changes reported in mammary tissues in virgin mice 

have not been satisfactorily established as precursors of 

breast cancer. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process given 

exposure uncertainties. 

Markey et al. (2003), supported by NIH and the 

Massachusetts Department of Public Health, examined 

the effects of prenatal bisphenol A exposure on development of the female reproductive system and mammary gland in mice. CD-1 mice were fed Purina Rodent 

Chow that tested as having negligible estrogenicity. 

Cages and bedding tested negative for estrogenicity in 

the E-SCREEN assay. Water was provided in glass 

bottles. Mice (n 5 6–10/group) were administered bisphenol A [purity not indicated in the manuscript; 

9772% per A. Soto, personal communication, March 2, 

2007] at 0 (DMSO vehicle), 0.000025, or 0.000250 mg/kg 

bw/day by s.c. pump from GD 9 through the remainder 

of pregnancy (GD 1 5 day of vaginal plug). [The dose 

levels were incorrect in the original and were corrected 

by an erratum (Markey et al., 2004).] Number of 

offspring, sex ratio, body weight, and age at vaginal 

opening were assessed. Beginning at 3 months of age and 

continuing for 2 weeks, estrous cyclicity was assessed by 

visual examination of the external vagina and confirmation by vaginal smears. Female offspring (6–10/group) 

were killed at 1, 3, 4, 6, 9, and 12 months of age on the 

afternoon of proestrus. Reproductive organs were 

grossly assessed, and morphometric measurements were 

obtained for ovary and mammary gland. [Although 

the methods section suggests that morphometric measurements were obtained at each time period of 

sacrifice, it does not appear that the measurements 

were taken at 12 months of age. The 1-month data 

were reported in a previous publication (Markey 

et al., 2001a).] A histopathological evaluation of the 

ovary was conducted at 3 months of age. Reproductive 

organ weights were obtained at 1, 3, and 6 months 

of age. [As in other studies reported from this 

laboratory, different litters were represented at each 

time period (A. Soto, personal communication 

March 2, 2007).] Statistical analyses included ANOVA, 

Kruskal–Wallis, and Mann–Whitney tests. [It was not 

clear if the litter or offspring was considered the 

statistical unit.] 

Bisphenol A exposure had no significant effect on litter 

size or sex ratio. A significant interaction between age for 

body weight and treatment was reported from 2–12 

months of age but the effect on body weight was not 

explained. No significant effects were observed for 

vaginal opening in treated mice. Significant increases 

were observed in percentages of 3-month-old mice with 

estrus/metestrus for Z4 or 8 days. At 6 months of age, 

the incidence of fluid-filled ovarian bursae was increased 

in both treatment groups. Reproductive organ weights 

were not affected at 1 or 6 months of age, but at 3 months 

of age, absolute and relative (to body weight) weights of 

vagina were decreased in the high-dose group. The 

percentage of ovary tissue consisting of antral follicles 

was increased in the high-dose group at 3 months of age. 

No significant differences were observed for mammary 

structures at 4 months of age. At 6 months of age, the 

percentage of alveolar buds/lobulo-alveoli was increased 

in both dose groups compared to the control group. The 

percentage of alveolar buds/lobulo-alveoli was decreased in the low-dose group compared to control 

group at 9 months of age. The study authors concluded 

that exposure of mice to environmentally relevant doses 

of bisphenol A during the development of estrogensensitive tissues results in effects that are manifested in 

adulthood. 

Strengths/Weaknesses: The examination of the mammary gland, a system not often studied, is a strength. A 

critical weakness is the uncertainty of the DMSO 

concentration as a vehicle and therefore pump 

performance. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process given 

exposure uncertainties. 

Vandenberg et al. (2007), supported by NIEHS and 

Tufts, examined the effects of prenatal bisphenol A 

exposure on mouse mammary gland development. CD-1 

mice were fed Harlan Teklad 2008, which was reported to 

contain 20 fmol/g estrogen equivalents. The type of 

caging and bedding used was not reported but they were 

stated to test negative for estrogenicity in the E-SCREEN. 

Water was supplied in glass bottles. On GD 8 (GD 

1 5 day of vaginal plug) mice were implanted [subcutaneous (A. Soto, personal communication, March 2, 

2007)] with osmotic pumps that delivered the 50% DMSO 

vehicle or bisphenol A [purity not reported in manuscript; 9772% per A. Soto, personal communication, 

March 2, 2007] at 0.000250 mg kg bw/day. The bisphenol 

A dose was selected because it was predicted (or 

estimated) to be environmentally relevant and shown 

to alter mammary endpoints (Markey et al., 2001a; 

Mun ̃oz-de-Toro et al., 2005). Pumps were left in place 

until dams were killed on GD 18. [The number of dams 

treated was not reported in the paper. The Expert Panel 

Birth Defects Research (Part B) 83:157–395, 2008
292 CHAPIN ET AL. 

has been informed that there were 20–30/group (A. 

Soto, personal communication, March 2, 2007).] Fetal 

mammary glands were mounted whole or sectioned to 

examine mammary gland development in 36–40 offspring/group. Immunohistochemistry techniques were 

used to measure expression of Ki67 and Bax in mammary 

structures from 4–8 offspring/group. Mammary collagen 

localization was assessed using Masson Trichrome stain 

in 6–17 mice/group. Expression of mRNA for ERa, ERb, 

adipocyte lipid binding protein, Col-l, and PPARg were 

measured by RT-PCR in mammary glands from 4–6 

offspring/group. Litter was accounted for in design and 

analyses by assigning 1 individual/litter to each group or 

endpoint. Statistical analyses included t-tests, ANOVA, 

Mann–Whitney U non-parametric tests, and w2 tests. 

Morphometric analysis revealed significantly higher 

ductal area and extension in the bisphenol A group than 

in controls. In the control group, females positioned next to 

two females in utero had significantly fewer branching 

points than females positioned next to 1 or 2 males; this 

difference was not observed in the bisphenol A group. In 

fetuses that were not positioned next to a male, significantly more branching points were observed in the 

bisphenol A than in the control group. Control females 

positioned next to 2 males had significantly larger 

epithelial duct area than control females not positioned 

next to a male; this difference was not observed in the 

bisphenol A group. In bisphenol A-treated females positioned next to 1 male, ductal extension was significantly 

greater than in control females positioned next to 1 male. 

In the bisphenol A group, epithelial cells were less 

rounded, more evenly spaced, and more dense than 

in controls. Bisphenol A did not significantly affect Ki67 

(a proliferation marker) expression in mammary epithelium. Lumen formation was observed in 6 of 16 control 

mice and 0 of 10 bisphenol A-exposed mice. Significantly 

decreased numbers of Bax-positive (apoptotic) cells were 

observed in the inner epithelial cord (not in contact with 

basement membrane) of bisphenol A-exposed than 

control mice. Optical density of histological staining 

was significantly lower in the fat pad of the bisphenol Aexposed than control group. Fat pads of the bisphenol A 

group compared to control group were found to be 

significantly less cellular, contain more Bax-positive cells, 

and have more vacuoles at a distance o1 mm from the 

epithelial compartment. Study authors interpreted the 

effect as increased epithelial penetration and advanced 

maturation of fat pads. No significant differences were 

observed for PPARg or adipocyte lipid binding protein 

mRNA expression. Density of collagen deposits was 

lower in the entire mammary gland but higher in the 

periductal stroma (within 10 mM of the epithelium) of the 

bisphenol A than the control group. Bisphenol A 

exposure did not affect collagen type I, ERa, or ERb

mRNA expression. ERa protein expression in the stroma 

was also unaffected by bisphenol A exposure. Study 

authors concluded that advanced maturation of fat pad 

and changes in extracellular matrix may be the cause of 

altered growth, cell size, and lumen formation in 

mammary epithelium of mouse fetuses exposed to 

bisphenol A. 

Strengths/Weaknesses: Strengths of this study are the 

rigor with which the measurements were made, and the 

fact that the authors were trying to quantify endpoints 

that are difficult to measure (e.g., the relationship of the 

stroma to the epithelium). The relevance of the endpoints 

is a strength as is the low dose used. The single dose and 

subcutaneous route of administration are weaknesses. A 

critical weakness is inappropriate statistical analysis of a 

complex study design that may have produced too many 

positive findings and a lack of statistical accounting for 

litter effects (i.e.,. 36–40 pups presented in Table 1 of 

study and only 20–30 litters treated). 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process 

because of insufficient control for litter effects. 

Honma et al. (2002), supported by the Japanese 

Ministry of Education, Culture, Sports, Sciences, and 

Table 79
 

Effects in Mice Exposed to Bisphenol A During Prenatal Developmenta
 

Dose (mg/kg bw/day) 

Endpoint 0.002 0.020 BMD10 BMDL10 BMD1SD BMDL1SD 

Female body weight 

Weaning k 10% k 7% 0.065 0.017 0.088 0.021 

PND 60 2 k 4% 0.054 0.021 0.11 0.021 

Male body weight 

Birth 2 k 5% 0.054 0.020 0.031 0.015 

PND 60 2 k 6% 0.048 0.020 0.044 0.020 

Anogenital distance 

Females at weaning m 6% 2

Males on PND 60 m 6% m 8% 0.035 0.020 0.035 0.020 

Age at vaginal openingb 2 k 1.3 days 

Body weight at vaginal openingb k 10% k 11% 

Age at 1st estrusb 2 k 1 day 

Estrous cycle length m 1.3 day m 1 day 0.021 0.007 0.12 0.021 

Cornified cells in vaginal smear m 3.1 days 2 0.17 0.020 0.44 0.021 

Lymphocytes in vaginal smear k 2.2 days 2 0.26 0.020 0.26 0.020 

a

Honma et al. (2002).
 

b

Value estimated from a graph by CERHR; data from graphs were not modeled.
 

m,k Statistically significant increase, decrease; 2 no significant effect.
 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 293 

Technology, examined the effect of prenatal bisphenol A 

exposure on the reproductive system of female mice. 

Mice were fed commercial diet (CE-2, CLEA, Tokyo, 

Japan). [No information was provided about bedding or 

caging materials.] Ten ICR/Jcl mice/group were s.c. 

injected with bisphenol A [purity not reported] in 

sesame oil at 0, 0.002, or 0.020 mg/kg bw/day on GD 

11–17 (GD 0 5 vaginal plug). Additional mice were 

injected with diethylstilbestrol at 0.02–2 mg/kg bw/day. 

Pups were sexed, counted, and weighed at birth. At 22 

days of age, offspring were weaned and litter sizes were 

adjusted to 8 pups. Male and female offspring were 

weighed during the postnatal period. Anogenital distance was measured in males and females at 22 and 60 

days of age. Females were monitored for vaginal opening. Vaginal smears were obtained for 30 days following 

vaginal opening. Female offspring were mated with 

untreated males from 90 to 120 days of age. F2 pups were 

counted and sexed at birth. The litter was considered the 

experimental until in statistical analyses. Data were 

analyzed by ANOVA and Student or Welch t-test. 

Statistically significant findings are summarized in 

Table 79. There were no effects on gestation duration, 

number of pups/litter, or sex ratio. Body weights were 

slightly lower in high-dose males at birth, both dose 

groups of females at weaning, and high-dose males and 

females at 60 days of age. Anogenital distance was 

increased in low-dose females at weaning and both dose 

groups of males at 60 days of age. Age of vaginal opening 

and first estrus was accelerated in the high-dose group, 

and body weight at vaginal opening was lower in both 

dose groups. Estrous cycle length was increased in both 

dose groups. Total days that cornified cells were present 

in vaginal smears was increased and total days that 

lymphocytes were detected was decreased in the lowdose group. In F1 offspring there were no significant 

effects on mating, number of F2 pups/litter, or sex ratio 

of F2 pups. Results in mice dosed with diethylstilbestrol 

were similar to those observed in mice dosed with 

bisphenol A. The study authors concluded that prenatal 

exposure to low doses of bisphenol A results in early 

vaginal opening in mice but did not affect female 

reproductive function. 

Strengths/Weaknesses: Strengths are that this study 

represents one of the few studies that appropriately 

examines the onset of puberty in the mouse as an 

endpoint, it uses low dose levels of bisphenol A, 

relatively large sample sizes, and effectively uses a 

positive control at 3 dose levels. The lack of AGD 

measurement at birth and difficulty of measurement at 

PND 60 are weaknesses. he Expert Panel was unable to 

confirm the statistical significance of the effects shown in 

Table 2 of the manuscript. 

Utility (Adequacy) for CERHR Evaluation Process: 

The study is adequate for inclusion but of limited utility 

due to statistical questions about body weight and AGD 

and subcutaneous route of exposure. 

Iwasaki and Totsukawa (2003), support not indicated, 

examined the effect of prenatal bisphenol A exposure on 

reproductive development of female mice. ICR mice 

were fed F1 diet (Funabashi, Chiba, Japan) and housed in 

polycarbonate cages containing an unspecified chip 

bedding. On GD 7–18 (GD 0 5 day of copulatory plug), 

6 dams/group received bisphenol A [purity not reported] at 0 (DMSO vehicle) 0.00025, 0.025, or 2.5 mg/kg 

bw/day by s.c. injection. A positive control group of 

mice received 100 mg/kg bw/day 17b-estradiol [route 

not specified]. Dams were weighed during the study. 

Pups were counted and sexed on PND 0, and pup 

viability was determined on PND 4. Pups were weaned 

on PND 21, and male pups were killed and discarded. 

Female pups (24–41/group) were observed for vaginal 

opening. On PND 21, 1 pup/litter(4/group) from the 

low- and mid-dose group was injected with 3 mg/kg bw/ 

day 17b-estradiol for 2 days and then killed. Uterine 

weights were assessed and expression of the ERa gene in 

uterus was determined using a colorimetric method. 

Statistical analyses included ANOVA, ANOVA on ranks 

(Kruskall–Wallis test), and Dunnett test. [It was not clear 

if the litter or offspring was considered the statistical 

unit.] 

Weight gain was described as increased in all treated 

dams compared to control dams, but there was no 

evidence of a dose–response relationship and statistical 

significance was not achieved. Pup birth weight was 

significantly lower [6%] in the low-dose group compared 

to the control group. There were no differences in litter 

size at birth. Pup viability on PND 4 was significantly 

reduced [by 26%] in the low-dose group. Age of vaginal 

opening was significantly delayed by 3 days in the lowdose group, but significantly accelerated by 2.2 days in 

the high-dose group. Following 17b-estradiol exposure, 

uterine weight was significantly decreased [by B85%] in 

the low-dose bisphenol A group and significantly 

increased [by B29%] in the mid-dose bisphenol A 

group. Although expression of ERa mRNA was observed 

at 132% of control levels in the mid-dose bisphenol A 

group following exposure to 17b-estradiol, the effect did 

not attain statistical significance. Expression of ERa gene 

was not detectable in the low-dose bisphenol A group 

following 17b-estradiol exposure. No significant effects 

were reported in mice treated with 17b-estradiol. The 

study authors concluded that ‘‘The levels tested in this 

study appear to be dangerous.’’ 

Strengths/Weaknesses: The use of 3 dose levels, 

including low doses, and the use of 17b-estradiol as a 

positive control are strengths of this study. Weaknesses 

include the use of DMSO as a vehicle, the subcutaneous 

route of administration, the small sample size, lack of 

significant effects detected in the 17b-estradiol positive 

control group, and the failure to account for litter in 

statistical analyses. 

Utility (Adequacy) for CERHR Evaluation Process: 

The study is inadequate for the evaluation process. 

Nakamura et al. (2006), supported by grants from the 

Japanese government, examined the effects of prenatal 

exposure to bisphenol A on the morphology and 

expression of certain genes related to brain development 

in the mouse neocortex. In the first experiment ICR/Jc1 

mouse dams were injected subcutaneous with either 0 

(sesame oil vehicle) or 20 m/kg bw/day bisphenol A 

[purity not indicated] daily from GD 0 (defined as the 

day that a vaginal plug was detected) until GD 10.5, GD 

12.5, GD 14.5 or GD 16.5. [No information was provided 

on feed, caging materials, bedding.] Dams were then 

given a single i.p. injection of 5-bromo-20

-deoxyuridine 

(BrdU). Fetuses were collected either 1 hr following BrdU 

treatment (to assess precursor cell proliferation) or 2 or 3 

days following BrdU treatment (to assess neuronal 

migration and differentiation). Brains were fixed in 4% 

Birth Defects Research (Part B) 83:157–395, 2008
294 CHAPIN ET AL. 

buffered paraformaldehyde for morphometry and immunohistochemical evaluation. The sections of the 

neocortex were sectioned into three zones: ventricular 

zone, intermediate zone, and cortical plate (the neocortex 

at GD 12.5 was divided into the ventricular zone and the 

primordial plexiform layer). Ten fetuses from two or 

more dams were collected at each time point. In the 

second study, ICR/Jc1 dams were treated as described 

above and fetal telencephalons were collected on GD 

12.5, GD 14.5, or GD 16.5 and frozen in liquid nitrogen 

and stored at -801C for mRNA expression analyses 

(n 5 10–15 fetuses in each group). 

There were no significant differences in the pattern of 

immunoreactivity for K1-67 (a marker for cell proliferation), nestin (a marker for neural progenitors), Musashi 

(another marker for neural progenitors), and histone H3. 

However, a marker for young neurons, Tuj1, was more 

prominent in the intermediate zone at GD 14.5 and GD 

16.5 in the bisphenol A group. The authors also looked at 

the immunoreactivity pattern for PDI, a microsomal 

enzyme that contains binding sites for T3 and estradiol. 

PDI is believed to act as a buffer for these hormones in 

cells. PDI is of interest because bisphenol A has been 

reported to bind to the T3 binding sites of PDI with 

10 to 100-fold lower affinity than T3 (Hiroi, 2006) and 

inhibit the binding of T3 to PDI when bound. PDI 

immunoreactivity was increased in the neocortex of 

bisphenol A treated fetuses from GD 12.5–GD 16.5 and in 

subplate cells at GD 14.5. 

There were no differences in BrdU labeled cells in any 

neocortical zone from brains collected 1 hr following 

BrdU treatment. However, the BrdU-labeled cells analyzed 2 days following BrdU injection were decreased in 

the ventricular zone of BPA-treated mice at GD 14.5 

(labeled at GD 12.5) and GD 16.5 (labeled at 14.5) and 

increased in the cortical plate at GD 14.5 (labeled at GD 

12.5). The authors used quantitative RT-PCR to examine 

the expression of several genes involved in brain 

development including those that help regulate the 

maintenance of neural stem cells and promote gliogenesis (Hes1 and Hes5), promote neurogenesis (Mash1, 

Math3, and Ngn2), and relate to thyroid hormone action 

(L1CAM, THR-a, and THR-b). The gene expression of 

Math3, Ngn2, Hes1, LICAM, and THR-a were upregulated 

significantly in the bisphenol A-treated group at GD 14.5 

(Hes1 and Hes5 were downregulated significantly at GD 

12.5). Overall, the authors interpreted these findings as 

suggesting that bisphenol A might disrupt normal 

neocortical development by accelerating neuronal differentiation and migration. 

Strengths/Weaknesses: The strengths of this study are 

that a reasonable sample size (10) for this type of study 

was used although the presumed dam effect was only 

partly controlled for by choosing 10 pups from two 

different dams. The study used a low dose (20 ug/kg) 

delivered s.c. to a pregnant mouse. The results revealed an 

effect on neocortical development in developing fetuses. 

Neurogenesis and gene expression were affected by BPA. 

Utility (Adequacy) for CERHR Evaluation Process: 

This is an adequate study for evaluation purposes but of 

limited utility because dam effects were only partly 

controlled for and because of the subcutaneous route of 

administration. 

Nikaido et al. (2004), supported by the Japanese 

Ministry of Health, Labor, and Welfare examined the 

effects of bisphenol A exposure on mammary glands and 

reproductive systems of mice. Outbred CD-1 (ICR) mice 

were fed NIH-07 (a low-phytoestrogen diet) and provided with water supplied in polycarbonate bottles with 

rubber stoppers. The mice were housed in polyisopentene cages with white pine chip bedding. Beginning on 

GD 15 (plug day not specified), mice were s.c. injected 

with 0 (DMSO vehicle), 0.5, or 10 mg/kg bw/day 

bisphenol A (Z99% purity) or 0.5 or10 mg/kg bw/day 

diethylstilbestrol for 4 days. [The control group contained 6 dams/group, but the number of dams in treated 

groups was not clear.] Additional groups of mice were 

treated with the same doses of genistein, resveratrol, or 

zearalenone. Female pups were weaned at 21 days of 

age. Onset of vaginal opening was monitored. 

Estrous cyclicity was monitored in 12 mice/group at 9– 

11 weeks of age. At 4, 8, 12, and 16 weeks of age, 6 

randomly selected mice/group were weighed and killed. 

Ovaries, uterus, vagina, and mammary glands were 

preserved in 10% formalin for histopathological evaluation. Differentiation of mammary structures was evaluated in whole mounts. Statistical analyses included 

homogeneity of variance tests followed by ANOVA or 

Kruskal–Wallis test. When P-values were o0.05, 

Fisher protected least significant difference test was 

conducted. [It appears that offspring were considered 

the statistical unit.] 

Body weight gain of offspring was increased by 

bisphenol A treatment, and at 16 weeks of age, body 

weight compared to controls was higher [by B50%] in the 

low-dose group and [by B23%] in the high-dose group. 

Vaginal opening was accelerated by 1.2 days at the highdose group. Estrous cycle length was increased by 2.8 

days in the low-dose group and 3 days in the high-dose 

group as a result of increased time spent in diestrus. 

Corpora lutea were observed in all control mice at each 

age. No corpora lutea were observed in 2 of 6 mice of the 

low-dose group and 3 of 6 mice of the high-dose group at 

4 weeks of age, but all mice had corpora lutea at 4, 8, 12, 

and 16 weeks of age. With the exception of vaginal 

cornification observed in mice lacking corpora lutea, no 

histopathological abnormalities were observed in the 

uterus or vagina. Two of three mice with corpora lutea 

in the high-dose bisphenol group had greater mammary 

alveolar differentiation compared to control mice at 4 

weeks of age. No differences in mammary differentiation 

were observed at later ages. The study authors concluded 

that both the high and low dose of bisphenol A produced 

transient changes in the mammary gland and reproductive tracts of mice. Transient effects on the reproductive 

tract and mammary gland were also observed with 

genistein and diethylstilbestrol, whereas prolonged effects 

were induced by zearalenone. 

Strengths/Weaknesses: The lack of clarity regarding 

sample size and the weak description of the histopathology findings are weaknesses, as are the use of DMSO as a 

vehicle, the subcutaneous route of administration, and 

statistical concerns. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process. 

Park et al. (2005b), support not indicated, treated ICR 

mice during pregnancy. Bisphenol A [purity not indicated] in corn oil was given i.p. at dose levels of 0, 0.05, 

0.5, or 5 mg/kg bw on the day of mating and every 3 

days for a total of six doses (n 5 12/group). Dams were 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 295 

killed on GD 18 (plug 5 GD 0) for determination of litter 

size, fetal weight, and sex ratio. The uterus and right 

ovary were removed from each dam, fixed in Bouin 

fluid, and sections were stained with hematoxylin and 

eosin for light microscopy. Results were analyzed with 

least significant difference test [apparently on a per 

fetus basis]. 

Maternal weight was not altered by treatment. Fetal 

body weight was decreased in the high-dose group by 

14% for males and 12% for females. There was no effect 

on litter size or sex ratio. There was no treatment effect 

on dam uterine or ovarian weight. Histopathology of the 

dam ovary was reportedly not affected by treatment. 

Histopathology of the dam uterus showed thickening of 

the endometrium in the 0.05 and 0.5 mg/kg bw groups 

and uterine muscle damage in the 5 mg/kg bw group. 

[The damage is not otherwise described. The photomicrographs available in the report were not interpretable due to poor reproduction quality.] The authors 

concluded that bisphenol A at low doses does not 

produce reproductive toxicity in mice. [This study was 

written in Korean with an English abstract and tables. 

A translation was provided to CERHR by the American 

Plastics Council.] 

Strengths/Weaknesses: The use of 3 dose levels is a 

strength. The lack of information on husbandry conditions, the i.p. dose route, failure to account for litter 

effects in statistical analyses, and the poor presentation 

of histopathology results are weaknesses. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process. 

Park et al. (2005a), support not indicated, treated ICR 

mice during pregnancy. Bisphenol A [purity not indicated] in corn oil was given i.p. at dose levels of 0, 0.05, 

0.5, or 5 mg/kg bw on the day of mating, and every 3 

days for a total of 6 doses (n 5 3–6/group). Offspring 

were evaluated on PND 45 for body weight, reproductive 

organ weight and histopathology, semen analysis, complete blood count, and serum chemistry. [There were 24 

female and male offspring evaluated per dose group 

(not indicated whether 12 of each sex). Litter of origin 

appears not to have been considered. No information 

was provided on standardization of litters, diet, or cage/ 

bedding materials.] Statistical analysis was performed 

using the least significant difference test. [It was not 

clear if the litter or offspring was considered the 

statistical unit.] 

There was a statistically significant 6% decrease in 

male body weight in the high-dose group; a comparable 

body weight decrement in female offspring was not 

statistically significant. There were no statistically significant treatment effects on the weights of the testis, 

epididymis, seminal vesicles, coagulating glands, uterus, 

or ovary. Sperm concentration, viability, motility, and 

morphology were not affected by treatment. Blood 

endpoints were not affected by treatment except for a 

statistically significant 6% increase in erythrocyte count 

in male offspring and a 2% decrease in serum albumin in 

female offspring. An 11% increase in blood urea nitrogen 

in mid-dose female offspring was not dose-related. 

Histopathology of the testis and ovaries was described 

as unaffected by treatment. Uterine intimal proliferation 

was described in the mid- and high-dose female offspring. [The histological methods were not described. 

The photomicrographs available in the report were not 

interpretable due to poor reproduction quality.] The 

authors concluded that bisphenol A at low doses does 

not produce reproductive toxicity in mice. [This study 

was written in Korean with an English abstract and 

tables. A translation was provided to CERHR by the 

American Plastics Council.] 

Strengths/Weaknesses: The inadequate description of 

methods, unacceptable small sample size, the i.p. dosing, 

inappropriate statistical analyses, and the poor presentation of histology results are weaknesses of this study. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process due 

to the reasons stated above. 

Sato et al. (2001), support not indicated, investigated 

the effects in mice of in utero exposure to bisphenol A on 

fetal growth, offspring reproductive and brain development, and behavior. Pregnant Jcl-ICR mice (n 5 20) were 

given s.c. injections of bisphenol A [purity not indicated] 

100 mg/kg bw/day, ethinyl estradiol 0.2 or 0.02 mg/kg 

bw/day, or olive oil vehicle on GD 11–19 [Plug day was 

not defined. Information regarding caging material, 

animals per cage, feed, culling, and weaning 

was not provided.] Pups were evaluated for onset of 

pivoting, righting, straight line walking, and grasp reflex. 

Open field testing was conducted at 40 days of age. 

Offspring were killed at 40 or 60 days of age and organs 

were weighed and processed for histology using hematoxylin and eosin [fixation method not given]. Brain 

myelin was evaluated using Klu ̈ ver-Barrera staining. 

Statistical analyses were performed using the Student ttest. [The pup appears to have been used as the 

statistical unit.] 

There were 11/93 stillborn fetuses after in utero 

exposure to bisphenol A, but no data were provided 

for the control group. There were no significant effects of 

bisphenol A treatment on litter size or offspring body 

weight at birth, 20, or 60 days of age. There were no 

significant effects of bisphenol A treatment on days at 

acquisition of pivoting, righting, straight-line walking, or 

grasp reflexes. In open field testing, mice in the 

bisphenol A-treated group showed significantly less 

defecation than controls [39% less]. There was no 

statistically significant difference between groups in 

grooming, rearing, line-crossing of inner and outer fields, 

or latency to first line crossing. At 60 days of age, 

seminiferous tubules from bisphenol A-exposed male 

offspring had a significant reduction in mean diameter 

[k16.6%] and cell layer thickness [k25%] compared to 

controls. There was no significant bisphenol A effect on 

brain myelination at 60 days of age or in mean diameter 

at 40 and 60 days of age of the tractus mamillothalamics. 

The authors suggest that in utero exposure to 100 mg/kg 

bw/day bisphenol A induces alterations in behavior 

similar to that seen at reduced plasma corticosterone 

levels and that bisphenol A exposure induces gross and 

cellular changes in seminiferous tubules, suggesting 

potential perturbation in hormone pathways involved 

in development. 

Strengths/Weaknesses: The use of multiple doses of 

estrogen as a positive control is a strength. Weaknesses 

include the evaluation of a single dose of BPA, 

subcutaneous dosing, and lack of details regarding 

husbandry. Behavioral methods were chosen from less 

sophisticated screening approaches and data were not 

analyzed appropriately using the litter as the statistical 

Birth Defects Research (Part B) 83:157–395, 2008
296 CHAPIN ET AL. 

unit. Further, there is no description of sex ratios in 

groups given behavioral testing, despite established sex 

differences in endpoints measured in the open field 

evaluation. As a result, behavioral findings are 

unreliable. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for inclusion in the evaluation 

process due to the reasons stated above. 

Rubin et al. (2006), supported by NIEHS, examined 

sexual differentiation in mice perinatally exposed to 

bisphenol A. Animals were fed rodent diet 2018 (Harlan 

Teklad, St. Louis, MO), which was reported to have 

negligible for estrogenicity (20 fmol 17b-estradiol equivalents/g). Caging and bedding materials were not 

indicated but were reported to have negligible estrogenic 

activity in the E-SCREEN assay. Water was supplied in 

glass bottles. On GD 8 (GD 1 5 day of vaginal plug) 

through Day 16 of lactation, CD-1 mice were s.c. dosed 

by osmotic pump with the 50% DMSO vehicle or 

bisphenol A [purity not reported] at 0.000025 or 

0.000250 mg/kg bw/day. [The numbers of dams exposed was not indicated.] Litters were culled to 8 pups 

(4/sex) on the day following birth. Litters were weaned 

on PND 22–24 (day of birth not defined). Anatomical 

examination and assessment of tyrosine hydroxylase 

neurons in the anteroventral periventricular preoptic 

area by an immunohistochemistry technique were conducted before puberty (PND 22–24) in 7 or 8 offspring/ 

sex/group (2/sex/litter). Open-field testing was conducted in 14–17 offspring/group (1 offspring/sex/litter) 

at 6–9 weeks of age. The study authors expressed concern 

about possible hormonal effects because their historical 

records indicated that regular estrous cycles are not 

observed in group-housed females at 6–9 weeks of age. 

Therefore, open-field testing was repeated in 27–29-dayold offspring (n 5 10–12/sex/group) exposed to 0 or 

0.000250 mg/kg bw/day bisphenol A. Statistical analyses 

included 2-way ANOVA, t-test, and ANOVA with 

Bonferroni post-hoc test. 

In control offspring, the total number of tissue sections 

through the anteroventral periventricular preoptic area 

was greater in females than males, but the sexually 

dimorphic difference was not observed in either treatment group. The number of sections through the 

anteroventral periventricular preoptic area was significantly lower in females from the high-dose bisphenol A 

than control group. In the control offspring, the number 

of tyrosine hydroxylase-positive neurons in the anteroventral periventricular preoptic area was higher in 

females and in males but this sexually dimorphic 

difference was not observed in the high-dose group. 

The number of tyrosine hydroxylase-positive neurons in 

the anteroventral periventricular preoptic area was lower 

in females in the high-dose bisphenol A than control 

group. The results for tyrosine hydroxylase-positive 

neurons were based on counting of all sections. When 

counting was limited to 7 sections or 4mid-sections, the 

sexually-dimorphic difference observed for tyrosine 

hydroxylase-positive neurons in the control group was 

not observed in either treatment group. When limited to 

3 caudal sections, the sexually dimorphic difference 

observed for tyrosine hydroxylase-positive neurons was 

maintained in the low-dose group and was borderline 

significant (P 5 0.06) in the high-dose group. Bisphenol A 

exposure had no significant effect on the number of 

tyrosine hydroxylase-positive neurons in the arcuate 

nucleus. In open-field testing of 6–9-week-old animals, 

significant effects in control females compared to control 

males included more rearing and time spent in the center 

and less time stopped. Sexually dimorphic differences in 

rearing and time spent in center were not observed in 

either bisphenol A treatment group and the sexually 

dimorphic difference in time stopped was not observed 

in the low-dose group. In open-field testing conducted at 

4 weeks of age, control females compared to males reared 

more times and spent less time stopped. The sexually 

dimorphic differences were not observed in animals 

exposed to 0.000250 mg/kg bw/day (the only dose tested 

in 4-week-old animals). The number of rearings was 

significantly lower in 4-week-old females in the 

0.000250 mg/kg bw/day group than in controls. The 

study authors concluded that bisphenol A may alter 

important events during critical periods of brain 

development. 

Strengths/Weaknesses: The strengths of this study are 

the care taken to control for extraneous estrogenic 

exposure, the delivery of BPA at 2 doses, both low, 

delivery from GD 1–PND 16, the reasonable sample 

sizes, and the inclusion as outcome measurements of 

behavior, anatomy, and an index of neurochemical effects 

in the brain. Significant weaknesses include the use of 

s.c. osmotic pumps, uncertainty about sample size, and 

whether litter effects were adequately controlled. 

Utility (Adequacy) for CERHR Evaluation Process: 

This is inadequate for the evaluation process due to the 

combination of route of administration and statistical 

concerns. 

Toyama (2005), supported in part by the Japanese 

Ministry of Education, Culture, Sports Science, and 

Technology, examined the effects of prenatal Bisphenol 

A exposure in CL/P mice, a strain with a high 

background rate of cleft lip/palate. The study was 

published in Japanese and a translation was provided 

by the American Plastics Council. Mice were fed CA-1 

(Japan CLEA, Inc.). [No information was provided 

about caging or bedding materials.] On GD 9.5 (GD 

0 5 day of vaginal plug), 25 dams/group were s.c. dosed 

with olive oil vehicle or bisphenol A [purity not 

reported] at 0.001, 0.01, 0.1, 1, or 10 mg/kg bw. Dams 

were killed on GD 18 and fetuses (169–184/group) were 

examined for cleft lip/palate or thymic anomaly (i.e., 

hypoplasia). Data were analyzed by Student t-test and w2 

test. [It appears that offspring were considered the 

statistical unit.] 

There were no significant differences for numbers of 

implantations or fetal survival. The incidence of cleft lip/ 

palate in fetuses from the control and each respective 

treatment group was 8.3, 8.0, 6.1, 1.8, 4.9, and 6.2%. There 

were no differences in the types of cleft palate observed 

in each group. Incidence of thymic anomaly in the 

control and each respective dose group was 11.8, 10.8, 

6.1, 1.8, 4.9, and 6.2%. Incidence of cleft/lip palate or 

thymus anomalies was lower in bisphenol A-treated than 

control groups and was lowest in the 0.1 mg/kg bw 

bisphenol A group. [Results of statistical analyses for 

cleft lip/palate and thymic anomaly were difficult to 

interpret.] A higher tendency for complication of cleft 

lip/palate and thymus hypoplasia [possibly fetuses 

with both types of defects] was observed in the 

bisphenol A groups; respective incidences in the control 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 297 

and each treatment group was 36, 57.1, 61.8, 100, 77.8, 

and 72.7%. The study authors concluded that U-shaped 

dose response curves were observed for cleft lip/palate 

and thymus hypoplasia and that complication of cleft 

lip/palate and thymus hypoplasia tended to be lower in 

the bisphenol A groups. 

Strengths/Weaknesses: Strengths of this study include 

that the authors explored a wide range of BPA doses. 

Time of dosing was appropriate with respect to palate 

development. The hypothesis that BPA administration is 

protective is interesting. Weaknesses include the route of 

administration, absence of exposure assessment, confusion on statistical analyses, absence of historical control 

perspective, and strain of mouse used. This strain of 

mouse has a high incidence of cleft palate making 

interpretation of these data challenging. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the CERHR evaluation 

process because of the combination of strain selection, 

confusion on statistical analyses, use of s.c. route of 

exposure, and use of offspring as the unit of analysis. 

Berger et al. (2007), supported by The Natural Sciences 

and Engineering Research Council of Canada, examined 

the effect of bisphenol A exposure on blastocyst 

implantation and pup survival in mice. CF-1 mice were 

housed in polypropylene cages mice and fed Harlan 

Teklad 22/5 rodent chow, a soy-containing feed. [No 

information was provided about bedding materials.] 

On GD 1–4 or 5 [inconsistently described in report], 6– 

15 mice/group were administered bisphenol A through a 

peanut butter supplement, or a mixture of feed and 

peanut butter. Mice were allowed to litter. Pups were 

counted on the day of parturition and observed for 

survival for 5 days. Pups were weaned at 28 days after 

birth and at that time, body weight and sex ratio were 

determined. Data were analyzed by w2 test. [It was not 

clear if offspring data were analyzed on a pup or litter 

basis.] 

In the study in which the diet was supplemented with 

peanut butter, bisphenol A was added to the peanut 

butter at 0, 0.11, 1, 3, or 9%. Based on weights of 

unconsumed peanut butter, the study authors estimated 

mean bisphenol A intake at 0, 1.08, 8.33, 16.50, or 

13.59 mg/day. [Assuming that the mice weighed 

0.02 kg at the start of gestation (USEPA, 1988), CERHR 

estimated bisphenol A intakes of 54, 417, 825, and 

680 mg/kg bw/day.] Peanut butter consumption was 

significantly decreased in the 9% group. There were no 

treatment effects on number of females delivering litters. 

Survival of pups from birth to weaning was lower in the 

9% group (76.1%) than in the control group (98.2%) and 2 

complete litters were lost in the 9% group. There was no 

significant difference in sex ratio of pups at weaning. 

There also did not appear to be an effect on pup weight 

at weaning. 

In the study in which feed was dosed, mice were fed 

one part feed to two parts peanut butter. The feed/ 

peanut butter mixture contained bisphenol A (97% 

purity) at 0, 3, or 6%. The study authors estimated 

bisphenol A intake at 0, 66.7, or 68.8 mg/day. [Assuming 

that the mice weighed 0.02 kg at the start of gestation 

(USEPA, 1988), CERHR estimated bisphenol A intakes 

of 0, 3335, or 3440 mg/kg bw/day.] Feed intake was 

significantly decreased in the 6% group. Controls were 

fed with the same quantity of food consumed by treated 

mice on the previous day. Delivery of litters in the 3% 

group was not affected but there were no litters 

delivered in the 6% group. Pup weight and sex ratio at 

weaning were not affected in the 3% group. Pregnancy 

disruption in the s.c. dosed mice is discussed in Section 

3.2.6. [It appears that with s.c. exposure, pregnancy 

disruption occurred at lower bisphenol A levels 

(10.125 mg/day, B500 mg/kg bw/day) than with oral 

exposure (68.8 mg/day, 3440 mg/kg bw/day).] The study 

authors concluded that the amount of bisphenol A 

required for pregnancy disruption was higher than 

typical environmental levels but that it is not known if 

bisphenol A could have additive or synergistic effects 

with other environmental estrogens. 

Strengths/Weaknesses: Major weaknesses include 

absence of key statistical information on the appropriate 

control for possible litter effects, absence of similar 

effects at the same estimated dose level, inability to 

discriminate between potential maternal toxicity and the 

findings in the offspring, and the absence of exposure 

data (i.e., does the matrix affect exposure?). 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the CERHR evaluation 

process. 

3.2.7 Mouse—oral exposure postnatally with or 

without prenatal exposure. Nagao et al. (2002), 

support not indicated, examined the effects of bisphenol 

A in mice following exposure during different life stages. 

An initial study compared the sensitivity of male juvenile 

C57BL/6N and ICR mice to 17b-estradiol. Following s.c. 

dosing of 10 mice/strain/group with 10 mg/kg bw/day 

17b-estradiol on PND 27–48, there were no weight 

changes or histopathological alterations in reproductive 

organs of ICR mice. In contrast, C57BL/6N mice exposed 

to 17b-estradiol experienced significant decreases in 

absolute and relative weights of testes, epididymides, 

and seminal vesicles. In addition, epididymal sperm was 

reduced and there was increased severity of seminal 

vesicle and Leydig cell atrophy. The study authors 

concluded that C57BL/6N mice are sensitive to estrogen 

and this strain of mice was used in the remaining 

experiments. 

Life stages examined in experiments with bisphenol A 

included prenatal development, adolescence, and adulthood. The studies conducted during prenatal development and adolescence are described here, and the study 

conducted during adulthood is described in Section 4.2. 

C57BL/6N mice were fed PLD (phytoestrogen-low diet, 

Oriental Japan). They were housed in polycarbonate 

cages with wood bedding. Daidzein and genistein levels 

were analyzed in the diet, tap water, and bedding and 

found to be o0.5 mg/100 g. Bisphenol A (stated to be 

99% pure in the study with adult mice) was administered 

to juvenile or pregnant mice by gavage at doses of 0.002, 

0.020, or 0.200 mg/kg bw/day. Control animals were 

gavaged with 0.5% carboxymethyl cellulose [assumed to 

be the vehicle]. Juvenile males (30/group, obtained from 

10 litters) were treated on PND 21–43 (day of birth not 

defined). At 6 weeks of age, 25 mice/group were 

necropsied. Ten pregnant C57BL/6N mice/group were 

treated on GD 11–17 (GD 0 5 day of vaginal plug). 

Fetuses were removed by cesarean section on GD 18 and 

that day was considered PND 0. Litters were fostered to 

untreated dams. On PND 4, females were disposed and 

litters were culled to 3 males. Males were weaned on 

Birth Defects Research (Part B) 83:157–395, 2008
298 CHAPIN ET AL. 

PND 21 and housed individually in polycarbonate cages. 

At 12 weeks of age, males were weighed and 25 males/ 

group were killed and necropsied. During necropsy of 

males that had been exposed during prenatal development or during adolescence, testes, epididymis, and 

seminal vesicles with coagulating glands were weighed. 

In the study conducted in adult mice, it was noted that 

ventral prostates were not weighed due to difficulties in 

obtaining only prostate and determining the precise 

weight of the organ. Epididymal sperm counts were 

obtained. Histopathological examinations were conducted for reproductive organs fixed in Bouin solution. 

For males exposed during gestation, the litter was 

considered a single sample. Data were analyzed by 

Bartlett’s test to determine homogeneity of variance, 

followed by ANOVA when homogeneity of variance was 

obtained or Wallace–Wallace analysis of ranks when 

variance was not homogenous. Dunnett test was used for 

multiple comparisons. 

There were no significant effects on embryo mortality 

after birth, body weight gain, or terminal body weight. 

[Data were not shown.] The only reproductive organ 

weight effect was a significant, but non-dose-related [6%] 

decrease in absolute seminal vesicle weight in the lowdose bisphenol A group. Organ weights were not affected 

in males exposed during adolescence. Sperm density was 

unaffected by bisphenol A exposure. No treatment-related 

lesions were observed in testes or other reproductive 

organs including ventral prostate. [Data were not shown.] 

The study authors concluded that low-dose bisphenol A 

exposure of mice did not reduce sperm density or disrupt 

male reproductive system development. 

Strengths/Weaknesses: Strengths are the use of three 

low dose levels, the oral route of administration, the 

careful description of methods, the use of a lowphytoestrogen diet, and the confirmation that the strain 

of mice used was estrogen sensitive. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

Kabuto et al. (2004), supported by the Kagawa 

Prefectural College of Health Sciences, examined the 

role of oxidative stress in bisphenol A-induced toxicity in 

mice. ICR mice were fed standard laboratory chow 

containing 24% protein (MF; Oriental Yeast Co.). [No 

information was provided about bedding or caging 

materials.] From 1 week before mating through gestation 

and lactation, 6 mice/group were given drinking water 

containing the 1% ethanol vehicle or bisphenol A [purity 

not reported] at 5 or 10 mg/L. [Based on the reported 

water intake of 5 mL/day and an assumed body weight 

of 0.02 kg (USEPA, 1988), it is estimated that bisphenol 

A intake in mice at the start of pregnancy was 0.0013 or 

0.0025 mg/kg bw/day.] Mice gave birth about 3 weeks 

following mating and pups were housed with dams for 4 

weeks. [Based on an assumed body weight of 0.0085 kg 

and assumed water intake rate of 0.003 L/day (USEPA, 

1988), it is estimated that intake of bisphenol A in 

weanling males was 0.0018 or 0.0035 mg/kg bw/day]. At 

4 weeks of age, male pups were killed and brain, kidney, 

liver, and testis were weighed in 8–13 mice/group. 

Tissues were homogenized to determine activities of 

superoxide dismutase, catalase, and glutathione peroxidase and concentrations of glutathione and L-ascorbic 

acid in 6–8 mice/group. Tissue level of thiobarbituric 

acid-reactive substance, a biogenic macromolecular 

peroxidation indicator, was measured in 6 mice/group. 

Data were analyzed by ANOVA followed by Scheffe F

test. [It appears that offspring were considered the 

statistical unit in some analyses.] 

Organ weight effects included decreased brain weight 

at the low dose, decreased kidney weight at the highdose, and decreased testis weight at both doses. [Relative 

organ weights were not determined.] In the high-dose 

group, thiobarbituric acid-reactive substance levels were 

increased in brain, kidney, and testis. Changes in 

antioxidant enzyme levels included decreased catalase 

activity in testis and increased glutathione oxidase 

activity in kidney. No significant effects were observed 

for superoxide dismutase activity or glutathione or 

ascorbic acid levels in any of the tissues examined. The 

study authors concluded that bisphenol A exposure 

during gestation and lactation results in oxidative stress 

and peroxidation in offspring that ultimately lead to 

underdevelopment of brain, kidney, and testis. 

Strengths/Weaknesses: The delivery of bisphenol A in 

drinking water at low dose levels is a strength. Weaknesses include small sample size of exposed dams 

(n 5 6), inappropriate use of the pup as the experimental 

unit in statistics, and mechanistic data without functional 

correlates. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process due 

to inappropriate statistical procedures and small sample 

size. 

Takao et al. (2003), support not indicated, examined 

the effects of bisphenol A exposure on expression of ERa

and ERb in the testis of young mice. [No information 

was provided about feed, caging, or bedding materials.] 

Three-week-old male C57BL/6 mice (n 5 7/group) were 

administered bisphenol A [purity not indicated] through 

drinking water at 0 (ethanol vehicle), 0.5, or 50 mg/L for 

8 weeks. [Assuming a weanling mouse drinks B0.35 L/ 

kg bw/day (USEPA, 1988), bisphenol A intake would 

have been B0, 0.175, or 17.5 mg/kg bw/day.] The 

stability of bisphenol A was not determined, but water 

bottles were changed two times a week to maintain a 

stable concentration of bisphenol A in drinking water. 

Mice were killed at an unspecified period following 

exposure, and the testis and spleen were weighed. The 

testis was examined for ERa- and ERb-positive cells 

using an immunohistochemistry method and ERa and 

ERb mRNA using a semi-quantitative RT-PCR technique. 

Data were analyzed by ANOVA followed by Fisher 

protected least significant difference test. 

Exposure to 50 mg/L bisphenol A resulted in a 

decreased number of ERb-positive cells and increased 

number of ERa-positive cells. Expression of ERb mRNA 

was decreased and expression of ERa mRNA was 

increased following exposure to 50 mg/L bisphenol A. 

There were no differences in body weight or absolute or 

relative weights of testis or spleen following bisphenol A 

treatment. The study authors concluded that differential 

modulation of ERa and ERb could be involved in effects 

observed following bisphenol A exposure. 

Strengths/Weaknesses: The delivery of bisphenol A in 

drinking water and the measurement of ER in the testis 

are strengths. The lack of clarity on age at sacrifice, 

limited number of endpoints assessed, and marginal 

sample size (n 5 7) are significant weaknesses. 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 299 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process based 

on the limitations noted above. 

Matsumoto et al. (2004), support not indicated, 

examined the effect of maternal bisphenol A exposure 

on growth of offspring in mice. Mice were fed standard 

rodent chow (CE-2, Japan Clea). [No information was 

provided on caging and bedding materials.] Mice of the 

ddY strain were exposed to bisphenol A (Z97% purity) 

through feed at 0 or 1% from GD 14–PND 7. The study 

authors stated that the bisphenol A dose was equivalent 

to 1000 mg/kg bw/day. [The number of dams treated 

was not indicated. Day of vaginal plug and day of birth 

were not defined]. Mice delivered pups on PND 21. 

During the postnatal period, body weight was monitored 

in 31 pups from the control group and 61–89 pups from 

the bisphenol A group. Serum prolactin levels were 

measured by RIA in 3 dams/group 4 days following 

delivery. Pups were killed on PND 7, and stomach 

weight was measured. Data were analyzed by Student ttest. [It was not clear if the litter or offspring was 

considered the statistical unit.] 

No differences were reported for live pups at birth. 

During the postnatal period, body weights of pups in the 

bisphenol A group were significantly lower [by B40%] 

than control group pups. No deaths were reported for 

pups in the control group, but 30% of pups in the 

bisphenol A group died before PND 7. On PND 1, milk 

could be seen in stomachs of pups from the control 

group, but not the bisphenol A group. [The number of 

pups evaluated for milk in stomach was not reported.] 

On PND 7, stomach weight was significantly lower [by 

40%] in pups from the bisphenol A than control group. 

Serum prolactin level was significantly reduced [by 46%] 

in dams from the bisphenol A group. The authors 

concluded that administration of a high bisphenol A 

dose to mice resulted in suppressed postnatal growth of 

offspring that probably resulted from an insufficient 

supply of milk, which might have been due to decreased 

prolactin secretion. [Because of the implications of this 

study for lactation competence, this study will be 

discussed again in Section 4.2.] 

Strengths/Weaknesses: Weaknesses of the study are 

the difficulty in calculating bisphenol A intake, the likely 

high exposure level, the lack of information on dam 

number and husbandry, and the high level of pup body 

weight decrement and mortality. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process due 

to the reasons stated above. 

Suzuki et al. (2003), supported by the Japanese 

Ministry of Health, Labor, and Welfare and the Ministry 

of Education, Culture, Sports, Science, and Technology 

conducted a study to determine the effect of prenatal 

bisphenol A exposure on dopamine-receptor mediated 

actions in mice. Female ddY mice were fed chow 

containing bisphenol A at 0.002, 0.5, or 2 mg/g feed 

from mating through weaning of offspring. [No information was provided on the number of dams 

treated, purity of bisphenol A, or the type of chow, 

bedding, or caging materials. Assuming a female 

mouse eats B0.2 kg feed/kg bw/day (USEPA, 1988), 

bisphenol A intake would have been 0.4, 100, or 400 mg/ 

kg bw/day.] Male offspring were subjected to a series of 

tests [age at testing not stated]. In a conditioned placepreference test, groups of 6–10 mice were injected with 

0.5 mg/kg bw methamphetamine and placed in either 

the dark or light area of the test apparatus for 3 days. On 

the other 3 days, males were injected with saline and 

placed in the other compartment of the testing apparatus. 

On Day 7, the divider in the apparatus was raised and 

the time spent in each compartment was measured. 

Activity was measured in groups of 9–10 mice for 3 hr 

following injection with saline or 2 mg/kg bw methamphetamine. Dopamine-induced binding of 35S-guanosine-50

[g-thio]-triphosphate in the limbic system was 

measured (n 5 3 samples/group). Protein levels of 

dopamine and vesicle monoamine transporters in brain 

were determined by Western blot (n 5 6 samples), and 

mRNA levels of dopamine receptor in brain were 

determined by RT-PCR. Data were analyzed by ANOVA 

with Bonferroni/Dunnett test. [It was not clear if the 

litter or offspring was considered the statistical unit.] 

In conditioned-preference testing, exposure to all 3 

bisphenol A doses resulted in a significant and doserelated increase in preference for compartments associated with methamphetamine exposure. [Control mice 

showed no compartment preferences while the times 

spent in the methamphetamine-associated compartment were B150, 200, and 275 sec by animals in each 

respective dose group.] Preference for the methamphetamine compartment was eliminated by injecting the 

animals with SCH23390, A dopamine D1 receptor 

antagonist. In mice exposed to the high-dose of bisphenol 

A, activity was significantly increased [by B80% at 

peak] compared to the control group following methamphetamine challenge, and sensitization to methamphetamine-induced activity was also enhanced. Dopamineinduced binding of 35S-guanosine-50

[g-thio]-triphosphate 

in the limbic system was potentiated [increased by 

B15%; not clear if statistically significant] and Gprotein activation was increased [by B75%] in mice 

exposed to the high bisphenol A dose. The effects on Gprotein activation were eliminated following injection 

with SCH23390 or sulpiride, a dopamine D2 receptor 

antagonist. No changes were observed for expression of 

dopamine and vesicle monoamine transporter proteins. 

Expression of dopamine D1 receptor mRNA was upregulated significantly to 130% of control levels in the highdose bisphenol A group. [For all endpoints except for 

conditioned preference, only the data from the highdose bisphenol A group was shown. It was not clear if 

that was the only dose tested for those endpoints or if 

the high-dose data were shown because it was the only 

dose that resulted in a statistically significant effect.] 

The study authors concluded that ‘‘prenatal and neonatal 

exposure to bisphenol A can potentiate central dopamine 

D1 receptor-dependent neurotransmission, resulting in 

supersensitivity of methamphetamine-induced pharmacological actions related to psychological dependence on 

psychostimulants.’’ 

Strengths/Weaknesses: Strengths include a wide range 

of doses administered orally. Weaknesses include absence of adequate experimental details, inappropriate 

statistical procedures that did not account for litter or 

repeated measurement, inadequate presentation of body 

weight data, and use of high-doses. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process due 

to the reasons stated above. 

Birth Defects Research (Part B) 83:157–395, 2008
300 CHAPIN ET AL. 

Tando et al. (2007), supported by the Japanese 

Ministries, investigated the effects of bisphenol A 

exposure in the maternal diet during the prenatal and 

lactational period on the long-term development of the 

cortex and substantia nigra. ddY mice were maintained 

under a 12-hr/12-hr light/dark cycle before mating. 

From GD 0 through weaning on PND 21, dams had free 

access to a diet containing bisphenol A (purity 499%) at 

0, 3, or 8000 mg/kg feed. Pups were weaned on PND 21 

to a diet without bisphenol A. [The basal feed, cage, and 

bedding were not specified. Daily feed consumption 

was not reported. Assuming a pregnant mouse eats 

B0.15 kg feed/kg bw/day and a lactating mouse eats 

B0.45 kg feed/kg bw/day, bisphenol A intake would 

have been B0, 4.5, or 1200 mg/kg bw/day during 

gestation and B0, 1.35, or 3600 mg/kg bw/day during 

lactation.] At 8–11 weeks of age, male and female 

offspring (n 5 4 and 5/sex/treatment group) were killed 

and formalin-perfused. Brains were harvested and 

embedded in paraffin. Immunohistochemical detection 

for tyrosine hydroxylase, calbindin D-28 K, calretinin, 

and parvalbumin proteins were performed. In situ 

TUNEL was also performed. Statistical analyses use 

ANOVA and post-hoc test using the Bonferroni/Dunn 

multiple comparison test. [It was not clear if the litter or 

offspring was considered the statistical unit.] 

No cytoarchitectural anomalies were seen in brain 

sections of either sex across treatment groups, based on 

hematoxylin-eosin and Kluver-Barrera stains. [Data were 

not shown.] The distribution and density of immunopositive staining for calbindin D-28 K, calretinin, and 

parvalbumin showed no statistically significant differences 

in low or high-dose bisphenol A exposed groups. Female 

offspring exposed to the lower dose level of bisphenol A 

exhibited a significant decrease in the volume of the 

substantia nigra. The number of tyrosine hydroxlyasepositive nuclei and fibers in this region was significantly 

reduced in low-bisphenol exposed female mice compared 

to control females and high-dose bisphenol A-exposed 

females [k18%, and 16%, respectively, estimated from a 

graph]. No significant differences in number of tyrosine 

hydroxylase positive cells were identified in bisphenol Aexposed males. Decreased values in immunopositive 

staining could not be attributed to apoptosis, based on 

TUNEL staining [data not shown]. 

The authors concluded that there were sex- and dosespecific sensitivities of the developing substantia nigra, 

in the DDY mice with females exposed to a low but not a 

high-dose of bisphenol A showing a significant reduction 

in the number of tyrosine hydroxylase-positive nuclei. 

They indicated that the functional significance of this 

reduction was unknown. The authors suggested a 

putative mechanism involving interaction of bisphenol 

A with ERb, which is abundantly present in the 

developing substantia nigra. 

Strengths/Weaknesses: Strengths of this study are that 

BPA was delivered orally to the dams during the 

gestational and lactational period and the use of 

appropriate methods for assay of the anatomical and 

some molecular aspects of brain development. Weaknesses include the lack of specification of the feed, broad 

range of the two doses used, small sample size given 

high variability of endpoints (4 and 5/sex/treatment 

group), and absence of expected sexually dimorphisms 

in measures in the controls. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate and not useful for the 

evaluation process for the reasons stated above. 

Mizuo et al. (2004), supported by the Japanese 

Ministry of Health, Labor, and Welfare and the Ministry 

of Education, Culture, Sports, Science, and Technology, 

examined the effect of perinatal bisphenol A exposure on 

morphine-induced rewarding effects and hyperlocomotion in mice. Testing was conducted in offspring of ddY 

mice that received chow containing 0, 0.002, 0.5, or 2 mg 

bisphenol A/g feed [0, 2, 500, or 2000 ppm] during 

gestation and the neonatal period of pup development. 

[No information was provided on the number of dams 

treated/group, purity of bisphenol A, or feed, caging, or 

bedding materials.] In place-conditioning testing, 6–10 

offspring/group were placed in one compartment of a 

testing apparatus following saline injection and in a 

second compartment of the apparatus following morphine injection; on the second day, mice were given free 

access to both compartments and the time spent in each 

compartment was measured. Locomotor activity was 

measured after injecting 9–10 mice/bisphenol A group 

with saline or 10 mg/kg bw morphine. Guanosine-50

­

diphosphate binding and expression of m-opioid receptor 

mRNA were measured in 3 independent samples/group. 

Statistical analyses included 2-way ANOVA with Bonferroni/Dunnett test. [No information was given on the 

ages that testing was conducted and the sex of mice 

tested. It was not clear if the litter or offspring was 

considered the statistical unit.] 

In place-preference conditioning testing, a dose-dependent increase was observed for the time spent in the 

compartment associated with morphine exposure and 

statistical significance was attained at the two highest 

dose levels. [The time spent in the morphine-associated 

compartment was B15 sec for controls, 150 sec for the 

mid-dose group, and 175 sec for the high-dose group.] 

Locomotion in the high-dose bisphenol A group was 

significantly increased following morphine injection 

[B130 compared to 10 activity counts in high-dose 

bisphenol A group compared to the control]. Bisphenol 

A treatment had no effect on guanosine-50

-diphosphate 

binding (i.e., m-opioid receptor mediated G-protein activation) or expression of m-opioid receptor mRNA. The 

study authors concluded that chronic exposure to 

bisphenol A induces morphine-induced rewarding effect 

and hyperlocomotion that does not occur through 

activation of the m-opioid receptor. 

Strengths/Weaknesses: Strengths of this study are that 

BPA was delivered orally to the dams during the 

gestational and lactational period. Weaknesses include 

the lack of specification of the feed, broad range of the 

two doses used, small sample size (n 5 6–10) and 

inappropriate statistics that do not account for litter or 

repeated measures. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate and not useful for the 

evaluation process. 

Miyatake et al. (2006), supported by the Japanese 

Ministry of Health, Labor, and Welfare and the Ministry 

of Education, examined the effects of developmental 

bisphenol A exposure on morphine-induced rewarding 

effects in male ddY mice. Maternal mice were orally 

exposed to olive oil vehicle, bisphenol A [purity not 

indicated] at 0.003 or 200 mg/kg bw/day, or 17bBirth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 301 

Table 80 

Behavior of Female Mice After Gestational and Lactational Exposuresa 

Bisphenol A, mg/kg bw/day 

Endpointb 2 200 Ethinyl estradiol 

Puberty onset 2 k 4.5 days k 6.25 days 

Time in open arms of plus maze 2 k 41% (P 5 0.06) k 73% 

Time in light part of light/dark preference box 2 k 52% k 69% 

Errors in radial arm and Barnes mazes 2 2 k

a

Ryan and Vandenbergh (2006).
 

b

The size of the difference from control was estimated from graphs.
 

k Statistically significant decrease from control value; 2 no statistical difference from control value, kdecrease identified by authors
 

although statistical difference from control not shown.
 

estradiol at 3 mg/kg bw/day by gavage. The compounds 

were administered 3 times a day from the mating period 

through weaning of offspring. Seven male offspring/ 

group were examined in a place-conditioning test at 7 

weeks of age. During the preconditioning period, mice 

were placed in one compartment of a cage following 

injection with saline and in another compartment of the 

cage following s.c. injection with morphine. During 

testing, the amount of time spent in each compartment 

of the cage was measured. Statistical analyses included 

ANOVA followed by Bonferroni/Dunnett test. [It was 

not clear if the litter or offspring was considered the 

statistical unit.] 

Developmental exposures to either bisphenol A dose 

resulted in a preference for the cage compartment 

associated with morphine exposure. Developmental 

exposure to 17b-estradiol at 3 mg/kg did not affect place 

preference. Based on the findings of this study and in 

vitro studies described in Section 3.2.1.1, the study 

authors concluded that bisphenol A alters dopamine 

responsiveness in mouse neurons and astrocytes, which 

could potentially contribute to development of psychological dependence on drugs of abuse. 

Strengths/Weaknesses: Strengths include the use of a 

positive control and corresponding measurement of in 

vitro and behavioral endpoints. Weaknesses include the 

use of only 2 doses, 1 very low and 1 high (both had 

similar effects), inadequate experimental details regarding exposure and numbers of dams, small sample size for 

behavioral endpoints, inappropriate statistical procedures that did not account for litter of origin or repeated 

behavioral measurements. 

Utility (Adequacy) for CERHR Evaluation Process: 

This report is inadequate and not useful for the 

evaluation process. 

Ryan and Vandenbergh (2006), supported by North 

Carolina State University and EPA, evaluated the effects 

in mice of prenatal and postnatal exposure to bisphenol 

A on sexually dimorphic behaviors. C57BL/6 mice were 

maintained in polycarbonate cages (checked frequently 

for condition) with chip bedding and were given Purina 

5001 chow. Females were mated and the day a vaginal 

plug was identified was considered GD 1. Beginning on 

GD 3, dams were treated with bisphenol A [purity not 

indicated] 2 or 200 mg/kg bw/day, ethinyl estradiol 5 mg/ 

kg bw/day, or the tocopherol-stripped corn oil vehicle. 

The dose was placed in the back of the throat with a 

gavage needle. Daily dosing was continued to PND 21, 

when pups were weaned. One female per litter was 

randomly selected for behavioral testing and was 

ovariectomized. Pup anogenital distance was 

measured at weaning. Non-ovariectomized mice were 

checked for vaginal opening and vaginal smears taken 

daily thereafter. Puberty was defined as the first 

day on which cornified cells were detected in 4–7 

females/group. Fourteen mice/treatment group were 

tested in an elevated plus maze and a light/dark 

preference chamber. Sixteen mice/treatment group were 

tested in a radial arm maze and a modified Barnes maze. 

Testing occurred 2 weeks after ovariectomy. Statistical 

analysis used ANOVA with post-hoc Student t-test. The 

radial arm and Barnes mazes were run for 5 consecutive 

days and a repeated measures design was added to the 

ANOVA. 

There was no effect of treatment on anogenital 

distance or anogenital distance divided by body weight. 

Other results are summarized in Table 80. Puberty was 

advanced by exposure to ethinyl estradiol or the highdose of bisphenol A. The results of the elevated plus and 

light/dark preference tests led the authors to conclude 

that bisphenol A and ethinyl estradiol increased anxiety. 

The improved performance in the radial arm and Barnes 

mazes led the authors to conclude that ethinyl estradiol 

masculinized spatial ability. [The results from the 

elevated plus maze also suggest masculinization of 

behavior, because males show more ‘‘anxiety’’ in this 

paradigm.] Bisphenol A 200 mg/kg bw/day resulted in a 

decrease in errors on earlier trials than the control in the 

radial arm maze, but this effect was not characterized by 

the authors as providing strong evidence of an alteration 

in spatial memory. 

Strengths/Weaknesses: Selection of established measurements of sexually dimorphic behaviors and replication of previous work by Howdeshell et al. (1999), the use 

of positive controls, the appropriate evaluation of 

pubertal onset, adequate sample sizes for behavioral 

methods, weight, and AGD measures are all strengths of 

this work. A weakness is the small sample size for 

evaluating pubertal onset. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process with the exception of the pubertal 

data. 

Tyl et al. (2006), sponsored by the American Plastics 

Council, conducted a two-generation GLP study of 

bisphenol A in CD-1 mice. [This study is discussed in 

detail in Section 4.2.3.2. Results relevant to developmental toxicity are presented here.] Mice were fed 

Birth Defects Research (Part B) 83:157–395, 2008
302 CHAPIN ET AL. 

Purina Certified Ground Rodent Diet No. 5002 containing 177–213 ppm genistein, 173–181 ppm daidzein, and 

39–55 ppm glycitein. Mice were housed in polypropylene 

cages with Sani-Chip bedding. F0 and F1 mice (28 sex/ 

group/generation) were fed diets containing bisphenol A 

(99.70–99.76% purity) at 0.018, 0.18, 1.8, 30, 300, or 

3500 ppm. Target intakes were 0.003, 0.03, 0.3, 5, 50, or 

600 mg/kg bw/day. The study authors estimated bisphenol A intake in males at 0.0024–0.0038, 0.024–0.037, 

0.24–0.37, 3.98–6.13, 39.1–60.8, or 529–782 mg/kg bw/ 

day. Bisphenol A intakes (in mg/kg bw/day) by females 

were estimated at 0.0030–0.0041, 0.030–0.042, 0.32–0.43, 

5.12–7.12, 54.2–67.8, 653–910 during the pre-mating 

period; 0.0027–0.0029, 0.027–0.028, 0.28–0.29, 4.65–4.80, 

47.0–48.6, 552–598 during the gestation period; and 

0.0087–0.0063, 0.062–0.091, 0.61–0.89, 10.4–15.1, 103.2– 

146.4, 1264–1667 during the lactation period. In each 

generation, there were 2 vehicle control groups with 28 

mice/sex/group. A positive control group was given 

feed containing 17b-estradiol at 0.5 ppm (target intake of 

0.08 mg/kg bw/day). [The Expert Panel notes that a 

separate 2-generation study was used to characterize 

the dose–response relationship for 17b-estradiol.] 

Homogeneity, stability, and concentration of bisphenol 

A in feed were verified. Exposure of F0 mice began at B6 

weeks of age. Exposure of F1 animals began at weaning, 

although it was noted that pups began eating the dosed 

feed in the late lactation period. F0 and F1 mice were fed 

the bisphenol A-containing diets for a minimum of 8 

weeks before mating and during a 2-week mating period. 

Exposures of females continued through the gestation 

and lactation period. 

Live F1 and F2 pups and litters at birth, sex ratio, and 

survival during the lactation period were not affected 

and there were no clinical or gross signs of toxicity in F1 

or F2 offspring. A non-dose-related decrease in PND 21 

survival index and lactational index (pups surviving on 

PND 21/PND 4) was described in F2 pups of the 300 ppm 

group. [The biological significance of the effect was not 

discussed by the study authors, but because the effect 

was not dose-related it is unlikely to be of biological 

significance.] In F1 pups from the 3500 ppm group, body 

weights were reduced during PND 7, 14, and 21 in F1 

females and both sexes combined and on PND 7 and 21 

in F1 males. An increase in male pup body weight 

observed on PND 7 in the 1.8 ppm group was not 

considered to be treatment-related by the study authors 

because no dose–response relationship was observed. 

There was no effect on anogenital distance in F1 or F2 

males or females on PND 0. Anogenital distance was also 

unaffected in F2 males and F1 and F2 females on PND 21. 

Anogenital distance adjusted for body weight was 

reduced in F1 males from the 300 and 3500 ppm groups 

on PND 21. Based on the lack of effect on anogenital 

distance at birth and inconsistencies between generations, the study authors did not consider the decreases in 

anogenital distance in F1 males to be treatment-related. 

An increase in anogenital distance in F2 females from the 

0.018 ppm group on PND 0 was not considered to be 

treatment-related by the study authors. Preputial separation (absolute age and adjusted for body weight on day 

of acquisition) was delayed in parental and retained F1 

males of the 3500 ppm group. When adjusted for PND 30 

body weight, preputial separation was delayed in 

retained but not parental F1 males from the 3500 ppm 

group. Body weights on day of vaginal opening were 

lower in F1 females from the 3500 ppm group. Day of 

vaginal opening was accelerated in the 3500 ppm group if 

adjusted for PND 21 body weight, but not body weight 

on the day of acquisition. Due to the lack of effect when 

adjusted for body weight on day of acquisition, the study 

authors did not consider effects on vaginal opening to be 

treatment-related. 

Dose-related organ weight changes in F1 weanlings 

that were considered to be treatment-related by study 

authors included decreased absolute and relative (to 

body or brain weight) spleen and paired testes weights at 

3500 ppm. Treatment-related absolute organ weight 

changes in F2 weanlings included decreased weights of 

spleen, paired testes, and seminal vesicles with coagulating glands in the 3500 ppm group. Changes in organ 

weights relative to body weight in F2 weanlings included 

decreased spleen weight in males and females and 

increased relative left kidney weight in 3500 ppm males. 

Treatment-related changes in organ weight relative to 

brain weight in F2 weanlings were decreased spleen 

weight in both sexes and decreased paired testes weight 

at 3500 ppm and seminal vesicles with coagulating 

glands at 300 and 3500 ppm. Other organ weight effects 

(e.g., affecting epididymides, thymus, brain, ovaries, 

and/or uterus with cervix and vagina weights) were not 

considered to be dose-related due to lack of dose– 

response relationships or no consistent effects across 

generations. The study authors reported no gross 

findings in F1 or F2 weanlings. [Although not clear 

because the number of animals examined for gross 

testicular effects was not reported in Tables 23 and 49 

of the study, it appeared that the incidence of 

undescended bilateral testes may have been increased 

in F1 and F2 weanling males of the 3500 ppm group.] 

The incidence of hepatic cytoplasm alteration (clear 

hepatocellular cytoplasm, slightly more basophilic cytoplasm, and/or minute vacuoles) was apparently increased in F1 males from the 300 and 3500 ppm groups 

and F1 females and F2 males from the 3500 ppm group. 

The incidence of seminiferous tubule hypoplasia was 

increased in F1 and F2 weanlings from the 3500 ppm 

group. [Another histopathological finding that appeared to be possibly increased in weanlings from 

the 3500 ppm group was unilateral hydronephrosis in 

F1 males. It did not appear that histopathological data 

were statistically analyzed.] 

The study authors identified bisphenol A NOELs of 

30 ppm (B5 mg/kg bw/day) for systemic effects and 

300 ppm (B50 mg/kg bw/day) for developmental toxicity. [The lowest benchmark doses were obtained from 

F1 body weight data on PND 21: BMD10 548 mg/kg bw/ 

day, BMDL10 267 mg/kg bw/day, BMD1 SD 580 mg/kg 

bw/day, BMDL1 SD 370 mg/kg bw/day.] 

Strengths/Weaknesses: Strengths include the large 

number and range of doses examined, the rigor with 

which the study was performed (including evaluation of 

phytoestrogen content of feed), the large sample size in 

each group, the number of additional animals per litter 

that were retained and examined, the use of a concurrent 

estrogenic positive control group, and the thoroughness 

of the histological evaluation. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 303 

3.2.8 Mouse—parenteral exposure postnatally 

with or without prenatal exposure. 

3.2.8.1 Female reproductive endpoints: Suzuki et al. 

(2002), supported by Japanese Ministry of Education, 

Culture, Sports, Sciences, and Technology, the Special 

Coordination Funds of Science and Technology Agency 

of the Japanese Government, and the Japanese Ministry 

of Health, Labor, and Welfare, conducted a study to 

examine the effects of bisphenol A exposure on the 

reproductive system of the female mouse. Two sets of 

studies were conducted, one with prenatal exposure, and 

one with postnatal exposure. In both studies, ICR/Jcl 

strain mice were fed a commercial diet (CE-2, CLEA, 

Tokyo, Japan). [No information was provided about 

bedding or caging materials.] Bisphenol A [purity not 

reported] was administered by s.c. injection in sesame oil 

vehicle. For histological examinations, organs were fixed 

in Bouin solution. Parametric data were analyzed by 

ANOVA, with post-hoc Student t-test or Welch t-test. 

Data expressed as proportions were analyzed by Fisher 

exact probability test. For exposures occurring in the 

prenatal period, the litter was maintained as the 

statistical unit. 

In the prenatal exposure study, mice were administered 

bisphenol A by s.c. injection at 0 (vehicle), 10, or 100 mg/ 

kg bw/day on GD 10–18 (day of vaginal plug 5 GD 0). 

Other groups of mice were treated with diethylstilbestrol 

at 0.0067–67 mg/kg bw/day during the same period. 

[Numbers of dams treated were not specified.] On GD 

19, fetuses were removed by cesarean section, weighed, 

adjusted to 7 pups/litter [numbers for each sex not 

indicated], and fostered to untreated mothers. Pups were 

weaned at 22 days of age. Some pups were ovariectomized at 30 days of age, and some were killed at 30 or 40 

days of age for histological examination of reproductive 

organs, polyovular follicle numbers, corpora lutea numbers, and mitotic index in uterine and vaginal cells. In the 

remaining pups, vaginal smears were examined from 41– 

70 days of age. Fertility was then assessed by mating the 

mice with untreated males (2 or 3 females/male). Offspring were counted and sexed. The authors stated that 2 

or 3 pups/litter were used in each analysis. Data tables list 

the sample size as 8–11/group/time period for the 

bisphenol A and control groups. 

Bisphenol A treatment did not affect the histology of 

the uterus or vagina in ovariectomized mice. The study 

authors stated there was no evidence of increased 

mitogenicity compared to controls in uterine cells of 

intact or ovariectomized mice exposed to bisphenol A. 

[Figure 3 of the study indicated a higher mitotic index 

in epithelial cells of ovariectomized mice of the highdose group.] Mitotic indices were significantly lower in 

stromal cells of intact mice of both dose groups and in 

glandular cells of the low-dose group. There was no 

increase in mitogenicity of vaginal cells compared to the 

control group; in intact mice, the mitotic index was lower 

than control values in vaginal epithelial cells of the highdose group and stromal cells of the low-dose group. 

Number of vaginal epithelial layers was increased in 

both bisphenol A dose groups of intact mice compared to 

control mice. No effect was reported for uterine or 

vaginal epithelial stratification. There were no effects on 

numbers of polyovular follicles. [Data were not shown 

by study authors.] The number of mice with corpora 

lutea at 30 days of age was significantly reduced in the 

low-dose group (4 of 9 mice in low dose group compared 

to 7 of 9 mice in control group). Estrous cyclicity was not 

affected by bisphenol A treatment. In mating studies, 

bisphenol A exposure did not affect the number of mice 

giving birth, number of fetuses/litter, or sex ratio. 

Several effects were observed in mice prenatally exposed 

to diethylstilbestrol, and most of the effects occurred at 

the high-dose of 67 mg/kg bw/day. In the high-dose 

diethylstilbestrol group, there were changes in vaginal 

and uterine histology, increases in mitotic indices in 

vaginal and uterine cells of ovariectomized animals, 

vaginal stratification and increased layers of epithelial 

cells in ovariectomized animals, disrupted estrous cycles, 

and complete infertility. The number of mice with 

corpora lutea at 30 days was decreased at the two 

highest diethylstilbestrol doses (Z6.7 at mg/kg bw/day). 

In the postnatal exposure experiment, female mice 

(1.5 g bw) were s.c. injected with bisphenol A at 0.015 or 

0.150 mg/pup/day or diethylstilbestrol at 0.3 or 3 mg/ 

pup/day for 5 days, beginning on the day of birth. [The 

number of animals treated was not stated. Based on 

body weights provided by authors, bisphenol A doses 

were estimated at 10 and 100 mg/kg bw/day; diethylstilbestrol doses were estimated at 200 and 2000 lg/kg 

bw/day.] Two-thirds of mice were ovariectomized at 30 

days of age and then killed at 30, 40, or 90 days of age for 

histological examination of reproductive organs. Numbers of polyovular follicles were determined at 30 days of 

age, and number of corpora lutea were counted at 30 and 

90 days of age. Estrous cyclicity was monitored in the 

remaining mice at 61–90 days of age. The 90-day-old 

mice were s.c. injected with 5 mg/kg bw colchicine and 

killed 5 hr later. Mitotic rates of uterine and vaginal cells 

were determined, and histological examinations of 

reproductive organs were conducted. Sample sizes were 

6–17/group/time period in analyses conducted in mice 

exposed postnatally. 

Vaginal stratification was observed at 40 days of age in 

4 of 7 ovariectomized mice of the high-dose bisphenol A 

group, which was higher than in the control. The 

incidence of vaginal stratification in 90-day-old ovariectomized mice of the high-dose group (4 of 10) did not 

attain statistical significance compared to control. In 

ovariectomized mice, significant increases in the 

mitotic rate compared to controls were observed in 

uterine stromal cells and vaginal epithelial cells at the 

high-dose. The number of vaginal epithelial layers was 

also increased in the high-dose bisphenol A group (B4 

layers in treated group compared to 3.5 layers in 

control group). There were no significant changes in 

estrous cycles or number of mice with corpora lutea. In 

30-day-old mice of the high-dose group, significant 

increases were observed in the number of mice with 

polyovular follicles (15 of 17 in exposed group 

compared to 6 of 15 in control group) and the numbers 

of polyovular follicles/mouse (mean7SE: 0.870.2 

in the exposed group and 0.270.1 in control group); 

polyovular follicles contained 2 oocytes in the 

control and bisphenol A groups. Effects observed in 

mice treated with both doses of diethylstilbestrol 

included increased stratification of vaginal cells in 

ovariectomized mice at 40 and 90 days of age, increased 

mitotic rates of vaginal and uterine cells in ovariectomized mice, disrupted estrous cycles, and increased 

polyovular follicles. The study authors concluded that 

Birth Defects Research (Part B) 83:157–395, 2008
304 CHAPIN ET AL. 

high-doses of bisphenol A induce ovary-independent 

vaginal stratification and polyovular follicles when 

administered during postnatal but not prenatal 

development. 

Strengths/Weaknesses: The use of diethylstilbestrol as 

a positive control is a strength as are an experimental 

design that appropriately examined litter effects. The use 

of relatively high-doses by s.c. injection and small sample 

sizes for ovarian histopathology are weaknesses. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate but of limited utility due to the 

route and dose level. 

Nikaido et al. (2005), supported by the Japanese 

Ministry of Health, Labor, and Welfare, examined the 

effects of bisphenol A exposure on the development of 

the reproductive system in female mice. Mice used in this 

study were housed in polyisopentene cages with white 

pine chip bedding. The mice were fed a low-phytoestrogen diet (NIH-07 PLD; Oriental Yeast Co.) and provided 

water in polycarbonate bottles with rubber stoppers. At 

15 days of age, 17–24 female CD-1 mice/group were s.c. 

injected with DMSO vehicle, 10 mg/kg bw/day 

bisphenol A (Z99% purity), or 10 mg/kg bw/day 

diethylstilbestrol for 4 days. Additional groups were 

dosed with other compounds, but those results will not 

be discussed. [No information was provided on the 

numbers of litters represented.] Mice were weaned at 21 

days of age. Body weights were measured weekly. Day of 

vaginal opening was determined and estrous cyclicity 

was assessed over 21-day periods beginning at 5, 9, and 

21 weeks of age. Six mice/group/time period were killed 

and necropsied at 4, 8, 12, and 24 weeks of age. [In 

contrast to the Materials and Methods section, there 

was no mention of animals killed at 12 weeks of age in 

the Abstract or Results section of the study.] Ovaries, 

uteri, vaginas, and inguinal mammary glands were fixed 

in 10% neutral buffered formalin. Histopathological 

analyses were conducted of the ovary, uterus, and 

vagina. Mammary glands were examined as wholemount preparations. It appears that all endpoints were 

assessed in every mouse. Statistical analyses included 

homogeneity of variance analysis and ANOVA or 

Kruskal–Wallis test. If statistical significance was obtained, data were further analyzed by Fisher protected 

least significant difference test. 

Exposure to bisphenol A resulted in no effects on body 

weight gain, age of vaginal opening, estrous cyclicity, 

histopathological changes in the uterus or vagina, or 

growth or development of the mammary gland. At 4 

weeks of age, 33% of mice in the control group, 83% of 

mice in the bisphenol A group, and 100% of mice in the 

diethylstilbestrol group lacked corpora lutea. [It appears 

that the study authors considered the lack of corpora 

lutea to be normal based on the age of mice.] No effects 

on corpora lutea numbers or numbers of polyovular 

follicles were observed at later ages. Mice treated with 

diethylstilbestrol experienced accelerated vaginal opening and increased time in estrus. In their conclusion, the 

study authors reiterated the lack of effects in the 

bisphenol A group. 

Strengths/Weaknesses: The use of diethylstilbestrol as 

a positive control is a strength, but the lack of information on sample size of dams, small sample size for 

postnatal endpoints, subcutaneous route, and high-dose 

level are weaknesses. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate and not useful in the evaluation. 

Markey et al. (2005), supported by NIH, examined the 

effects of perinatal bisphenol A exposure on reproductive development in mice. CD-1 mice were fed Purina 

rodent chow that tested ‘‘negligible for estrogenicity in 

the E-SCREEN assay.’’ Cages and bedding tested 

negative for estrogenicity in the E-SCREEN assay. Tap 

water was supplied in glass bottles. From GD 9 (GD 

1 5 day of vaginal plug) through PND 4, 6–10 mice/ 

group were exposed to bisphenol A [purity not reported 

in the manuscript; 9772% per A. Soto, personal 

communication, March 2, 2007] at 0 (DMSO vehicle), 

0.000025, or 0.000250 mg/kg bw/day through a s.c. 

pump. Offspring were culled to 10/litter on PND 7 and 

weaned on PND 20. One pup/litter from 6–10 litters/ 

treatment group was killed on the day of proestrus at 3 

months of age. The uterus and vagina were weighed and 

subjected to morphometric analysis. The uterus was also 

assessed for cell proliferation by bromodeoxyuridine 

(BrdU) incorporation, apoptosis by TUNEL method, and 

expression of ERa and progesterone receptor by an 

immunostaining procedure. Data that were normally 

distributed and showed homogeneity of variance were 

analyzed by ANOVA and least significant difference test. 

Other data were analyzed by Kruskall–Wallis and Mann– 

Whitney U test. 

Significant effects observed in 3-month-old offspring 

exposed to the high-dose included decreased absolute 

and relative (to body weight) vaginal weight, decreased 

volume of uterine lamina propria, and increased percentage of proliferating uterine glandular epithelial cells. In 

mice of both dose groups, there were significant 

increases in expression of ERa and progesterone receptor 

in uterine luminal epithelial cells; levels of both receptors 

were also increased in the subepithelial stroma. No 

treatment effects were observed for apoptosis in uterine 

luminal and glandular epithelial cells. No treatment 

effects were observed for vaginal morphometry or cell 

proliferation. The study authors concluded that environmentally relevant doses of bisphenol A affect the 

development of the genital tract at the gross and cellular 

level in the female offspring of mice exposed during 

pregnancy. 

Strengths/Weaknesses: The administration of very 

low doses is a strength. A critical weakness is the use 

of DMSO as a vehicle which is known to degrade the 

pump apparatus, and is inappropriate as a vehicle for in 

vivo studies. A critical weakness is the uncertainty of the 

DMSO concentration as a vehicle and therefore pump 

performance. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process given 

exposure uncertainties. 

Mun ̃ oz-de-Toro et al. (2005), supported by NIH and 

National University of Litoral (Argentina), examined the 

effect of perinatal bisphenol exposure on mammary 

gland development in mice. Food, caging, and bedding 

material were reported to test negligible for estrogenicity 

in the E-SCREEN. Water was provided in glass bottles. 

CD-1 mice (n 5 6–10/group) were implanted with 

osmotic pumps designed to deliver bisphenol A [purity 

not indicated] at 0 (DMSO vehicle), 0.000025, or 

0.000250 mg/kg bw/day from GD 9 (GD 1 5 day of 

vaginal plug) through PND 3 (not defined). Offspring 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 305 

were culled to 10 pups/litter on PND 7. One female 

offspring/litter, from 6–10 litters/group, was killed on 

PND 20 and 30 and at 4 months of age. The 4-month-old 

mice were killed on proestrus. Another group of mice 

[number not specified] was killed on the first proestrus. 

Mammary glands were collected for evaluation of 

mammary structures at 20 and 30 days and 4 months 

of age and day of first proestrus. Mammary glands were 

also collected from 30-day-old mice for analysis of DNA 

synthesis by BrdU incorporation, expression of ERa and 

progesterone receptor using immunohistochemistry 

techniques, apoptosis by TUNEL method, and Wnt4

mRNA by RT-PCR. Plasma 17b-estradiol levels were 

measured in mice killed at first proestrus. In an 

experiment to monitor response to 17b-estradiol, 1 

pup/litter (n 5 10/group) was ovariectomized at 25 days 

of age and implanted with a s.c. pump supplying vehicle 

or 0.5 mg 17b-estradiol/kg bw/day on PND 25–35. Mice 

were killed following 17b-estradiol treatment for examination of mammary structures. Statistical analyses 

included ANOVA and Dunn post-hoc test. If the data 

were not normally distributed, statistical analyses were 

done by Kruskall–Wallis and Mann–Whitney test. 

In 30-day-old mice, bisphenol A exposure increased 

numbers of terminal end buds at both doses and area of 

terminal end buds at the high-dose. Percentages of 

apoptotic cells were decreased on PND 30 in mice from 

both bisphenol A dose groups. The percentage of stromal 

cells undergoing proliferation on PND 30 was reduced in 

the high-dose bisphenol A group. The number of 

epithelial cells expressing progesterone receptors was 

increased in both dose groups on PND 30, but there were 

no treatment-related changes in ERa receptor expression. 

Clusters of progesterone receptors were often observed 

in the ductal epithelium of bisphenol A-treated mice. 

Slopes of the correlation between age of first proestrus 

and mammary length were significantly reduced in the 

high-dose group, suggesting slower ductal invasion of 

stroma. There were no significant differences in plasma 

17b-estradiol levels in mice killed at first proestrus. 

Trends for increasing expression of mRNA for Wnt4,a 

mediator of lateral branching downstream from progesterone receptors, did not attain statistical significance. 

The number of lateral branches in mammary gland at 4 

months of age was significantly increased at the low but 

not the high-dose. In mice exposed to the high-dose of 

bisphenol A during perinatal development and 17bestradiol during postnatal development compared to 

mice who were exposed to 17b-estradiol but not bisphenol A, there were increases in numbers, area, and 

size of terminal end buds, terminal end bud numbers/ 

ductal area, and terminal end bud area/ductal area. 

The study authors concluded that ‘‘yperinatal exposure 

to environmentally relevant [bisphenol A] doses 

results in persistent alterations in mammary gland 

morphogenesis.’’ 

Strengths/Weaknesses: This study was a follow-up on 

the study of Markey et al. (2005) and tested the same 

doses using a similar schedule for effects on mammary 

tissue. The administration of very low doses is a strength. 

The statistics appear to be inappropriate in not accounting for the significant number of comparisons made. A 

critical weakness is the uncertainty of the DMSO 

concentration as a vehicle and therefore pump 

performance. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process given 

exposure uncertainties. 

3.2.8.2 Male reproductive endpoints:: Nakahashi 

et al. (2001), supported by the Japanese Ministry of 

Education, Science, Sports, and Culture, examined the 

effect of neonatal bisphenol A exposure on adult sperm 

count in mice. On the first 5 days of life, 10–15 neonatal 

SHN mice/group were injected [route not indicated] 

with sesame oil/DMSO vehicle or with bisphenol A 

[purity not reported] in sesame oil at 0.0005 or 0.050 mg/ 

day. [Assuming a neonatal mouse weights 2 g, the mice 

received doses of 0.25 and 25 mg/kg bw/day.] A group of 

12 mice received 0.050 mg/day bisphenol A in sesame oil 

in combination with 100 IU retinol acetate in DMSO 

vehicle. In a second exposure protocol, pregnant mice 

were fed a vitamin A-deficient diet (Low Vitamin A diet; 

Clea Japan) from 3 days before gestation to PND 5. After 

PND 5, the dams were fed commercial diet (CE-7, Clea 

Japan). On the first 5 days of life, their pups (n 5 7–9/ 

group) were injected with bisphenol A at 0 (sesame oil) 

or 0.0005 mg/day. Male offspring from both studies were 

weaned at 20 days of age and fed the CE-7 diet. Mice 

were killed at 14 weeks of age and epididymal sperm 

counts were obtained. [No information was provided 

about caging and bedding materials. Numbers of litter 

represented were not indicated. Procedures for statistical analyses were not discussed.] 

A 35% reduction in sperm counts was observed in 

mice from the 0.050 mg/day group compared to the 

control group. A significant reduction in sperm counts 

was not observed in the group co-treated with 0.050 mg/ 

day bisphenol A and retinol acetate. Administration of a 

vitamin A-deficient diet to dams had no effect on sperm 

counts in their offspring, but sperm counts were reduced 

in mice born to mothers fed a vitamin A-deficient diet 

and injected with 0.0005 mg/day bisphenol A in the 

neonatal period. The study authors concluded that 

vitamin protects infants from the effects of environmental xenoestrogens. 

Strengths/Weaknesses: The subcutaneous route of 

administration, lack of clarity on exposure issues, lack 

of husbandry and statistical information are weaknesses. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for inclusion and not useful. 

Aikawa et al. (2004), supported by the Japanese 

Ministry of Education, Science, Sports, and Culture, 

examined the effects of neonatal bisphenol A exposure 

on sperm endpoints in adult mice. Unless otherwise 

specified, dams were fed CE-7 and CA-1 (Clea Japan 

Inc.). [No information was provided about caging or 

bedding materials.] In the first experiment, SHN mice 

were s.c. injected with bisphenol A, bisphenol A plus 

retinol acetate, or vehicle for 5 days beginning on the day 

of birth. Doses of each compound were 0.5 or 50 mg/day 

bisphenol A [purity not reported] (n 5 10–14/group), 

50 mg bisphenol A plus 100 IU retinol acetate/day (n 5 5), 

and vehicle control (sesame oil for bisphenol A and or 

DMSO for retinol acetate; n 5 11). [Assuming a neonatal 

mouse weighs 2 g, these bisphenol A doses would be 

0.25 and 25 mg/kg bw/day.] In another group, pregnant 

mice were fed a low vitamin A diet from 3 days before 

gestation to PND 5 and were fed a normal vitamin Acontaining diet (CE-7 and CA-1) beginning on Day 6 

following parturition [number/group not stated]. Pups 

Birth Defects Research (Part B) 83:157–395, 2008
306 CHAPIN ET AL. 

born to those dams (n 5 7–8/group) were s.c. injected 

with 0.5 mg/day bisphenol A or vehicle for 5 days, 

beginning on the day of birth. In all groups, mice were 

weaned at 3 weeks of age, individually housed at 8 

weeks of age, and killed at 10 weeks of age. Sperm were 

collected for analysis of motility and abnormalities. In 

pups not born to vitamin A-deprived dams, testes were 

fixed in formalin for histopathological evaluation. Data 

were analyzed by ANOVA and Fisher least significant 

difference test. 

Sperm motility was significantly reduced in mice 

injected with 50 mg/day bisphenol A (B25 vs. 50% in 

controls) but was not affected in mice exposed to 50 mg/ 

day bisphenol A plus retinol acetate. Sperm motility was 

not affected in mice born to mothers fed a normal diet and 

exposed to 0.5 mg/day bisphenol. Compared to the 

vehicle control group born to mothers fed a normal 

diet, the mice born to mothers fed a vitamin A-deficient 

diet and injected with 0.5 mg/day bisphenol A had 

significant reductions in sperm motility [B19 compared 

to 50% in vehicle controls]. Sperm motility was also 

reduced in the mice born to mothers fed a vitamin Adeficient diet but not exposed to bisphenol A. In groups 

born to mothers fed a vitamin A-deficient diet, there were 

no differences in sperm motility following exposure to 

vehicle or bisphenol A. Percentage abnormal sperm was 

6.8% in the vehicle control group and was significantly 

increased in mice exposed to 0.5 mg/day bisphenol A 

[B45%], 50 mg/day bisphenol A (78.2%), 50 mg/day bisphenol A plus retinol acetate (27.8%), vehicle following 

birth to vitamin A-deficient mothers [B45%], or bisphenol 

A following birth to vitamin A-deficient mother [B70%]. 

No histopathological alterations were reported in testes of 

mice exposed to 0.5 or 50 mg/day bisphenol A or 50 mg/ 

day bisphenol A plus retinol acetate. The study authors 

concluded that neonatal exposure to a relatively large 

dose of bisphenol A damages sperm motility and 

morphology, effects that are inhibited by vitamin A and 

enhanced by vitamin A-deficient diets. 

In a second experiment, 3 pups/group were s.c. 

injected with 20 mg 17b-estradiol/day, 20 mg 17b-estradiol 

plus 100 IU acetate retinol acetate/day, 50 mg bisphenol 

A/day, or vehicle (sesame oil for bisphenol A and 17bestradiol or DMSO for retinol acetate) for 5 days 

beginning on the day of birth. Mice were killed at 18 

days of age. Testis, efferent duct, epididymis, and vas 

deferens were fixed in formalin and analyzed for ERa

using an immunohistochemical method. Data were 

analyzed by ANOVA and Fisher least significant 

difference test. 

In a second experiment, 3 pups/group were s.c. 

injected with 20 mg 17b-estradiol/day, 20 mg 17b-estradiol 

plus 100 IU acetate retinol acetate/day, 50 mg bisphenol 

A/day, or vehicle (sesame oil for bisphenol A and 17bestradiol or DMSO for retinol acetate) for 5 days 

beginning on the day of birth. Mice were killed at 18 

days of age. Testis, efferent duct, epididymis, and vas 

deferens were fixed in formalin and analyzed for ERa

using an immunohistochemical method. Data were 

analyzed by ANOVA and Fisher least significant 

difference test. Bisphenol A exposure had no effect on 

ERa expression in male reproductive organs. Exposure to 

17b-estradiol increased the numbers of ER-positive cells 

in vas deferens epithelium, but there was no increase 

when mice were treated with acetate retinol in addition 

to 17b-estradiol. The study authors concluded that the 

lack of effect of bisphenol A may be due to its weak 

estrogenic activity. 

Strengths/Weaknesses: This study provided follow-up 

information to that of Nakahashi et al. (2001). The use of 

17b-estradiol as a positive control in the testis histology 

study is a strength. Weaknesses include subcutaneous 

route of administration, lack of clarity on exposure 

issues, small sample sizes, lack of husbandry and 

statistical information. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate and not useful based on small 

sample sizes and inadequate presentation of statistical 

methods of analysis. 

Toyama and Yuasa (2004), supported in part by the 

Japanese Ministry of Environment and Ministry of 

Education, Science, Sports, and Culture, examined the 

effects of neonatal bisphenol A [purity not reported] 

exposure on spermatogenesis during puberty and adulthood in rats and mice. [No information was provided 

about chow or bedding and caging materials. The rat 

data are reported in Section 3.2.4.] ICR mice were s.c. 

injected with bisphenol A in a DMSO and olive oil 

vehicle on PND 1, 3, 5, 7, 9, and 11 (PND 0 5 day of 

birth). Bisphenol A doses were 0.0001, 0.001, 0.005, and 

0.010 mg/kg bw in mice. Additional animals were 

treated with 17b-estradiol and estradiol benzoate. Animals were killed weekly at 2–10 weeks of age and some 

pups were also killed at 24 and 31 days of age. There 

were 5 animals/dose/time point in bisphenol groups A 

groups and apparently 3–4 vehicle control mice. Testes 

were examined by light and electron microscopy. Males 

from each experimental group (a total of 12 mice) were 

mated with 2 females [numbers tested in each dose 

group not reported]. A total of 12 mouse dams were 

allowed to complete pregnancy. [It does not appear that 

any statistical analyses were conducted.] 

In mature spermatids of 7-week-old mice in the vehicle 

control group, incidences of deformed acrosome, deformed nucleus, and abnormal ectoplasmic specialization were o0.3%. In 7-week-old mice treated with 

Z0.001 mg/kg bw bisphenol A, the incidence of deformed acrosome was 450–60%, the incidence of 

deformed nucleus was 440%, and the incidence of 

abnormal ectoplasmic specialization was 460–70%. 

[Data were not shown for individual dose levels.] 

Similar effects were observed in the groups treated with 

17b-estradiol and estradiol benzoate. No effects were 

reported at other ages. [Data were not shown by study 

authors.] The blood–testis barrier remained intact based 

on histologic observations. All tested males from the 

bisphenol A group were fertile, and sex ratio, litter sizes, 

and pup weights were reported to be normal. [No results 

were shown for individual dose levels. Fertility 

data were presented in Table 4 and 5 of the study, but 

it is not clear which dose level(s) were represented.] 

The study authors concluded that bisphenol A acts 

as an estrogen and induces transient changes in 

the male reproductive system of rodents that resolve in 

adulthood. 

Strengths/Weaknesses: The strengths include the use 

of multiple doses of bisphenol A and the use of both rats 

and mice, allowing interspecies comparisons. Weaknesses include small sample size, unclear data analyses, 

and s.c. route of administration. 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 307 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate and not useful due to critically 

small sample size, route of administration, lack of clarity 

of design, and inappropriate statistical procedures. 

3.2.9 Sheep. Evans et al. (2004), supported by the 

British Council, Irish Health Research Board, and the 

Royal Society, examined the effects of bisphenol A 

exposure on gonadotropin secretion on prepubertal 

female lambs. [No information was provided about 

feed or composition of bedding or caging materials.] 

Starting at 3 weeks of age, female Poll Dorset lambs were 

weighed weekly, and blood samples were collected 2 

times/week for measurement of LH and FSH levels. At 4 

weeks of age, lambs were assigned to treatment groups 

according to body weight. From 4–11 weeks of age, 6 

lambs/group received biweekly i.m. injections with the 

10:1 corn oil/alcohol vehicle, 3.5 mg/kg bw bisphenol A 

[purity not reported], 0.175 mg/kg bw diethylstilbestrol 

[listed as 0.0175 in the legend for Figure 1 of the study], 

or 3.5 mg/kg bw octylphenol. Lambs were ovariectomized at 9 weeks of age. [The text of the methods 

sections reported ovariectomy at the beginning of 

treatment, but that statement appears to be an error 

because it is not indicated elsewhere in the study.] On 

the last day of treatment, blood was collected every 

15 min for 6 hr to assess pulsatile LH secretion. All lambs 

were then killed. Adrenal glands, kidneys, and ovaries 

were weighed. Uteri were examined as discussed in 

Morrison et al. (2003). Data were analyzed by ANOVA, 

Dunnett multiple comparison post-hoc test, regression 

analysis, Munro algorithm, and paired t-tests. 

Compared to the control group, the bisphenol A group 

did not experience significant changes in body, kidney, 

adrenal, or ovarian weights. [No data were shown for 

body, kidney, and ovarian weights in the control vs. 

bisphenol A group.] Uteri from the bisphenol A group 

were reported to be visually larger, but no uterine 

weights were provided. Over the 7-week treatment 

period, bisphenol A did not significantly affect blood 

LH or FSH levels compared to controls. Compared to 

controls, the bisphenol A group experienced significant 

decreases [% change compared to controls] in concentration [48%], amplitude [77%], and frequency [66%] of 

pulsatile LH secretion. Octylphenol did not have any 

effect on the endpoints examined. Diethylstilbestrol 

treatment resulted in decreased blood levels of LH and 

FSH over the treatment period, including the period 

following ovariectomy. Concentration, amplitude, and 

frequency of pulsatile LH secretion were also lower in 

the diethylstilbestrol group, with a greater magnitude of 

effect compared to bisphenol A. The study authors 

concluded that the bisphenol A dose tested can inhibit 

LH secretion in lambs. 

Strengths/Weaknesses: The unique animal model and 

the use of LH pulsatile response are uncommon but 

interesting. The high-dose level via i.m. injection is a 

weakness as are small sample sizes (n 5 6). The statistical 

tests for LH trends did not appear to take into account 

the repeated nature of the sampling leading to over 

stating the significance of trend effects. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate for inclusion but of limited utility 

for the evaluation process. 

Morrison et al. (2003), supported by the Wellcome 

Trust, Dr. Ferranti, and the Irish Health Research Board, 

examined the effects of bisphenol A exposure on the 

lamb uterus. [No information was provided on feed or 

composition of bedding or caging materials.] At 4 

weeks of age, female Poll Dorsett lambs were randomly 

assigned to treatment groups according to body weight. 

Beginning at 4 weeks of age and continuing for 7 weeks, 

6 lambs/group received biweekly i.m. injections with the 

10:1 corn oil:alcohol vehicle, 3.5 mg/kg bw bisphenol A 

[purity not reported], 0.175 mg/kg bw diethylstilbestrol, 

or 3.5 mg/kg bw octylphenol. Lambs were ovariectomized during the fifth week of exposure. Throughout the 

study, blood was collected for measurement of gonadotropin levels and the results of those analyses were 

reported in the study by Evans et al. (2004). Lambs were 

killed following 7 weeks of exposure. Uteri and cervices 

were fixed in Bouin solution for histopathological 

examination, morphometric measurement, and immunohistochemical detection of ERa and ERb. Statistical 

analyses included ANOVA with Fisher protected least 

significant difference. 

Significant effects observed with bisphenol A treatment [% change compared to controls] were increased 

uterine/cervical tract weight [87%], endometrial area 

[154%], and endometrial/myometrial ratio [65%]. Qualitative histopathological observations in uteri from 

bisphenol A-treated lambs included endometrial edema, 

decreased endometrial gland density compared to controls, and crowding of cells in the uterine epithelium, 

which contained substantial amounts of eosinophilic, 

non-vacuolated cytoplasm. In contrast to uteri from 

control lambs, mononuclear cell exocytosis was not a 

common observation in uteri from the bisphenol A 

group. The cervical epithelium was keratinized in the 

bisphenol A group. Qualitative analyses revealed that 

diffuse intracellular staining for ERa and ERb in the 

uterine subepithelium was most pronounced in the 

bisphenol A and diethylstilbestrol groups. Similar to 

animals treated with bisphenol A, the diethylstilbestrol 

group had increased uterine weight, keratinized cervical 

epithelium, changes in uterine histology, and keratinized 

cervical epithelium, but there was no change in endometrial/myometrial ratios. No changes were observed 

following exposure to octylphenol. The study authors 

concluded that bisphenol A exposure altered the uterocervical environment of lambs. 

Strengths/Weaknesses: This is a companion to the 

study of Evans et al. (2004) with similar strengths. The 

single high-dose level via i.m. injection is a weakness as 

is the exclusion of data from 2 lambs based on responses 

for E/M ratio endpoints, thus reducing the n to 5 and 

potentially biasing the data. The statistical analyses do 

not appropriately account for the number of multiple 

comparison made which can increase the probability of 

detecting an effect by chance. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate for inclusion but of limited utility 

in the evaluation process. 

Savabieasfahani et al. (2006), supported by the U.S. 

Public Health Service, NIH, and the University of 

Michigan, used Suffolk ewes to investigate the effects 

of maternal exposure to bisphenol A or methoxyclor [not 

discussed here] during gestation. Pregnant Suffolk ewes 

used in this experiment were exposed to a natural 

photoperiod in the same pasture and fed a diet of 1.25 kg 

alfalfa/grass hay. Pregnant ewes (n 5 10) of similar 

Birth Defects Research (Part B) 83:157–395, 2008
308 CHAPIN ET AL. 

average weight were injected s.c. on GD 30–90 with 

5 mg/kg bw day bisphenol A (991% purity) dissolved in 

cottonseed oil. Control pregnant ewes (n 5 16) were 

administered vehicle injections. Lambs were born over 

about a 1-month interval in early spring. Birth outcome 

measurements included number and gender of offspring, weight, height, chest circumference, genital 

development, and measurement of blood insulin and 

insulin-like growth factor-1. Lambs were cross-fostered 

and group housed on PND 3. Lactating ewes were fed a 

diet of corn and alfalfa hay. Lambs had free access to 

standardized Shur Gain feed pellets. [The authors note 

the presence of phytoestrogens in the feed but did not 

provide quantification.] At weaning, female were 

separated from male offspring, and the females were 

housed in open air pens under natural photoperiod with 

free access to feed pellets, as described above. 

Maternal blood samples were taken on GD 50, 70, and 

90 for measurement of bisphenol A using HPLC. The 

number and sex of offspring in each treatment group, 

weight, height, chest circumference, and genital development were noted. Blood levels of insulin and insulinlike growth factor 1 were assayed by RIA on PND 1. In 

female offspring [n not indicated], blood was drawn 

biweekly during the first 2 postnatal months for 

determination of LH by RIA. Timing of puberty onset 

was estimated through twice weekly blood draws for 

progesterone (n 5 11/group). Estrus cycling patterns 

were determined by frequent measurement of FSH, LH, 

and progesterone by RIA in 3 female offspring/group 

after synchronization with prostaglandin F2a at 40 weeks 

of age. Statistical analyses were performed using 

ANOVA, repeated measures ANOVA, or a linear mixed 

model. A cluster algorithm was used to identify LH 

pulses, with Student t-test to determine LH nadirs. 

Blood levels of bisphenol A were significantly higher 

in exposed pregnant ewes than controls at all sampling 

times. The levels reached (37.473.3 mg/L) were compared to exposure levels reported in pregnant women 

[0.3–18.9 mg/L (Scho ̈nfelder et al., 2002b)]. No statistical 

difference was reported in gestation length, number of 

offspring, or sex. There were no significant differences in 

female lambs in anogenital distance, insulin, or insulinlike growth factor levels on PND 1. In female offspring, 

prenatal bisphenol treatment significantly decreased 

birth weight [by B11%], height [by B5%], and chest 

circumferences [by B7%, all comparisons estimated 

from a graph]. In male offspring exposed to bisphenol A, 

there were no significant differences from control in birth 

weight, height, chest circumference, or anogenital distance, but anoscrotal:anonavel ratio was increased 

significantly [by 21%]. Bisphenol A treatment increased 

significantly levels of circulating LH [by B89%, estimated from a graph] during the first 2 months of life in 

female offspring. Onset of puberty was not affected by 

treatment in bisphenol A-exposed female offspring, but 

these females had a significantly longer first breeding 

season [by B2 weeks] and larger number of cycles 

during the first breeding season). Estrous cycle length 

and progesterone levels were not different from controls. 

The bisphenol A group had significantly lower peak and 

total LH, and the amplitude of LH pulses was increased 

significantly, whereas frequency showed no difference 

from control group. No differences in FSH were seen 

between groups. Progesterone secretion pattern showed 

no difference between groups, despite perturbations in 

LH patterns. 

The authors concluded that prenatal exposure to 

bisphenol A impairs growth in female fetuses and is 

associated with dampening of the LH surge. Although 

there was no apparent effect on progesterone production, 

the authors suggested that the changes induced by 

prenatal exposure of females could interfere with 

fertility. 

Strengths/Weaknesses: This study appears to have 

been well-conducted with the utilization of multiple 

endpoints in sheep. Weaknesses are the use of a single 

dose level and the relatively small sample size. The 

single time point for bisphenol A plasma determination 

at an unknown time relative to s.c. injection is a 

weakness. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate though of limited utility. 

3.2.10 Non-mammalian species. Although these 

studies in non-mammalian species can be quite useful for 

understanding mechanisms and environmental impacts, 

the studies are not considered useful for the evaluation 

process, because of the uncertain relationship between 

human biology and that of the model species. 

3.2.10.1 Invertebrates: Hill et al. (2002) supported by 

the Council on Undergraduate Research and the Association for Biological Laboratory Education, examined 

the effects of bisphenol A on the development of 2 

freshwater sponge species. (Heteromyenia sp. and Eunapius fragilis). Sponge gemmules were incubated in 

tissue culture wells containing bisphenol A [purity not 

indicated] at 0, 0.16, 16, 80, or 160 ppm [mg/L]. The 

control group was incubated in the spring water vehicle. 

There were 5 replicates/treatment. Nonylphenol and 

ethylbenzene were also examined. Growth was measured on Days 3, 6, and 9. Because growth patterns were 

similar at all 3 evaluation periods, statistical analyses 

were conducted only for Day 6 data. Data were analyzed 

by ANOVA and Tukey multiple comparison test. In both 

species, abnormal development or malformation of the 

water vascular system was observed at a bisphenol A 

dose of 16 ppm and germination was completely inhibited at 80 and 160 ppm. Significantly reduced growth 

rates were observed in Heteromyenia sp. at 160 ppm. 

Similar effects were observed with nonylphenol and 

ethylbenzene. The study authors stated that sponges may 

prove useful for examining endocrine-disrupting 

compounds. 

Strengths/Weaknesses: This study used a unique 

model with a focus on the aquatic system. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study may have utility for environmental assessment, but is not useful for human risk assessment. 

Roepke et al. (2005), supported by the National 

Oceanic and Atmospheric Administration, examined 

the effects of bisphenol A exposure on development of 

two species of sea urchin, Strongylocentrotus purpuratus

and Lytechinus anamesus. In dose–response studies, sea 

urchin embryos were incubated from 1–96 hr postfertilization in media containing bisphenol A [purity 

not indicated] at 0, 250, 500, 750, or 1000 mg/L [culture 

ware not discussed]. Development toxicity was assessed 

at 96 hr by examining larvae at the pluteus stage. The 

larvae were categorized as normal, delayed, abnormal, 

elongated, or hatched. Data were obtained in 3 replicates. 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 309 

Results were reported to be similar for the 2 species, and 

unless otherwise indicated, data were shown for S.

purpuratus. In additional studies, sea urchin embryos 

were incubated in bisphenol A at 0–500 mg/L with and 

without addition of tamoxifen or bisphenol A at 0– 

750 mg/L with and without the addition of ICI 182,780. 

Data were analyzed by ANOVA followed by Tukey– 

Kramer test or Tukey or Student-Newman–Keuls tests 

for pair-wise multiple comparison. An EC50 of 226.6 mg/ 

L (lower limit: 121.6, upper limit: 323.5 mg/L) was 

estimated for developmental toxicity associated with 

bisphenol A exposure. Based on EC50 values, 17bestradiol was B15 times more potent than bisphenol A. 

Tamoxifen inhibited developmental toxicity, and ICI 

182,780 enhanced the developmental toxicity induced 

by bisphenol A; similar results were obtained for 17bestradiol. The study authors concluded that bisphenol A 

induced developmental toxicity in sea urchins through a 

tamoxifen-sensitive mechanism at levels exceeding environmentally relevant concentrations. 

Strengths/Weaknesses: The use of 2 species and 

multiple concentrations are strengths. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study may have utility for environmental assessment, but is not useful for human risk assessment. 

Andersen et al. (1999b), supported by the Danish 

Strategic Environmental Research Program, evaluated 

the effects of bisphenol A on female sexual maturation in 

the zoo planktonic crustacean Acartia tonsa. Eggs were 

grown in the presence of the algal food source for the 

organism after exposure of the algae to bisphenol A 

(499% purity) for 3 hr to promote sorption by the algae 

of the test chemical [culture ware not discussed]. The 

treated algae were added to Acartia tonsa eggs to give 

nominal bisphenol A concentrations of 0.2, 2, and 20 mg/ 

L. [Actual concentrations were not reported. An 

untreated or vehicle-treated control appears to have 

been used.] 17b-Estradiol 23 mg/L was used as a positive 

control, and 2,3-dichlorophenol 13.6 mg/L was used as a 

negative control. On Day 8 of incubation, 10–25 juvenile 

Acartia tonsa/group were transformed to an egg-collection apparatus, in which exposure to treated algae 

continued. Eggs were collected daily and counted until 

Day 12, at which time a stable adult level of egg 

production was established. Egg production by group 

was compared using Student t-test. [A repeated-measures test appears not to have been used.] A significant 

increase in egg production was shown on Day 10 in 

animals treated with bisphenol A 20 mg/L and 17bestradiol 23 mg/L compared to control. The authors 

concluded that bisphenol A accelerated female reproductive maturation in Acartia tonsa and that the effect 

appeared to be estrogenic. 

Strengths/Weaknesses: Strengths are the use of multiple exposure levels, the inventive method of feeding 

bisphenol A to the test organisms, and the use of 17bestradiol as a positive control. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study may have utility for environmental assessment, but is not useful for human risk assessment. 

Watts et al. (2001), supported by the European Union, 

examined development and reproduction in 2 generations of non-biting midges (Chironomus riparius) exposed 

to bisphenol A. The study began with incubation of 4 egg 

ropes/group in media containing vehicle, bisphenol A, 

or ethinyl estradiol [apparently at the same concentrations described below]. Twenty first-instar larvae from 

the appropriate media were added to each exposure 

glass jar containing dechlorinated water and sediment 

spiked with bisphenol A [purity not indicated] at 

concentrations of 0 (ethanol vehicle control and dechlorinated tap water control), o0.010, 0.078, 0.55, 77, 750, or 

10,400 mg/L. Four replicate jars were prepared for 

each dose level. Concentrations in sediment 

were verified. Numbers and sexes of adults emerging 

from each replicate jar were determined. Egg ropes 

produced by the first generation were counted and 

placed in media containing test solutions or vehicle 

controls. Four egg ropes/group were selected and used 

to reseed the sediments with the second generation of 

larvae. Adults emerging from the second generation 

were counted. Statistical significance was determined by 

ANOVA. In the first generation, adult emergence was 

delayed in females from the o0.010, 0.55, and 77 mg/L 

bisphenol A groups but was not affected in males. Males 

were reported to emerge significantly earlier than 

females. In the second generation, emergence of males 

and female adults was significantly delayed at 

Z0.078 mg/L bisphenol A. At concentrations of 0.010– 

750 mg/L, there were no significant differences in the 

percentage of adults emerging in either generation. No 

second-generation adults emerged in the group exposed 

to 10,400 mg/L. There were no effects on sex ratio. 

Exposure to bisphenol A did not significantly affect the 

number of eggs produced by the first generation. In 

contrast to bisphenol A, exposure to ethinyl estradiol 

accelerated adult emergence. The study authors concluded that the endpoints evaluated indicated general 

sediment toxicity but were not useful for detecting 

estrogenic effects. 

Strengths/Weaknesses: The wide range of exposure 

levels and the use of ethinyl estradiol as a positive 

control are strengths. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study may have utility for environmental assessment, but is not useful for human risk assessment. 

Watts et al. (2003), supported by the European Union, 

examined the effects of bisphenol A exposure on 

moulting and mouthpart deformities in non-biting 

midge (Chironomus riparius) larvae. Four egg-ropes/ 

group were incubated in glass jars in media containing 

bisphenol A [purity not indicated] at 0 (ethanol 

vehicle or dechlorinated water group), 0.010, 0.1, 1, 10, 

100, or 1000 mg/L. Concentrations of bisphenol A were 

verified in the 1000 mg/L group. On hatching, 

exposures were continued in 10 larvae/group. Endpoints 

examined included survival, time of moulting to 

successive instars, wet weight 2 days after moulting to 

fourth instar, and mouthpart morphology in fourthinstar head capsules. Statistical analyses included ANOVA, Tukey–Kramer multiple comparison test, and Kruskal–Wallis test. [Effects were similar in ethanol and 

water controls.] Moulting was delayed and larval 

weights were significantly decreased in the 1000 mg/L 

bisphenol A group. Deformities of the mentum were 

significantly increased in the range of 0.010–1 mg/L 

bisphenol A. The effects of ethinyl estradiol were also 

examined, and the study authors noted similar patterns 

of malformations, with greater incidence following 

exposure to ethinyl estradiol than bisphenol A. The 

Birth Defects Research (Part B) 83:157–395, 2008
310 CHAPIN ET AL. 

study authors concluded that exposure to bisphenol A 

delayed moulting and increased mouth part deformities 

at concentrations that were at opposite ends of the 

exposure range. 

Strengths/Weaknesses: This study is similar in its 

strengths to that of Watts et al. (2001). 

Utility (Adequacy) for CERHR Evaluation Process: 

This study may have utility for environmental assessment, but is not useful for human risk assessment. 

3.2.10.2 Frog: Iwamuro et al. (2003), support not 

indicated, conducted a series of studies to examine the 

effects of bisphenol A exposure on development of the 

frog Xenopus laevis. In a study to assess survival and 

morphological abnormalities, 60–100 Stage 7 embryos/ 

group were exposed to bisphenol A [purity not 

indicated] at 0 (ethanol vehicle), 10, 20, 25, 30, 50, or 

100 mM [0, 2.3, 4.6, 5.7, 6.8, 11, or 23 mg/L; culture 

ware not discussed]. Siblings were randomly distributed 

among different treatment groups. Survival was assessed 

at 48, 96, and 120 hr. At least 3 embryos/group were 

examined for malformations at 5–7 days following 

fertilization. Data were analyzed by w2 test. Survival of 

embryos was significantly reduced following exposure 

to Z25 mM [5.7 mg/L] bisphenol A for 96 or 120 hr. 

Complete mortality was observed at concentrations 

Z50 mM [11 mg/L]. The study authors calculated a 

median LD50 for survival of 21 mM [4.8 mg/L]. 

The malformation rate was reported for the 10 and 

25 mM [2.3 and 4.6 mg/L] group, and significant 

increases in malformations occurred in the 25 mM 

[4.6 mg/L] group. The types of malformations were 

reported as scoliosis, swollen head, and shortened 

distance between eyes. The effects of 17b-estradiol were 

also examined. An increase in malformations was 

observed with exposure to 10 mM 17b-estradiol, but there 

was no effect on survival. 

In a second study, metamorphosis was observed in 10– 

12 tadpoles (Stage 52) placed in solutions containing 10 

or 25 mM [2.3 or 5.7 mg/L] bisphenol A [purity not 

indicated] with and without the addition of 0.1 mM 

thyroxin for 21 days. Expression of thyroid hormone 

receptor-b gene was measured by RT-PCR in three 

regions (head, trunk, and tail) of tadpoles that were 

exposed to 10 or 100 mM [2.3 or 23 mg/L] bisphenol A 

with and without the addition of 0.1 mM triiodothyronine 

or thyroxin. Negative controls were exposed to ethanol/ 

DMSO vehicle. Metamorphosis data were analyzed by 

Duncan new multiple range test. Bisphenol A inhibited 

significantly both spontaneous and thyroxine-induced 

metamorphosis. All concentrations of bisphenol A 

reduced expression of thyroid hormone receptor-b

hormone and inhibited increases in thyroxine- and 

triiodothyronine-induced expression. 

In a third study, tails were removed from 4 tadpoles/ 

group and cultured for 4 days in media containing 10 or 

100 mM [2.3 or 23 mg/L] bisphenol A with and without the 

addition of 0.1 mM triiodothyronine. Negative controls 

were exposed to ethanol/DMSO vehicles. Data were 

analyzed by Duncan new multiple range test. Growth of 

the tails was measured over a 4-day period. Neither 

bisphenol A dose significantly affected tail growth. Both 

bisphenol A doses blocked tail shortening that was 

induced by triiodothyronine. The study authors concluded that high-doses of bisphenol A adversely affect 

development of Xenopus laevis embryos and larvae. 

Strengths/Weaknesses: The wide range of exposure 

levels is a strength. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study may have utility for environmental assessment, but is not useful for human risk assessment. 

Oka et al. (2003), support not indicated, examined the 

effects of bisphenol A exposure on development of the 

frog Xenopus laevis. Embryos were exposed to the ethanol 

vehicle or 10–100 mM [2.3–23 mg/L] bisphenol A from 

developmental stage 6 until the early tadpole stage (late 

stage 10) [purity not indicated, and culture ware not 

discussed]. Embryos were harvested at Stages 19, 23, 33/ 

34, and 40 and prepared for histological examination to 

determine the presence of apoptotic cells. Apoptosis was 

also assessed using a TUNEL staining method. Ten 

embryos were killed at the tail bud stage (Stage 35/36, 

37/38, and 40), and genomic DNA was isolated and 

examined by electrophoreses to determine if 180 base 

pair ladders indicative of apoptosis were present. [No 

information was provided on the number of individual 

doses examined or the number of embryos exposed/ 

dose. No quantitative data were presented by authors, 

and it does not appear that data were statistically 

analyzed.] Embryos exposed to 40–100 mM [9.1–23 mg/L] 

bisphenol A died during the gastrula stage. Developmental abnormalities were observed in embryos exposed 

to 20 mM [4.6 mg/L] bisphenol A. The abnormalities 

included open neural tubes at Stage 19, morphological 

defects at Stages 23 and 33/34, and crooked vertebrate, 

swollen abdomen, and malformed head at Stage 40. 

Malformations persisted following Stage 40, and 

death occurred during the tadpole stage. In Stage 33/34 

and 40 embryos of the 20 mM [4.6 mg/L] group, 

apoptotic cells were observed in the prosencephalon, 

mesencephalon, rhombencephalon, and spinal cord. 

Apoptosis was confirmed using the TUNEL staining 

method. Using the DNA ladder method, it was found 

that apoptosis also occurred at Stages 35/36, 37/38, and 

40. The authors briefly stated that they tested Stage 10, 

19, or 23 embryos and found normal development 

following bisphenol A exposure. [No additional details 

were provided.] The effects of 17b-estradiol were also 

examined. Malformations were observed in embryos 

exposed to 10 mM 17b-estradiol, but apoptotic cells were 

not observed in the nervous system. A very brief 

description was provided of a study in which embryos 

were exposed simultaneously to 20 mM [4.6 mg/L] bisphenol A and 1–10 mM 17b-estradiol. Co-exposure with 

17b-estradiol did not inhibit bisphenol A-induced apoptosis. The study authors concluded that bisphenol A 

induced malformations and apoptosis in Xenopus laevis at 

concentrations exceeding environmental levels and that 

the effects did not appear to occur through an estrogenic 

mechanism. 

Strengths/Weaknesses: The use of 17b-estradiol exposure to suggest a non-estrogenic mechanism of bisphenol A toxicity is a strength. The omission of some 

important details and the high concentrations are 

weaknesses. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study may have utility for environmental assessment, but is not useful for human risk assessment. 

Sone et al. (2004), supported by the Japanese Ministry 

of Environment and Ministry of Education, Culture, 

Sports, Science, and Technology, examined the effects of 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 311 

bisphenol A exposure on the development of Xenopus

laevis embryos. Three different sets of experiments were 

conducted. Data were analyzed by ANOVA followed by 

Fisher protected least significant difference test. From 3– 

96 hr following fertilization, embryos were exposed to 

bisphenol A [purity not indicated] at 1, 2.5, 5, 10, 15, 20, 

25, or 30 mM (0.3, 0.6, 1.1, 2.3, 3.4, 4.6, 5.7, or 6.8 mg/L). 

Each exposure was replicated 3 times. Negative control 

groups consisted of the ethanol vehicle, medium alone, 

or dilution medium. Rates of normal embryo development were equivalent in the 3 different negative control 

groups. In groups exposed to Z20 mM bisphenol A, there 

was a significant decrease in normal embryos and a nonsignificant increase in mortality rate. Teratogenicity was 

characterized by short body length, microcephaly, flexure, edema, and abnormal gut coiling. Increases in 

embryo abnormalities were also observed following 

exposure to Z10 mM 17b-estradiol or nonylphenol. 

To determine sensitive stages, embryos were exposed 

to control media or 20 mM [4.6 mg/L] bisphenol A for 45– 

48-hour periods ranging from 3–48 hr post-fertilization, 

12–60 hr post-fertilization, 24–72 hr post-fertilization, 36– 

84 hr post-fertilization, or 48–96 hr post-fertilization. 

Body length, gross malformations, and distance between 

eyes were measured at 96 hr following exposure. [The 

Methods section indicated that 59–71 embryos were 

examined in the bisphenol A group for each time 

period of exposure. However, a figure in the study 

reported the sample size as 3/time period.] During the 

period of 3–48 hr following fertilization, statistically 

significant effects in the bisphenol A group included 

decreased body length and increased incidences of 

microcephaly, flexure, edema, and abnormal gut coiling. 

No increases in abnormal effects were observed following exposure at later time periods. Abnormalities were 

observed following exposure to 17b-estradiol or nonylphenol at early or late stages. 

In the third part of the study, embryos were exposed to 

20 mM [4.6 mg/L] bisphenol A from 3–96 hr following 

fertilization. RNA was isolated from whole embryos and 

subjected to analysis by cDNA microarray. Results 

obtained in microarray analyses were confirmed by 

PCR analysis. The sample size was reported as 2. The 

microarray analysis revealed 179 upregulated and 103 

downregulated genes following exposure of embryos to 

bisphenol A. The study authors identified 27 genes in 

which expression was changed following exposure to 

bisphenol A, nonylphenol, or 17b-estradiol. The identified genes included: KNP-Ia, CmaB, XIRG, a-skeletal 

tropomyosin, apelin, cyclin G1, Ube213, HGF, toponin C2, 

ribosomal protein L9, and Rattus norvegicus similar to 

CG10042-PA. The other genes were not identified. The 

study authors concluded that these findings might 

provide clues to deciphering mechanisms of teratogenic 

effects associated with bisphenol A and the other 

compounds examined in this study. 

Strengths/Weaknesses: The inclusion of 17b-estradiol 

as a comparator was a strength and the high bisphenol A 

concentration is a weakness. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful for the evaluation process. 

Pickford et al. (2003), supported by the Bisphenol A 

Global Industry group, the Society of the Plastics 

Industry, the Bisphenol A Sector Group of the European 

Chemical Industry Council, and the Japan Chemical 

Industry Association, examined the effects of bisphenol 

A exposure on development of frog gonads. Beginning at 

Stage 43/45 (B2 days post-hatching, 4 days postfertilization, exposure day 0) and continuing through 

Stage 66, Xenopus laevis larvae were exposed to bisphenol 

A [purity not indicated] at nominal concentrations of 0 

(water control), 1.0, 2.3, 10, 23, 100, or 500 mg/L in a flowthrough test system [culture ware not discussed]. Actual 

concentrations were verified as 0.83, 2.1, 9.5, 23.8, 100, 

and 497 mg/L. A positive control group was exposed to 

2.7 mg/L 17b-estradiol. There were 4 replicate test 

vessels/dose, with each containing 40 larvae (i.e., 160 

larvae/test condition). Larvae were observed daily for 

mortality, behavior, and appearance. Growth and development were assessed on all larvae of a replicate tank on 

exposure days 32 and 62 (36 and 68 [66?] days postfertilization). Froglets were killed and observed at 

completion of metamorphosis (Stage 66). Total length 

was measured, sex was determined, and testes and 

ovaries were assessed for abnormalities such as asymmetry, complete absence, presence of melanocytes, 

irregular shape, segmentation or fragmentation, vacuoles, and ambiguous sexual morphology. Data were 

analyzed by Fisher exact test, ANOVA, Wilcoxon rank 

sum test, G test, and w2 test. Following exposure to 

bisphenol A, there were no significant differences in 

survival, distribution of developmental stages on Day 32 

or 62, time to completion of metamorphosis (Stage 66), or 

length of Stage 66 froglets. Bisphenol A exposure 

did not affect sex ratio or abnormalities in testis or ovary 

[data were not shown by authors for testis and 

ovary effects]. In contrast, exposure to 17b-estradiol 

resulted in an increase in ratio of females to males and 

testicular and ovarian abnormalities. The study authors 

identified a no-observed-effect concentration of 500 mg/L 

for bisphenol A. 

Strengths/Weaknesses: The use of a wide range of 

exposure levels is a strength, but the incomplete data 

presentation with missing organ weight data and the lack 

of histological evaluations are weaknesses. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study may have utility for environmental assessment, but is not useful for human risk assessment. 

Levy et al. (2004), supported by the Ministry of 

Environment and Traffic of Baden-Wu ̈rttemberg, evaluated the effect of bisphenol A on gonad development in 

Xenopus laevis tadpoles. Tadpoles (n 5 40/group) were 

exposed beginning at Stages 42/43 to ethanol vehicle or 

to bisphenol A (499% purity) or 17b-estradiol, both at 

concentrations of 10 -8 or 10 -7 M [bisphenol A concentrations 2.3 and 23 lg/L. Actual concentrations were 

90–105% of target concentrations after addition of 

bisphenol A to the media but decreased to low levels 

by the end of the 48-hr period between media changes. 

Culture ware was not discussed.] After completion of 

metamorphosis, froglets were killed for examination of 

gonads. Tadpoles not completing metamorphosis were 

killed after 120 days of chemical exposure for examination of gonads. In a second experiment, bisphenol A 

concentrations were 10 -8

, 10 -7, or 10 -6 M [2.3, 23, or 

228 lg/L] and the 17b-estradiol positive control used a 

concentration of 10 -7 M. In a third experiment, 50 

tadpoles/group were treated for 2 weeks with ethanol 

vehicle, bisphenol A 10 -7 M [23 lg/L], or 10 -7 M 17bestradiol after which whole-body homogenates were 

Birth Defects Research (Part B) 83:157–395, 2008
312 CHAPIN ET AL. 

used for extraction of RNA and determination of ER by 

RT-PCR. Statistical analyses were performed with Kruskal–Wallis H test followed by Mann–Whitney U test. The 

gonadal sex of control animals was 56% male and 44% 

female. 17b-Estradiol treatment increased the female 

ratio to 81% at 10 -7 M and 84% at 10 -8 M. Bisphenol A 

treatment resulted in a significant increase in females 

(69%) at 10 -7 M [23 lg/L]. At 10 -8 M bisphenol A, there 

were 65% females, which did not reach statistical 

significance. In the second experiment, a significant 

increase in females was seen after treatment with 10 -7 M 

[23 lg/L] (70%, compared to 48% in controls and 96% 

with 17b-estradiol treatment). There was no significant 

effect of bisphenol A at 10 -8 M [2.3 lg/L] (51% female) or 

10 -6 M [228 lg/L] (53% female). Bisphenol A and 17bestradiol both resulted in increased ER mRNA. The 

authors concluded that bisphenol A affects the sexual 

development of Xenopus laevis, probably through an 

estrogenic mechanism. 

Strengths/Weaknesses: The measurement of bisphenol 

A in the media is a strength, but its lack of stability is a 

weakness. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful for the evaluation process. 

Yang et al. (2005), supported by the Chinese Ministry 

of Science and Technology, examined the effects of 

bisphenol A exposure in black-spotted pond frog tadpoles. Thirty tadpoles/tank were exposed in duplicate 

to bisphenol A (Z95% purity) at concentrations of 0, 0 

(1DMSO vehicle), 2, 20, or 200 mg/L [ppb] for up to 60 

days [culture ware not discussed]. Tadpoles were 

also exposed to mixtures containing bisphenol A1

nonylphenol at 212, 20120, or 2001200 mg/L. 

Additional tadpoles were exposed to mixtures containing 

the same bisphenol A/nonylphenol mixtures in addition 

to p,p0

-DDE 21210.5, 2012015, or 2001200150 mg/L. 

Five tadpoles/tank were pooled at 15, 30, 45, and 60 

days. The tadpoles were homogenized for measurement 

of testosterone and thyroxin levels by radioimmunoassay. Alkaline-labile phosphate was measured as a 

biomarker for vitellogenin. Data were analyzed by 

ANOVA. 

Malformations of tail flexure were observed in 10% of 

tadpoles exposed to 200 mg/L bisphenol for 45 days, and 

similar rates of malformation (13.3%) were observed in 

the mixtures containing 200 mg/L bisphenol A. A 

‘‘decrease’’ (not statistically significant) in thyroxine 

levels was observed following 60 days of exposure to 

all bisphenol A doses (Z2 mg/L). ‘‘Increases’’ (not 

statistically significant) in testosterone levels were 

reported with all bisphenol A doses at 30 days of 

exposure. p,p0

-DDE at Z5 mg/L inhibited increases in 

testosterone level observed with mixtures of bisphenol A 

and nonylphenol [not statistically analyzed]. ‘‘Increases’’ (not statistically significant) in alkaline-labile 

phosphate levels were reported following 30 or more 

days of exposure to all bisphenol A doses. In animals 

exposed to bisphenol A and nonylphenol in combination 

compared to either compound alone, alkaline-labile 

phosphate levels were increased at 15 days of exposure 

but decreased at 60 days of exposure [not statistically 

analyzed]. p,p0

-DDE inhibited the increase in alkalinelabile phosphate levels induced by the bisphenol A1

nonylphenol mixture on Day 15 of exposure [not 

statistically analyzed]. 

Strengths/Weaknesses: The lack of attention to statistical analysis is a weakness and makes the authors’ 

conclusions unreliable. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful in the evaluation process. 

Imaoka et al. (2007), supported by the Japanese 

Ministry of Education, Science, Culture, Sports, and 

Technology, evaluated the effects of bisphenol A on 

development of the African clawed frog, Xenopus laevis. 

Embryos were cultured with bisphenol A from Stage 

10.5, formation of the neural plate, to Stage 35 at a 

bisphenol A (in DMSO) concentration of 25, 50, or 100 mM 

[5.8, 11, or 23 mg/L]. Tadpoles were morphologically 

evaluated at Stages 28–35. Total RNA was extracted and 

reversed transcribed and RT-PCR used to quantify the 

expression of specific genes. Expression levels relative to 

b-actin or histone H4 were compared with Student t-test. 

Abnormalities in the head and eye region were described 

with a ‘‘minor effect’’ at 25 mM and a ‘‘major effect’’ at 

50 mM bisphenol A. [Data were not shown.] There were 

no treatment-related effects on expression of sox-2, nrp-1, 

myoD, sox17a, or notch. Relative expression levels of pax6 declined in a concentration-related manner to about 

56% if control at the high concentration [estimated from 

a graph]. Relative expression levels of esr-1 decreased in 

a concentration-dependent manner to about 22% of 

control at the high concentration [estimated from a 

graph]. Microinjection into blastomeres of plasmids 

containing NICD (the intracellular domain of notch), 

but not of X-delta-1 (a notch ligand) corrected the 

decreased expression of esr-1. The authors concluded 

that bisphenol A decreased esr-1 expression by disrupting notch signaling. 

Strengths/Weaknesses: This is an interesting study on 

the molecular alterations induced in frog embryos 

exposed to BPA. The study demonstrated alterations in 

several key developmental genes and malformed development at high concentrations. The high concentrations 

are, however, weaknesses and the effects of uncertain 

concern to human health because humans would not be 

exposed in this manner. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful in the evaluation process. 

3.2.10.3 Fish: Kishida et al. (2001), supported by the 

National Science Foundation and U.S. EPA, included 

bisphenol A in a study to test the utility of changes in 

CYP450 aromatase mRNA expression as a marker of 

xenoestrogen effects in the CNS of zebrafish (Danio rerio). 

Fish embryos were incubated in solutions containing 

bisphenol A [purity not indicated] at 0 (DMSO vehicle), 

0.01, 0.1, or 10 mM [0, 2.3, 23, or 228 lg/L] from 2–48 hr 

post-fertilization [culture ware not discussed]. Expression of the CYP450 aromatase gene was determined in 50 

embryos/treatment group using an RT-PCR/Southern 

blot technique. [There was no mention of statistical 

analyses of data.] The Southern blot analysis revealed a 

B3-fold increase in the band intensity of CYP450 

aromatase at the high concentration (10 mM) of bisphenol 

A. The potency of bisphenol A was determined to be 

lower than those of 17b-estradiol and diethylstilbestrol, 

which induced B3–4-fold increases in band intensity at 

concentrations up to 3 orders of magnitude lower than 

bisphenol A. In additional experiments with exposure to 

bisphenol at 2–48 hr post-fertilization, embryo mortality 

was increased by exposure to 10 and 20 mM [228 and 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 313 

457 lg/L] bisphenol A and malformations (curved tails) 

were increased by exposure to 20 mM. The effects were 

similar to those observed with 17b-estradiol, but bisphenol A was less potent. [Very few protocol details 

were provided, and no data were shown by study 

authors for mortality and malformation endpoints.] The 

study authors concluded that bisphenol A could act as a 

developmental neurotoxicant by upregulating CYP450 

aromatase expression but that further studies were 

needed to determine if there are changes in neural 

estrogen biosynthesis or CNS development. 

Strengths/Weaknesses: A weakness of this study for 

the current evaluation is the lack of morphometric data. 

The significance of the observed change in aromatase is 

not clear. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful in the evaluation process. 

Segner et al. (2003), supported by the European 

Commission, examined estrogenicity responses and in 

vivo life cycle effects in zebrafish exposed to bisphenol 

A. Estrogenicity studies are discussed in Section 2. One 

hundred fertilized eggs/vessel were exposed to bisphenol A (98% purity) at 0, 94, 188, 375, 750, or 

1500 mg/L under semistatic conditions [culture ware 

not discussed]. Exposures were continued until fish 

became sexually mature. The numbers of fish/vessel 

were adjusted to 50 following 42 days of exposure and 30 

following 75–78 days of exposure. Two replicates were 

examined. Bisphenol A concentrations were confirmed 

by GC/MS. Endpoints evaluated included survival, 

behavior, growth, time to first spawning, egg production, 

and fertilization success (percent fertilized eggs/vessel/ 

day). Statistical analyses included ANOVA and William 

test. EC50-values were calculated by probit analysis and 

analyzed by Kruskal–Wallis and Mann–Whitney U tests. 

17b-Estradiol, ethinyl estradiol, and 4-tert-octylphenol 

were also examined using similar protocols. The authors 

only discussed results for reproductive success because 

they stated that it was the most consistent and reproducible effect following exposure of the fish to estrogenic 

substances. An EC50-value of 6140 nM [1.4 mg/L] bisphenol A was obtained for fertilization success, and the 

study authors stated that the value exceeded concentrations typically found in the environment. Bisphenol A had 

a relative potency of 0.0000006 compared to17b-estradiol 

and was 45 times less potent than 4-tert-octyl-phenol. The 

study authors concluded that the in vivo potency of the 

compounds was overestimated by in vitro estrogenicity 

assays (described in Section 2). 

Strengths/Weaknesses: This study was well 

performed. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is useful in showing a lack of effect on 

fertilization at environmentally relevant concentrations 

of bisphenol A, but not useful to the evaluation process. 

Metcalfe et al. (2001), supported by the Environmental 

Science and Technology Alliance Canada, the Natural 

Sciences and Engineering Research Council of Canada, 

and Health Canada, in glass jars, exposed medaka 

(Oryzias latipes) from 1 day after hatching until 85–110 

days after hatching to bisphenol A [purity not indicated] 

at 0, 10, 50, 100, or 200 mg/L (n 5 60 fish/treatment). Over 

the 48 hr between media change, actual concentrations 

were a mean 59.6% of nominal concentrations. Fish were 

killed and embedded in paraffin for section. Gonads 

were evaluated to determine the sex of the fish and 

whether testes contained ova, an intersex condition. 

Length and weight of the animals and sex ratio were not 

altered by treatment [statistical methods not reported]. 

There were 2 instances of intersex gonads in males 

exposed to bisphenol A 10 mg/L and no instances at 

higher concentrations. Histologic changes in testes 

including a reduction in germ cells were noted at 

50 mg/L and higher. At 200 mg/L, oogenesis in females 

was more advanced than in controls. 

Strengths/Weaknesses: Strengths of this study are the 

step-sectioning of gonads and the use of several positive 

control estrogens, which worked as expected. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful to the evaluation process. 

Yokota et al. (2000), supported by the Japanese 

Environment Agency, exposed medaka (Oryzias latipes) 

to bisphenol A (499% purity) at 0, 3.2, 16, 80, 400, or 

2000 mg/L from fertilization until 60 days after hatching 

(n 5 60/treatment) [culture ware not discussed]. Actual 

bisphenol A concentrations were generally within 3% of 

nominal concentrations before hatching. After hatching, 

the lower 2 concentrations were B70–80% of nominal 

and the higher concentrations were B90% of nominal. 

Fish were assessed for survival, time to hatching, and 

growth. Sixty days after hatching, 19 or 20 fish/treatment 

were killed and sectioned for examination of the gonads 

using hematoxylin and eosin staining of fixed specimens. Statistical analysis was performed using ANOVA 

and nonlinear regression. Hatchability was 490% in all 

treatment groups. Time to hatch and mortality were not 

affected by treatment, although there was a nonconcentration dependent delay in hatching at 13 mg/L. 

Body length and weight 60 days after hatching were 

negatively correlated with bisphenol A concentration, 

and length and weight at 2000 mg/L were significantly 

lower than control values on pair-wise comparison. 

Based on external appearance and gonad examination, 

there were more females than males at 400 mg/L and 

there were no males at 2000 mg/L. Control sex ratio was 

2:1 (male:female). There were 6 fish with intersex gonads 

among the 19 examined in the 2000 mg/L group. The 

authors concluded that bisphenol A adversely affects the 

early life stage of medaka with alteration of sexual 

differentiation. 

Strengths/Weaknesses: This study was well 

performed. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful to the evaluation process. 

Pastva et al. (2001), support not indicated, examined 

the effects of bisphenol A exposure on development of 

medaka (Oryzias latipes). In a study examining abnormalities in embryos, 5 eggs were placed in individual glass 

vials containing bisphenol A [purity not indicated] at 0, 

20, or 200 mg/L. There were 5 glass vials/exposure 

concentration, for a total of 25 embryos/group. The 

exposure period began 5 hr following fertilization and 

was continued for 9 days. Embryos were examined for 

malformations daily by observing them through the clear 

protective membrane of the egg. The severity of 

malformations was scored and severity indices were 

determined. In a second study examining mortality, 

newly hatched larvae were exposed for 96 hr to a method 

control solution, ethanol vehicle control solution, or 

200 mg/L bisphenol A. Ten larvae were added to each 

Birth Defects Research (Part B) 83:157–395, 2008
314 CHAPIN ET AL. 

jar, and there were 3 replicates/test solution (i.e., 30 

larvae/concentration). Data were analyzed by t-test. The 

malformation severity index was increased significantly 

at 5–8 days following fertilization in embryos exposed to 

200 mg/L bisphenol A, but the severity index did differ 

significantly from the control value on Day 9. Abnormalities consisted of pericardial edema, hemorrhage, and 

hemostasis. Larval mortality was not affected by 

exposure to 200 mg/L bisphenol A. The study authors 

concluded that exposure to environmentally relevant 

concentrations of bisphenol A resulted in embryonic 

deformities in medaka, but that the embryos were able to 

repair the abnormalities before hatching. 

Strengths/Weaknesses: This study using medaka is 

similar in design to the FETAX assay, which uses 

Xenopus. These types of assays have not been demonstrated to have relevance for human risk assessment. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful in the evaluation process. 

Lee et al. (2003b), supported by Jeonnam Regional 

Environment Technology Development Center, exposed 

51-day-old Korean rockfish (Sebastes schlegeli) fry to 

bisphenol A in feed at 0, 0.05, 0.5, 5, 50, and 100 mg/kg 

diet for 29 days [purity of bisphenol A, stability in feed, 

and culture ware not indicated]. At the end of the 

experiment, gonads were removed and sex determined 

by light microscopy of stained sections. There was no 

effect of bisphenol A on sex ratio compared to controls. 

[The data presentation and statistical analysis are 

unclear: the number of female fish and number of 

male fish in each dose group are presented as averages 

with an unspecified error and analyzed by Student ttest. Whole numbers would have been expected with v2 

analysis.] The authors concluded that there was no 

estrogenic effect of bisphenol A on sex differentiation in 

the Korean rockfish. 

Strengths/Weaknesses: The use of a positive control, 

which worked as expected, is a strength of this study. The 

inadequate presentation of data and statistical analysis is 

a weakness. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful in the evaluation process. 

Honkanen et al. (2004), supported by the Finnish 

Graduate School of Environmental Science and Technology and the Academy of Finland, examined the effects of 

bisphenol A exposure on yolk-sac fry of landlocked 

salmon. Ten 8-day-old fry/beaker were exposed to 

bisphenol A [99% pure] at concentrations of 0, 10, 100, 

or 1000 mg/L for 42 days, in glass beakers. The ethanol 

vehicle and pure tap water were used as negative 

controls. There were 3–4 replicates/dose. One fry/beaker 

was photographed and killed following 6 days of 

exposure. After 6 weeks of exposure, all remaining fry 

were blotted and weighed. Three fry/beaker were 

photographed and 3 fry/beaker were examined 

histologically. Statistical analyses included ANOVA and 

Tukey test. Effects observed in fry exposed to the 

highest bisphenol A concentration included: yolk sac 

edema and hemorrhaging around gill arches and the 

front part of the yolk sac at 6 days of exposure; 

phlegmatic behavior (lack of activity during 

siphoning to renew solutions) on Day 8 of exposure; 

and darkening of color at Day 17 of exposure. No 

increases in mortality were observed. At the end of the 

exposure period, wet weights were increased in fry 

exposed to the highest concentration, and the 

study authors stated that the effect was due to fluid 

accumulation. In fry exposed to the mid- and highconcentration of bisphenol A, strongly stained fragments 

were observed in nuclei and storage substances in liver 

were decreased. No abnormalities were observed in 

histological examinations of heart, kidney, and thyroid 

gland. The study authors concluded that bisphenol A 

induced toxicity in fry at concentrations rarely found in 

the environment. 

Strengths/Weaknesses: The range of concentrations 

used in this study is a strength. 

Utility (Adequacy) for CERHR Evaluation Process: 

The finding of an effect only at a high concentration of 

bisphenol A may have importance for environmental 

assessments but is not of utility in the current evaluation 

process. 

3.2.10.4 Reptile and bird: Stoker et al. (2003), 

supported by the Argentine National Agency for the 

Promotion of Science and Technology and Argentina 

Ministry of Health, examined the effects of in ovo 

bisphenol A exposure on sexual development of the 

crocodilian reptile Caiman latirostris. A preliminary 

experiment was conducted to determine the effects of 

temperature on sex determination, and it was established 

that incubation at 301C resulted in production of females 

while incubation at 331C resulted in the production of 

males. In the main experiment, eggs were collected from 

5 nests in Argentina. Half the eggs were incubated at 

301C and the other half at 331C. Care was taken to avoid 

exposing eggs to putative sources of estrogens such as 

spray paint, plastic, and nesting materials. At each 

incubation temperature, eggs from each nest were 

distributed among treatment groups. Twenty days 

following collection, 1 egg/nest/incubation temperature 

was opened for stage determination. At developmental 

stage 20, bisphenol A [purity not indicated] was 

applied topically to the eggshell at concentrations of 1.4 

or 140 ppm (0.09 or 9 mg/egg). Other eggs were 

treated with 0.014 or 1.4 ppm 17b-estradiol. Control eggs 

were left untreated or exposed to the ethanol vehicle. 

Hatchlings were weighed and measured at birth. 

At 10 days of age, 4 animals/group/incubation temperature were killed for determination of sex by 

examination of internal genitalia. Sex determination 

was confirmed by histological evaluation of organs, 

which were fixed in 10% buffered formalin. Morphometric analysis of seminiferous tubules was also conducted in 10-day-old animals. The remaining animals (6– 

11/group/incubation temperature) were raised until 6 

months of age, at which time they were killed, measured, 

and sexed by examination of external genitalia. Evaluators were blinded to treatment conditions. Statistical 

analyses included Kruskal–Wallis ANOVA and Mann– 

Whitney U test. 

At 331C, there was 100% sex reversal in the high-dose 

bisphenol A and high-dose 17b-estradiol groups at 10 

days and 6 months of age. Whereas 100% of control and 

low-dose animals in the 331C group were male, 100% of 

animals in the high-dose bisphenol A and 17b-estradiol 

group were female. Although there was no sex reversal 

in the low-dose bisphenol A or 17b-estradiol groups 

incubated at 331C, morphometric evaluations at 10 days 

of age revealed significantly increased perimeter of 

seminiferous tubules, which had empty lumens. There 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 315 

were no significant effects reported for bisphenol A 

following incubation at 301C. The study authors concluded that bisphenol A induced estrogenic effects in 

caiman as evidenced by reversed gonadal sex and 

disrupted gonadal histoarchitecture. 

Strengths/Weaknesses: This study appears to have 

been well performed and the use of a positive control is a 

strength. A weakness is the expression of exposure level 

in terms of total egg weight, which precludes easy 

comparison to human exposure levels. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study has no utility in the evaluation process. 

Berg et al. (2001), supported by the Foundation for 

Strategic Environmental Research and the Swedish 

Council for Forestry and Agricultural Research, examined the effects of bisphenol A exposure on development 

of sex organs in quail and chicken embryos. The effects 

of tetrabromobisphenol A were also examined but will 

not be discussed. Bisphenol A (99.4% purity) was 

injected into yolk of Japanese quail eggs on the third 

day of incubation and into chicken (domestic fowl) eggs 

on the fourth day of incubation at doses of 0 (propylene 

glycol vehicle), 67, and 200 mg/g egg. Eggs were also 

injected with diethylstilbestrol at doses of 2, 20, and 

200 ng/g egg [culture ware not discussed]. Two days 

before the anticipated hatching date, embryos were 

examined for mortality (32–43 quail embryos and 34–91 

chicken embryos/group examined) and mu ̈ llerian duct 

abnormality or testicular histopathology (8–15 quail 

embryos/group and 7–30 chicken embryos/group examined). Testes were fixed in 4% formalin. Data were 

analyzed by Fisher exact probability test. 

Exposure to bisphenol A did not increase mortality in 

quail embryos. Incidence of females with abnormal 

mu ̈ llerian ducts was increased in quail embryos exposed 

to the high bisphenol A dose but the incidence of 

ovotestis in males was not increased by bisphenol A 

exposure. Mortality of chicken embryos was increased 

following exposure to both bisphenol A dose levels. The 

incidence of male chicken embryos with ovotestis was 

increased at the high-dose of bisphenol A but there was 

no effect on females with abnormal mu ̈llerian ducts. 

Effects observed in one or more diethylstilbestrol groups 

included increased incidence of females with abnormal 

mu ̈ llerian ducts in quail embryos and males with 

ovotestis in quail and chicken embryos. Based on study 

findings, the study authors concluded that bisphenol A 

can cause estrogen-like malformations in reproductive 

organs of birds. 

Strengths/Weaknesses: The detailed evaluation of 

genital tract morphology is a strength, but the expression 

of exposure level in mg per g egg makes it difficult to 

compare to human exposure levels. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful to the evaluation process. 

Halldin et al. (2001, 2005), supported by the European 

Union and numerous Swedish agencies, examined the 

effect of in ovo exposure to bisphenol A on sexual 

behavior of male Japanese quail. On Day 3 of incubation, 

the yolks of an unspecified number of quail eggs were 

injected with vehicle (emulsion of peanut oil, lecithin, 

and propylene glycol) or Bisphenol A (499% purity) 

at 67 or 200 mg/g egg, and eggs were incubated at 37.51C 

at 60% relative humidity. After hatching, male and 

female chicks were housed together. Males were 

individually housed at 7 weeks of age. At 9 weeks of 

age, 17 control and 4–7 treated males/group were 

examined for sexual behavior. Behavior with a sexually 

receptive female was evaluated by observing actions 

such as neck grab, mount attempt, mounts, and cloacal 

contact movement. Testing was conducted for 2 min/day 

over 5 consecutive days. At the completion of testing, 

testis weight was measured, gonado-somatic index was 

determined, and plasma testosterone levels were measured by RIA. Females exposed in ovo (n 5 5–8/group) 

were evaluated for numbers of eggs laid over 5 days and 

oviduct morphology. Statistical analyses included Kruskal–Wallis test, or w2 test for trend. No effects of 

bisphenol A exposure were reported for any of the 

effects examined including sexual behavior of males, 

testicular weight, gonado-somatic index in males, plasma 

testosterone levels, or numbers of eggs produced. 

Numbers of females with retained right oviduct were 

increased in the bisphenol A groups (2 of 5 and 4 of 7 in 

each respective bisphenol A group vs. 1 of 8 in controls) 

but the effect did not achieve statistical significance. 

Sexual behavior was reportedly affected at an ethinyl 

estradiol dose of 0.006 mg/g egg and diethylstilbestrol 

doses of 0.019 and 0.057 mg/g egg. The study authors 

concluded that, with the possible exception of a trend for 

retained right oviduct in females exposed to 200 mg/g 

egg, bisphenol A was not shown to affect any of the 

endpoints examined in Japanese quail, which were 

demonstrated to be a well-suited model for studying 

effects of estrogenic compounds. 

Strengths/Weaknesses: The use of 2 positive controls 

and the attention to sexual behavior are strengths. 

Weaknesses are the expression of exposure level in mg 

per g egg, making it difficult to compare to human 

exposure levels, the lack of detail in the reporting of 

methods and results, and the lack of apparent statistical 

analysis. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful to the evaluation process. 

Panzica et al. (2005), supported by the University of 

Torino and Region Piemonte, conducted a study that 

intended to examine the effects of in ovo bisphenol A 

exposure on the vasotocin system and sexual behavior of 

Japanese quail. In 2 sets of experiments, quail eggs were 

injected with bisphenol A [purity not indicated] at 50, 

100, or 200 mg/egg following 3 days of incubation 

[culture ware not discussed]. Exposure to bisphenol A 

resulted in a dramatic decrease in the number of live 

chicks hatching (8–11% vs. 55–60% in controls). Chicks 

that hatched survived less than a week. Dissection of 

non-hatched embryos indicated that development was 

blocked immediately following injection in most embryos. A high rate of malformations was observed in 

chicks that died following hatching. [No further information was presented for methods, and no data were 

presented for individual doses.] 

Strengths/Weaknesses: Weaknesses are the expression 

of exposure level in mg per g egg, making it difficult to 

compare to human exposure levels, and the lack of data 

presentation. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful in the evaluation process. 

Furuya et al. (2002), supported by the Japanese 

Ministry of Education, Science, Sports, and Culture, 

examined the effects of bisphenol A exposure on growth 

Birth Defects Research (Part B) 83:157–395, 2008
316 CHAPIN ET AL. 

of testes and combs of male chickens. Beginning at 2 

weeks of age, male white Leghorn chicks were orally 

dosed weekly with corn oil vehicle (n 5 5) or 200 mg 

bisphenol A [purity not indicated] (n 5 12). [The 

specific method of oral dosing was not reported. It is 

assumed that birds were dosed until they were killed.] 

Chickens were killed at 16 weeks of age. Combs and 

testes were weighed. Testes were fixed in 4% paraformaldehyde and examined histologically. [Statistical 

methods were not discussed, and the levels of statistical significance were not reported.] Bisphenol treatment did not affect body weight, but comb and testis 

weight were significantly lower in the chickens exposed 

to bisphenol A. Spermatogenesis was disturbed in the 

chickens of the bisphenol A group, as observed by small 

seminiferous lumen and scarcity of spermatids and 

mature sperm. Diameter of seminiferous tubules and 

incidence of seminiferous tubules with mature sperm 

were significantly lower in the bisphenol A group. The 

study authors concluded that bisphenol A might disturb 

the growth of comb and testes in male chickens, possibly 

through an endocrine mechanism. 

Strengths/Weaknesses: The study of male puberty in 

chickens is a strength. Weaknesses are the use of a single 

dose level and the lack of information on dosing and 

statistical analysis. The study would have been strengthened by measurement of hormone levels. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful to the evaluation process. 

Sashihara et al. (2001), supported by the Japan Ministry 

of Education, Science, and Culture and the Uehara 

Memorial Foundation, examined the effects of early life 

exposure to bisphenol A on growth and behavior in male 

chicks. Layer type (Julia) chicks were obtained from a local 

hatchery, housed in windowless rooms [no further 

housing details provided], given ad lib access to water 

and feed (Toyohashi Feed and Mills Co.), and provided 

continuous lighting. Birds were group housed based on 

weight. At 4 days of age, 0, 100, or 200 mg of bisphenol A 

[purity not given] dissolved in 10% ethanol and sesame 

oil, was injected into the brain (n 5 12 or 13 per group). 

Chicks were followed for growth up to 20 days after 

treatment. A subset of 7 chicks/group was used for 

behavioral testing 8 days after treatment. Birds were 

placed under isolation distress condition and for a 5-min 

period were observed in a cage for motor activity and 

vocalization. At 20 days of age, birds were killed and liver, 

kidney, testis, and brain were weighed. Statistical analyses 

were performed using ANOVA and Duncan multiple 

range tests. 

There were no treatment effects on food intake 6 hr 

after injection or on body weight gain measured 3 days 

after exposure. In the behavioral test, there were no 

treatment effects on jumping, locomotor activity, and 

duration of crouching. There was a statistically significant dose-dependent increase in the frequency of distress 

vocalizations. There were no treatment effects at 20 days 

on body or organ weights. The authors concluded that an 

acute early life exposure of the chick brain to 100 or 

200 mg bisphenol A may affect stress-induced behavior, 

which may involve an estrogen-mediated pathway. 

Strengths/Weaknesses: The rationale for the selection 

of the test animal and dosing procedures are not 

provided. Given that acute doses were injected directly 

into the brain, specific rationale for the method and 

selection of dose are critical to understanding the 

relevance of the study to human health or to wildlife or 

livestock concerns. This provides a vacuum for the 

interpretation of the dose-related increase in vocalizations that were reported. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful to the evaluation process. 

Furuya et al. (2006), supported by the Japanese 

Ministry of Education, Science, Sports, and Culture, 

examined the effects of bisphenol A exposure on 

development of male chicks. Beginning at 2 weeks of 

age, male white Leghorn chicks were orally dosed every 

2 days with bisphenol A at 0 (alcohol/corn oil vehicle) 

0.002, 0.020, 0.200, 2, or 200 mg/kg bw. The high-dose 

level was considered to be a positive control based on 

previous observations in the laboratory. [No information 

was provided about the specific method of oral dosing, 

number of birds treated, purity of bisphenol A, or the 

type of feed or caging and bedding materials used. It 

was implied but not clearly stated that exposures were 

continued until the birds were killed.] The birds were 

killed at 5, 10, 15, 20, and 25 weeks of age. The comb, 

wattle, and testes were weighed. Part of the testicular 

tissue was used to isolate mRNA for evaluation of ERa

and aromatase expression by RT-PCR. Additional testicular tissue was fixed in 10% buffered formalin for 

histopathology analysis and assessment of spermatogenesis by using immunohistochemistry techniques to 

measure proliferating cell nuclear antigen levels. [Methods for statistical analyses were not reported.] 

Although responses were not dose-related, significant 

decreases in weight (doses at which effects were 

observed) were reported for comb and wattle at 10 

weeks of age (Z0.002 mg/kg bw), testis at 10 weeks of 

age (200 mg/kg bw), comb and testis at 15 weeks of age 

(Z0.020 mg/kg bw), wattle at 15 weeks of age (Z

0.2 mg/kg bw), comb at 20 weeks of age (Z0.200 mg/ 

kg bw), testis at 20 weeks of age (200 mg/kg bw), and 

comb and testis at 25 weeks of age (200 mg/kg bw). 

There were no effects on body weight. Histopathological 

observations in testis (doses at which effects were 

observed) included significant and dose-related reductions in the number of spermatogonia at 5 weeks of age 

(Z 2 mg/kg bw) and number of spermatogonia, spermatocytes, and spermatids at 10–25 weeks of age 

(Z0.02 mg/kg bw, except for decreases in spermatocytes 

at 10 weeks of age, which occurred at Z0.200 mg/kg 

bw). Seminiferous tubule diameter was significantly 

reduced at all ages in groups exposed to Z0.020 mg/kg 

bw. Significant and dose-related reductions in testicular 

proliferating cell nuclear antigen levels were observed at 

Z0.200 mg/kg bw at 10 weeks of age and Z0.020 mg/kg 

bw at 15–25 weeks of age. ERa mRNA was significantly 

increased according to dose (doses at which effects were 

observed) at 10 weeks of age (Z0.020 mg/kg bw), 15 and 

20 weeks of age (Z0.200 mg/kg bw/day), and 25 weeks 

of age (200 mg/kg bw). Significant and dose-related 

increases were also observed for aromatase mRNA 

expression (doses at which effects were observed) at 5 

weeks of age (Z0.002 mg/kg bw), 10 weeks of age 

(0.200 mg/kg bw), and 15 weeks of age (200 mg/kg bw). 

The study authors concluded that exposure to bisphenol 

A at environmentally relevant levels may affect male 

chicken phenotypes and result in unbalanced gene 

expression in the testis. 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 317 

Strengths/Weaknesses: This study is a more detailed 

follow-up of the previous study by these authors (Furuya 

et al., 2002), and replication of these results is a strength. 

Additional strengths are the use of multiple exposure 

levels and the oral route of administration. The lack of 

information on statistical methods is a weakness. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful to the evaluation process. While 

the in vitro studies are useful for mechanistic insights, 

cellular evaluation, and endpoint identification, inter alia, 

the studies as a group were considered not useful for the 

evaluation process. 

3.2.11 In vitro. Takai et al. (2000), supported by the 

Japanese Ministry of Education, Science, and Culture, the 

Ministry of Health and Welfare, and the Science and 

Technology Agency, examined the effects of in vitro 

bisphenol A exposure on preimplantation mouse embryos. Two-cell embryos were obtained from B6C3F1 

mice and incubated for 48 hr in media containing 

bisphenol A [purity not indicated] at concentrations 

ranging from 100 pM to 100 mM [23 ng/L to 23 mg/L] 

[culture ware not discussed]. A negative control group 

was exposed to the ethanol vehicle and the effects of 

tamoxifen were also tested. Cell numbers were counted, 

and trophoblast spreading was evaluated in blastocysts. 

Statistical analyses included w2

, Fisher post-hoc, and 

Student t-tests. The number of embryos or samples/ 

group ranged from 14–400 for each endpoint evaluated. 

Significant effects observed with bisphenol A exposure 

(percent change vs. control) included increased rate of 

development from 2- to 8-cell embryos following 24 hr 

exposure to 3 nM [0.68 lg/L] (94% vs. 88%), increased 

development to the blastocyst stage following 48 hr 

exposure to 1 and 3 nM [0.23 and 0.68 lg/L] (69% in both 

dose groups vs. 58.7%), and decreased development to 

the blastocyst stage following 48 hr exposure to 100 mM 

[23 mg/L] bisphenol A (31.2 vs. 58.7%). No effects were 

observed at concentrations between 10 nM and 10 mM 

[23 lg/L and 2.3 mg/L] bisphenol A. [Data were not 

shown by study authors.] Addition of 100 nM tamoxifen 

to cultures decreased development to the blastocyst stage 

at 1 and 3 nM [0.23 and 0.68 lg/L] bisphenol A and 

increased development to blastocyst stage at 100 mM 

[23 mg/L] bisphenol A. Trophoblast spreading was 

increased in blastocysts exposed to 100 mM [23 mg/L] 

bisphenol A. Bisphenol A exposure did not affect 

morphology of or cell numbers in blastocysts. The study 

authors concluded that environmentally relevant concentrations of bisphenol A may affect early embryonic 

development through the ER and may also affect 

subsequent development. 

Strengths/Weaknesses: The wide range of bisphenol A 

concentrations is a strength. The postulated involvement 

of the ER in bisphenol A activity could have been 

more convincingly demonstrated with a positive 

control such as 17b-estradiol and with a more 

specific estrogen antagonist than tamoxifen. The 

use of serum-free and phenol red-free media is an 

appropriate way to avoid estrogenic contamination 

but is an artificial environment compared to the 

estrogen-rich milieu in which preimplantation embryos 

normally develop. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study provides some mechanistic information but is 

not useful in the evaluation process. 

Takai et al. (2001), supported by the Japanese Ministry 

of Education, Science, Sports, and Culture, the Ministry 

of Health and Welfare, and the National Institute for 

Environmental Studies, examined the effects of in vitro 

preimplantation exposure of mice to bisphenol A. Twocell embryos were obtained from B6C3F1 mice and 

incubated for 48 hr in media containing bisphenol A 

[purity not indicated] at 0 (ethanol vehicle), 1 nM 

[0.23 lg/L] or 100 mM [23 mg/L] [culture ware not 

discussed]. Embryos were assessed for number developing to the blastocyst stage, and then blastocysts were 

transferred to uterine horns of pseudopregnant mice (7/ 

mouse). The dams were allowed to deliver and nurse the 

litters until weaning on PND 21 (day of birth not 

defined). Pups were randomly culled to maintain litter 

sizes at no more than 6. Body weight of pups was 

measured at birth and at weaning. Litters and pups were 

considered the experimental unit for statistical analyses. 

Statistical analyses included w2 and Fischer protected 

least significant difference tests. The number of embryos 

developing to the blastocyst stage was significantly 

increased by exposure to bisphenol A at 1 nM [0.23 lg/ 

L] but decreased by exposure to 100 mM [23 mg/L] (72.2 

and 33.3% at each respective concentration vs. 62.1% in 

controls). Developing embryos appeared morphologically normal and there were no significant differences in 

the numbers of cells. Birth weight, number of pups/litter, 

and sex ratio were not affected by treatment. At 

weaning, pups in both dose groups weighed more than 

controls (34–39% greater) and the effect was significant 

on a litter and pup basis. The study authors concluded 

that bisphenol A may affect early embryonic and 

postnatal development at low, environmentally relevant 

concentrations. 

Strengths/Weaknesses: This study was cleverly 

designed as a follow-up to the previous study and 

appears to show that a low concentration of bisphenol A 

stimulates early embryo development while a high 

concentration inhibits early embryo development. 

This study did not evaluate the effect of exogenous 

bisphenol A under physiologic conditions. The use of 

serum-free and phenol red-free media is an appropriate 

way to avoid estrogenic contamination but is an artificial 

environment compared to the estrogen-rich milieu in 

which preimplantation embryos normally develop. The 

trophic effects of bisphenol A at low concentration may 

have been compensating for the estrogen deprivation of 

the control culture. It would have been interesting to 

compare physiologic concentrations of 17b-estradiol to 

the control culture conditions. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful in the evaluation process. 

Li et al. (2003), support not indicated, examined the 

effect of in vitro bisphenol A exposure on postimplantation mouse and rat embryos. A limited 

amount of information was available for the study, 

which was published in Chinese, but included an 

abstract and data tables presented in English. GD 8.5 

mouse embryos and GD 9.5 rat embryos were cultured 

for 48 hr in media containing bisphenol A [purity not 

indicated] at 0, 40, 60, 80, or 100 mg/L [culture 

ware not discussed]. Exposure of rat embryos to 

bisphenol A concentrations Z60 mg/L resulted in 

reduced crown-rump length and yolk sac diameter 

and affected yolk sac circulation and morphologic 

Birth Defects Research (Part B) 83:157–395, 2008
318 CHAPIN ET AL. 

differentiation of the nervous system, heart, and 

forelimbs. Additional effects observed in rats at 

Z80 mg/L included reductions in head length, number 

of somites, and flexion and changes in morphologic 

differentiation of the otic and optic system and tail. 

Exposure of mouse embryos to Z60 mg/L bisphenol A 

resulted in reductions in flexion, yolk sac diameter, 

and yolk sac circulation and changes in morphologic 

differentiation of the olfactory system and branchial 

arches. In mouse embryos exposed to Z80 mg/L 

bisphenol A, there were reductions in head and 

crown-rump length and number of somites and changes 

in morphologic differentiation of the visual system, 

heart, brain, auditory system, and fore- and hindlimb 

buds. The study authors concluded that high concentrations of bisphenol A are toxic to rat and mouse embryos 

in vitro. 

Strengths/Weaknesses: The use of excessively high 

concentrations of bisphenol A is a weakness. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful in the evaluation process. 

Monsees et al. (2000), supported by the Federal 

Environmental Agency of Germany, examined the effects 

of bisphenol A exposure on rat Sertoli cell cultures. 

Sertoli cell cultures were prepared using testes from 18– 

21-day-old Sprague–Dawley rats. The cultures were 

exposed for 24 hr to bisphenol A or ethinyl estradiol at 

0 or 10–50 mM [2.3–11 mg/L] [culture ware not discussed]. The effects of pesticides and heavy metals were 

also examined but will not be discussed. Endpoints 

assessed following the incubation period included 

viability by measurement of mitochondrial enzyme 

activity and lactate and inhibin B production. There 

were 8 replicates/experiment, and the experiment was 

repeated 3 times. Data were analyzed by Student t-test or 

unpaired Mann–Whitney test. Exposure of cells to 

bisphenol A resulted in increased lactate production 

(up to 30%) at B25 mM [5.7 mg/L] bisphenol A and 

increased inhibin B production at B10 mM [2.3 mg/L] and 

greater. There was no effect on cell viability following 

exposure to bisphenol A. Effects of ethinyl estradiol 

included increased mitochondrial dehydrogenase activity and a biphasic effect on inhibin B production, with an 

increase at B10 mM and decreases at higher doses. The 

study authors concluded that secretion of lactate and 

inhibin B by Sertoli cells appeared to be sensitive 

markers for exploring possible Sertoli cell toxicants. 

Strengths/Weaknesses: The use of high concentrations 

of bisphenol A is a weakness. It is not clear how the 

increased lactate and inhibin B production would 

correlate with reproductive capacity. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful in the evaluation process. 

Iida et al. (2003), supported by an unnamed grantor 

and by Takeda Science Foundation, examined the effects 

of in vitro bisphenol A exposure on cultured rat Sertoli 

cells. The cell cultures were prepared using testes of 18­

day-old rats and were exposed for up to 48 hr to 

bisphenol A [purity not indicated] at concentrations 

ranging from 50–100 mM [11–23 mg/L] [culture ware not 

discussed]. Control cells were incubated in the 

DMSO-containing media. Morphology was examined 

by phase-contrast microscopy, and viability was assessed 

using the CellTiter 96 system in cells exposed to 0, 50, 

100, 150, 200, and 300 mM [0, 11, 23, 34, 46, and 68 mg/L]. 

Immunochemistry analyses were conducted to detect 

transferrin and caspase-3 and apoptosis was assessed 

using a TUNEL method in cells exposed to 0, 100, and 

200 mM [0, 23, and 46 mg/L] bisphenol A for 48 hr. A 

fluorescence staining technique was used to examine 

actin structure in cells incubated with 200 mM [46 mg/L] 

bisphenol A. Experiments were performed in triplicate 

and repeated at least three times. Data were analyzed by 

ANOVA. 

Bisphenol A concentrations of Z150 mM [34 mg/L] 

increased detachment of Sertoli cells from substrate and 

reduced viability. In a time-response study, cell viability 

was reduced following exposure to 200 mM [46 mg/L] 

bisphenol A for Z12 hr. Transferrin secretion by Sertoli 

cells was decreased following incubation with bisphenol 

A [apparently at Z100 mM (23 mg/L); statistical significance not indicated]. Following incubation with 200 mM 

[46 mg/L] bisphenol A, observations included solitary 

cells with a cortical ring of actin filaments and underdeveloped stress fibers, cells with membrane blebs 

consisting of protruding actin filaments, and round cells 

with a disorganized actin cytoskeleton and chromatin 

condensation. The study authors indicated that the 

observations were consistent with apoptosis. Expression 

of capsase-3 was observed in the round Sertoli cells. 

Capsase-3-positive cells were rarely observed in control 

cells, but were observed at incidences of o1% in the 

100 mM [23 mg/L] group and B9% in the 200 mM group. 

Further examinations revealed that most and possibly all 

of TUNEL-positive cells were stained with the caspase-3 

antibody. The study authors concluded that decreased 

viability of Sertoli cells was most likely due to apoptosis 

and not necrosis. 

Strengths/Weaknesses: The evaluation of multiple 

endpoints is a strength; however, the concentrations of 

bisphenol A were much higher than are likely to be 

achieved with human exposures. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful for the evaluation process. 

Miyatake et al. (2006), supported by the Japanese 

Ministry of Health, Labor, and Welfare, and the 

Ministry of Education, Culture, Sports, Science, and 

Technology, conducted a series of studies to examine the 

effect of bisphenol A exposure on cultures of mouse 

neuron/glia cells and astrocytes. Cell cultures were 

obtained from midbrains of ICR mice on PND 1. 

Statistical analyses included ANOVA followed by Student t-test. 

In the first two studies, astrocyte and neuron/glia 

cultures were incubated for 24 hr in media containing 

bisphenol A [purity not indicated] or 17b-estradiol at 0 

or 10 fM to 1 mM [bisphenol A concentrations of 2.3 pg/ 

L–0.23 mg/L] for 24 hr, and intensity of glial fibrillary 

acidic protein immunoreactivity was measured [culture 

ware not discussed]. In astrocyte cultures activation of 

cells, as determined by stellate morphology and significantly increased glial fibrillary acidic protein, occurred with exposure to bisphenol A at 100 fM [23 pg/L], 

1 pM [0.23 ng/L], 10 pM [2.3 ng/L], 10 nM [2.3 lg/L], 

100 nM [23 lg/L], and 1 mM [0.23 mg/L], but the effect was 

not observed in cells exposed to bisphenol A at 10 fM [2.3 

pg/L], 100 pM [23 ng/L], or 1 nM [0.23 lg/L]. In neuron/ 

glia cultures, a significant increase in glia fibrillary acidic 

protein was observed at bisphenol A concentrations of 

100 fM [23 pg/L], 1 pM [0.23 ng/L], 10 pM [2.3 ng/L], 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 319 

100 nM [23 ng/L], and 1 mM [0.23 mg/L], but not at 

bisphenol A concentrations of 10 fM [2.3 pg/L], 100 pM 

[23 ng/L], 1 nM [0.23 lg/L] or 10 nM [2.3 lg/L]. 

Increases in glial fibrillary acidic protein immunoreactivity were not observed in astrocyte or neuron/glia 

cultures following treatment with 17b-estradiol. 

The study authors concluded that exposure of cell 

cultures to bisphenol A results in biphasic activation of 

astrocytes. 

In a third study, the role of steroid hormone receptors in 

bisphenol A-induced astrocyte activation was examined. 

Astrocyte and neuron/glia cell cultures were pretreated 

with an ER antagonist (ICI 182,780), an ER agonist/ 

antagonist (tamoxifen), a progesterone receptor antagonist 

(mifepristone), or an androgen receptor antagonist (flutamide) for 24 hr. The cultures were then incubated with 

bisphenol A at 0, 1 pM [0.23 ng/L], or 1 mM [0.23 mg/L], 

with and without the receptor ligands, for another 24 hr. 

None of the ligands attenuated astrocyte activation, and 

the study authors concluded that bisphenol A-induced 

activation of astrocytes was not mediated by estrogen, 

progesterone, or androgen receptors. 

In a fourth study, mouse midbrain astrocyte or neuron 

cultures were incubated for 24 hr in media containing 

bisphenol A at 0, 1 pM [0.23 ng/L], 1 nM [0.23 lg/L], or 

1 mM [0.23 mg/L]. A fluorescent technique was used to 

measure calcium levels following treatment of cells with 

1–100 mM dopamine. In astrocyte and neuron cultures, 

dopamine-induced increases in intracellular calcium 

were enhanced following pretreatment with bisphenol 

A at 1 pM [0.23 ng/L], but not at 1 nM [0.23 mg/L] or 1 mM 

[0.23 mg/L]. In neuron cells, pretreatment with 1 mM 

[0.23 lg/L] bisphenol A suppressed dopamine-induced 

increases in intracellular calcium. The study authors 

concluded that in vitro bisphenol A exposure results in 

altered dopamine responsiveness in astrocytes and 

neurons. 

In a fifth study, neuron/glia cultures were incubated in 

media containing bisphenol A or 17b-estradiol at 1 pM, 

1 nM, or 1 mM for 24 hr [bisphenol A concentrations of 

0.23 ng/L, 0.23 lg/L, and 0.23 mg/L]. An immunohistochemistry technique was used to identify apoptotic cells 

by the presence of caspase-3. Treatment with 1 mM 

[0.23 lg/L] bisphenol A activated caspase-3 in neurons. 

No increase in caspase 3 was observed following 

exposure to cells to 17b-estradiol. The study authors 

concluded that high in vitro exposures to bisphenol A 

may result in toxicity to neurons. 

Strengths/Weaknesses: The use of multiple concentrations of bisphenol A over a wide range, the evaluation of 

multiple endpoints, and the comparison to known 

receptor ligands are strengths. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is interesting in suggesting a non-hormonal 

mechanism of bisphenol A activity. Although the study 

contains suggestive mechanistic information, it is not 

useful for the evaluation process. 

Yamaguchi et al. (2006), supported by the Promotion 

and Mutual Aid Corporation for Private Schools of 

Japan, examined the effects of low-level bisphenol A 

exposure on the differentiation of serum-free mouse 

embryo astrocyte progenitor cells into astrocytes. Astrocyte progenitor cells were grown on fibronectin-coated 

Petri dishes under standard incubator conditions. Differentiation of astrocyte progenitor cells was induced with 

leukemia inhibitory factor (LIF) and bone morphogenetic 

protein-2 (BMP2) [culture ware not discussed]. Cells 

were additionally exposed to bisphenol A [purity not 

provided] at concentrations of 0.1 ng/L to 100 mg/L with 

or without tamoxifen for 24, 48, 72, or 120 hr, to establish 

optimal experimental parameters. A tetrazolium salt 

based colorimetric assay was used to assess cell viability 

and dot-blot or Western blot detection of glial fibrillary 

acidic protein production was used as a marker of 

differentiated astrocytes. Subsequent assays were performed using bisphenol A treatments of 0.1 ng/L [4 pM] 

or 1 mg/L [40 mM]. Controls were treated with LIF and 

BMP-2 for 48 hr. ANOVA and Tukey test were used for 

statistical analyses. 

Bisphenol A 0.11 ng/L had no effect on astrocyte 

progenitor differentiation; However, bisphenol A at 1, 10, 

and 100 ng/L induced significant differentiation compared to controls based on dot-blot assays of glial 

fibrillary acidic protein production. The highest glial 

fibrillary acidic protein levels were induced with 10 ng/L 

bisphenol A exposure. At bisphenol A concentrations 

Z1mg/L, there were no differences in astrocyte progenitor differentiation compared to control. Bisphenol A 

10 ng/L induced significantly higher levels of phosphorylated signaling transducer and activator protein 3 

(pSTAT3) and phosphorylated mothers against decapentaplegic homolog 1 (pSmad1), the activated forms of both 

proteins, which are induced to form a protein complex 

by BMP-2 and LIF, and in turn, promote glial fibrillary 

acidic protein expression. Addition of 10 -6 M 

tamoxifen resulted in glial fibrillary acidic protein, 

pSTAT3, and pSmad1 comparable to control levels. 

Bisphenol A at 10 ng/L and 1 mg/L only marginally 

increased levels of Smad6 and oligodendrocyte lineage 

transcription factor 2, inhibitors of pSTAT3-p300 and 

pSmad1–Smad4 protein complex formation, which induce glial fibrillary acidic protein expression. 

The authors suggested that low levels of bisphenol A 

may alter brain development through a mode of action 

involving elevated levels of glial fibrillary acidic protein 

production through estrogen receptor regulation of glial 

fibrillary acidic protein expression and through a 

stimulatory BMP-2/LIF signaling pathway that induces 

the formation of pSmad and pSTAT3 coactivator complexes of glial fibrillary acidic protein expression. 

Strengths/Weaknesses: This study is interesting, but 

the in vitro system is not useful for predicting in vivo 

effects in humans. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful in the evaluation process. 

3.3 Utility of Developmental Toxicity Data 

3.3.1 Human. There are no human data on developmental effects of bisphenol A. 

3.3.2 Experimental animals. There are 21 studies 

in which bisphenol A was given at a single dose level to 

rats and six studies in which bisphenol A was given at a 

single dose level to mice. These studies explored various 

aspects of bisphenol A developmental effects but are not 

useful in establishing dose–response relationships. The 

lowest dose level evaluated in these studies was 

0.0024 mg/kg bw/day in rats (Akingbemi et al., 2004) 

and 0.002 mg/kg bw/day in mice (Nishizawa et al., 

2003). There are 25 rat and 30 mouse studies in which 

Birth Defects Research (Part B) 83:157–395, 2008
320 CHAPIN ET AL. 

Table 81 

Summary of High Utility Developmental Toxicity Studies (Single Dose Level) 

Dose and dosing period 

Model (route) (mg/kg bw/day) Significant developmental findings Reference 

Rat 

Sprague–Dawley (oral by 0.04, PND 23–30 and animals m ERa expression in females vs. males in medial Ceccarelli et al. 

pipette) evaluated at PND 37 or 90 pre-optic area (also seen with positive control)k (2007) 

Testosterone in males at PND 37 but not PND 90 

Sprague–Dawley males 0.040, PND 23–30 k Investigation of new object, k intromission Della Seta et al. 

(oral by pipette) latency, kserum testosterone (2006) 

F344/N dams (gavage) 0.1, GD 3–PND 20 k Correct avoidance responses and m failure of Negishi et al. 

avoidance in active avoidance testing; no m in (2004a) 

locomotion following trans-2­

phenylcyclopropylamine hydrochloride 

challenge in males 

Sprague–Dawley males 100, PND 23–53 m Age of preputial separation; m kidney and Tan et al. (2003) 

(gavage) thyroid weights; k liver weight; k cortical 

thickness of the kidney; m hydronephrosis; m

multinucleated giant cells in seminiferous 

tubules; kno. undergoing spermatogenesis 

Mouse 

CD-1 dam (oral) 0.050, GD 16–18 m Anogenital distance adjusted for body weight Gupta (2000) 

on PND60; m prostate weights on PND 3, 21, 

and 60; k relative (to body weight) epididymis 

weight in the bisphenol A group on PND 60; m

androgen receptor binding on PND 21 and 60 

CD-1 dam (oral from 0.010, GD 11–18 k Place preference associated with d- Laviola et al. 

syringe) amphetamine in females (2005) 

CD-1 dam (oral by pipette) 0.010, GD 14–18; offspring In mice exposed only during gestational Palanza et al. 

mated and dosed with 0 or development or in adulthood during (2002) 

0.010 on GD 14–18 pregnancy: k time nursing and in nest and m

time nest building, resting alone, grooming, and 

out of nest In mice exposed during both 

gestational development and in adulthood 

during pregnancy: m time resting alone 

CD-1 dam (oral by pipette) 0.010, GD 14–18 m No. of prostate ducts and proliferating cell Timms et al. 

nuclear antigen staining in dorsolateral (2005) 

prostate; m prostate duct volume in dorsolateral 

and ventral prostate 

m, k Statistically significant increase, decrease compared to controls; 2no statistically significant effects compared to controls. 

bisphenol A was given at multiple dose levels. These 

studies included oral and subcutaneous administration 

routes; due to pharmacokinetic considerations, studies 

using the oral route are of greater utility in estimating 

human risk. 

3.4 Summary of Developmental Toxicity Data 

The studies summarized here are those considered by 

the Panel to be the most important and relevant for the 

assessment of the effects of Bisphenol A on the human 

population. Evaluation of the scientific literature was 

made on the scientific quality of the study and also on its 

relevance to the assessment of the level the concern about 

potential effects of BPA on human health. The judgment 

was based on the criteria the Panel adopted which 

focused on the potential for providing information for 

the evaluation process. Several excellent studies have 

been placed in the ‘‘adequate-but-limited-utility’’ category with regard to the evaluation process. The Panel did 

not consider the source of funding of any of the studies 

in any of their deliberations. 

It is highly unlikely that humans would ever experience the very high internal levels of bisphenol A that are 

produced after an injection of bisphenol A. While it 

would be possible to measure levels of parent compound 

and metabolite after injections, no parenteral exposure 

studies in this data set have done so. Section 1 and 

(Wilson et al., 2006) indicate that ca. 99% of human 

exposure comes from dietary sources, and bisphenol A is 

subject to efficient first-pass metabolic conversion in the 

gut and liver to the inactive glucuronide conjugate in 

humans and rats (Pottenger et al., 2000; Vo ̈lkel et al., 2002; 

Inoue et al., 2003b). In contrast, bisphenol A injected 

subcutaneous or i.p. circulates as much higher proportion of the unconjugated parent compound (Pottenger 

et al., 2000). Because oral exposure is so relevant to the 

human situation, and the uncertainties associated with 

the altered internal metabolite profile and the abundant 

data from oral studies, the Panel puts greater weight on 

studies using the oral route of exposure for formulating 

levels of concern about human exposures. 

The hypothesis has been advanced that the Charles 

River SD rat is insensitive to estrogens and other EDCs 

and therefore it should not be used for developmental 

studies of potential endocrine disruptors, and the studies 

of the effects of BPA that used this strain should be 

discounted. In order to address this important issue the 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 321 

Table 82 

Summary of High Utility Developmental Toxicity Studies (Multiple Dose Levels) 

Bisphenol A dose level (mg/kg bw/day)	 Reference 

Model (treatment)	 Endpoint NOAEL LOAEL BMD10 BMDL10 BMD1SD BMDL1SD 

Rat 

Han–Wistar (drinking Male reproductive organ Z0.775–4.022 Cagen et al. 

water from before weights, sperm production, (high dose) (1999b) 

mating through testicular histopathology 

gestation and lactation) 

CD (gavage, 2-generations Prenatal or postnatal growth Z0.2 Ema et al. 

exposure including or survival, developmental (high dose) (2001) 

pre-and postnatal landmarks, anogenital distance, 

development periods) age of puberty, fertility, 

estrous cyclicity, or 

sperm counts
 

Sprague–Dawley dam k Live fetuses/litter 300 1000 929 348 982 713 Kim et al.
 

(gavage GD 1–20) (2001b)
 

k Male body weight 100 300 456 339 694 497 

k Female body weight 300 1000 439 328 682 490 

k Ossification 300 1000 

Sprague–Dawley dam Volume of SDN-POA, age or Z320	 Kwon et al. 

(gavage GD 11–PND 20)	 weight at vaginal opening (high dose) (2000)
 

or first estrous, estrous cyclicity,
 

mean lordosis intensity, prostate
 

weight, or histopathology in
 

ventral prostate, ovary, or uterus
 

CD dams (gavage Implantation sites, resorptions, Z640 Morrissey et al. 

GD 6–15) body weight, viability, sex ratio, (high dose) (1987) 

and malformations 

Sprague–Dawley m Uterine epithelial cell nuclei 0.1 Scho ̈nfelder 

dam (gavage (low dose) et al. (2004) 

GD6–PND 21) 

m Uterine epithelial nuclei with 0.1 

condensed chromatin (low dose) 

m Uterine epithelial cells 0.1 

with cavities (low dose) 

k ERb-positive cells in 0.1 

uterine tissue (low dose) 

kThickness of uterine 0.1 50 

luminal epithelium 

m ERa-positive cells in 0.1 50 

uterine epithelium 

Wistar-derived Delayed vaginal opening 0.1 50 68 51 35 16 Tinwell et al. 

Alderley–Park (2002) 

dams (gavage GD 6–21) 

k Sperm count/testis 0.1 50 55 30 57 31 

k Sperm count/g testis 0.1 50 81 41 68 34 

k Daily sperm count/testis 0.1 50 56 31 59 31 

k Daily sperm count/g testis 0.1 50 83 42 70 34 

Sprague–Dawley (dietary, Live F1 pups/litter 47.5 475 268 192 559 394 Tyl et al.
 

multiple generations (2000b, 2002b)
 

with exposure during
 

pre-and post natal
 

development)
 

Live F2 pups/litter 47.5 475 422 152 459 294 

Live F3 pups/litter 47.5 475 236 174 376 286 

F1 body weight, PND 4 47.5 475 406 283 561 400 

F1, F2, or F2 body weight, PND 7 47.5 475 217–328 183–257 265–410 218–313 

F1, F2, or F2 body weight, PND 14 47.5 475 183–243 163–209 177–227 153–191 

F1, F2, or F2 body weight, PND 21 47.5 475 208–252 166–226 223–267 175–220 

m Age at F1 vaginal opening 47.5 475 394 343 206 176 

m Age at F2 vaginal opening 47.5 475 404 336 277 228 

m Age at F3 vaginal opening 47.5 475 471 401 396 203 

m Age at F1 preputial separation 4.75 47.5 466 411 188 163 

m Age at F2 preputial separation 47.5 475 300 255 241 203 

m Age at F3 preputial separation 47.5 475 547 473 222 189 

Mating, fertility, pregnancy, or Z475 

gestational indices; (high dose)
 

precoital interval,
 

postimplantation loss,
 

estrous cyclicity,
 

and reproductive
 

organ histopathology;
 

sperm count, morphology
 

or motility;
 

Birth Defects Research (Part B) 83:157–395, 2008
322 CHAPIN ET AL. 

Table 82 

Continued 

Bisphenol A dose level (mg/kg bw/day) Reference 

Model (treatment) Endpoint NOAEL LOAEL BMD10 BMDL10 BMD1SD BMDL1SD 

anogenital distance 

in males or females; 

areolas/nipples in males 

Mouse 

CD-1 dam m Resorptions/litter 1000 1250 817 377 1245 1162 Morrissey 

(gavage GD 6–15) et al. (1987) 

k Fetal body weight/litter 1000 1250 1079 785 1249 1024 

C57BL/6N males Sperm density or lesions Z0.200 Nagao et al. 

(gavage GD 11–17 in reproductive organs (high dose) (2002) 

or PND 21–43) 

k Absolute seminal r0.002 

vesicle weight in (low dose)a,b 

group exposed 

during gestation 

CF-1 (oral by pipette, m Prostate weight r0.002 Nagel et al. 

GD 11–17) (low dose) (1997) 

C57BL/6 dam (gavage no effect AGD or Z0.2 Ryan and 

GD 3–PND 21) AGD corrected (high dose) Vandenbergh 

for body weight (2006) 

No effect on errors in Z0.2 

radial arm and (high dose) 

Barnes mazes 

k Time in open arms 0.002 0.2 

of plus maze 

k Time in light part 0.002 0.2 

of light/dark 

preference box 

CD-1 (dietary, multiple k F1 body weight 50 600 548–560 267–313 580–617 370–506 Tyl et al. (2006) 

generations with on PND 7, 14, and 21 

exposure during 

pre- and post-natal 

development) 

kF1 male body weight 50 600 564 313 640 599 

at PND 21 necropsy 

kF1 female body weight 50 600 387 254 776 598 

at PND 21 necropsy 

Hepatic cytoplasmic 5 50 124 92.5 

variation, F1 male 

Hepatic cytoplasmic 50 600 224 178 

variation, F2 male 

Hepatic cytoplasmic 5 50 333 200 

variation, F1 female 

Seminiferous tubule 50 600 591 406 

hypoplasia, F1 male 

Seminiferous tubule 5 50 283 233 

hypoplasia, F2 male 

Age of preputial separation, 50 600 727–754 572–576 491–551 364–414 

F1 parental or 

non-mated males 

Anogenital distance 5 50 1373 607 1769 616 

per body weight, 

F1 male on PND 21 

Postnatal survival; daily Z600 

sperm production; efficiency (high dose) 

of daily sperm production; 

sperm motility or 

morphology; 

estrous cyclicity; 

numbers of 

ovarian primordial follicles; 

mating or fertility indices; 

or adult prostate weight 

a

There was little to no evidence of a dose-response relationship. 

b

No effects were observed at one or more higher dose levels. 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 323 

Table 83 

Summary of Limited Utility Developmental Toxicity Studies (Single Dose Level) 

Dose and dosing period 

Model (route) (mg/kg bw/day) Significant developmental findings Reference 

Rat 

Long–Evans male 0.0024, PND 21–90 m Serum LH level; k weight of seminal vesicles; k Testicular Akingbemi et al. 

offspring (gavage) testosterone level; and k basal and LH-induced ex vivo (2004) 

Experiment 3 testosterone production 

Wistar male pup (s.c. 100, PND 2–12 Advanced testicular lumen formation, mtestis weight, m Atanassova et al. 

injection) Sertoli cell volume/testis, m spermatocyte nuclear volume/ (2000) 

unit Sertoli cell, and m plasma FSH on PND 18; m plasma 

FSH on PND 25; m testicular weight in adulthood 

Wistar dam (drinking B2.5, gestationa

–PND 21 In rats 4–7 months of age: no effect on the number of Funabashi et al. 

water) corticotropin-releasing hormone neurons in the preoptic (2004a) 

areas of males, a loss in sex difference in the anterior and 

posterior bed nuclei of the stria terminalis 

Sprague–Dawley 0.010, PND 1, 3, and 5; half In rats with no 17b-estradiol and testosterone exposure in Ho et al. (2006b) 

male pup (s.c. the rats exposed to 17b­ adulthood: no effects on dorsal prostate weight, 

injection) estradiol and testosterone histopathology alterations, proliferation index, or apoptotic 

in adulthood index In rats with 17b-estradiol and testosterone exposure 

in adulthood: m incidence and severity of prostatic 

intraepithelial neoplasia; m proliferation and apoptosis in 

regions of prostatic intraepithelial neoplasia 

Sprague–Dawley 300, PND 1–5 No effects on age of vaginal opening or preputial separation, Nagao et al. (1999) 

pup (s.c injection) copulation or fertility indices, sexual behavior of males, 

histopathologic alterations in males, or female reproductive 

organs, or effects on SDN-POA. [Panel noted possible m

number of apically located nuclei in prostate, but a 

definitive conclusion could not be made based on 1 

photograph] 

Wistar. male pup (s.c 50, PND 22–32 m Serum prolactin levels on PND 29 but not PND 120; m lateral Stoker et al. (1999) 

injection) but not ventral prostate weight; m focal luminal 

polymorphonuclear cellular infiltrate in prostate No 

histological evidence of prostate inflammation 

Mouse 

CF1 (oral) 0.0024, GD 11–17 m Body weight at weaning; k postnatal pup survival; k period Howdeshell et al. 

between vaginal opening and first estrus No effect on age (1999) 

of vaginal opening 

ICR/Jc1 mouse dams 0.02, GD 0 to GD 10.5, GD m Tuj1 in the intermediate zone at GD 14.5 and GD 16.5; m PDI Nakamura et al. 

(s.c. injection) 12.5, GD 14.5, or GD 16.5 immunoreactivity in the neocortex from GD12.5 until (2006) 

GD16.5 and in subplate cells at GD 14.5; variable changes in 

BrdU labeling depending on when labeled and location; m

gene expression of Math3, Ngn2, Hes1, LICAM, and THRalpha at GD14.5; k gene expression of Hes1 and Hes5 at GD 

12.5 No effect on immunoreactivity pattern for KI-67, 

nestin, Musashi and histone H3. 

ICR (oral) 0.002 mg/kg bw/day from Variable changes in retinoic acid retinoid X receptors a ́ mRNA Nishizawa et al. 

6.5–11.5, 6.5–13.5, 6.5–15.5, expression in brain, ovary, and testis, depending on brain (2003) 

and 6.5–17.5 days post- region and day of exposure 

coitum 

Other 

Prepubertal Poll 3.5 biweekly, at 4–11 weeks 2 On blood levels during treatment; 2 on body, kidney, Evans et al. (2004) 

Dorset female lambs of age (ovariectomy at 9 adrenal, or ovarian weights; k pulsatile LH secretion 

(i.m. injection) weeks of age) 

Prepubertal Poll 3.5 biweekly, at 4–11 weeks m Uterine/cervical tract weight, endometrial area, and Morrison et al. 

Dorset female lambs of age (ovariectomy at 9 endometrial/myometrial ratio Qualitative observations (2003) 

(i.m. injection) weeks of age) included endometrial edema, decreased endometrial gland 

density, crowding of cells in the uterine epithelium, 

keratinized cervical epithelium, m intracellular staining for 

ERa and ERb in the uterine subepithelium 

Suffolk ewes (s.c. 5 GD 30–GD 90 k Birth weight, height and chest circumference in female Savabieasfahani 

injection) offspring at birth m Anoscrotal:anonavel ratio in male et al. (2006) 

offspring at birth m LH and first breeding season in female 

offspring at PND 60 

a

Implied but not stated that exposure occurred during the entire gestation period.
 

m,k Statistically significant increase, decrease compared to controls; 2 no statistically significant effects compared to controls.
 

Panel members reviewed the literature on estrogen- Different strains of rats show clear, robust reproducisensitivity across rat strains and suppliers, the following ble differences in response to potent estrogens and 

is a summary of our findings. antiandrogens. Several traits have been shown to be 

Birth Defects Research (Part B) 83:157–395, 2008
324 CHAPIN ET AL. 

Birth Defects Research (Part B) 83:157–395, 2008 

Table 84


Summary of Limited Utility Developmental Toxicity Studies (Multiple Dose Levels)


Bisphenol A dose level (mg/kg bw/day)

Model (treatment) Endpoint NOAEL LOAEL BMD10 BMDL10 BMD1SD BMDL1SD Reference 

Rat

Long–Evans males (gavage k Serum 17b-estradiol 0.0024 (low dose)a,b Akingbemi et al.PND 21–35) Experiment 1 (2004) 

k Serum LH and testosterone 0.0024 (low dose)a,b 

Sprague–Dawley (dietary for k pup weight at weaning (PND 70 200 General Electric,

17 weeks) 21) (1976) 

Sprague–Dawley (dietary for No adverse effects reported 60 (high dose) General Electric18 weeks) (1978) Sprague–Dawley female pups kBody weight in lactation period 105 427 286 200 233 156 Kato et al. (2003) 

(sc injection PND 0–9) 

m Age of vaginal opening 26 105 345 267 159 116 

k No. with normal estrous cycles 105 427 81 28 

m No. with cleft clitoris 26 105 299 failed 

k Ovary weight 105 427 85 59 140 93 

k Uterus, wet weight 105 427 66 55 128 96 

k Uterus, blotted weight 105 427 273 128 318 168 

k Uterine fluid weight 26 105 42 34 139 104 

m No. with polycystic ovaries r105 (lowest dose 81 24 

examined) 

k No. corpora lutea 105 427 238 90 

k No. with corpora lutea 105 427 65 38 137 83 

kCorpora lutea area r105 (lowest dose 42 37 84 66 

examined) 

Sprague–Dawley female pups No adverse effects reported Z97 (high dose) Kato et al. (2006) 

(sc injection PND 0–9) Sprague–Dawley (feed GD15– No adverse effects reported 3000 ppm c Masutomi et al.

PND 10) (2004) 

Mouse

CF-1 (oral by pipette, GD 11– Prostate weight and sperm Z0.020 (high dose) Ashby et al. (1999) 

17) production ICR/Jcl (s.c. GD 11–17) k Female body weight at r0.002 (low dose)a 0.065 0.017 0.088 0.021 Honma et al. (2002) 

weaning 

k Male body weight at birth 0.002 0.020 0.054 0.020 0.031 0.015 

m Anogenital distance of females r0.002 (low

at weaning dose)a,b 

mAnogenital distance of males on r0.002 (low dose) 0.035 0.020 0.035 0.020 

PND 60 
325 

Birth Defects Research (Part B) 83:157–395, 2008 

BISPHENOL A 

k Age at vaginal opening 0.002 0.020 k Body weight at vaginal

r0.002 (low dose) 

opening 

k Age at 1st estrus 0.002 0.020 

m Estrous cycle length 

r0.002 (low dose)a 0.021 0.007 0.12 0.021 

m Cornified cells 

r0.002 (low dose)b 0.17 0.020 0.44 0.021 

k Lymphocytes in vaginal smear 

r0.002 (low dose)b 0.26 0.020 0.26 0.020 

ICR (oral GD 6.5–13.5 or 6.5– m mRNA expression for

r0.00002 (low Nishizawa et al.

17.5) arylhydrocarbon receptor in dose)b (2005b) 

brain, testis, and ovary 

m mRNA expression for retinoic

r0.00002 (low


acid a receptor in brain and dose)b
 

ovary
 

m mRNA expression for retinoic 0.20 20
 

acid a receptor in testis
 

m mRNA expression for retinoid

r0.00002 (low


X a receptors in brain dose)b
 

m mRNA expression for retinoid 0.002 0.020b
 

X a receptor in testis and ovary
 

ICR (oral GD 6.5–13.5 or 6.5– m mRNA expression for

r0.00002 (low	 Nishizawa et al.

17.5)	 arylhydrocarbon receptor, dose)b (2005a) 

arylhydrocarbon receptorrepressor, and arylhydrocarbonreceptor nuclear translocator inbrain, testis, and ovary 

ICR/Jcl (s.c. GD 10–18; female m No. of vaginal epithelial layers 

r10 (low dose) Suzuki et al. (2002) 

offspring ovariectomized) 

k No. with corpora lutea 

r10 (low dose)b 

ICR/Jcl (s.c. for 5 days m Mitotic rate in uterine stromal 10 100 Suzuki et al. (2002) 

beginning at birth; mice later cells and vaginal epithelial cells ovariectomized except thoseused to monitor estrous cycles) 

m Vaginal epithelial layers 10 100 

m No. with polyovular follicles 10 100
 

and no. polyovular follicles/
mouse


Estrous cyclicity 100 (high dose)

CF-1 (oral by pipette, GD 11– k Body weight 

r0.002 (low dose)b vom Saal et al.

17) (1998) 

aThere was little-to-no evidence of a dose-response relationship.bNo effects were observed at one or more higher dose levels.cFeed consumption and dam weight not reported-dose not calculable. 
326 CHAPIN ET AL. 

Table 85 

Summary of Behavioral Studies in Rats and Mice Treated With Bisphenol A 

Treatment, mg/kg 

bw/day Treatment age Age at assessment Results Reference 

High Utility 

Rat 

Treatment of dam

3.2, 32, or 320, GD 11–PND 20 6 months Lordosis behavior not affected by Kwon et al. (2000) 

gavage treatment 

0.1, gavage GD 3–PND 20 Open field: 8 weeks Open field: No treatment effect Negishi et al. (2004a) 

Spontaneous motor Spontaneous motor activity: No 

activity: 12 weeks treatment effect 

Passive avoidance: 13 Passive avoidance: No treatment effect 

weeks 

Elevated plus maze: 14 Elevated plus maze: No treatment 

weeks effect 

Active avoidance: 15 Active avoidan: Fewer correct 

weeks avoidance responses 

Treatment of offspring

0.04, micropipette PND 23–30 45 days No treatment effect on environmental Della Seta et al. (2006) 

exploration, social investigation, play, 

or social interaction 

k Response to novel object 

k Intromission latency 

Mouse 

0.010, syringe GD 11–18 60 days k Conditioned place preference Laviola et al. (2005) 

feeding (reinforced with amphetamine) in 

females 

0.010, micropipette GD 14–18 Maternal behavior of Altered maternal behaviors when Palanza et al. (2002) 

(treatment of F0 F1 assessed exposure was either prenatal or as an 

and F1 females) adult; however, exposure prenatally 

plus as an adult was not effective 

2 or 200, placed in GD 3–PND 21 5 weeks, No effect on errors in radial arm and Ryan and 

back of dam’s ovariectomized Barnes mazes Vandenbergh (2006) 

throat female offspring 

Effects in high dose group:Puberty 

advanced 

k Time in open arms of plus maze 

k Time in light part of light/dark 

preference box 

m,k Statistically significant increase, decrease compared to controls; 2 no statistically significant effects compared to controls. 

estrogen sensitive in rats including prolactin regulation 

in the pituitary, thymic involution, uterine pyometra, and 

liver carcinogenesis to name a few. It is evident that there 

are strain differences in respect to specific estrogen 

induced endpoints. However, there is no clear pattern in 

which one strain can be considered to be more or less 

sensitive than another. The results of BPA studies with 

the SD rat cannot therefore be ignored. 

3.4.1 Human. There are no human data on developmental effects of bisphenol A. A study of the 

association between miscarriage and mean serum bisphenol A levels is discussed in Section 4.4.1. 

3.4.2 Experimental animal. Studies considered by 

Expert Panel members to be of utility in evaluating 

developmental toxicity in mice are summarized in 

Tables 81, 82, 83, 84. Rat and mouse studies with 

behavioral endpoints are summarized in Table 85. The 

discussion of developmental toxicity is arranged according to general endpoints evaluated. 

General developmental toxicity (growth, survival, 

malformations). 

Rat Studies: Prenatal studies with oral dosing of rats 

consistently demonstrated an absence of malformations 

at doses up to 1000 mg/kg bw/day (Morrissey et al., 

1987; Kim et al., 2001b). Reduced fetal survival and body 

weights at birth or during the postnatal period were 

reported in studies with oral exposures occurring 

throughout the entire gestation and/or lactation periods 

(Tyl et al., 2000a, 2002b; Kim et al., 2001b). LOAELs for 

decreased numbers of live fetuses or pups ranged from 

475–1000 mg/kg bw/day (Tyl et al., 2000a, 2002b; Kim 

et al., 2001b). LOAELs for decreased pup body weight at 

birth were estimated at 300–1000 mg/kg bw/day (Tyl 

et al., 2000a, 2002b; Kim et al., 2001b). The LOAEL for 

reduced body weight during the postnatal period was 

475 mg/kg bw/day (Tyl et al., 2000a, 2002b). 

Mouse Studies: No increase in malformations was 

observed in mice with oral gavage of bisphenol A at 

doses of r1250 mg/kg bw/day (Morrissey et al., 1987). 

Prenatal developmental toxicity reported for mice included increased resorptions (LOAEL 1250 mg/kg bw/ 

day) and decreased fetal body weight (LOAEL 1250 mg/ 

kg bw/day) (Morrissey et al., 1987). Decreased body 

weight during the postnatal period was also reported in 

offspring of mouse dams exposed to bisphenol A during 

the entire gestation and lactation period (LOAEL 

Birth Defects Research (Part B) 83:157–395, 2008
327 

Table 86

Adult Body Weights of Offspring Exposed During Gestation or Lactation

Dose (mg/ Measured

Strain Period of dosing Route kg/day) on PND Finding Weights SE Sample size Reference 

CR:Long– GD 12–PND 21 Gavage 0.0, 2.4 90 M: m 90 days at 2.4 450, 494 

B14 12–14/group Akingbemi

Evans et al. (2004)

CR:Long– GD 21–PND 90 Gavage 0.0, 2.4 90 NE 407, 412 

B11 12–14/group Akingbemi

Evans et al. (2004)

F344/N GD 10–PND 20 Gavage 0.0, 4.0, (7, 21, M: k 7 days, 28 days at 40, M 303, 303, 303, 297

B3 27

1, 27

1, Negishi

40.0, 400.0 28, 56), 84	 k 7 days, 21 days, F: 186, 187, 185, 184 27

1, 15

1, 9

1 et al. (2003) 

28 days, 56 days at400 F: k 7 days, 28 days

at 4 and 40, k 7 days,21 days, 28 days at 400 

Fisher GD 1–PND 21 Gavage 0.0, 7.5, 120 (23, 28) 91 NE 259, 267, 259 

B9 5, 5, 5 Yoshino

et al. (2002)

Fisher GD 1–PND 21 Gavage 0.0, 0.05, 7.5, 455 NE 427, 427, 420, 428 

B21 12,12, 12, 12 Ichihara

30, 120 et al. (2003)

SD GD 6–GD 21 Gavage 0.0, 0.1, 50 44, 50, 44 F: k B47 at 0.1 F: 246, 227, 242 

B15 20, 20, 20 Talsness

et al. (2000)

SD GD 6–PND 21 Water 0.0, 0.1, 1.2 (22, 28, 37, M: m 28 days, 37 days, 56 M: 

B510, 

B540, 

B540; M: 

B12 M: 27, 19, 19 Rubin

56, 87) 110 days at 0.1 and 28 days, F: 

B310, 

B325, 

B310 F: 

B12 F: 23, 18, 19 et al. (2001) 

37 days, 560 days at 1.2 (from graph) 

F: m 22 days, 28 days,

37 days, 56 days,87 days, 110 days at 0.1 

SD GD 6–PND 20	 Gavage 0.0, 4.0, 40.0 63 NE M: 

B380, 

B385, 

B381 M: 

B4 M: 5, 4, 4, 0 Kobayashi

F: 

B250, 

B245, 

B237 F: 

B3 F: 5, 5, 5, 0 et al. (2002)

SD GD 6–GD 21 Gavage 0.0, 0.023, 98 NE F: 260, 261, 258, 250 

B17.5 31, 21, 25, 25 Tinwell

0.049, 0.108 et al. (2002)

SD CD All F1 Mating 1–PND 21 Diet 0.0, 0.015, 0.3,

4168 M: k 84 days at 7500 M: 501, 505, 493, 506, 518, M: 

B8 30,10,10,10, Tyl et al.

generations 4.5, 75, 750, F: k 84 days at 7500 476, 369 F: 290, 383, F: 

B5 10,10,10 (2002b) 

7500 287, 294, 295, 283, 234 

SD Crj IGS- F1 GD 11–GD 17 Gavage 0.0, 0.0002, 0.002, M: 40; F: 30 NE M: 241,237, 245, 228, 236 M: 

B18 25, 25, 25, Ema

generation 0.02, 0.2 F: 114, 120, 113, 114, 113 F: 

B12 25, 25 et al. (2001)

SD Crj IGS- F2 GD 11–GD 17 Gavage 0.0, 0.0002, 0.002, M: 41; F: 31 NE M: 240, 241, 237, 236, 237 M: 

B18 25, 25, 25, Ema

generation 0.02, 0.2 F: 116, 115, 113, 113, 117 F: 

B13 25, 25 et al. (2001)

SD Crj:CD IGS GD 15–PND 10 Diet 0.0, 60, 3000 77 M: k 77 days at 3000 M: 465, 452, 468, 421 M: 

B33 8 litters Takagi et al.

279, 272, 299, 254 F: F: 

B25 (2004) 

SD Crl:CD BR GD 11–PND 20 Gavage 0.0, 3.2, 320 

B47 NE F: 691, 736, 683, 668, 697 

B19 44, 51, 47, Kwon et al.

28, 38 (2000) 

Wistar–Hans 14 days before Water 0.0, 0.01, 0.1, (22, 29, 36, 43, NE 331, 321, 328, 328,

B20 51, 26, 26, 28, Cagen et al.

mating–PND 21 1.0, 10.0 50, 57, 64, 71, 328, 332 27, 25 (1999b) 

78, 85) 90 (litters) 

Wistar GD 1–PND 21 Gavage 0.0, 0.03, 0.3 

B87 NE 455.2, 460.8, 454.1 

B7 13, 15, 13 Kubo et al.

(2003) 

Wistar–AP GD 6–GD 21 Gavage 0.0, 0.024, 0.051, 98 NE F: 228, 241, 237, 237 

B15 26, 26, 27, 26 Tinwell et al.

0.109	 (2002) 

Birth Defects Research (Part B) 83:157–395, 2008 

BISPHENOL A 

NE, no effect. 
328 CHAPIN ET AL. 

600 mg/kg bw/day), but the effect was not observed in a 

second generation exposed according to the same 

protocol (Tyl et al., 2006). An increase in hepatic 

histopathologic findings (cytoplasmic variation) at weaning was also observed in offspring of mouse dams 

exposed during gestation and lactation (LOAEL 50– 

600 mg/kg bw/day) (Tyl et al., 2006). A single dose level 

study with gestational exposure in mice reported 

increased lactational body weight gain and decreased 

postnatal pup survival at 0.0024 mg/kg bw/day 

(Howdeshell et al., 1999). 

Reproductive system development. 

Rat studies: Delays in vaginal opening were observed 

in offspring of rat dams receiving high oral doses of 

bisphenol A on GD 6–15 or during the entire gestational 

and lactational period (Tyl et al., 2000a, 2002b; Tinwell 

et al., 2002). No delays in vaginal opening were observed 

with doses of bisphenol A r1.2 mg/kg bw/day administered to dams during gestation or lactation (Tyl et al., 

2000a, 2002b; Ema et al., 2001). 

Estrous cycle alterations were not reported in rat oral 

exposure studies covering a wide range of doses (o1– 

475 mg/kg bw/day) administered during all or part of 

the gestational or lactational periods (Kwon et al., 2000; 

Tyl et al., 2000a, 2002b; Ema et al., 2001). 

Studies suggest that preputial separation is delayed 

following oral administration of high bisphenol A doses 

(LOAELs 5 47.5–475) to male rat offspring in the post 

weaning period (Tyl et al., 2000a, 2002b; Tan et al., 2003). 

No effects on preputial separation were observed when 

treatment of rat dams with high-doses (50–384 mg/kg 

bw/day) ended during the gestation or lactation period 

(Tinwell et al., 2002) Oral doses of bisphenol A r1 mg/ 

kg bw/day also had no effect on preputial separation 

(Ema et al., 2001; Tyl et al., 2000a, 2002b). 

Effects on rat sperm parameters were inconsistent. 

Decreased sperm count and daily sperm production 

were reported in offspring of dams exposed during 

gestation (LOAEL 50 mg/kg bw/day for sperm count/g 

testis, LOAEL 50 mg/kg bw/day for daily sperm count/ 

g testis) (Tinwell et al., 2002). A single dose level study 

reported decreased numbers of rats undergoing spermatogenesis following post-weaning exposure of males to 

100 mg/kg bw/day (Tan et al., 2003). In contrast, no 

consistent effects on sperm parameters were observed in 

rats following exposures with up to 475 mg/kg bw/day 

during the prenatal, lactational, and post-weaning 

periods (Tyl et al., 2000a, 2002b). Other rat studies with 

gestational and lactational doses ranging from o1–4 mg/ 

kg bw/day also reported no effects on sperm parameters 

(Cagen et al., 1999b; Ema et al., 2001). Testicular 

histopathology (multinucleated giant cells in seminiferous tubules and absent spermatogenesis) was only 

reported in a single dose level study at a bisphenol A 

dose of 100 mg/kg bw/day administered in the postweaning period (Tan et al., 2003). 

Although some sporadic effects were reported for 

anogenital distance in male and female rats, study 

authors concluded that the endpoint was not affected 

by prenatal, lactational, and/or post-weaning exposure 

to bisphenol A (Ema et al., 2001; Tinwell et al., 2002; Tyl 

et al., 2000b, 2002b). 

No effects on rat prostate weight were observed with 

bisphenol A doses of o1–475 mg/kg bw/day administered during the gestational, lactational, and/or postweaning periods (Cagen et al., 1999b; Kwon et al., 2000; 

Tyl et al., 2000b, 2002b; Tinwell et al., 2002). The study of 

Timms et al. (2005) in mice raise a level of concern. 

Mouse studies: Exposure of mice to bisphenol A 

during pre- and post-natal development delayed preputial separation (LOAEL 600 mg/kg bw/day) (Tyl et al., 

2006). Effects reported for anogenital distance were 

inconsistent. A single dose study reported an increase 

in anogenital distance in male mice at 0.050 mg/kg bw/ 

day (Gupta, 2000). A second study with a wide dose 

range (0.003–600 mg/kg bw/day) reported no consistent 

or dose-related effects on anogenital distance (Tyl et al., 

2006). 

One group of investigators reported increased prostate 

weight at 0.002 and 0.020 mg/kg bw/day in offspring of 

mouse dams exposed during pregnancy (Nagel et al., 

1997). These prostate effects were consistent with 

findings in single dose level studies with gestational 

exposure of mice, however, it is noted that the studies 

had differing periods of exposure and ages of evaluation. 

One of these studies demonstrated increased prostate 

weight at 0.050 mg/kg bw/day (Gupta, 2000). Another 

study demonstrated increased numbers of prostate ducts 

and proliferating cell nuclear antigen staining in dorsolateral prostate and increased prostate duct volume in 

dorsolateral and ventral prostate at 0.010 mg/kg bw/day 

(Timms et al., 2005). However, no effects on prostate or 

sperm production were observed in more robust studies 

with multiple dose levels and larger group sizes. A third 

mouse study with exposures occurring during gestation, 

lactation, and post-lactational periods also reported no 

effects on prostate weight, daily sperm production, or 

efficiency of daily sperm production at doses of 0.003– 

600 mg/kg bw/day (Tyl et al., 2006). A fourth 

mouse study demonstrated no effect on sperm density 

following low-dose exposure (r0.200 mg/kg bw/day) 

during gestation or the post-weaning period (Nagao 

et al., 2002). 

Seminiferous tubule hypoplasia in association with 

undescended testes in mouse weanlings was reported 

following exposure during pre- and post-natal 

development (LOAEL 50–600 mg/kg bw/day; BMD10 

283–591 mg/kg bw/day) but the effect was not observed 

in mice examined in adulthood (Tyl et al., 2006). The 

findings were similar to those in studies reporting no 

testicular histopathology or lesions in reproductive 

organs following pre- and post-natal exposure to bisphenol A at r0.2 mg/kg bw/day (Nagao et al., 2002). 

Following exposure of mice during pre- and postnatal 

development; no effect on age of vaginal opening, 

estrous cyclicity, or numbers of ovarian primordial 

follicles were observed at doses ranging from 0.003– 

600 mg/kg bw/day (Tyl et al., 2006). No effect on age of 

vaginal opening was reported but there was a shortened 

period between vaginal opening and first estrus following gestational exposure to 0.0024 mg/kg bw/day in a 

single dose level study (Howdeshell et al., 1999). 

Body Weight: All rat and mouse multigenerational 

studies have measured body weight as an endpoint. No 

consistent differences have been detected in the weights 

of offspring of animals exposed to low to moderate doses 

of BPA (Table 86). 

Hormone Levels: Several studies have measured testosterone and LH levels in rats, there have also been 

investigations of thyroid hormone (T4) levels. No 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 329 

consistent effects on the levels of these hormones have 

been seen (Ema et al., 2001). 

Fertility and ability to raise pups to weaning following

developmental exposure: Multigenerational studies in 

both rats and mice have shown that BPA over a 

wide dose range does not compromise the ability of 

animals exposed during development to successfully 

produce offspring, raise them to weaning and for those 

offspring to successfully give rise to a subsequent 

generation of animals (Ema et al., 2001; Tyl et al., 

2002b, 2006). 

Neural and Behavioral Endpoints Following Oral

Administration: Several studies addressing effects on 

neural and behavioral endpoints have been conducted 

following gestational and lactational exposure 

[rats: (Funabashi et al., 2004a; Negishi et al., 2004a; 

Della Seta et al., 2005)]; mice: [(Palanza et al., 2002; 

Nishizawa et al., 2003, 2005b; Laviola et al., 2005; Ryan 

and Vandenbergh, 2006)], pubertal exposure [rat: (Akingbemi et al., 2004; Della Seta et al., 2006; Ceccarelli et al., 

2007)], and exposure during adulthood [gerbils: (Razzoli 

et al., 2005)]. 

Gestational and lactational exposures in rats have 

reported subtle effects on sexually-dimorphic brain 

nuclei (Funabashi et al., 2004a), hormonal receptors in 

brain (Nishizawa et al., 2003, 2005b), and certain 

sexually-dimorphic or reproductively relevant behaviors 

(Negishi et al., 2004a; Ryan and Vandenbergh, 2006). 

Most of this work has utilized single doses across the 

range of 2–40 mg/kg, and none has been confirmed or 

linked to other functional or clearly adverse effects. No 

effects on the volume of the SDN-POA of the hypothalamus were observed in offspring of rats orally exposed 

to bisphenol A doses ranging from 3.2–320 mg/kg bw/ 

day during the gestation and lactation period (Kwon 

et al., 2000). Single dose level rat studies demonstrated 

reduced sexually dimorphic difference in corticotropinreleasing hormone neurons in anterior stria terminalis at 

2.5 mg/kg bw/day (Funabashi et al., 2004a). No changes 

in sexual behavior were reported for female rats exposed 

to 0.3–320 mg/kg bw/day or males exposed to r0.3 mg/ 

kg bw/day during the gestation and/or lactation period 

(Kwon et al., 2000). 

Maternal behavior of dams has also been suggested to 

be altered in two studies of dams exposed during 

gestation and lactation (Palanza et al., 2002; Della Seta 

et al., 2005). 

One study involving exposures during puberty (Della 

Seta et al., 2005) suggested alterations in exploratory and 

sexual behavior of males following 40 mg/kg on PND 

23–30. Certain changes in hypothalamic estrogen receptors (Ceccarelli et al., 2007) following 40 mg/kg exposures 

on PND 23–30 have been reported. Akingbemi et al. 

(2004) reported effects on gonadal hormonal and 

receptor endpoints in the pituitary following 2.4 mg/kg/ 

day on PND 21–35. 

Other endpoints: Following oral exposure of mice 

to bisphenol A during gestation, changes were 

observed for mRNA expression of arylhydrocarbon 

receptors, receptor repressor, or nuclear translocator 

and retinoic acid and retinoid X receptors in brain, 

testes, and/or ovary at 0.00002–20 mg/kg bw/day 

(Nishizawa et al., 2003, 2005a,b). The strongest 

effects were found at the lowest doses following 

exposures during organogenesis (GD 6.5–13.5 or 

6.5–17.5) (Nishizawa et al., 2005a,b). The study authors 

suggested those changes as possible mechanisms for 

bisphenol A-induced toxicity. 

A summary of LH and testosterone effects observed in 

humans and in bisphenol A-exposed experimental 

animals is included in Section 4.4. 

Summary and Conclusion of Developmental 

Hazards: There are sufficient data to conclude that 

bisphenol A does not cause malformations or birth 

defects in fetuses exposed during gestation at levels up to 

640 mg/kg/day (rats) and 1000 mg/kg/day (mice) (Morrissey et al., 1987). This is consistent with the lack of 

malformations seen in offspring in multigenerational 

studies (Tyl et al., 2002b, 2006). 

There are sufficient data to conclude that bisphenol A 

does not alter male or female fertility in rats or mice after 

gestational exposure up to doses of 450 mg/kg/day 

(Cagen et al., 1999b; Tyl et al., 2000a, 2002b; Ema et al., 

2001). 

There are sufficient data to conclude that bisphenol A 

does not change the age of puberty in male or female rats 

[NOAELs of 0.2 mg/kg/day (Ema et al., 2001) and 

1823 mg/kg/day (Tyl et al., 2002b)]. While limited data 

available suggest an effect on the onset of female puberty 

in mice [LOAEL 0.2 mg/kg/day (Ryan and Vandenbergh, 2006), 0.002 mg/kg/day, (Howdeshell et al., 

1999)], the data are insufficient to conclude that bisphenol A accelerates puberty in female mice. The 

limited data available suggest, but are insufficient to 

conclude, that bisphenol A slightly delays the age of 

puberty in male mice at a LOAEL of ca. 550–800 mg/kg/ 

day (Tyl et al., 2006). 

There are sufficient data to conclude that bisphenol A 

exposure during development does not permanently 

affect prostate weight in adult rats or mice [NOAELs of: 

1823 mg/kg/day (Tyl et al., 2002b), 600 mg/kg/day (Tyl 

et al., 2006), 4 mg/kg/day (Cagen et al., 1999b), 0.2 mg/ 

kg/day (Ema et al., 2001), 50 mg/kg/day (Tinwell et al., 

2002), and 320 mg/kg/day (Kwon et al., 2000). There are 

sufficient data to conclude that bisphenol A does not 

cause prostate cancer in rats or mice after adult 

exposure [calculated dose ranges of 25–400 mg/kg/day 

for rats, 600–3000 mg/kg/day, mice (NTP, 1982)]. There 

are slight suggestions, but insufficient data to conclude, 

that bisphenol A might predispose toward prostate 

cancer in rats in later life following developmental 

exposure [at 10 mg/kg (Ho et al., 2006a)]. There are 

slight suggestions, but insufficient evidence to conclude, 

that fetal exposure to bisphenol A can contribute to 

urinary tract deformations in mice [10 mg/kg (Timms 

et al., 2005)]. 

There are sufficient data to suggest that developmental 

exposure to bisphenol A causes neural and behavioral 

alterations related to sexual dimorphism in rats and mice 

(ca. 2.5 mg/kg/day, gestation and lactation in rats, 

(Funabashi et al., 2004a); LOEL 0.00002 mg/kg/day, fetal 

mice, (Nishizawa et al., 2005a); 0.0002 mg/kg/day, fetal 

mice, (Nishizawa et al., 2003), 0.04 mg/kg/day, weaning 

to puberty, rats, (Ceccarelli et al., 2007); 0.1 mg/kg/day, 

GD 3–PND 20, rats, (Negishi et al., 2004a); 0.2 mg/kg/ 

day, GD 3–PND 20, mice, (Ryan and Vandenbergh, 2006); 

0.01 mg/kg/day, GD 11–18, mice, (Laviola et al., 2005), 

although other studies report no change in a related 

measure, the size of the sexually dimorphic nucleus of 

the pre-optic area (SDN-POA) [300 mg/kg/day, rats 

Birth Defects Research (Part B) 83:157–395, 2008
330 CHAPIN ET AL. 

(Nagao et al., 1999); NOEL of 320 mg/kg/day, rats, 

(Kwon et al., 2000)]. 

4.0 REPRODUCTIVE TOXICITY DATA 

4.1 Human 

4.1.1 Female. Takeuchi and Tsutsumi (2002), supported by the Japanese Ministry of Education, Science, 

Sports, and Culture, the Ministry of Health and Welfare, 

and the Science and Technology Agency, measured 

bisphenol A in the blood serum of 14 healthy women, 

11 healthy men, and 16 women with polycystic 

ovary syndrome [diagnostic criteria not discussed]. 

The healthy women were evaluated in the mid-follicular 

phase of the menstrual cycle. Bisphenol A was 

measured using a competitive ELISA. Serum was also 

evaluated for total and free testosterone, 17b-estradiol, 

androstenedione, dehydroepiandrosterone sulfate, LH, 

FSH, and prolactin. Statistical analysis was by ANOVA. 

Correlation coefficients were obtained from a linear 

regression analysis. Mean7SEM bisphenol A serum 

concentrations (ng/mL) were 0.6470.10 in normal 

women, 1.4970.11 in normal men, and 1.0470.10 in 

women with polycystic ovary syndrome. Bisphenol A 

serum concentrations were correlated significantly with 

total testosterone (r 5 0.595) and free testosterone 

(r 5 0.609) in all subjects and in all female subjects 

(r 5 0.559 for total testosterone and 0.598 for free 

testosterone). Bisphenol A serum concentrations were 

not significantly correlated with any other hormone 

measures. The authors concluded that either bisphenol A 

stimulates testosterone production or metabolism of 

bisphenol A is inhibited by testosterone. They further 

suggested that displacement of sex steroids from sexhormone binding globulin by bisphenol A might disrupt 

the estrogen-androgen balance. 

Strengths/Weaknesses: Quality assurance for the 

hormone radioimmunoassays appeared adequate; however, there was no standardization for time of day for the 

serum samples, which may result in variable testosterone 

levels. ELISA has not been standardized for human sera, 

and may overestimate bisphenol A due to nonspecific 

binding (see Section 1.1.5). Very little descriptive information was given on any of the groups beyond mean age 

and body-mass index. No information was given on 

recruitment methods and participation rates/exclusions. 

The lack of diagnostic criteria for polycystic ovary 

syndrome is a weakness. No potential confounders or 

effect modifiers were identified except mean age and 

body-mass index. Mean values appear to have been 

similar between groups. The positive correlations between bisphenol A level and total/free testosterone 

levels in all women and in entire study group were 

noted, but these analyses were not adjusted for potential 

confounders or effect modifiers. No information was 

given on whether the data were normally or lognormally 

distributed. The study was limited by small numbers in 

each group and the results should be regarded as 

descriptive epidemiology. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate but has limited utility given its 

small size, and limited design. The study provides some 

insight for potential mechanisms affecting the levels of 

bisphenol A in the body. 

Takeuchi et al. (2004a), supported by the Japanese 

Ministry of Education, Science, Sports, and Culture, the 

Ministry of Health, Labor, and Welfare, the National 

Institute for Environmental Studies, and the Science and 

Technology Agency, examined relationships between 

serum sex hormone and bisphenol A concentrations in 

women with ovarian dysfunction and obesity. Fasting 

blood samples were collected during the midfollicular 

phase from 19 non-obese and 7 obese healthy women 

with normal menstrual cycles. Blood samples were also 

obtained from 7 women with hyperprolactinemia, 21 

patients with hypothalamic amenorrhea, and 13 nonobese and 6 obese patients with polycystic ovary 

syndrome. [It not known whether any of these subjects 

were the same as those reported earlier by this group 

(Takeuchi and Tsutsumi, 2002).] Mean ages for the 

subgroups ranged from 25–29 years old. Blood serum 

was analyzed for bisphenol A levels using an ELISA 

technique, and total and free testosterone, 17b-estradiol, 

androstenedione, dehydroepiandrosterone sulfate, LH, 

FSH, prolactin, and insulin levels were measured using 

by RIA. Statistical analyses included ANOVA and linear 

regression analysis. 

Compared to non-obese healthy women, concentrations of bisphenol A in serum were significantly higher 

in non-obese women with polycystic ovary syndrome 

[48% higher], obese women with polycystic ovary 

syndrome [65% higher], and obese healthy women 

[46% higher]. Statistically significant positive correlations were found between bisphenol A level in serum 

and body mass index (r 5 0.500) and serum levels of total 

testosterone (r 5 0.391), free testosterone (r 5 0.504), 

androstenedione (r 5 0.684), and dehydroepiandrosterone sulfate (r 5 0.514). The study authors concluded that 

there is a strong relationship between serum levels of 

bisphenol A and androgens, possibly due to androgen 

effects on metabolism of bisphenol A. 

Strengths/Weaknesses: Quality assurance for the 

hormone radioimmunoassays appears adequate. In contrast to the 2002 article by these authors (Takeuchi and 

Tsutsumi, 2002), blood draws were time-standardized to 

9:00–10:00 AM after overnight fasting. As noted in 

Section 1.1.5, ELISA may overestimate bisphenol A. It 

was not clear whether any of the women in this study 

were also included in their 2002 publication. No potential 

confounders or effect-modifiers were identified except 

mean age and body-mass index, and neither of these was 

controlled in the analyses. Positive correlations were 

observed for bisphenol A level with body-mass index, 

total testosterone, free testosterone, androstenedione, 

and dehydroepiandrosterone sulfate for all study 

groups. These correlations are also found (with the 

exception of total testosterone) in the control (‘‘normal 

women’’) group as well. Normality of the distributions of 

the hormones were not reported, and not transformed 

before analysis. The study was limited by small numbers 

and results should be regarded as descriptive 

epidemiology. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate but has limited utility in assessing 

possible relationships of bisphenol A with androgens 

(testosterone, free testosterone, androstenedione, dehydroepiandrosterone sulfate) and conditions that may 

promote hyperandrogenism (obesity, polycystic ovarian 

syndrome). 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 331 

Hiroi et al. (2004), supported by the Japanese Ministry 

of Health, Labor, and Welfare, the National Institute for 

Environmental studies, and the Japan Science and 

Technology Agency, compared blood bisphenol A levels 

in women with and without endometrial hyperplasia. 

Volunteers were recruited from an outpatient clinic in 

Japan. Women included in the study consisted of 11 

controls with normal endometrium, 19 with endometrial 

hyperplasia, and 7 with endometrial carcinoma. The 

hyperplasia group was further divided according to 

severity: 10 with simple hyperplasia and 9 with complex 

hyperplasia. Mean ages were 48.4–48.9 years in groups 

without cancer, and the mean age was 63.1 years in the 

group with endometrial cancer. Blood samples were 

collected at the time of endometrial examination. Serum 

bisphenol A levels were measured by ELISA. Data were 

analyzed by Student t-test, with the exception of 

gravidity and parity, which were analyzed by w2 test. 

There were no significant differences in age, gravidity, 

parity, or body height, weight, or mass index between the 

groups without endometrial cancer. Women with endometrial cancer were significantly older and had 

significantly lower values for gravidity, parity, height, 

and weight. Mean7SD serum bisphenol A levels were 

reported at 2.571.5 ng/mL in controls, 2.271.6 ng/mL 

in women with hyperplasia, and 1.470.5 ng/mL in 

women with endometrial cancer. When the group with 

hyperplasia was divided according to severity, serum 

bisphenol A blood levels were reported at 2.972.0 ng/ 

mL in the group with simple hyperplasia and 

1.470.4 ng/mL in the group with complex hyperplasia. 

Serum bisphenol A levels were significantly lower in 

women with complex endometrial hyperplasia or endometrial cancer than in controls. The study authors 

concluded that their preliminary findings demonstrated 

a possible link between bisphenol A exposure and 

endometrial hyperplasia or cancer. It was noted that 

modes of action for bisphenol A may be more complex 

than expected and that these contradictory results might 

provide a clue about mechanisms of production of 

estrogen-dependent diseases. 

Strengths/Weaknesses: Because this was a small, 

cross-sectional study, it is not possible to determine 

whether this association preceded disease, or could have 

been associated with the disease process. As noted in 

Section 1.1.5, ELISA may over estimate bisphenol A. 

Utility (Adequacy) for CERHR Evaluation Process: 

The cross-sectional study design is adequate but of 

limited utility for this evaluation, but raises research 

questions regarding mechanisms of production of estrogen-dependent diseases. 

Sugiura-Ogasawara et al. (2005), supported by the 

Japanese Ministry of Health, Labor, and Welfare, conducted a study to determine if there is an association 

between recurrent miscarriage and bisphenol A levels in 

blood. The cases in this study were 45 patients with a 

history of 3 or more (3–11) consecutive first trimester 

miscarriages. Mean7SD age of the cases was 31.674.4. 

None of the cases had a history of live birth. All were 

seen at a Japanese hospital between August, 2001– 

December, 2002. Half of the cases were housewives and 

half were employed in various occupations. A hysterosalpingography analyses was conducted in cases, and 

chromosome analyses were conducted for both cases and 

their partners. Women were excluded from the study if 

uterine anomalies were observed or chromosomal 

abnormalities were detected in either partner. Serum 

bisphenol A levels were determined by ELISA. Immunological endpoints examined included antinuclear antibodies, antiphospholipid antibodies, and natural killer 

cell activity. Blood testing for hypothyroidism, diabetes 

mellitus, and hyperprolactinemia was conducted. Blood 

samples were obtained 5–9 days following ovulation in at 

least 2 cycles. Blood samples to determine progesterone 

and prolactin levels were taken at 3 months following the 

last miscarriage and before the next conception. For 

subsequent pregnancies, ultrasounds were conducted, 

and spontaneously aborted embryos/fetuses were karyotyped. Serum levels of bisphenol A in cases were 

compared to those of 32 healthy non-pregnant hospital 

employees with no history of live birth, infertility, or 

miscarriage. Mean7SD age of controls was 32.074.8. 

None were taking oral contraceptives. Like the cases, the 

controls lived near Nagoya City. Statistical analyses 

included Welch test, Mann–Whitney test, and Pearson 

correlation coefficient. 

Bisphenol A levels (mean7SD) were reported to be 

significantly higher in women with recurrent miscarriages (2.5975.23 ng/mL) compared to healthy controls 

(0.7770.38 ng/mL). In the 45 cases, incidences of 

abnormal conditions were 15.6% for hypothyroidism, 

13.3% for antiphospholipid antibodies, 22.2% for antinuclear antibodies, 11.1% for hyperprolactinemia, and 

20.5% for luteal phase defect. Serum levels of bisphenol 

A were significantly higher in patients who tested 

positive versus negative for antinuclear antibodies 

(mean7SD 5 7.38279.761 vs. 1.22271.54 ng/mL). 

Thirty-five of the patients became pregnant and 48.6% 

had another miscarriage. Serum bisphenol A levels in 

patients who miscarried were 4.3978.08 ng/mL, and 

serum bisphenol A in patients with successful pregnancies were 1.2271.07 ng/mL (not statistically significant). 

The study authors concluded that exposure to bisphenol 

A is associated with recurrent miscarriage. 

In a letter to the editor, Berkowitz (2006) stated that this 

study did not support an association between bisphenol 

A blood levels and recurrent miscarriage. Several 

limitations were noted for the study. Timing and 

numbers of blood samples collected were not defined 

clearly. It was noted that because bisphenol A has a short 

half-life, it would be critical to know if blood samples 

were obtained in a timeframe relevant to the occurrence 

of miscarriage. Although differences in serum bisphenol 

A levels in cases compared to controls achieved statistical 

significance, it was noted that median levels of bisphenol 

A in serum were nearly identical in patients with 

recurring miscarriages (0.71 ng/mL) and controls 

(0.705). The similarities in median values suggested there 

were no differences between the two groups, and it was 

suggested that apparent differences in mean serum 

levels of bisphenol A were due to a few individuals, as 

was demonstrated in Figure 1 of the Sugiura-Ogasawara 

et al. (2005) report. Berkowitz (2006) stated that the Welch 

test was inappropriate for statistical analyses and noted 

that the two evaluation groups could not be considered 

comparable because of differences in occupation (housewives compared to medical workers) and unknown 

fertility of controls. Because the controls were not 

evaluated for factors such as hypothyroidism and 

systemic lupus erythematosus (associated with 

Birth Defects Research (Part B) 83:157–395, 2008
332 CHAPIN ET AL. 

antinuclear antibodies), the conditions may have been 

over represented in cases and may have been the cause of 

the reported differences between the 2 groups. Although 

mean bisphenol A levels were (non-significantly) lower in 

women who subsequently became pregnant and had a 

successful pregnancy compared to those who miscarried, 

Berkowitz (2006) noted that the median level of bisphenol 

A was actually higher in women with the successful 

pregnancies. Last, the ELISA method for measuring 

bisphenol A levels has not been validated and is subject 

to inaccuracy due to extensive cross-reactivity. 

In a response to the comments by Berkowitz (2006), 

Sugiura-Ogasawara (2006) stated that although measurement of bisphenol A levels at various time points would 

have been ideal, obtaining samples every day during 

pregnancy would have been difficult. Sugiura-Ogasawara (2006) clarified that bisphenol A values were based 

on a single sample in each individual, but that similar 

tendencies were observed for a second blood sample. 

With respect to the use of women with live births as 

controls, Sugiura-Ogasawara (2006) explained that the 

same blood samples were used for measurements of 

other environmental compounds, some of which are 

known to decrease after delivery. It was noted that none 

of the cases had systemic lupus erythematosus, and that 

use of controls with hypothyroidism or antinuclear 

antibodies was not considered important for the study. 

Superiority of the HPLC method compared to the ELISA 

method for measuring serum bisphenol A levels was 

acknowledged, but the authors stated that the ELISA 

method was used because of limited funding, reiterated 

that the study was preliminary and used a small number 

of volunteers, and that additional studies using a larger 

sample and more appropriate analytical methods were 

needed. 

Strengths/Weaknesses: The letter from Berkowitz (2006) 

summarizes many of the weaknesses of this study. No 

quality assurance information was given for the biomarker/hormone measurements. As the Berkowitz letter 

points out, the ELISA method is not standardized for 

human sera (and may overestimate bisphenol A due to 

nonspecific binding), the distribution of exposure was not 

normal, and median values of the two groups were similar, 

with two women skewing the mean. Little information 

was provided on the characteristics of the two study 

groups or response rates. Age and body-mass index were 

controlled in the analyses, but other potential confounders 

and effect modifiers were not. The time between exposure 

and observation was not appropriate. Spontaneous abortions have been associated with many factors which have 

not been addressed here. The authors’ conclusions require 

the assumption that bisphenol A measurement levels 

represent those present during the important time frame 

for the spontaneous abortion. The authors do not report 

the time frame for collection of the blood samples. Nonnormal data were not appropriately transformed for 

analysis. Welch’s test was used ‘‘yto compare bisphenol 

A levelsybecause the distribution of the two groups 

might have differed.’’ Welch’s test is a t-test for groups 

with unequal variance, not different distributions (both 

should be normal, which was probably not the case). 

Utility (Adequacy) for CERHR Evaluation Process: 

Because of limitations in the design and analysis of this 

work, this study is inadequate has no utility in this 

evaluation. 

Yang et al. (2006), supported by the Korean FDA, 

measured urine bisphenol A in 172 Korean men and 

women and evaluated the relationship of these values 

with UDP-glucuronosyl- and sulfotransferase polymorphisms, with sister-chromatid exchange testing, and 

with self-reported symptoms of possible endocrine 

origin. First-morning urine samples were collected at 

the time of a routine physical examination, as was a 

blood sample, and a questionnaire was completed. Urine 

bisphenol A was measured using reverse phase HPLC. 

DNA was isolated from blood samples and polymorphisms were determined at SULT1A1 and UGT1A6. Sister 

chromatid exchange in response to N-methyl-N0

-nitro-Nnitrosoguanidine (MNNG) was evaluated in blood cells 

[not otherwise specified]. The relationship between 

urine bisphenol A and continuous variables was assessed 

with simple or multiple regression analysis and the 

relationship with categorical variables assessed with the 

Wilcoxon test. 

None of the subjects reported occupational exposure to 

bisphenol A. The median urine bisphenol A concentration was 7.86 mg/L. Urine bisphenol A was not different 

in men and women. Urine bisphenol A was associated 

with body-mass index (P 5 0.06) and self-reported 

frequency of alcohol consumption (P 5 0.08). SULT1A1

and UGT1A6 polymorphisms were not associated significantly with urine bisphenol A concentrations. No 

significant associations were observed between urine 

bisphenol A and MNNG-induced sister-chromatid exchange, although they were associated when lower levels 

of MNNG were used. There were no significant associations between urine bisphenol A and self-reported 

symptoms of possible endocrine origin, including 

thirst/frequent urination, dizziness, neck mass, heat 

intolerance, sweating, hot flashes, swelling of lymph 

nodes, dysmenorrhea, menstrual irregularity, or menorrhagia. The authors concluded that even though they had 

been unable to associate an endocrine disorder with 

urine bisphenol A, continuous biologic monitoring of 

bisphenol A would be prudent. 

Strengths/Weaknesses: Bisphenol A was measured in 

urine using HPLC. No information was given regarding 

any selection criteria or response rates and some outcome 

measures were self-reported. 

Utility (Adequacy) for CERHR Evaluation Process: 

While small, this study is useful for providing descriptive exposure information on BPA urinary levels (see 

Section I). This study does not have utility for evaluation 

of reproductive endpoints. 

4.1.2 Male. Luconi et al. (2001), supported by the 

Italian Public Health Project, examined the effects of in 

vitro exposure of human spermatozoa to bisphenol A. 

Semen was collected from normozoospermic men, and 

spermatozoa were separated. Intracellular calcium was 

measured using a spectrofluorometric method in cells 

treated with 1 mM bisphenol A, 1 mM 17b-estradiol, 10 mM 

progesterone, [17 b-estradiol is noted as 10lM in Figure 

6, text states 1lM] or the same concentrations of 

bisphenol A in combination with 17b-estradiol or 

progesterone. Effects on acrosome reaction were examined using a fluorescent staining method in cells exposed 

to 1 mM [0.23 mg/mL] bisphenol A for 2 hr, with and 

without exposure to 10 mM progesterone. [In the study 

figures summarizing results, sample numbers in 

studies involving bisphenol A were listed at 5–11. It 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 333 

is not known if the sample numbers represented total 

numbers of sperm donors. Very few protocol details 

were provided in the Methods section and many of the 

limited details presented above were obtained from the 

Results section.] Data were analyzed by Student t-test 

and 1-way ANOVA. Treatment of spermatozoa with 

bisphenol A resulted in a modest influx of calcium, but 

bisphenol A had no effect on calcium responses induced 

by 17b-estradiol or progesterone. Bisphenol A exposure 

did not affect basal acrosome reaction or acrosome 

reaction induced by progesterone. Results were in 

contrast to those observed with 17b-estradiol, which 

inhibited the acrosome reaction induced by progesterone. 

The study authors concluded, BPA did not exert any 

direct effect on calcium fluxes and acrosomal reaction in 

human spermatozoa either in basal conditions or in 

response to progesterone challenge. 

Strengths/Weaknesses: Strengths of this study include 

examining human spermatozoa and use of a concurrent 

control (E2) to demonstrate the responsiveness of the 

system. Weaknesses include limited information on the 

spermatozoa samples, the single concentration of BPA 

used, and lack of clarity of concentrations of E2 versus 

bisphenol A administered. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study did not demonstrate that BPA-altered 

progesterone-mediated acrosomal reaction and is not 

useful in the evaluation process. 

Hanaoka et al. (2002), supported by the Japanese 

Ministry of Health and Welfare and Ministry of Education, Science, Sports, and Culture, examined possible 

relationships between bisphenol A exposure and hormone levels in male workers. Exposed workers included 

42 men in 3 Japanese plants who sprayed an epoxy 

hardening agent consisting of a mixture of bisphenol 

A diglycidyl ether (10–30%), toluene (0–30%), xylene 

(0–20%), 2-ethoxyethanol (0–20%), 2-butoxyethanol (0– 

20%), and methyl isobutyl ketone (0–30%). The workers 

were said to wear ‘‘protection devices’’ during spraying. 

Controls consisted of 42 male assembly workers from the 

same plants who did not use bisphenol A diglycidyl 

ether, were within 3 years of age to exposed workers (37 

years vs. 38 years), and smoked the same number of 

cigarettes/day as exposed workers (21/day). Percentages 

of smokers were 86% in both groups, but percentages of 

alcohol drinkers were significantly lower in the exposed 

workers (43%) than in controls (57%) (P 5 0.03). Urine 

and blood samples were obtained during periodic health 

examinations performed in June and July, 1999. Urinary 

bisphenol A was measured by HPLC, and urinary 

organic solvent metabolites were measured by GC or 

HPLC. Plasma LH, FSH, and free testosterone levels were 

measured by immunosolvent assay in a commercial 

laboratory. Data were log transformed and compared by 

paired t-test, Pearson correlation coefficient, and w2 test. 

Adjustments were made by linear regression for age and 

drinking habits, which were considered possible 

confounders. 

Urinary bisphenol A concentrations were significantly 

higher in exposed workers (median: 1.06 mmol/mol 

creatinine [0.043 lg/kg bw]; range: o0.05 pmol to 

11.2 mmol/mol creatinine) than in controls (median: 

0.52 mmol/mol creatinine [0.021 lg/kg bw]; range: 

o0.05 pmol to 11.0 mmol/mol creatinine). Average 

difference was reported as 2.5 (95% CI 5 1.4–4.7; 

P 5 0.002). Bisphenol A was not detected in 3 exposed 

workers and 1 control. Urinary solvent metabolites were 

detected more frequently in exposed workers than 

controls. No differences in plasma testosterone or LH 

concentrations were observed between exposed workers 

and controls. Plasma FSH concentrations were significantly lower in exposed workers (median 5 5.3 mIU/mL; 

range 5 4.0–8.3 mIU/mL) than in controls (median 5 7.6 ­

mIU/mL; range 5 5.4–11.0 mIU/mL; average difference 5 1.3; 95% CI 5 -1.5 to -1.0). A ‘‘mild correlation’’ 

was reported between urinary bisphenol A and FSH 

(r 5 -0.20, P 5 0.071) but was not observed for urinary 

solvent levels. A statistically significant relationship was 

observed between FSH and bisphenol A following 

adjustment for alcohol intake (r 5 -0.23; P 5 0.045). The 

study authors concluded that bisphenol A may be 

generated endogenously following exposure to bisphenol A diglycidyl ether, and bisphenol A may disrupt 

gonadotropic hormone secretion in men. 

Strengths/Weaknesses: Quality assurance for the 

hormone radioimmunoassays appeared adequate. Blood 

draws and urine samples were time standardized 

between 10:00 AM and 12:00 PM. Reference values were 

given and population values were considered in the 

discussion. Use of HPLC for bisphenol A and standard 

methods for the other urinary metabolites with creatinine-adjustment are strengths. The epoxy sprayer workers were matched to coworkers from other parts of the 

process. All selected workers participated in the study. 

Analyses were adjusted for age and alcohol use, and 

workers were matched on age (73 years) and cigarette 

use. A plausible (P 5 0.07) correlation between bisphenol 

A and decreasing FSH was reported. The authors took 

care to note that all FSH levels were within the clinical 

normal range. Correlations between other workplace 

exposures and hormones were not observed. Blood and 

urine samples were collected concurrently, but not on the 

first day of the week. Statistical methods were appropriate to the study size and distribution of the data. Nonnormal distributions were transformed or treated as nonnormal. Biomarker data were handled appropriately in 

analysis. 

Utility (Adequacy) for CERHR Evaluation Process: 

This survey was methodologically sound and mechanistically thoughtful. This study is adequate and of high 

utility for the evaluation. 

4.2 Experimental Animal 

Studies in this section examine reproductive endpoints 

after administration of bisphenol A to sexually mature 

animals. Reproductive endpoints after administration of 

bisphenol A during pregnancy, the neonatal period, or 

puberty are discussed in Section 3.2. 

4.2.1 Female 

4.2.1.1 Rat: Goloubkova et al. (2000), supported by 

the Brazilian National Council of Scientific and Technological Development and the National University of Rio 

Grande Do Sul, examined the effects of bisphenol A 

exposure on the uterus and pituitary of ovariectomized 

rats. Wistar rats (60–67 days old) were fed a standard 

certified rodent diet. [No information was provided on 

housing or bedding materials.] Rats were subjected to 

bilateral ovariectomy or sham surgery. At 14 days postsurgery, rats were randomly assigned to groups of at 

Birth Defects Research (Part B) 83:157–395, 2008
334 CHAPIN ET AL. 

least 6 animals. Rats were s.c. injected with bisphenol A 

in DMSO vehicle (499% purity) at doses of 11, 78, 128, or 

250 mg/kg bw/day for 7 days. An ovariectomized 

vehicle control group was exposed to the 50% DMSO 

vehicle. A sham-operated control group was not exposed 

to the vehicle. Rats were killed following the dosing 

period, and body and uterine weight were measured. 

Trunk blood was collected for measurement of serum 

prolactin level by RIA. The anterior pituitary was 

weighed and preserved in 10% formalin. An immunohistochemical technique was used to identify pituitary 

cells expressing prolactin. A total of 3 or 4 rats per group 

were evaluated for prolactin-positive cells in the pituitary and 6–8 rats were evaluated for the other endpoints. 

Data were analyzed by ANOVA followed by post-hoc 

Student-Neuman–Keuls test or Kruskal–Wallis ANOVA 

followed by post-hoc Dunn test. 

In the 250 mg/kg bw/day group, final body weight 

was 7% lower than in the ovariectomized vehicle control 

group, and body weight gain was lower compared to the 

ovariectomized vehicle and sham controls. There was no 

effect of treatment on food intake. A dose-related 

increase in uterine weight occurred in all groups of rats 

exposed to bisphenol A compared to the ovariectomized 

vehicle controls, but uterine weight in the bisphenol A 

groups was lower than in the sham controls. Ovariectomy resulted in decreased pituitary weight in ovariectomized vehicle controls and in the bisphenol A 11 and 

78 mg/kg bw/day dose groups compared to sham 

controls. Pituitary weight did not differ from sham 

controls after 128 mg/kg bw/day bisphenol A and was 

greater than in sham controls after 250 mg/kg bw/day 

bisphenol A. Basal prolactin levels did not differ between 

the sham and ovariectomized vehicle controls. Serum 

prolactin levels were increased in the 128 and 250 mg/kg 

bw/day bisphenol A groups compared to the ovariectomized vehicle controls. Ovariectomy reduced the numbers of prolactin-positive cells in the pituitary. The 

number of prolactin positive cells in the pituitary was 

increased by 64% in the 250 mg/kg bw/day group 

compared to the ovariectomized controls. The study 

authors concluded that the reproductive tract and 

neuroendocrine axis of Wistar rats can respond to 

bisphenol A. 

Strengths/Weaknesses: This study represents a comprehensive neuroendocrine assessment across multiple 

doses. Weaknesses are the absence of a positive control to 

demonstrate maximal response in endpoints examined, 

high-dose levels required to induce response, and the s.c. 

route of administration. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate but of limited utility for the 

evaluation process. 

Funabashi et al. (2001), supported by Yokoyama City 

University, examined the effects of bisphenol A exposure 

on expression of progesterone receptor mRNA in the 

brain of ovariectomized rats. The effects of butylbenzyl 

phthalate were also examined but will not be discussed. 

[No information was provided on feed, caging, or 

bedding materials.] Wistar rats were ovariectomized at 

7–8 weeks of age. Ten days following ovariectomy, 6 

rats/group were s.c. injected with sesame oil vehicle, 

10 mg bisphenol A [purity not reported], or 10 mg 17bestradiol. Rats were killed 24 hr later and the preoptic 

area, medial basal hypothalamus, and anterior pituitary 

were removed. Expression of mRNA for progesterone 

receptor, preproenkephalin, and neurotensin were assessed by Northern blot. Data were analyzed by ANOVA 

followed by Fisher protected least significant difference 

test. Exposure to bisphenol A resulted in increased 

expression of progesterone receptor mRNA in the 

preoptic area and anterior pituitary. Bisphenol A did 

not affect expression of mRNA for neurotensin in the 

preoptic area or preproenkephalin in medial basal 

hypothalamus. 17b-Estradiol increased expression of 

mRNA for progesterone receptor in the preoptic area, 

medial basal hypothalamus, and anterior pituitary and 

increased preproenkephalin mRNA expression in medial 

basal hypothalamus. The study authors concluded that 

bisphenol A increases expression of progesterone receptor mRNA in the preoptic area of adult ovariectomized 

rats. 

Strengths/Weaknesses: Strengths are the use of a 

positive control and the biological plausibility of the 

model. Weaknesses include subcutaneous administration 

of a single high-dose level. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate for inclusion but of limited utility. 

Yamasaki et al. (2002a) conducted a 28-day exposure 

study that provided some information on the reproductive organs of male and female rats. [Complete details of 

this study are included in Section 2. Results for females 

are discussed in this section, and results for males are 

discussed in Section 4.2.2.1.] CD rats were fed a 

commercial diet (MF Oriental Yeast Co.) and housed in 

stainless steel wire mesh cages. Ten 7-week-old rats/sex/ 

group were gavaged with bisphenol A [98% purity] at 0 

(olive oil vehicle), 40, 200, or 1000 mg/kg bw/day 

for 28 days. Due to the death of 1 animal exhibiting 

clinical signs in the 1000 mg/kg bw/day group, the 

high-dose was reduced to 600 mg/kg bw/day on 

Day 8 of the study. In an additional study, rats 

were exposed to ethinyl estradiol at 0, 10, 50, or 

200 mg/kg bw/day for 28 days. There were no treatment-related alterations in blood levels of thyroid 

hormones, FSH, LH, 17b-estradiol, prolactin, or 

testosterone. The numbers of females with diestrus 

lasting 4 or more days was increased in the high-dose 

group. Relative weights of ovary and uterus were 

unaffected. No gross or histopathological alterations 

were reported for reproductive organs. The study 

authors concluded that change in estrous cyclicity was 

the only useful endpoint for evaluating the endocrinemediated effects of bisphenol A. In comparison, females 

from the mid- and/or high-dose ethinyl estradiol group 

experienced alterations in estrous cyclicity, decreased 

ovarian weight, increased uterine weight, and histopathological changes in the ovary, uterus, and vagina. 

Strengths/Weaknesses: This study was well-conducted, used an appropriate route of administration, a 

positive control group, adequate sample sizes, a range of 

doses, and evaluations of both sexes. Weaknesses include 

failure to define the criteria for an abnormal estrous 

cycle, female necropsy at a point unrelated to stage of 

estrous. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

Spencer et al. (2002), supported by NIH, evaluated the 

uterine response to bisphenol A before and after 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 335 

deciduoma formation in pseudopregnant Sprague– 

Dawley rats. [Cage and bedding materials and feed 

were not indicated.] Adult females underwent mechanical cervical stimulation to induce pseudopregnancy 

[pseudopregnancy day not indicated]. On pseudopregnancy day 4, deciduoma formation was induced under 

ether anesthesia by antimesometrial uterine epithelial 

trauma, applied through a laparotomy under ether 

anesthesia. Rats were treated with s.c. bisphenol A 

[97% purity] 0 or 200 mg/kg bw in alcohol/saline on 

pseudopregnancy days 1–4 and killed on pseudopregnancy day 5, or treated on pseudopregnancy days 5–8 

and killed on pseudopregnancy day 9. Uteri and 

pseudopregnancy day 9 endometria were harvested. 

Uteri were weighed and homogenized for measurement 

of protein and DNA content. Inducible nitric oxide 

synthase activity, decidual prolactin-related protein 

mRNA, ER mRNA, and cytosolic ER binding sites were 

measured in uteri and/or endometria. Blood was 

obtained for determination of serum 17b-estradiol and 

progesterone. [n 5 5 was indicated for some of the data 

presentations.] Results are summarized in Table 87. The 

authors called attention to the difference in bisphenol A 

effect depending on whether exposure was before or 

after deciduoma induction. They concluded that there 

was a decrease in proliferation when bisphenol A was 

given during deciduoma induction, with a decrease in 

decidual proteins, in spite of a lack of differential effect 

on ER mRNA or cytosolic ER binding sites. The authors 

also concluded that bisphenol A activity appeared to be 

antagonized by progesterone [although they probably 

meant that bisphenol A antagonized the action of 

progesterone]. 

Strengths/Weaknesses: These data are intriguing, but 

the functional consequences of bisphenol A administration on decidual formation were not assessed and the s.c. 

route of administration and the use of a single high-dose 

are a weakness. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate but of limited utility to the 

evaluation process. 

Funabashi et al. (2003), supported by Yokohama City 

University, examined the effects of bisphenol A exposure 

on sexual behavior and progesterone receptor expression 

in adult rats. Wistar rats were ovariectomized at 7–8 

weeks of age. [No information was provided on feed, 

caging, or bedding materials.] In two sets of experiments, an immunohistochemistry technique was used to 

measure expression of progesterone receptor in the 

preoptic area and ventromedial hypothalamus following 

bisphenol A exposure. In the first experiment, 3–5 rats/ 

group were s.c. injected with sesame oil vehicle, 10 mg 

bisphenol A (B40 mg/kg bw) [purity not reported], or 

10 mg 17b-estradiol (B40 mg/kg bw) 2 weeks following 

ovariectomy. In the second experiment, ovariectomized 

rats (3–4/group) were s.c. injected with bisphenol A at 

0.001, 0.010, 0.1, or 1 mg (B0.004, 0.040, 0.4, or 4 mg/kg 

bw). Rats were killed the day following dosing, 

and brains were removed and fixed in 2% paraformaldehyde. Statistical analyses included ANOVA followed 

by Scheffe ́ post-hoc test and Kruskall–Wallis test. 

Sexual behavior was examined in a third experiment. 

Ovariectomized rats were s.c. injected with sesame oil 

vehicle, 10 mg bisphenol A, or 10 mg 17b-estradiol. 

The next day, rats were injected with 1 mg progesterone 

Table 87
 

Bisphenol A Effects on Pseudopregnant Ratsa
 

Treatment period, 

pseudopregnancy day 

Endpoint 1–4 5–8 

Uterus 

Wet weight m 1.4-fold k 63% 

Protein content m 1.4-fold k 64% 

DNA content 2 k 53% 

Decidual prolactin- 2 k 44% 

related protein mRNAb 

ER mRNAb k 29% k 50% 

Cystosolic ER-binding k 57% k 37% 

sites 

Nitric oxide synthase 2 k 50% 

activityb 

Pseudopregnancy day 9 

endometrium 

Decidual prolactin-related Not applicable k 48% 

protein mRNAb 

ER mRNAb Not applicable k 43% 

Nitric oxide synthase Not applicable k 40% 

activityb 

Serum 

17b-Estradiol 2 2

Progesterone 2 k 49% 

a

Spencer et al. (2002).
 

b

Estimated from a study graph by CERHR.
 

m, k, 2 Statistically significant increase, decrease, or no change
 

compared to vehicle control.
 

or vehicle to generate 4 treatment groups: sesame 

oil1progesterone (n 5 5), bisphenol A1sesame oil 

(n 5 5), bisphenol A1progesterone (n 5 8), or estradiol1

progesterone (n 5 6). Examination of behavior with a 

sexually receptive male was conducted 5–7 hr 

following progesterone or vehicle injection. Statistical 

analyses included ANOVA followed by Scheffe ́ 

post-hoc test. 

In the first experiment, injection of rats with 10 mg 

bisphenol A increased progesterone-positive cells in both 

the preoptic area and ventromedial hypothalamus. The 

dose–response experiment demonstrated that dose-related increases in progesterone-positive cells in both 

brain regions occurred following exposure to Z0.1 mg 

bisphenol A. In sexual behavior testing, treatment with 

bisphenol A had no effect on lordosis quotient. Rejection 

quotient was significantly higher in rats exposed to 

10 mg bisphenol A and primed with 1 mg progesterone 

than in the vehicle control rats primed with progesterone. Treatment with 17b-estradiol resulted in increased 

numbers of progesterone positive cells in the preoptic 

area and ventral medial hypothalamus and increased 

lordosis quotient. The study authors concluded that the 

findings suggest that bisphenol A influences sexual 

behavior by altering the progesterone receptor system 

in the hypothalamus. 

Strengths/Weaknesses: This study appears to have 

been relatively well conducted with the incorporation 

of a positive control group and examination of 

anatomical and functional endpoints. The number 

of animals per group is sufficient given the nature 

of this study design. However, the route of administration was sc. 

Birth Defects Research (Part B) 83:157–395, 2008
336 CHAPIN ET AL. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate for the evaluation process but of 

limited utility due to the route of administration. 

Funabashi et al. (2004b), supported by Yokohama City 

University and the Japanese Ministry of Education, 

Culture, Sports, Science, and Technology, examined the 

effects of bisphenol A exposure on expression of 

progesterone receptor mRNA in brain of adult ovariectomized rats. p-Nonylphenol and 4-tert-octyl phenol 

were also examined, but will not be discussed. [No 

information was provided on feed, housing, or bedding 

materials.] Wistar rats were ovariectomized at 7 weeks of 

age, and experiments were conducted 10 days following 

ovariectomy. In the first experiment, 6 rats/group were 

s.c. injected with sesame oil vehicle or 10 mg bisphenol A 

(B40 mg/kg bw) [purity not reported]. Rats were killed 

24 hr following injection, and frontal, parietal, and 

temporal cortex were removed. In a second experiment, 

frontal, temporal, and occipital cortex were collected 

from rats at 0, 6, 12, or 24 hr following injection with 

10 mg bisphenol A; 5–6 rats were killed and examined at 

each time point. In both experiments, progesterone 

receptor mRNA expression was determined by Northern 

blot in each area of the cortex. Data were analyzed by 

ANOVA followed by Fisher protected least significant 

difference post-hoc test. At 24 hr following bisphenol A 

exposure, expression of progesterone receptor mRNA 

was increased in the frontal cortex and decreased in the 

temporal cortex. In the time-course experiments, expression of progesterone receptor mRNA was increased in 

the frontal cortex and decreased in the temporal cortex 

from 6–24 hr following exposure. Bisphenol A had no 

effect on expression of progesterone receptor mRNA in 

the parietal or occipital cortex. The study authors 

concluded that bisphenol A can alter the neocortical 

function through the progesterone receptor in adult rats, 

but the physiological significance of the effect is not 

known. 

Strengths/Weaknesses: This study links relatively 

high single-dose (10 mg) s.c. bisphenol A administration 

to the induction of progesterone receptor mRNA, an 

estrogenic response. Weaknesses is the absence of a 

positive control to demonstrate maximal response in 

estrogen-mediated increases in progesterone mRNA and 

the failure to examine any physiological or functional 

endpoints. It was also not determined if increases in 

mRNA were associated with increases in progesterone 

receptor protein. There was only one dose level administered at a single time point. The s.c. route of dose 

administration is a weakness. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate for inclusion but of limited utility. 

Della Seta et al. (2005), supported by a grant from 

MURST, Italy, examined the effects of bisphenol A 

exposure on maternal behavior in rats. [No information 

was provided in the manuscript on the type of chow, 

bedding, and caging used. The Expert Panel has been 

informed that Harlan Teklad 2018 chow, Lignocel 

bedding, and polysulfone cages were used (F. Farabolli 

et al., personal communication, March 1, 2007).] Female 

Sprague–Dawley rats were trained to ingest peanut oil 

from a micropipette. At 14 weeks of age, female rats were 

mated for 48 hr. On the day following mating, females 

were randomly assigned to groups administered peanut 

oil (n 5 23) or 0.040 mg/kg bw/day bisphenol A [purity 

not indicated in the manuscript; Z95% according to the 

authors (F. Farabollini et al., personal communication, 

March 1, 2007)] (n 5 17) through a micropipette. Dosing 

was continued through the gestation and lactation 

periods. Two days following delivery, litters were culled 

to 4 male and 4female pups and were cross-fostered 

within treatment groups. Pups were weighed on Days 2, 

7, and 21 following birth. Maternal behavior was tested 

at 3 and 4 days and at 8 and 9 days following delivery. In 

30-min test sessions, frequency, duration, and latency of 

behaviors such as retrieving pups, licking pups, 

postures, and nest building were evaluated with pups 

of the same sex. Behavior with pups of the opposite sex 

was evaluated on the second day of the test period, and 

the order of testing with male and female pups was 

reversed during each testing period (Days 3–4 and 8–9). 

Data were analyzed by general linear model, Duncan 

multiple range test, and/or Mann–Whitney U test. The 

numbers of females giving birth were 9 of 17 in the 

bisphenol A group and 18 of 23 in the control group. 

Nine dams in the control group and 7 in the bisphenol A 

group were evaluated for maternal behavior. The only 

significant effect reported for bisphenol A was reduced 

duration of licking-grooming pups, which occurred with 

both sexes of pups during both observation periods 

[B25–50 % decrease as estimated from a graph]. Effects 

reported to be marginally significant were decreased 

frequencies of licking-grooming of pups (Po0.09), 

anogenital licking of pups (Po0.08), and arched back 

posture (Po0.07). The study authors concluded that 

maternal behavior in rats is influenced by prolonged 

exposure to low bisphenol A doses during pregnancy 

and lactation. 

This behavioral study suggested that a low, oral dose 

of bisphenol A (0.040 mg/kg bw/day) affects pregnancy 

and maternal behavior. 

Strengths/Weaknesses: Weaknesses include the use of 

a single dose level and an unusually low pregnancy rate 

in the controls (18/23) as well as the authors emphasis on 

marginally significant bisphenol A effects. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

4.2.1.2 Mouse: Park et al. (2004), support not 

indicated, examined the effects of bisphenol A exposure 

on the reproductive and hematological systems of male 

and female mice. [Results for females are discussed 

here, and results for males are discussed in 

Section 4.2.2.2.] Adult ICR mice were fed mouse 

formulation feed (Cheil Feed). [No information was 

provided about caging or bedding materials.] Fifteen 

mice/sex/group were i.p. injected with bisphenol A 

[purity unknown] in an ethanol/corn oil vehicle at 0.05, 

0.5, or 5.0 mg/kg bw on 5 occasions (every 3 days over a 

14-day period). One control group received no treatment 

and a second control group was i.p. injected with corn oil. 

Females were examined 7 days following administration. 

Reproductive organs were weighed and fixed in Bouin 

solution, and histopathological examination was conducted. Hematological and clinical chemistry endpoints 

were also assessed. Data were analyzed by least significant difference test. 

Exposure to bisphenol A had no effect on body 

weight. Significant decreases were observed for right 

ovary weight in the mid- and high-dose group 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 337 

and left ovary weight in the mid-dose group 

[25–27% lower]. No treatment effects were observed for 

uterine or ovarian histology. There were no effects of 

bisphenol A treatment on hematological endpoints in 

females. Blood urea nitrogen levels were decreased 

significantly [by 28–32%] in females of all dose groups. 

The study authors did not report conclusions regarding 

study findings. 

Strengths/Weaknesses: The study design regarding 

frequency and route of administration and the lack of an 

appropriate positive control are weaknesses. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate though of limited utility for the 

evaluation process. 

Berger et al. (2007), supported by the Natural Sciences 

and Engineering Research Council of Canada, examined 

the effect of bisphenol A exposure on blastocyst 

implantation in mice. CF-1 mice were housed in 

polypropylene cages and were fed Harlan Teklad 22/5 

rodent chow, which was stated to contain soy. [No 

information was provided about bedding materials.] 

On GD 1–4 or 5 [described as GD 1–5 in Methods 

section and GD 1–4 in study figures and tables] (GD 

0 5 day of vaginal plug), 8–9 mice/group were s.c. 

injected with peanut oil vehicle or bisphenol A (97% 

purity) at 10.125 mg/animal/day. [Assuming that the 

mice weighed 0.02 kg at the start of gestation (USEPA, 

1988), CERHR estimated bisphenol A intake at 500 mg/ 

kg bw/day.] Mice were killed on GD 6 for an examination 

of implantation sites. Data were analyzed by w2 test or 2­

sample t-test. The number of implantation sites was 

reduced significantly in the treated animals (mean of 

B2.5 compared to B15 in controls). Implantation sites 

were observed in 8 of 8 control females at a range of 12– 

17/female. Six of 9 females in the bisphenol group had 

no implantation sites. The study authors concluded that 

pregnancy disruption occurred during the period of 

implantation. 

Strengths/Weaknesses: Weaknesses include lack of 

experimental details for examining the uteri, use of a 

single high-dose, number of corpora lutea were not 

recorded. 

Utility (Adequacy) for CERHR Evaluation Process: 

Due to the absence of key information and faulty 

methodology, this study is inadequate for evaluation 

process. 

Al-Hiyasat et al. (2004), supported by Jordan University of Science and Technology, examined the effect of 

bisphenol A and dental composite leachate on fertility of 

female mice. In this study, Swiss mice were fed a 

standard laboratory feed containing soy protein. [No 

information was provided on caging and bedding 

materials.] At 60 days of age, 11 mice/group were 

gavaged with distilled water or composite leachate for 28 

days. Components of the composite leachate were 

identified by HPLC and included tri-(ethylene glycol)­

dimethacrylate (5945 mg/L), bisphenol A glycerolate 

dimethacrylate (2097 mg/L), and bisphenol A (78 mg/ 

L). [Based on the reported volume of administration 

of 0.2 mL and a body weight of 34.4 g, CERHR 

estimated bisphenol A intake from leachate at 

0.45 mg/kg bw/day.] Additional 60-day-old mice 

(n 5 15/group) were gavaged with bisphenol A (97% 

purity), at doses of 0 (ethanol/distilled water vehicle), 

0.005, 0.025, or 0.1 mg/kg bw/day for 28 days. Five 

mice/group in the bisphenol A study were killed at the 

end of the dosing period for measurement of body, 

uterus, and ovary weights. All mice in the leachate 

study and 10 mice/group in the bisphenol A study were 

mated to untreated males (2 females to 1 male) for 10 

days. One week following the end of the mating period, 

the mice were killed and examined for pregnancy, 

implantations, viable fetuses, and resorptions. Body, 

ovary, and uterus weights were measured in mice from 

the leachate study. Data were analyzed by Student t-test 

or Fisher exact test. 

Effects in the leachate group included increased 

relative (to body weight) ovarian weight and decreased 

percentages of pregnant mice. In mice exposed to 

bisphenol A, body weights were decreased at all dose 

levels. Effects observed in mice exposed to the mid- and 

high-dose of bisphenol A included increased uterine 

weight, increased percentages of resorptions/implantations, and increased percentages of mice with resorptions. Ovarian weight was increased in mice of the highdose bisphenol A group. [Although the effects were not 

statistically significant, the percentages of pregnant 

females were 90, 77.7, 80, and 60% pregnant mice in the 

control and each respective dose group.] In both the 

composite leachate and bisphenol A groups, there were 

no statistically significant effects on implantations or 

viable fetuses. The study authors concluded that bisphenol A and components leached from dental composite have adverse effect on fertility and the reproductive 

system of mice. 

Strengths/Weaknesses: With only 5–10/group, 

this study was underpowered for determination of 

potential bisphenol A-related effects on fertility and 

other endpoints. Confirmation of mating was not 

performed (cohabitation was for 10 days; if the mice 

mated on day 10, the necropsy would have been 

performed on GD 7. Mean body weight and reproductive 

organ weights of bisphenol A-treated animals were 

only collected from 5 mice/dose level. Moreover, 

the normal body weight range for 10-week-old female 

Swiss mice is 28–35 g. Given that there are only 5 mice/ 

group, it is hard to draw any meaningful conclusions 

from these data. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation based on 

small sample size. 

Matsumoto et al. (2004), support not indicated, 

examined the effect of maternal bisphenol A exposure 

on growth of offspring in mice; this study was discussed 

in Section 3.2.7. Because the results of this study bear on 

lactation competence in treated dams, the study will also 

be considered here. Mice were fed standard rodent chow 

(CE-2, Japan Clea). [No information was provided on 

caging and bedding materials.] Mice of the ddY strain 

were exposed to bisphenol A (Z97% purity) through 

feed at 0 or 1% from GD 14–PND 7. The study authors 

stated that the bisphenol A dose was equivalent to 

1000 mg/kg bw/day. [The numbers of dams treated was 

not indicated. Day of vaginal plug and day of birth 

were not defined.] Mice delivered pups on PND 21. 

Body weight of pups were monitored during the 

postnatal period in 31 pups from the control group and 

61–89 pups from the bisphenol A group. Serum prolactin 

levels were measured by RIA in 3 dams/group 4 days 

following delivery. Pups were killed on PND 7, and 

Birth Defects Research (Part B) 83:157–395, 2008
338 CHAPIN ET AL. 

stomach weight was measured. Data were analyzed by 

Student t-test. 

No differences were reported for live pups at birth. 

During the postnatal period, body weights of pups in the 

bisphenol A group were significantly lower [by B40%] 

than control group pups. No deaths were reported for 

pups in the control group, but 30% of pups in the 

bisphenol A group died before PND 7. On PND 1, milk 

could be seen in stomachs of pups from the control 

group, but not the bisphenol A group. [The number of 

pups evaluated for milk in stomach was not reported]. 

On PND 7, stomach weight was significantly lower [by 

40%] in pups from the bisphenol A compared to control 

group. Serum prolactin level was significantly reduced 

[by 46%] in dams from the bisphenol A group. The 

authors concluded that administration of a high bisphenol A dose to mice resulted in suppressed postnatal 

growth of offspring which probably resulted from an 

insufficient supply of milk, which might have been due 

to decreased prolactin secretion. 

Strengths/Weaknesses: This study was conducted at a 

single high-dose that likely induced maternal toxicity 

(which was not assessed); therefore, it is difficult to 

delineate if the findings in the mouse pups are the result 

of potential bisphenol A-related effects of maternal 

toxicity or an effect on the pup. 

Utility (Adequacy) for CERHR Evaluation Process: 

Given the likely confounding effects of maternal toxicity, 

this study is considered inadequate and of no utility. 

4.2.1.3 Other mammals: Nieminen et al. (2002a), 

support not indicated, examined the effects of bisphenol 

A exposure on hormone levels in the European polecat 

(Mustela putorius). Five animals/group/sex [age not 

reported] were administered bisphenol A [purity not 

reported] in feed at concentrations providing doses of 0, 

10, 50, or 250 mg/kg bw/day for 2 weeks. Body weight 

and length were measured during the study. Animals 

were killed at the end of the exposure period, with 

sampling conducted in random double-blinded order. 

Liver and kidney were weighed. Blood samples were 

obtained for measurement of hormone levels by RIA. 

Microsomal enzyme activities were determined. Statistical analyses included ANOVA, post-hoc Duncan test, 

Student t-test, Spearman correlation coefficient, Kolmogorov–Smirnov test, and/or Levene test. 

There were no clinical signs of toxicity and no effects 

on body weight or body mass index following bisphenol 

A exposure. Absolute and relative liver weight were 

significantly increased in females of the high-dose group. 

Plasma cortisol levels were significantly reduced in 

females of the mid-dose group. Bisphenol A exposure 

had no significant effects on plasma levels of testosterone, estradiol, FSH, or thyroid hormones. Glutathione-Stransferase (GST) activity was significantly increased in 

females of the high-dose group. UDPGT activity was 

significantly higher in females of the mid- and high-dose 

group and males of the high-dose group. There was no 

effect on 7-ethoxyresorufin O-deethylase (EROD) activity. The study authors concluded that the endocrine 

effects in this study were not as remarkable as the effects 

on liver enzymes. 

Strengths/Weaknesses: A strength of this study is the 

use of a non-rodent species and multiple doses. Weaknesses include small sample size and absence of 

reproductive endpoints. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate and not useful due to small 

sample size and absence of reproductive endpoints. 

Nieminen et al. (2002b), support not indicated, 

examined the effects of bisphenol A exposure on 

endocrine endpoints in field voles (Microtus agrestis). 

Animals were housed in plastic cages with wood 

shavings and fed R36 diet (Lactamin, Sweden). Sexually 

mature field voles were randomly assigned to groups 

that received bisphenol A [purity not reported] in 

propylene glycol by s.c. injection for 4 days. Doses of 

bisphenol A (numbers of females in each group) were 0 

(n 5 5), 10 (n 5 7), 50 (n 5 5), and 250 (n 5 8) mg/kg bw/ 

day. Animals were killed the day following the last dose. 

Body and liver weights were measured. Blood was 

drawn for measurement of sex steroids, thyroxine, and 

weight-regulating hormone levels in plasma using RIA 

or immunoradiometry methods. The activities of EROD, 

UDPGT, and GST were measured in hepatic and renal 

microsomes using appropriate substrates. Statistical 

analyses included ANOVA, post-hoc Duncan test, Student t-test, Kolmogorov–Smirnov test, Levene test, 

Mann–Whitney U test, w2 test, and Spearman correlation. 

[Results for males are discussed in Section 4.2.2.3.] 

Mortality was significantly increased by bisphenol A 

treatment, with incidences of 18, 36, and 20% in the lowto high-dose groups. No mortality was observed in the 

control group. Bisphenol A treatment did not significantly affect body or liver weight. Plasma testosterone 

levels increased with dose, and statistical significance 

was attained in high-dose females compared to control 

females. 17b-Estradiol levels decreased with dose in 

females. Pooled (male1female) LH levels were not 

significantly altered by treatment. Liver EROD activity 

[apparently combined for males and females] was 

significantly decreased at the mid- and high-dose, and 

liver GST activities [not clear if for males or females or 

both] was significantly decreased at the highest dose 

level. There were no other significant effects on microsomal enzymes examined. The study authors concluded 

that wild mammals such as field voles could be more 

susceptible to bisphenol A-induced toxicity than laboratory rodents. 

Strengths/Weaknesses: A strength is the use of 

another species. The small number of voles/dose level, 

the subcutaneous route of administration, and questionable statistical procedures are weaknesses. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process. 

Razzoli et al. (2005), supported by the Ministry of 

University Education and Research and the University of 

Parma, examined the effects of bisphenol A on sociosexual and exploratory behavior in female Mongolian 

gerbils, a monogamous species. Animals were fed Mil 

Morini Rodent Chow (Reggio Emilia, Italy) and housed 

in Plexiglas cages with wood shaving/cotton nesting 

material. At 11–12 weeks of age, female gerbils were 

trained to drink corn oil from a syringe, and 1 week later, 

they were paired with a male. From Days 1–21 of 

cohabitation, 12 females/group were fed 0 (corn oil 

vehicle), 0.002, or 0.020 mg/kg bw/day bisphenol A 

[purity not indicated] from a syringe. A group of 12 

females received ethinyl estradiol, the positive control, 

0.04 mg/kg bw/day during the same time period. During 

the cohabitation period, social behavior (e.g., agonism, 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 339 

social investigation, huddling, and nest sharing) was 

observed and body weights of females were measured. A 

free exploratory test, which measured the amount of time 

females spent in an area of a cage with home nesting 

material compared to the time spent in an unfamiliar 

area of a cage, was conducted following the 21-day 

cohabitation period. Exploratory behavior was evaluated 

by an observer blinded to treatment groups. Statistical 

analyses included ANOVA and Duncan test for multiple 

comparisons. 

Bisphenol A treatment did not affect body weight. 

Social sniffing was increased significantly [by 60%] in 

the low-dose bisphenol A group. Significant effects 

[percent changes compared to control] observed in the 

exploratory test were decreased time in the unfamiliar 

area at the low [60%] and high [44%] dose, fewer 

transitions to the unfamiliar area at the low [60%] and 

high [50%] dose, fewer transitions to the home cage at 

the high-dose [29%], and less time in the unfamiliar area 

at the low dose [46%]. Similar results for both social 

sniffing and exploratory behavior were observed in the 

positive control group. According to the study authors, 

this study demonstrated that chronic exposure of adult 

female gerbils to environmentally relevant doses of 

bisphenol A during the hormonally sensitive period of 

cohabitation resulted in subtly altered social and exploratory behavior. 

Strengths/Weaknesses: This study examined behavioral endpoints in gerbils, and included a positive 

control (ethinyl estradiol) and 2 doses of bisphenol A. It 

appears to be well conducted using oral dosing, 

respectable sample size (given study complexity), and 

use of a positive control. Weaknesses include failure to 

account for temporally repeated measures in statistical 

analyses. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate for inclusion but of limited utility 

for the evaluation process. 

4.2.1.4 Invertebrates: Although studies in invertebrates may be important for understanding mechanisms 

of action and environmental impact, the Panel views 

these studies as not useful for the evaluation process. 

Oehlmann et al. (2000), supported by the Berlin Federal 

Environmental Agency, reported the effects of bisphenol 

A on reproductive organs in the freshwater ramshorn 

snail (Marisa cornuarietis) and the marine dog whelk 

(Nucella lapillus). In the first experiment, adult ramshorn 

snails were exposed for 5 months to bisphenol A in 

ethanol at 0, 1, 5, 25, or 100 mg/L. Thirty snails/group 

were removed every month for evaluation of reproductive 

organs. [Culture ware type not indicated. The purity of 

bisphenol A and its stability during the exposure period 

were not reported. The snails removed for evaluation 

were adults; this species requires 8 months to attain 

sexual maturity. Octylphenol was also evaluated, but is 

not discussed here.] In the second experiment, ramshorn 

snails were exposed to bisphenol A in ethanol at 0, 1, or 

100 mg/L for 1 year. Thirty F1 snails per time point were 

removed for evaluation at 6, 8, and 12 months. In the third 

experiment, dog whelk were exposed to bisphenol A in 

glacial acetic acid at 0, 1, 25, or 100 mg/L for 3 months. 

Thirty specimens were removed for evaluation each 

month. Evaluations included measurements of sex organs 

and the identification of sperm or oocytes in the genital 

tract. Statistical analyses included ANCOVA followed by 

Tukey or Student-Newman–Keuls test, Kruskal–Wallis 

test, w2 test, and Weir test. 

Adult ramshorn snails were reported to show increases in volume of the capsule and albumen glands 

(portions of the oviduct). [Apparently, the increase in 

volume was based on appearance rather than measurements. The measured lengths of the sex organs were 

not affected by treatment.] Occasional specimens that 

had been exposed to bisphenol A showed rupture of the 

oviduct with protrusion of the egg mass. Enumeration of 

spawning masses and eggs showed statistically significant time-dependent increases in all bisphenol A groups. 

Histologic examination of the gonads did not suggest 

abnormalities of spermatogenesis or oogenesis. The F1 

snails also demonstrated a statistically significant increase in spawning mass and oocyte production at the 

100 mg/L bisphenol A concentration, and some specimens showed rupture of the oviduct at 12 months of age 

in both bisphenol A groups. An increase in imposex [the 

presence of vas deferens tissue] was noted significantly 

more often in snails exposed to bisphenol A 100 mg/L 

than controls. Adult dog whelk demonstrated a significant increase in the length and weight of the sex glands 

and an increase in number of females with oocytes in the 

oviduct. The authors concluded that invertebrates are 

sensitive to bisphenol A toxicity at environmentally 

relevant concentrations. 

Strengths/Weaknesses: The study appears to be well 

conducted and suggests that bisphenol A has stimulatory 

(17b-estradiol-like) effects on the spawning masses and 

eggs of snails. These changes occurred in the absence of a 

histological correlate. The potential stability/biotransformation was discussed in the introduction but not 

determined during the exposure period. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not considered useful for the evaluation 

process. 

Forbes et al. (2007), supported by the Bisphenol A 

Global Industry Group, evaluated the effects of bisphenol A on reproduction in the freshwater ramshorn 

snail (Marisa cornuarietis). Bisphenol A [purity not 

indicated] concentrations in test water were 0, 0.10, 1.0, 

16, 160, and 640 mg/L. Concentrations were periodically 

checked. Thirty breeding pairs per treatment level were 

observed for a 12-week period. The number of egg 

masses and number of eggs/egg mass were recorded. 

Hatchability was evaluated using 5 consecutive egg 

masses collected from 5females/replicate (75 egg 

masses/treatment). Juvenile growth rates were calculated for a subset of the offspring. Nested ANOVAs were 

used for data analysis. All snails survived. There were no 

significant treatment-related differences in adult egg 

production, hatchability, or juvenile growth rate. Interindividual variability in these parameters was prominent, and the authors concluded that a large number of 

replicates would be necessary using this animal model to 

detect reproductive effects. 

Strengths/Weaknesses: This study examined dose 

response over a 12-week exposure of freshwater snails 

to bisphenol A with egg masses and number of eggs/egg 

mass as endpoints. Although no treatment-related effects 

were observed, interindividual variability was high. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not considered useful for the evaluation 

process. 

Birth Defects Research (Part B) 83:157–395, 2008
340 CHAPIN ET AL. 

Schirling et al. (2006), supported by the county of 

Baden-Wu ̈rttemberg, examined the effects of bisphenol 

A on embryo development in the apple snail, Marisa

cornuarietis. Stocks of 150 adult snails were maintained in 

a glass aquarium containing tap water and sea salt, 

exposed to a 12/12 hr light/dark cycle, and fed fish flake 

food, carrots, and cucumbers. Fifteen to twenty eggs/ 

exposure group were placed in a glass Petri dish with 

bisphenol A [purity not indicated] 50 or 100 mg/L [11.4 

or 22.8 mM], ethinyl estradiol 10 mg/L, DMSO 0.005%

(solvent for ethinyl estradiol), or water (solvent for 

bisphenol A). From embryo visibility (B3.5 days after 

egg laying) to Bday 14, eggs were evaluated daily for 

formation of eyes, tentacles, heart rate, and hatching. 

Statistical analyses were performed using Student t-test 

or Kruskal–Wallis test. 

There were no differences in formation of eyes 

and tentacles between treatments groups Heart rate 

was significantly decreased on Day 9 for bisphenol 

A 100 mg/liter compared to the water control group 

with description of ‘‘a similar trend’’ in hatching. 

[The data figure does not show a statistically significant effect of bisphenol A treatment on hatching.] 

There was a significantly higher hatching weight in the 

100 mg/L bisphenol A group compared to the 

water control group. Ethinyl estradiol treatment significantly decreased embryo heart rate compared to the 

water control group but not compared to the DMSO 

control. No statistically significant effects of ethinyl 

estradiol on time to hatch or hatching weight were 

demonstrated. The authors concluded that bisphenol A 

and ethinyl estradiol had similar effects on snail 

development. 

Strengths/Weaknesses: Weaknesses include the lack of 

evaluation of the achieved concentration and stability of 

bisphenol A in water and the comparison of ethinyl 

estradiol to the water control instead of the DMSO 

control. The authors’ conclusions are weakened by the 

lack of statistical significance of most of their findings. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not considered useful for the evaluation 

process. 

4.2.1.5 In vitro: Although in vitro studies may be 

important for understanding mechanisms of action and 

cellular and subcellular events, the Panel views these 

studies as not useful for the evaluation process. 

Xu et al. (2002), supported by the Japan Society for the 

Promotion of Science, examined the effects of bisphenol 

A exposure on mouse ovarian granulosa cells in a series 

of experiments. Ovarian granulosa cells were obtained 

from 4-week-old B6C3F1 mice. Following incubation of 

cells with 0 or 100 fM [23 pg/L] to 100 mM [23 mg/L] 

bisphenol A [purity not indicated] in ethanol vehicle for 

72 hr, the CellTiter 96 assay was used to evaluate cell 

viability, and the TUNEL assay and 40

,6-diamidino-2­

phenyllindole staining were used to evaluate apoptosis. 

In cells that were incubated in 100 mM [23 mg/L] bisphenol A for 24, 48, or 72 hr, the TUNEL method was 

used to evaluate apoptosis and a flow cytometry 

technique was used to assess apoptosis and the cell 

cycle. Bcl2 and Bax protein expression was examined by 

Western blot, and mRNA expression was assessed by RTPCR in cells that were exposed to 100 mM [23 mg/L] 

bisphenol A for 72 hr. Experiments were repeated a 

minimum of 3 times. Statistical analyses included 

ANOVA followed by Fisher protected least significant 

difference test. [Statistical significance was not clearly 

indicated for some endpoints.] 

A dose-related reduction in cell viability was observed 

at bisphenol A concentrations Z100 pM [23 ng/L]. 

Examination of cells by the TUNEL method indicated a 

concentration-related increase in apoptosis at bisphenol 

A concentrations Z100 pM [23 ng/L]. Features noted in 

apoptotic cells included cellular shrinkage, membrane 

blebbing, and nuclear condensation. Apoptotic cells, as 

determined by TUNEL and the presence of sub-G1 cells 

were increased in a time-related manner following 

incubation with 100 mM [23 mg/L] bisphenol A from 24 

to 72 hr. An increase in G2-M arrest was also observed 

and reached a maximum value following a 48-hr 

incubation of cells with 100 mM [23 mg/L] bisphenol A 

(18 vs. 12% in controls). Expression of Bax protein was 

increased and Bcl2 protein was decreased following 

incubation with 100 mM [23 mg/L] bisphenol A for 

72 hr. Similar expression patterns were observed for Bax

and Bcl2 mRNA expression [data were not shown by 

study authors]. The study authors concluded that 

bisphenol A at doses of 100 pM [23 ng/L] and higher, 

presumably relevant to environmental concentrations, 

decreases viability and increases apoptosis in granulosa 

cells. The study authors postulated that apoptosis may 

have been induced by decreases in the anti-apoptotic 

protein Bcl2 and increases in the pro-apoptotic protein 

Bax. 

Strengths/Weaknesses: Because this study used in 

vitro study PMSG-stimulated murine cells, metabolism is 

likely to have been minimal (if present at all) and the in 

vitro dosimetry of bisphenol A is difficult to extrapolate 

to in vivo dosimetry. Bisphenol A is known to induce 

reactive oxygen species, which may influence the 

tetrazolium salt-based assay. Moreover, based on the 

data presented the mechanism by which bisphenol A 

may be inducing cell cytotoxicity/apoptosis is likely not 

‘‘endocrine disruptor’’ mediated. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not considered useful for the evaluation 

process. 

Mlynarcı ́kova ́ et al. (2005), supported by the European 

Union, examined the effects of bisphenol A exposure on 

hormone production by porcine ovarian granulosa cells. 

Granulosa cell cultures were prepared from porcine 

ovaries collected from a slaughter house. The cells were 

incubated for 72 hr in media containing bisphenol A 

[purity not indicated] at 10 -8

–10 -4 M [2.3 lg/L to 23 mg/ 

L] or the DMSO vehicle, with or without addition of 

1 mg/mL FSH or LH. Following the incubation period, 

media were collected for measurement of progesterone 

and 17b-estradiol concentrations by RIA. Experiments 

were replicated 5–8 times. Data were analyzed by 

ANOVA and Bonferroni post test. Significant changes 

in progesterone production, included an increase at 10 -5 

M [2.3 mg/L] and decrease at 10 -4 M [23 mg/L] bisphenol 

A. Bisphenol A significantly increased FSH-stimulated 

progesterone synthesis at 10 -6 M [0.23 mg/L] and 

inhibited FSH-stimulated progesterone production at 

10 -4 M [23 mg/L]. LH-induced progesterone production 

was inhibited by 10 -4 [23 mg/L] bisphenol A. FSHinduced 17b-estradiol production was also inhibited by 

bisphenol A at all concentrations tested, but statistical 

significance was only attained at doses Z10 -6 M 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 341 

[0.23 mg/L]. Bisphenol A dimethylacrylate was also 

tested, and most results were similar to those observed 

with bisphenol A. The study authors concluded that 

ovarian steroidogenesis might be a target of bisphenol A 

toxicity. 

Strengths/Weaknesses: Potential estrogenic effects 

were observed at 10 -5 M bisphenol A. Decreases in 

responses observed at the 10 -4 M concentration are likely 

due to nonspecific cytotoxicity. Bisphenol A-mediated 

responses in progesterone endpoints appeared to reach a 

near maximum at the lowest dose level examined. There 

was no mention of whether phenol red-free media were 

used or whether fetal bovine serum was charcoalstripped. The serum likely contained steroids, which 

would have been potential confounding factors. Also, it 

appears that cell viability was not examined after the 

incubation period. With exception of the highest dose 

level, there was no dose response (inconsistent trends); 

the statistical flags are potentially due to random chance. 

Since this was an in vitro study, the potential effects of 

metabolism could not be assessed. 

Utility (Adequacy) for CERHR Evaluation Process: 

Due the weaknesses and limitation in the experimental 

design, this study is considered inadequate. 

Mohri and Yoshida (2005), supported by the Japanese 

Ministry of Education, Science, Sports, and Culture, 

examined the effects of bisphenol A and 17b-estradiol 

exposure on calcium oscillations in immature mouse 

oocytes. Immature oocytes with intact germinal vesicles 

were obtained from 8–12-week-old CD-1/ICR mice and 

incubated in bisphenol A [purity not indicated] in a 

DMSO vehicle at concentrations of 0 or 10 nM [2.3 lg/L] 

to 100 mM [23 mg/L] for 60 min. Calcium oscillations 

were measured using a Fura-2 dye and image analyzer. 

Data were analyzed by Student t-test. At 100 mM [23 mg/ 

L] bisphenol A, the duration of calcium oscillations 

was significantly shortened and the oscillations 

became irregular. The same findings were observed 

following exposure to 17b-estradiol at concentrations that 

were 10,000-fold lower than that of bisphenol A, 

producing the same effect. The study authors stated that 

estrogens may affect the oocyte by regulating calcium 

oscillations and that bisphenol A could affect oocyte 

maturation. 

Strengths/Weaknesses: This study appears to have 

been well conducted; however, because this study used 

an in vitro system, metabolism could not be assessed. It is 

unclear if calcium oscillations play a role in oocyte 

maturation in other species, including humans. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not considered useful for the evaluation 

process. 

4.2.2 Male.. Studies on the androgenicity of bisphenol A, including Hershberger assays, are discussed 

in Section 2.2.3. 

4.2.2.1 Rat: Yamasaki et al. (2002a), support not 

indicated, conducted a 28-day exposure study that 

provided some information on the reproductive organs 

of male and female rats. [Complete details of this study 

are included in Section 2. Results for males are 

discussed in this section, and results for females are 

discussed in Section 4.2.1.1.] CD rats were fed a 

commercial diet (MF Oriental Yeast Co.) and housed in 

stainless steel wire mesh cages. Ten 7-week-old rats/sex/ 

group were gavaged with bisphenol A [98% purity] at 0 

(olive oil vehicle), 40, 200, or 1000 mg/kg bw/day for 28 

days. Due to the death of 1 animal exhibiting clinical 

signs in the 1000 mg/kg bw/day group, the high-dose 

was reduced to 600 mg/kg bw/day on Day 8 of the 

study. In an additional study, rats were exposed to 

ethinyl estradiol at 0, 10, 50, or 200 mg/kg bw/day for 28 

days. There were no treatment-related abnormalities in 

sperm or alterations in blood levels of thyroid hormones, 

FSH, LH, 17b-estradiol, prolactin, or testosterone. 

Changes in relative reproductive organ weights [assumed to be relative to body weight] included a 28% 

decrease in relative ventral prostate weight and 21% 

increase in relative testis weight in the high-dose group. 

No gross or histopathological alterations were reported 

for reproductive organs. The study authors concluded 

that change in estrous cyclicity was the only useful 

endpoint for evaluating the endocrine-mediated effects 

of bisphenol A. In comparison, male rats exposed to the 

mid and/or high doses of ethinyl estradiol experienced 

decreased prostate, seminal vesicle, and pituitary 

weights; increased testis weight; and histopathological 

alterations in prostate, seminal vesicle, mammary gland, 

and testis. 

Strengths/Weaknesses: This study was well-conducted, used an appropriate route of administration, a 

positive control group, adequate sample sizes, a range of 

doses, and evaluations of both sexes. A weaknesses 

include an insufficient duration of exposure to examine 

the full spermatogenic cycle. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

Takahashi and Oishi (2001), support not indicated, 

examined the effects of bisphenol A exposure on testis of 

rats. F344 rats were fed standard, soy-containing diet 

(CE-2; Clea Japan) and housed in stainless steel 

suspended cages. Four-week-old male rats (n 5 8/group) 

were administered bisphenol A (99.0% purity) through 

diet at concentrations of 0, 0.25, 0.5, or 1.0% for 44 days. 

The study authors estimated bisphenol A intake at 235, 

466, and 950 mg/kg bw/day. The stability of bisphenol A 

in the diet was verified. Food intake was measured, and 

animals were weighed and observed daily for clinical 

signs. Rats were killed when mean body weight of 

controls reached B200 g. Testosterone levels were measured in serum using an ELISA method. Preputial gland, 

testes, epididymides, prostate, seminal vesicles, kidneys, 

and liver were weighed. The testis was fixed in buffered 

6% formaldehyde and examined histologically. Statistical 

analyses included Bartlett test, ANOVA, Dunnett or 

Scheffe ́ parametric test, Kruskall–Wallis test, Dunnett 

non-parametric test, Wilcoxon rank sum test, w2 test, 

Mantel–Haenzel test, and Fisher exact test. 

Statistically significant findings are summarized in 

Table 88. Body weight gain and terminal body weights 

were reduced in males of the mid- and high-dose groups. 

Food intake was said to be slightly decreased according 

to dose. Absolute organ weight effects included decreased weight of preputial glands at all doses; liver in 

the mid- and high-dose group; and seminal vesicles with 

coagulation glands, dorsal and lateral prostate, and 

hypophysis at the high-dose. [The Expert Panel assumes 

that by coagulation gland, the authors mean the 

anterior prostate or coagulating gland.] Significant 

organ weight effects relative to body weights are 

Birth Defects Research (Part B) 83:157–395, 2008
342 CHAPIN ET AL. 

Table 88 

Effects Observed in Male Rats Exposed to Bisphenol A Through Dieta 

Endpoint 

Dose, % in diet [mg/kg bw/day] 

0.25 0.5 1.0 BMD10 BMDL10 BMD1SD BMDL1SD 

Terminal body weight 2 k 13% k 18% 0.55 [522] 0.42 [399] 0.41 [389] 0.30 [285] 

Relative weight 

Dorsal and lateral prostate 2 2 k 32% 0.29 [276] 0.22 [209] 0.52 [494] 0.36 [342] 

Preputial glandb k 22% k 26% k 25% 0.13 [124] 0.09 [86] 0.18 [171] 0.12 [114] 

Liver 2 k 10% k 14% 0.69 [656] 0.56 [532] 0.30 [285] 0.23 [218] 

Kidney m 8% m 8% m 12% 0.99 [940] 0.69 [656] 0.50 [475] 0.34 [323] 

No. rats with: 

Seminiferous tubule degenerationc 2 m to 6/8 m to 5/8 

Disorganization of Stage I–VI 

spermatids (1 severity)c 

m to 4 of 8 2 2

Disorganization of Stage I–VI 

spermatids (21 severity)c 

2 2 m to 6 of 8 0.36 [342] 0.22 [209] 

% Seminiferous tubules in stages 

I–VI k 59% k 70% k 53% 

IX–XI m 3.4-fold m 5.2-fold m 4-fold 

XII–XIV m 3.2-fold m 3.6-fold m 3-fold 

a

Takahashi and Oishi (2001).
 

b

Benchmark doses were estimated using a polynomial model.
 

c

Control value 5 0 of 8. 
 

m,k Statistically significant increase, decrease compared to controls; 2no statistically significant effect compared to controls.
 

summarized in Table 88. Changes in relative organ 

weights included decreased preputial gland weight and 

increased kidney weights at all doses, decreased liver 

weight at the mid- and high-dose, and decreased dorsal 

and lateral prostate weight at the high-dose. Testicular 

lesions observed with bisphenol A treatment included 

seminiferous tubule degeneration at the mid and highdose, disorganized spermatids at all dose levels, and 

differences in percentages of seminiferous tubules in 

spermatogenic stages at all dose levels. Although it does 

not appear that statistical significance was attained, 

dose-related increases in arrested spermatogenesis and 

disappearance of elongated spermatids were also reported. There were no significant effects on serum 

testosterone concentrations. The study authors concluded that bisphenol A was toxic to the testis and 

accessory sex organs of F344 rats at a minimum toxic 

dose of 235 mg/kg bw/day. 

Findings suggest a hormonal effect on hormonedependent reproductive tissues at all doses examined. 

The lowest dose level, 0.25% in diet, exhibited histopathological changes in the testes, most strikingly 

described as a large alteration in the relative frequency 

of the different stages of the seminiferous epithelium. 

Due to techniques used for fixation and embedding of 

the testes, the histopathological analyses may be of 

limited value. 

Strengths/Weaknesses: This study reports a relatively 

well conducted study with a relevant route of administration. General toxicity was demonstrated. Formalin 

produces excessive shrinkage of testes when followed by 

paraffin embedding and is inappropriate especially 

when staging will be conducted. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

Sakaue et al. (2001), supported by the Japanese 

Science and Technology Agency, examined the effect 

of bisphenol A exposure on spermatogenesis in the 

adult rat. Animals were fed CE-2 chow (CLEA Japan) 

and housed in stainless steel wire caging. Thirteen-week 

old male Sprague–Dawley rats (5/group) were 

gavaged for 6 days with the ethanol/corn oil vehicle or 

bisphenol A (99.6% purity) at doses 0.020, 0.200, 2, 20, or 

200 mg/kg bw/day. The high-dose was based on a 

preliminary experiment that demonstrated reduced 

daily sperm production in a Holtzman rat gavaged 

with 200 mg/kg bw/day bisphenol A for 6 days. In this 

study, rats were killed 2 days following dosing (at 14 

weeks of age) or at 18 weeks of age. Testes were 

weighed. Sperm endpoints were measured from 

one testis. Histopathological examinations were 

conducted on the other testis after fixation in Bouin 

fluid, paraffin embedding, and staining with hematoxylin and eosin. Statistical analyses included Student ttest, ANOVA, and Fisher protected least significant 

difference test. 

There were no changes in daily sperm production/g 

testis at 14 compared to 18 weeks of age. [No data were 

shown for 14-week-old rats, and results of bisphenol A 

treatment were not discussed.] Bisphenol A did not 

significantly affect body or testis weight at 18 weeks of 

age. In the 18-week-old rats, daily sperm production and 

daily sperm production/g tissue were significantly 

reduced [by B25%] in all bisphenol A treatment groups. 

The study authors noted the lack of a dose–response 

relationship and that daily sperm production in treated 

groups at 18 weeks of age was comparable to that of 14­

week-old controls. Histopathological evaluations of testis 

revealed no evidence of atrophy or disrupted spermatogenesis in the seminiferous tubules. [Data were not 

shown by study authors.] 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 343 

Table 89 

Conditions Used in Experiments to Study Bisphenol A Effects on Sperm Production in Ratsa 

Experiment 

Bisphenol A doses 

mg/kg bw/day No. rats/group Diet/water Caging 

1 

2 

3 

4 

5 

0, 0.020, 2, or 200 

0, 0.020, 2, or 200 

0, 0.020, 2, or 200 

0, 0.020, 2, or 200 

0 

10 

10 

10/bisphenol A 

group 20/control group 

10/bisphenol A 

group 20/control group 

10 

RM3/Automatic system 

5002/Glass bottles 

5002/ Glass bottles 

CE2/ Glass bottles 

RM3, 5002, or CE2/not 

specified 

Stainless steel, unspecified bedding 

Stainless steel, no bedding 

Stainless steel, no bedding 

Stainless steel, no bedding 

Not specified 

a

Ashby et al. (2003). 

To obtain more dose–response information, Sakaue 

et al. (2001) repeated the study in 8 rats/group dosed 

[assumed by gavage as in the first study] with 0.000002, 

0.00002, 0.0002, 0.002, 0.020, 0.200, or 2 mg/kg bw/day 

bisphenol A. [It is assumed that ages of rats, treatment 

period, and observation periods were the same as in the 

first study.] Body and testis weights were not affected by 

bisphenol A treatment at Week 18. At Week 18, 

significant decreases in daily sperm production and 

daily sperm production/g tissue were observed at 0.020, 

0.200, and 2 mg/kg bw/day. [The decrease compared to 

control was estimated from a graph. For daily sperm 

production, the decreases were B30% at 0.020 mg/kg 

bw/day, B34% at 0.200 mg/kg bw/day, and B32% at 

2 mg/kg bw/day. For daily sperm production/g tissue, 

the decreases were B24% at 0.020 mg/kg bw/day, B32% 

at 0.200 mg/kg bw/day, and B28% at 2 mg/kg bw/day.] 

In a third experiment, rats were given a single oral 

dose of 0.020 mg/kg bw bisphenol A. Six hours later, the 

rats were killed, the right testis was homogenized, and 

the cytosol was examined for protein expression using 

two-dimensional polyacrylamide gel electrophoresis. 

Changes in intensity and mobility were noted for 3 

unidentified proteins. The study authors concluded that 

the dose–response curve for bisphenol A affects on 

spermatogenesis in the adult rat was monotonic rather 

than having an inverted U-shape. 

Strengths/Weaknesses: This study used a relevant 

route of administration and multiple doses. A weakness 

is the brief exposure period. Variability in control daily 

sperm production between the first and second study is 

disturbing; given the small sample (5 or 8/group), this 

variability severely decreases confidence in the data. No 

histopathologic correlate was presented. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate but of limited utility due to small 

sample and variable control values between experiments. 

Ashby et al. (2003), support not indicated, examined 

the effects of bisphenol A exposure on sperm production 

in rats. The study attempted to replicate earlier findings 

from Sakaue et al. (2001). Five independent experiments 

were conducted, and the conditions for each experiment 

are summarized in Table 89. Some of the experiments 

used the same conditions as the Sakaue et al. (2001) 

study, including stainless steel cages with no bedding, 

CE2 diet (CLEA, Tokyo, Japan), and glass water bottles. 

In the first 4 studies, 10–20 adult (B13-week-old) 

Sprague–Dawley rats/group were gavaged with bisphenol A (99% purity) at 0 (ethanol/corn oil vehicle), 0.020, 

2, or 200 mg/kg bw/day for 6 days. Concentrations of 

dosing solutions were verified. In the fifth study, rats fed 

different diets were gavaged with vehicle for 6 days. Rats 

were fed 1 of 3 diets as indicated in Table 89. Phytoestrogen aglycone content of the feed was measured. 

Respective concentrations of daidzein, genistein, and 

coumestrol in each feed were reported at 94, 62, and 

0.6 mg/g diet for Rat and Mouse No. 3 (RM3; Special Diet 

Services Ltd.); 40, 23, and 0.1 mg/g diet for 5002 (Purina 

Mills); and 157, 106, and 2.2 mg/g CE2 diet. Ten rats were 

used in each group, except in third and fourth studies, 

where 20 control rats were split into 2 groups before 

dosing. Rats were administered drinking water through 

an automatic system in the first study and via glass 

bottles in the other studies. In the first study, rats were 

housed 3/cage at the start of the study and 2/cage later 

in the study. In the other 4 studies, rats were housed 2/ 

cage. Rats were weighed during the study. Animals were 

killed at 18 weeks of age, 5 weeks after the start of 

dosing. Liver, kidney, and reproductive organs were 

weighed, and sperm counts were obtained. In the first 4 

studies, data were analyzed by ANOVA, ANCOVA for 

organ and body weights, and Dunnett test. Results from 

all 4 studies were also analyzed by ANOVA in an attempt 

to increase study power. Data from the fifth study were 

analyzed by Fisher least significant difference test. 

In the 4 studies that compared the effects of bisphenol 

A exposure to a vehicle control group, there were no 

significant effects of bisphenol A exposure on sperm 

count, daily sperm production, or weights of body, liver, 

kidney, testis, prostate, epididymis, or seminal vesicle. 

One animal exposed to 200 mg/kg bw/day bisphenol A 

in the third study was reported to have unexpectedly 

small testes and epididymides, but the study authors 

indicated that inclusion of this animal in later statistical 

analyses had no effect on outcome. One animal in the 

200 mg/kg bw/day group in the fourth study had a 

small testis. No significant effects were observed when 

data from the first 4 experiments were pooled and 

analyzed. The study authors noted that some endpoints 

were variable from one experiment to the other. It was 

noted that prostate weights were 10% lower in animals 

from Experiment 1 than from Experiments 2–4. Sperm 

counts and daily sperm production were reportedly 

different in control animals from Experiment 1 compared 

to Experiment 2. It was noted that rats were fed different 

diets in Experiment 1 (RM3) and Experiment 2 (5002), 

and a study to examine the effects of feed was 

conducted. In the study examining effects in rats fed 

Birth Defects Research (Part B) 83:157–395, 2008
344 CHAPIN ET AL. 

different diets but exposed to vehicle, no effects of diet 

on daily sperm production were observed. The only 

significant effect reported was a 9% lower weight of right 

epididymis in rats fed CE2 compared to RM3 or 5002 

feed. The study authors stated that the effect was likely 

spurious due to lack of effect on other endpoints, no 

effect on left or total epididymis weight, and lack of the 

effect in the first 4 experiments. The study authors 

concluded that there was no evidence in their study that 

bisphenol A affected reproductive organ weights or daily 

sperm production. Lack of bisphenol A-induced effect on 

daily sperm production was in contrast to observations 

of the Sakaue et al. (2001) study, which reported a 

decrease in this endpoint. Subtle genetic differences in 

the rats were suggested as a possible reason for 

differences in results between the 2 studies. 

Given the robustness and comprehensiveness of this 

study, it is highly useful. It strongly suggests that the 

NOAEL for potential bisphenol A-mediated effects on 

the adult rat reproductive system exceeds 200 mg/kg/ 

day. Absence of confirmation of the work of Sakaue et al. 

(2001) led to an extensive study of the potential variables 

(e.g., diet, housing, etc.) that might account for the 

discrepancies. These data suggests that subtle changes in 

study endpoints, especially daily sperm production and 

organ weights, may occur by random chance or genetic 

differences in the respective lab’s supplier of rats may 

play a role. These data also strongly suggest bisphenol A 

administered orally has no effect on sperm production 

albeit following only 6 days of administration. 

Strengths/Weaknesses: This study reports a well 

conducted, comprehensive assessment of the potential 

effects of bisphenol A delivered by 6 daily doses on daily 

sperm production. The 6-day treatment period is a 

(understandable) weakness. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and useful for the evaluation 

process. 

Tohei et al. (2001), supported in part by the Japan 

Society for the Promotion of Science, examined the 

effects of bisphenol A exposure on testicular function 

of Wistar–Imamichi rats. [No information was provided 

about composition of chow, bedding, or caging.] In a 

series of studies, rats were dosed with bisphenol A 

[purity not indicated] in sesame oil by s.c. injection for 2 

weeks. Bisphenol A doses were 0.1 or 1 mg/day [B0.3 or 

3 mg/kg bw/day based on the reported body weights of 

300–350 g]. The dose of 1 mg/day bisphenol A was stated 

to be similar to the highest exposures reported in 

humans, which were based on saliva levels measured 

in patients receiving composite dental sealants. Doses 

and exposure duration were based on results of 

preliminary studies. Five or 6 animals/dose group were 

used in each experiment. Statistical analyses included 

ANOVA, Fisher protected least significant difference 

test, and Mann–Whitney U test. 

In the first study conducted to examine testicular and 

pituitary function, LH, FSH, prolactin, testosterone, and 

inhibin were measured in plasma, pituitary, and/or testis 

by RIA in rats s.c. dosed with 1 mg/day bisphenol A for 2 

weeks. Statistically significant effects [percent differences compared to controls, as estimated from a graph] 

included increases in plasma levels of LH [150%] and 

prolactin [1067%] and decreases in levels of plasma 

testosterone [29%] and testicular inhibin [36%]. 

In a second experiment to examine testicular response, 

rats were s.c. dosed with 0.1 or 1 mg/day bisphenol A for 

2 weeks. The rats then received 10 IU hCG through an 

atrial cannula. Blood samples were drawn for measurement of progesterone and testosterone levels before and at 

various time intervals between 30 and 180 min following 

the hCG challenge. Plasma progesterone and testosterone 

levels were increased following the hCG challenge in 

control rats. In the bisphenol A-treated rats, only a slight 

increase in progesterone levels occurred 30 min following 

challenge, and plasma progesterone levels were significantly lower compared to the control group at 60–150 min 

following challenge. There was an increase in plasma 

testosterone level following challenge of the bisphenol A 

group, but values were significantly lower than control 

values at 90–120 min following the challenge. 

In a third experiment examining pituitary response, 

adult male rats were castrated 5 days before bisphenol A 

treatment. Castrated rats were s.c. injected with 1 mg/ 

day bisphenol A and 75 mg/day testosterone propionate 

for 2 weeks. The rats then received 250 ng gonadotropinreleasing hormone by s.c. injection. Plasma LH was 

measured before and at various time intervals between 

0.25 and 4 hr following the gonadotropin-releasing 

hormone challenge. No statistically significant effects 

were observed. 

In a fourth study, males were dosed with 1 mg/day 

bisphenol A for 2 weeks and then paired with females in 

proestrus. Sexual function was evaluated by scoring 

mounts, intromissions, and ejaculations. No significant 

effects were observed for sexual function. Based on the 

findings reported in all studies, the study authors 

concluded that ‘‘The testis is probably a more sensitive 

site for [bisphenol A] action than the hypothalamuspituitary axis.’’ 

Strengths/Weaknesses: RIAs appear to have been 

conducted competently. Subcutaneous is not a relevant 

route of exposure, and the sample size was limited. 

Blood collection via decapitation is not appropriate, 

because decapitation stress affects plasma prolactin and 

LH secretion. No mention is made of the order of killing. 

If controls were killed first and the guillotine was not 

cleaned between uses (and animals were not in separate 

rooms), there may be serious confounding of the data. 

Because rat plasma testosterone levels are normally 

highly variable, the low degree of variability in this 

study, given the small sample size, is remarkable 

(B70.12 ng/mL). No functional consequence of the 

alterations in hormone levels were described. Weaknesses include use of two doses delivered subcutaneously, critically small sample sizes, use of an 

inappropriate method of plasma collection, the stressful 

nature of cannula insertion just one day before measurement, and inappropriate statistical analyses that did not 

account for temporally repeated measures. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation process. 

Kim et al. (2002b), supported by the Korean 

Ministry of Health and Social Welfare, examined the 

effects of bisphenol A exposure on the male reproductive 

system. A translation of the study was provided 

by the American Plastics Council. Four-week-old 

male F344 rats (7/group) were given bisphenol A in 

drinking water at 0 (ethanol vehicle), 0.1, 1, 10, or 

100 ppm for 13 weeks. According to the study authors, 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 345 

Table 90 

Reproductive Effects in Male Rats Orally Dosed With Bisphenol Aa 

Endpoint 

Dose, mg/kg bw/day 

0.0002 0.002 0.020 BMD10 BMDL10 BMD1SD BMD1SD 

Relative organ weight 

Testis 

Epididymis 

Ventral prostate 

Epididymal sperm motilityb 

Epididymal sperm count 

k 5% 

k 13% 

m 13% 

k 23% 

2

k

k

m

k

k

6% 

17% 

34% 

37% 

18% 

k

k

m

k

k

7% 

26% 

29% 

41% 

27% 

0.056 

0.011 

0.014 

0.021 

0.0082 

0.0083 

0.014 

0.0069 

0.015 

0.0087 

0.0050 

0.0089 

a

Chitra et al. (2003a).
 

b

Values estimated from a graph by CERHR; data estimated from graphs were not modeled.
 

m,k Statistically significant increase, decrease; 2 no statistically significant effect.
 

these values were equivalent to 0.011, 0.116, 1.094, and 

11.846 mg/kg bw/day. [No information was provided 

about bisphenol A purity, or feed, caging, or bedding 

materials.] Body weight and food and water consumption were measured during the study. Urine was 

collected for 24 hr following completion of dosing, and 

then animals were killed. Blood was collected. Organs, 

including those of the male reproductive system, were 

weighed. Parts of organs were preserved in formalin and 

examined histologically. Testes and epididymides were 

preserved in liquid nitrogen to obtain sperm counts and 

for measurement of levels of testicular enzymes. Data 

were analyzed by ANOVA. 

Bisphenol A treatment had no significant effect on 

body weight or food or water intake. There were no 

effects on absolute or relative weights of the testis, 

epididymis, prostate, seminal vesicle, liver, kidney, heart, 

lung, spleen, or brain. Daily sperm production and 

number of sperm heads were unaffected by bisphenol A 

treatment. No significant effects were observed for 

activities of testicular g-glutamyl transpeptidase, sorbitol 

dehydrogenase, acid phosphatase, or b glucuronidase. 

No histopathological alterations were reported for the 

testis, epididymis, seminal vesicle, prostate, spleen, or 

brain. Bisphenol A levels in urine are reported in Section 

2. The study authors concluded that sperm density and 

the male reproductive system do not appear to be 

affected in F344 rats exposed to bisphenol A. 

Strengths/Weaknesses: Strengths include a wide range 

of doses, use of an appropriate route of exposure, and the 

use of Fischer 344 rats. Weaknesses include marginal 

sample size and the absence of information about certain 

study design features. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility in the 

evaluation process. 

Chitra et al. (2003a), supported by the Lady Tata 

Memorial Trust, Indian Council of Medical Research, and 

the Population Council, examined the effects of bisphenol 

A on the reproductive system of male rats. Animals were 

given ‘‘standard commercial laboratory chow.’’ [Bedding 

and caging materials were not reported.] Six 45-day-old 

male Wistar rats/group were orally dosed [gavage 

assumed] with bisphenol A (97% purity) in olive oil at 

0, 0.0002, 0.002, and 0.020 mg/kg bw/day for 45 days. 

Rats were killed 24 hr following the last treatment. Testes, 

epididymides, seminal vesicles, and ventral prostate were 

weighed. Epididymal sperm counts and motility were 

assessed. Antioxidant enzyme activities were measured 

in sperm. Statistical analyses included ANOVA followed 

by Student t-test. Significant effects on organ weights and 

sperm endpoints are summarized in Table 90. Bisphenol 

A treatment did not affect body weight. Absolute and 

relative (to body weight) weights of testis and epididymis 

and were reduced, and absolute and relative ventral 

prostate weights were increased at all dose levels. Effects 

on relative organ weights are summarized in Table 90. 

Sperm motility was decreased at all dose levels, and 

sperm counts were reduced at the mid and high-dose. 

There were dose-related decreases in activity of superoxide dismutase, catalase, glutathione reductase, and 

glutathione peroxidase in sperm at all dose levels. 

Hydrogen peroxide generation and lipid peroxidation in 

sperm increased dose-dependently at all dose levels. The 

study authors concluded that adverse effects of bisphenol 

A on the male reproductive system may be due to 

oxidative stress. 

Although these studies have a limited number of 

animals per group, they appear to be relatively well 

conducted, and there are apparently consistent dosedependent changes in testis and epididymis weights and 

sperm parameters. The epididymal (portion not mentioned) sperm numbers measured in this study are 

consistent with the daily sperm production measured 

by Sakaue et al. (2001). A potential significant concern in 

this study is the use of olive oil as the vehicle. The 

stability/reactivity of bisphenol A was not determined 

and it is possible that bisphenol A interacted with olive 

oil, resulting in the observed findings. This study 

provides suggestive data that bisphenol A induces 

oxidative stress in epididymal sperm and alters testis 

and epididymis weights at low doses. 

Strengths/Weaknesses: Strengths include the use of 

oral and low multiple doses and appropriate measures. 

A weakness includes the marginal sample size. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate for inclusion but of limited utility 

based on small group size. 

Chitra et al. (2003b), supported by the Population 

Council, New York, examined the effects of bisphenol A 

and vitamin C exposure on epididymis and sperm 

counts in rats. Wistar rats (45-days old) were fed 

standard commercial laboratory chow and housed in 

plastic cages. [No information was provided about 

bedding.] Four rats/group were orally dosed with 

bisphenol A (97% purity) at 0 (olive oil vehicle), 0.0002, 

0.002, or 0.020 mg/kg bw/day for 60 days. Additional 

rats received the same bisphenol A doses in conjunction 

Birth Defects Research (Part B) 83:157–395, 2008
346 CHAPIN ET AL. 

with 40 mg vitamin C. [The specific method of oral 

dosing was not stated. A vehicle control group 

administered vitamin C was not included.] Rats were 

killed 24 hr following the last dose. Epididymides were 

fixed in Bouin solution and examined histologically. 

Sperm were counted and examined for viability and 

motility. Levels of antioxidant enzymes were measured 

in sperm and epididymis. Data were analyzed by 

ANOVA followed by Student t-test. 

There was no effect on sperm viability, but significant 

dose-related reductions were observed in sperm motility 

and count in all dose groups. [In the low- to high-dose 

group, sperm motility was reduced to B70, 60, and 55% 

of control levels. Sperm counts in the low to high-dose 

group were B12, 30, and 40% lower than control 

values.] Complete degeneration of epithelia of caput, 

corpus, and cauda epididymis was reported at all dose 

levels. [It was not clear if the effect occurred in every rat 

of each dose group.] Significant dose-related decreases 

in glutathione peroxidase and superoxide dismutase 

activity and increased lipid peroxidation were observed 

in sperm and epididymis of rats from each bisphenol A 

treatment group. No changes in sperm motility, sperm 

count, antioxidant enzyme activity, or lipid peroxidation 

were observed when bisphenol A was administered with 

vitamin C. The study authors concluded that bisphenol A 

induced oxidative stress and degeneration of epididymal 

epithelium, and vitamin C protected against those 

effects. 

Strengths/Weaknesses: A critical weakness is the use 

of only 4 animals per dose group. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for inclusion due to concerns 

with group size. 

Saito et al. (2003a), support not indicated, examined 

the effects of bisphenol A exposure on sex hormone 

levels in male rats. Wistar rats were fed MF feed (Oriental 

Yeast Co.). [No information was provided about bedding and caging materials.] Eight or 9 rats/group were 

s.c. injected with bisphenol A [purity not reported] at 0 

(corn oil vehicle), 0.005, or 5 mg every 2 days from 3–11 

weeks of age. [Based on a graph showing body weights 

of B50 g at the beginning of treatment and B300 g at 

the end of treatment, the bisphenol A doses would 

have been 0.1 and 100 mg/kg bw at the beginning of the 

treatment period and 0.017 and 17 mg/kg bw at the end 

of the treatment period.] Additional groups of 8–9 rats 

were injected with 5 mg/day 17b-estradiol or diethylstilbestrol. Rats were killed at 13 weeks of age, 2 weeks 

following the last treatment. Body, testes, and other 

reproductive organs were weighed. Levels of 17bestradiol and testosterone were measured in plasma by 

RIA. Data were analyzed by Student t-test and Dunnett 

test. No clinical signs of toxicity or changes in behavior 

were observed. Exposure to bisphenol A did not affect 

body weight gain or absolute or relative testis weight. No 

effects were observed for weights of prostate, preputial 

gland, or epididymis. [Data were not shown by study 

authors.] Plasma testosterone levels were significantly 

reduced in the low bisphenol A group [by B1.5 fold] 

and plasma estradiol levels were significantly increased 

in the high bisphenol A dose group [by B8-fold]. Effects 

observed with 17b-estradiol and diethylstilbestrol exposure included decreased body weight gain, reduced 

absolute and relative testis weight, decreased plasma 

testosterone levels, and increased plasma 17b-estradiol 

levels. The study authors concluded that bisphenol A 

disturbed sex steroid production in male rats. 

Single point testosterone measurements are normally 

highly variable; the apparent significant decrease in 

testosterone observed in this study may be spurious and 

due to the small group size, an unusual low variability in 

testosterone, and the use of the Student t-test, an 

inappropriate statistical test for this analysis. There is 

some concern with the dynamic range of the 17bestradiol RIA as 17b-estradiol is normally measured in 

pg/mL. 

Strengths/Weaknesses: Weaknesses include the s.c. 

route of exposure, the use of an inappropriate method of 

anesthesia when measuring hormone levels, inadequate 

sample sizes for highly variable testosterone endpoint, 

and inappropriate statistical tests on hormone data. 

Utility (Adequacy) for CERHR Evaluation Process: 

Based on experimental design concerns, this study is 

inadequate for the evaluation. 

Takahashi and Oishi (2003), support not indicated, 

examined species, strain, and route differences in 

reproductive systems of male rodents exposed to bisphenol A. The studies in rats are discussed in this 

section, and the studies in mice are discussed in Section 

4.2.2.2. Animals were housed in stainless steel suspended 

cages or ‘‘chip-bedded’’ plastic cages. [No information 

was provided about the type of chow used.] Animals 

used in this study were 4 weeks old at the start of dosing. 

In the dietary portion of the study, male Wistar rats or 

Holtzman SD rats were given feed containing 0 or 0.25% 

bisphenol A (499.0% purity) for 2 months. There were 8 

animals in each dose group. The 0.25% dose group was 

reported to produce minimal testicular effects in a 

previous study. Mean bisphenol A intakes were estimated by study authors at B200 mg/kg bw/day in rats. 

In parenteral exposure studies, 4-week-old male Wistar 

rats were s.c. dosed with bisphenol A in propylene glycol 

at 0 or 200 mg/kg bw on 4 days/week for 1 month. 

Additional male Wistar rats were given i.p. injections of 

bisphenol A in propylene glycol at 0, 2, or 20 mg/kg bw 4 

days/week for 1 month. An i.p. dose of 200 mg/kg bw 

was originally administered but resulted in death. There 

were 5–6 animals/group in the parenteral exposure 

studies. In both the dietary and parenteral exposure 

studies, animals were observed daily for clinical signs, 

and body weight and food intake were measured. 

Animals were killed at the end of the dosing period. 

Liver, kidney, and reproductive organs were weighed. 

Testes were fixed in formaldehyde solution and examined histologically. The study authors noted that the 

appropriate fixative for the testis is Bouin solution but 

that obvious and severe injuries could be detected with 

the method used in the present study. Testosterone was 

measured in serum by ELISA. Daily sperm production 

and efficiency and epididymal sperm reserves were 

evaluated. Statistical analyses included F test, Student ttest, Aspin–Welch test, Bartlett test, ANOVA, Dunnett 

test, Kruskall–Wallis test, Dunnett non-parametric test, 

Wilcoxon rank-sum test, w2 test, Mantel–Haenzel test, 

and Fisher exact test. 

In rats exposed through diet, there was no effect on 

body weight or absolute organ weight. Relative liver 

weight was significantly increased in Wistar rats exposed 

to bisphenol A. [Data were not shown by study 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 347 

Table 91 

Effects in Rats Given Bisphenol A by Intraperitoneal 

Injectiona 

Dose, mg/kg bw 

Endpoint 2 20 BMD10 BMDL10 BMD1SD BMDLSD 

Weight 

Terminal 2 k12% 19 12 17 5 

body 

Ventral 2 k29% 7 5 9 6 

prostate 

Liver 2 k18% 14 8 12 6 

Kidney 2 k12% 20 11 19 6 

Serum 2 k69% 3 2 16 9 

testosterone 

a

Takahashi and Oishi (2003).
 

m,k Statistically significant increase, decrease; 2 no statistically
 

significant effect.
 

authors.] The study authors indicated that they forgot to 

weigh seminal vesicles and prostate glands. No effects 

were reported for reproductive organ histopathology, 

daily sperm production or efficiency of production, 

epididymal sperm reserves, or serum testosterone levels 

in rats exposed to bisphenol A through diet. [Data were 

not shown by study authors.] 

In the portion of the study where rats were administered 200 mg/kg bw bisphenol A, stiffness was observed 

at the injection site. Terminal body weight was lower [by 

20%] in treated rats. Treatment resulted in [B20%] 

decreases in absolute liver, kidney, preputial gland, and 

testis weight and [B40–80%] decreases in epididymis, 

seminal vesicle, and prostate weight. The study authors 

also reported decreases in relative weights of epididymis, 

seminal vesicle and coagulation gland, and prostate. 

[Data were not shown. The Expert Panel assumes that 

by coagulation gland, the authors mean the anterior 

prostate or coagulating gland.] No histopathological 

alterations were observed in the seminiferous tubules of 

control animals. In the bisphenol A group, histopathological observations (incidence) in seminiferous tubules 

included focal atrophy (60%), exfoliation (60%), detachment (20%), missing Stage VII/VIII spermatids (40%), 

retention of Stage IX/XI spermatids (60%), and loss of 

basement membrane (20%). Bisphenol A treatment 

reduced daily sperm production [by B25%, as estimated 

from a graph for total production but not per g testis.] 

Reserves in head and body of the epididymis and the 

cauda epididymis were also reduced/g of tissue in 

bisphenol A-treated rats [by B43 % in the head and 

body of epididymis and 63% in the cauda epididymis, 

as estimated from a graph]. There was no significant 

effect on serum testosterone level. 

Effects in rats administered bisphenol A by i.p. 

injection are summarized in Table 91. At 20 mg/kg bw, 

terminal body weight and prostate, liver, and kidney 

weight were reduced. Serum testosterone levels were 

also reduced in rats from the 20 mg/kg bw/day group. 

There were no effects on testicular histopathology or 

sperm endpoints. [Data were not shown by study 

authors.] Enlarged ileum was observed at necropsy in 

the 20 mg/kg bw group and histopathological examination revealed mucosal degeneration and hyperplastic 

duodenum, jejunum, ileum, and cecum. The study 

authors concluded that bisphenol A is more toxic 

through s.c. and i.p. exposure routes than by oral 

exposure in the diet. 

This study reports a comprehensive study comparing 2 

mouse and 2 rat strains using minimal numbers of 

animals per group. The data suggest that systemic 

exposure is necessary for bisphenol A estrogenic activity 

to be exhibited and strongly indicate that route of 

administration (oral vs. i.p.) is an important consideration. A minimal exposure range; the study did not 

explore low doses. 

Due to differences in strain sensitivities, a NOAEL was 

not established. Nevertheless, it is likely to be near 0.25% 

in the diet. 

Strengths/Weaknesses: Strengths include multiple 

routes of exposure, use of two strains of mice and rats, 

and a comparison of the oral, i.p., and subcutaneous 

routes. Weaknesses include use of single high doses 

administered for different durations across groups using 

minimal sample sizes. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate but of limited utility. 

Herath et al. (2004), supported by Japan Society for 

Promotion of Science and the Japanese Ministry of 

Education, Culture, Sports, Science, and Technology, 

examined the effects of bisphenol A exposure on 

reproductive hormones and sperm endpoints in male 

rats. Octylphenol was also examined in this study, but 

results will not be discussed. Wistar–Imamichi rats were 

fed a soy-containing commercial feed (Nosan, Japan) and 

housed in metal cages. Rats were randomly assigned to 

groups and beginning at 50 days of age, 10–11 rats/ 

group were s.c. injected with bisphenol A (Z95% purity) 

at 0 (DMSO vehicle) or 3 mg/kg bw/day for 5 weeks. 

Rats were weighed during the study. LH, testosterone, 

and progesterone concentration were measured in blood 

after 2 weeks of treatment and on the following day, 1 hr 

after a challenge with gonadotropin-releasing hormone. 

Rats were killed after 5 weeks of treatment. Blood was 

obtained for measurement by RIA of LH, progesterone, 

testosterone, immunoreactive inhibin, and insulin 

growth factor 1 levels. The testis, seminal vesicle, 

epididymis, and prostate were weighed, and sperm 

counts and motility were determined. A total of 5–11 

rats/group were examined for each endpoint. Statistical 

analyses included ANOVA and Duncan Multiple Range 

test. 

No statistically significant effects on baseline LH, 

testosterone, or progesterone levels were observed 

following 2 weeks of bisphenol A treatment. Following 

injection with gonadotropin-releasing hormone, LH 

levels were significantly increased in the bisphenol A 

group and progesterone levels were significantly increased in the vehicle control group. In the bisphenol A 

group compared to the control group, incremental 

increases following injection with gonadotropin-releasing hormone were smaller for testosterone [B410 vs. 

875%] and progesterone [B75 vs. 510%]; statistical 

significance was reported for the progesterone effect. 

Following 5 weeks of bisphenol A treatment, significant 

effects on plasma hormone levels compared to controls 

included decreased testosterone [by B55%] and increased insulin-like growth factor 1 [by B20%]. Ventral 

prostate weight was significantly higher [by B29%] in 

Birth Defects Research (Part B) 83:157–395, 2008
348 CHAPIN ET AL. 

the bisphenol A versus control group, but there were no 

effects on testis, seminal vesicle, or right epididymis 

weight. [Relative reproductive organ weights were not 

reported.] Epididymal sperm counts were reduced 

significantly [by B10%] in the bisphenol A group, but 

there was no significant effect on sperm motility. The 

study authors concluded that bisphenol A exposure can 

affect basal and gonadotropin-releasing hormone-stimulated LH production and reduced daily sperm production in rats. 

Strengths/Weaknesses: This study appears to have 

been relatively well conducted. A major weakness of this 

study is the inconsistency in the hormone data (control 

data after 2 weeks were dramatically different than after 

5 weeks even though both are from sexually mature rats). 

The subcutaneous route of administration with the use of 

DMSO as vehicle are weaknesses. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate and not useful for the evaluation 

process primarily due to the significant inconsistencies in 

the hormone data from control animals. 

Toyama et al. (2004), supported in part by the Japanese 

Ministry of Environment and Ministry of Education, 

Science, Sports, and Culture, examined the effects of 

bisphenol A exposure on the reproductive system of 

male rats and mice. [No information was provided 

about feed, caging, or bedding materials. The mouse 

portion of the study is discussed in Section 4.2.2.2.] 

Adult male Wistar rats (n 5 12/group) were s.c. injected 

with bisphenol A [purity not indicated] at 0.020 or 

0.200 mg/kg bw/day for 6 consecutive days. Three 

control animals were s.c. injected with the DMSO/olive 

oil vehicle for 6 days. Ten animals/bisphenol A group 

and 2 controls were killed the day following treatment 

and perfused with glutaraldehyde. Testes were weighed 

and examined by light and electron microscopy. Epididymis, preputial gland, ventral prostate, and seminal 

vesicle with coagulating glands were also weighed. The 

remaining animals, 2 in each bisphenol A group and 1 in 

the control group, were held an additional 2 months and 

then subjected to fertility tests. In fertility testing, each 

male was mated to 2 untreated females. One of the 2 

mated females was kept until parturition. [The males 

were apparently killed for an examination of reproductive organs following fertility testing.] Results were 

qualitatively reported, and statistical analyses were not 

conducted. 

The description of the results was limited primarily to 

rats in the 0.020 mg/kg bw/day group. Body and male 

accessory reproductive organ weights were not affected 

by bisphenol A treatment. [Data were not shown by 

study authors.] In the bisphenol A group, examination 

by light microscopy revealed exfoliation of round 

spermatids, deformed heads of mature spermatids, and 

multinucleated giant cells in seminiferous epithelium. 

Testicular effects observed by electron microscopy 

included abnormal acrosomal caps and invagination 

and/or vacuole formation in nuclei of spermatids 

beyond Step 1. Ectoplasmic specialization around Sertoli 

cells was also affected by bisphenol A treatment. No 

histological or ultrastructural abnormalities were observed in the testis 2 months following exposure. Sexual 

behavior was observed to be normal in treated males. 

Females delivered normal pups and litter sizes were 

similar between groups. The study authors concluded 

that bisphenol A exposure did not affect fertility in rats 

and that adverse effects were transient. 

Strengths/Weaknesses: Definite conclusions cannot be 

drawn from such a limited data set; the fertility 

assessment was not meaningful due to the sample size 

(2/group). The background incidence of the electron 

microscope findings was not discussed. Another weakness is the subcutaneous route with DMSO as a vehicle. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate and not useful in the evaluation. 

4.2.2.2 Mouse: Takao et al. (1999), support not 

indicated, examined the effects of bisphenol A exposure 

on the reproductive system of mice. Five-week-old male 

C57BL/6 mice were exposed to bisphenol A [purity not 

indicated] in drinking water at 0 (0.005% ethanol in 

water vehicle), 0.0005, or 0.050 g/L for 4 or 8 weeks. 

[Based on daily water intakes and body weights 

reported in the study, bisphenol A intake was estimated by CERHR at 0.14 and 13 mg/kg bw/day.] To 

maintain bisphenol A at a stable concentration, drinking 

water was changed twice a week, but the stability of 

bisphenol A was not verified. Mice were killed, and both 

testes and spleen were removed and weighed. One testis 

was processed for histopathological evaluation. Plasma 

testosterone, corticosterone, and LH levels were measured in 7 mice/group using RIA or enzyme immunoassay. [No information was provided on the purity of 

bisphenol A, time between last dose and sacrifice, or 

the type of chow, caging, or bedding materials used. 

Very few details were provided on the methods, 

including histopathological evaluation.] Statistical analyses included ANOVA followed by Fisher protected 

least significant difference test. 

Water intake was reduced significantly [by 8%] in the 

high-dose group exposed for 4 weeks. There were no 

effects on body weight or absolute or relative (to body 

weight) testis or spleen weight. Plasma testosterone 

levels were reduced [by 87–89%] in the high-dose group, 

but statistical significance was attained only in the group 

exposed for 8 weeks. No statistically significant changes 

were reported for plasma corticosterone or LH levels. 

The number of multinucleated cells in the seminiferous 

tubules was increased in high-dose mice treated for 8 

weeks. The study authors concluded that exposure to 

bisphenol A around the peripubertal period may disrupt 

the reproductive tracts of male mice. 

Strengths/Weaknesses: This study lacks important 

experimental details on methodology, including 

numbers of treated animals. Although it appears that 

bisphenol A in the drinking water results in a doserelated decrease in plasma testosterone, this endpoint is 

highly variable because testosterone is secreted in a 

pulsatile manner, and controls for Weeks 4 and 8 varied 

by B30%. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate and not useful for the 

evaluation process due to the paucity of important 

experimental details and the variability of the testosterone data. 

Al-Hiyasat et al. (2002), supported by the Deanship of 

Scientific Research at Jordan University of Science and 

Technology, examined the effect of bisphenol A exposure 

on fertility of male mice. [No information was provided 

about composition of chow, bedding, or caging.] Ten 60­

day-old male Swiss mice/group were gavaged with the 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 349 

Table 92 

Effects Observed Following Gavage of Male Mice with Bisphenol A and Mating With Untreated Femalesa 

Dose, mg/kg bw/day 

Endpoint 0.005 0.025 0.1 BMD10 BMDL10 BMD1SD BMDL1SD 

Body weight k18% k 21% k13% 

Relative weight 

Testis 2 m 26% 2

Seminal vesicle 2 k 27% 2

No. sperm/testis 2 k 17% k29% 0.035 0.029 0.036 0.028 

No. sperm/mg testis 2 k 16% k37% 0.027 0.023 0.029 0.023 

Daily sperm production 2 k 17% k29% 0.035 0.029 0.036 0.028 

Efficiency of sperm production 2 k 16% k37% 0.027 0.023 0.029 0.023 

No. sperm/epididymis k 14% k 25% k35% 0.033 0.026 0.040 0.030 

Sperm/mg epididymis 2 k 17% k31% 0.033 0.025 0.053 0.038 

Percent pregnant females 2 k 40% k33% 

Resorptions/implantation site (3% control rate) 13% 15% 13% 

Percent females with resorption sites m 2.5-fold m 3.8-fold m 3.4-fold 

a

Al-Hiyasat et al. (2002).
 

m,k Statistically significant increase, decrease, 2 no statistically significant effect.
 

ethanol/distilled water vehicle or bisphenol A (97% 

purity) for 30 days. [The study listed the bisphenol A 

doses as 5, 25, and 100 ng/kg bw. An erratum was later 

released that indicated the correct units were lg/kg bw 

(0.005, 0.025, and 0.1 mg/kg bw/day).] Following the 

dosing period, each male was mated for 10 days with 2 

untreated female mice, who were placed inside the cage 

of the male during the same time period. The males were 

then killed for an evaluation of testes, seminal vesicles, 

and preputial gland weights. Sperm counts and daily 

sperm production were determined. Mated females were 

killed 10 days later to determine numbers of pregnancies, 

implantation sites, viable fetuses, total resorptions, and 

females with resorptions. [There was no indication that 

mating was confirmed by checking for sperm in the 

vagina.] Data were analyzed by Student t-test or Fisher 

exact test. 

Results that obtained statistical significance are summarized in Table 92. Body weights were lower in all dose 

groups compared to controls. There were no evident 

dose–response relationships for organ weights. Absolute 

testis weight was decreased at the low dose, and absolute 

seminal vesicle weight was reduced at the mid and highdose. Effects on relative organ weights are summarized 

in Table 92. Decreases in testicular sperm counts and 

daily sperm production were observed at the mid and 

high-dose. Total sperm counts in the epididymis were 

decreased at all dose levels, and sperm counts/mg 

epididymis were decreased at the mid and high-dose. 

The total number of resorptions and females with 

resorptions were increased at all dose levels. The 

percentage of pregnant females was reduced at the mid 

and high-dose. The study authors concluded that bisphenol A could adversely affect fertility and reproduction of adult male mice. 

The number of animals per group was too small 

(n 5 10) for a definitive assessment of study endpoints. 

The method of randomization (or initial body weights) 

was not presented. There is also an absence of a dose 

response in several of the endpoints assessed. Given that 

mice usually have poor (relative to rats) fertility rates, the 

confidence in control data is limited. The male mice were 

killed shortly after the mating period, which may have 

influenced/confounded the number of sperm in the 

epididymis. Student t-test is an inappropriate analysis 

for organ weights (ANOVA with appropriate post-hoc 

test would be appropriate). Statistical significance is 

suspect, and the changes in organ weights are minimal in 

magnitude. 

Strengths/Weaknesses: Weaknesses include small 

sample sizes for endpoints, inadequate coverage of the 

full spermatogenesis cycle in dosing duration, measurement of sperm counts without allowing adequate time 

following mating, and inappropriate accounting of sire 

influences on resorption rates in statistical analyses. 

Sample sizes are small for fertility assessments. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate for inclusion. Data on tissue 

weights are of limited utility for the evaluation process, 

however fertility data are not useful. 

Nagao et al. (2002), support not indicated, examined 

the effects of bisphenol A in mice following exposure 

during different life stages. An initial experiment, 

described in more detail in Section 3.2.7, found that 

C57BL/6N mice were more sensitive to 17b-estradiol 

than ICR mice, and the study authors therefore used 

C57BL/6N mice to examine the effects of bisphenol A. 

Life stages examined included prenatal development, 

adolescence, and adulthood. The study conducted in 

adult mice is described here, while the studies conducted 

during prenatal development and adolescence are 

described in Section 3.2.7. C57BL/6N mice were fed 

PLD (phytoestrogen-low diet; Oriental, Japan). They 

were housed in polycarbonate cages with wood bedding. 

Daidzein and genistein levels were analyzed in diet, tap 

water, and bedding and found to be below 0.5 mg/100 g. 

At 10 weeks of age, 20 male mice/group were gavaged 

with bisphenol A (99.0% purity) at 0.002, 0.020, or 

0.200 mg/kg bw/day for 6 days. Twenty control males/ 

group were given 0.5% carboxymethyl cellulose 

[assumed to be the vehicle]. Six weeks after the final 

dose was administered, the mice were weighed and 15 

males/group were killed and necropsied. The testis, 

epididymis, and seminal vesicles with coagulating 

glands were weighed. The ventral prostate was not 

weighed due to difficulties in obtaining only prostate 

Birth Defects Research (Part B) 83:157–395, 2008
350 CHAPIN ET AL. 

and determining the precise weight. Epididymal sperm 

counts were obtained. Histopathological examinations 

were conducted for organs fixed in Bouin solution. Data 

were analyzed by Bartlett test to determine homogeneity 

of variance, followed by ANOVA when homogeneity of 

variance was confirmed or Kruskal–Wallis analysis of 

ranks when variance was not homogenous. Dunnett test 

was used for multiple comparisons. 

In the bisphenol A group, there were no significant 

differences in body weight gain or terminal body 

weights. [Data were not shown.] There were no 

significant differences in absolute or relative (to body 

weight) weights of the testis, epididymis, or seminal 

vesicles. There were no significant effects on sperm 

count. No histopathological alterations in reproductive 

organs were reported. The study authors concluded that 

low-dose bisphenol A exposure of mice did not reduce 

sperm density. 

Strengths/Weaknesses: This study was well conducted and adds to the understanding of the potential 

effects of low doses of bisphenol A administered by a 

relevant route of exposure. Strengths are an appropriate 

number of mice per group, the use of response to 17bestradiol in 2 strains of mice to identify the most sensitive 

strain, and the presentation of sperm data in light of 

historical control data. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

Peknicova ́ et al. (2002), supported by the Czech 

Republic and EU, examined the effects of bisphenol A 

exposure on mouse sperm. CD-1 mice were given ST1 

feed (Velaz a.s., Prague). Three generations of mice were 

exposed to bisphenol A [purity not indicated] through 

drinking water at doses of 0.000002 and 0.000020 mg ‘‘/ 

animal’s weight/day. ’’ It was stated that there were 6 

pairs of mice in the control group. Litter size was 

evaluated in 3 generations; 1 litter was examined in the 

first and second generation and 2 litters were examined 

in the third generation. In each generation, samples of 

sperm were collected from all males and a histopathological investigation of testes was conducted in Z3 

males/group. Sperm acrosomal status was assessed 

using an immunohistochemical and Western blot method. Statistical analyses included ANOVA and Newman– 

Keuls test. [Very few experimental details were provided. No information was provided on bedding and 

caging materials, bisphenol A purity, the numbers of 

mice in each treatment group, treatment of the control 

group, ages of mice during treatment, durations of 

treatment, sample sizes and litter representation for 

sperm effects, and mating procedures. It was not clear 

if female rats were also treated.] Litter sizes were 

significantly reduced in the first and second generation 

of mice treated with the low dose (5–6.7 pups/litter vs. 

11.5–12 pups/litter in controls). There were no effects of 

bisphenol A treatment on testes weight. [Data were not 

shown by authors.] Pathological changes observed in 

testes from the low-dose group included damaged 

seminiferous tubule and reduced spermatogenesis. Acrosome integrity, evaluated as percent cells binding 

monoclonal antibodies to acrosin and intra-acrosomal 

proteins, was significantly reduced in all 3 generations of 

the low-dose group (48.5–57.7 compared to 93.3–95% 

integrity in controls) and the third generation of the highdose group (62.5 compared to 93.3% integrity in 

controls). [While the text of the study stated that 

acrosomal integrity was significantly affected only in 

the third generation of the high-dose group, the caption 

for Figure 7 of the study stated that both the second and 

third generations were significantly affected. Based on 

findings reported in the figure, it appears that the 

description in the text is correct.] The study authors 

concluded that bisphenol A exposure negatively impacts 

fertility, spermatogenesis, and sperm quality in mice. 

Strengths/Weaknesses: Although potentially interesting findings are presented, the study lacks many 

important details and sample sizes are critically 

inadequate. 

Utility (Adequacy) for CERHR Evaluation Process: 

Due to study design concerns, this study is inadequate 

and has no utility for the evaluation. 

Takahashi and Oishi (2003), support not indicated, 

examined species, strain, and route differences in 

reproductive systems of male rodents exposed to bisphenol A. Studies on mice are discussed here, and 

studies on rats are discussed in Section 4.2.2.1. Animals 

were housed in stainless steel suspended cages or ‘‘chipbedded’’ plastic cages. [No information was provided 

about the type of chow used.] Animals used in this 

study were 4 weeks old at the start of dosing. In the 

dietary portion of the study, CD-1 (ICR) mice and 

C57BL/6CrSlc mice were given feed containing 0 or 

0.25% bisphenol A (499.0% purity) for 2 months. There 

were 8 animals in each dose group. The 0.25% dose was 

reported to produce minimal testicular effects in a 

previous study. Mean bisphenol A intakes were estimated by study authors at B400 mg/kg bw/day in mice. 

The parenteral exposure studies were performed only in 

rats. Animals were observed daily for clinical signs, and 

body weight and food intake were measured. Animals 

were killed at the end of the dosing period. Liver, kidney, 

and reproductive organs were weighed. Testes were 

fixed in formaldehyde solution and examined histologically. The study authors noted that the appropriate 

fixative for the testis is Bouin solution, but that obvious 

and severe injuries could be detected with the method 

used in the present study. Testosterone was measured in 

serum by ELISA. Daily sperm production and efficiency 

and epididymal sperm reserves were evaluated. Statistical analyses included F test, Student t-test, Aspin– 

Welch test, Bartlett test, ANOVA, Dunnett test, Kruskall– 

Wallis test, Dunnett non-parametric test, Wilcoxon ranksum test, w2 test, Mantel–Haenzel test, and Fisher exact 

test. 

There were no significant effects on organ or body 

weights in C57BL/6CrSlc mice exposed through 

diet. In CD-1 (ICR) mice exposed through diet, there 

were increases in absolute testis [16%], liver [12%], 

and kidney [20%] weights and a decrease in absolute 

epididymis [12%] weight. The study authors 

reported that relative testis weight was not significantly 

affected, but when the value from 1 mouse with a high 

relative testis weight was deleted, the effect attained 

statistical significance. [Data were not shown by study 

authors.] No effects were reported for testis histopathology, daily sperm production or efficiency of production, 

epididymal sperm reserves, or serum testosterone levels 

in mice exposed to bisphenol A through diet. [Data were 

not shown by study authors.] The study authors 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 351 

concluded that the testicular toxicity of bisphenol A is 

‘‘relatively weak,’’ based on the co-occurrence of liver 

and kidney toxicity at exposure levels causing testicular 

effects. 

Strengths/Weaknesses: A strength is the use of dietary 

exposure and the examination of strain differences in 

mice. Weaknesses include use of a single very high-dose 

level. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate but of limited utility. 

Park et al. (2004), support not indicated, examined the 

effects of bisphenol A exposure on the reproductive and 

hematological systems of male and female mice. [Results 

for males are discussed here, and results for females are 

discussed in Section 4.2.1.2.] Adult ICR mice were fed 

mouse formulation feed (Cheil Feed). [No information 

was provided about caging or bedding materials.] 

Fifteen mice/sex/group were i.p. injected with bisphenol 

A [purity not indicated] in an ethanol/corn oil vehicle at 

0.05, 0.5, or 5.0 mg/kg bw on 5 occasions (every 3 days 

over a 14-day period). One control group received no 

treatment, and a second control group was i.p. injected 

with corn oil. Males were examined 2 days following 

administration. Semen was collected and assessed for 

sperm number, viability, and motility. Reproductive 

organs were weighed and fixed in Bouin solution, and 

histopathological examination was conducted. Hematological and clinical chemistry endpoints were also 

assessed. Data were analyzed by least significant 

difference test. 

Exposure to bisphenol A had no effect on body weight 

or on weights of male reproductive organs including 

testis, epididymis, vesicular gland, or coagulating gland. 

Reductions in sperm concentrations [by 18%] and 

increases in sperm abnormalities [by 28%] were significant in the high-dose group. There were no treatment 

effects on testicular histology. There were no significant 

effects on hematological or clinical chemistry endpoints 

in males treated with bisphenol A. The study authors did 

not report conclusions regarding study findings. 

Strengths/Weaknesses: Weaknesses include the i.p. 

route. Frequency of administration was every 3 days and, 

given the half-life of the chemical, it is unlikely that 

sufficient blood chemical levels were sustained to induce 

‘‘maximal’’ bisphenol A-mediated responses. 

Utility (Adequacy) for CERHR Evaluation Process: 

Given the dosing paradigm (i.p. injection every 3 days) 

this study is adequate but of limited utility in the 

evaluation process. 

Toyama et al. (2004), supported in part by the Japanese 

Ministry of Environment and Ministry of Education, 

Science, Sports, and Culture, examined the effects of 

bisphenol A exposure on the reproductive system of 

male rats and mice. [No information was provided 

about feed, caging, or bedding materials. The mouse 

study is discussed here, and the rat study is discussed 

in Section 4.2.2.1.] Adult male ICR mice (n 5 12/group) 

were s.c. injected with bisphenol A [purity not indicated] 

at 0.020 or 0.200 mg/kg bw/day for 6 consecutive days. 

Three control animals were s.c. injected with the DMSO/ 

olive oil vehicle for 6 days. Ten animals/bisphenol A 

group and 2 controls were killed the day following 

treatment and perfused with glutaraldehyde. Testes 

were weighed and examined by light and electron 

microscopy. Epididymis, preputial gland, ventral 

prostate, and seminal vesicle with coagulating 

glands were also weighed. The remaining animals,2 

males in each bisphenol A treatment group and 1 control 

male, were held an additional 2 months and then 

subjected to fertility tests. In fertility testing, each male 

was mated to 2untreated females. One of the 2 mated 

females was kept until parturition. [The males were 

apparently killed for an examination of reproductive 

organs following fertility testing.] Results were 

qualitatively reported, and statistical analyses were not 

conducted. 

The study authors noted that all effects were similar 

between rats and mice and between dose groups, and 

their description of results was primarily limited to rats 

in the 0.020 mg/kg bw/day group. Body and male 

accessory reproductive organ weights were not affected 

by bisphenol A treatment. [Data were not shown by 

study authors.] In the bisphenol A group, examination 

by light microscopy revealed exfoliation of round 

spermatids, deformed heads of mature spermatids, and 

multinucleated giant cells in seminiferous epithelium. 

Testicular effects observed by electron microscopy 

included abnormal acrosomal caps and invagination 

and/or vacuole formation in nuclei of spermatids 

beyond Step 1. Ectoplasmic specialization around Sertoli 

cells was also affected by bisphenol A treatment. No 

histological or ultrastructural abnormalities were observed in testes 2 months following exposure. Sexual 

behavior was observed to be normal in treated males. 

Females delivered normal pups and litter sizes were 

similar between groups. The study authors concluded 

that bisphenol A exposure did not affect fertility in mice 

and that adverse effects were transient. 

Strengths/Weaknesses: It is not possible to draw 

definite conclusions from such a limited data set; the 

fertility assessment was not meaningful due to the small 

sample size (2/group). The background incidence of the 

electron microscopy findings was not discussed. An 

additional weakness is the subcutaneous route with the 

use of DMSO as vehicle. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate and not useful due to the 

limited number of animals per group. 

Anahara et al. (2006), supported by the Japanese 

Ministry of Environment and Ministry of Education, 

Culture, Sports, Science, and Technology, examined the 

effects of bisphenol A exposure on expression of 

cortactin protein in the mouse testis. Cortactin is an actin 

binding protein that makes up the apical ectoplasmic 

specialization between Sertoli cells and spermatids and 

the basal ectoplasmic specialization between Sertoli cells. 

Cortactin is one of several proteins that control spermatid 

development. Adult (12-week-old) male ICR mice (n 5 5– 

7/group) were s.c. injected with corn oil vehicle, 

0.0024 mg/kg bw/day bisphenol A, 2.5 mg/kg bw/day 

diethylstilbestrol, or 1.2 mg/kg bw/day 17b-estradiol for 

5 days. [No information was provided on purity of 

bisphenol A or the types of feed, caging, or bedding 

used.] Animals were killed on the day following the last 

injection. Testes were homogenized and expression of 

cortactin protein was determined in testes from 5–7 rats/ 

group by Western blot, immunohistochemistry, and 

immuno electron microscopy techniques. Data were 

analyzed by t-test. Exposure to bisphenol A resulted in 

a significant decrease in testicular cortactin protein 

Birth Defects Research (Part B) 83:157–395, 2008
352 CHAPIN ET AL. 

expression [to B60% of control levels]. Immunohistochemical analysis revealed that cortactin staining was 

reduced in the apical ectoplasmic specialization but not 

in the basal ectoplasmic specialization. Examination by 

immuno electron microscopy revealed no expression of 

cortactin around heads of spermatid and deformation of 

nuclei and acrosomes. Effects observed with 17b-estradiol and diethylstilbestrol were similar to those observed 

with bisphenol A, with the exception that diethylstilbestrol also reduced cortactin protein expression in the basal 

ectoplasmic specialization and did not result in deformation of spermatids. The authors concluded that exogenous chemicals can damage junctional proteins like 

cortactin and have adverse effects on Sertoli cell protein 

regulation. 

Strengths/Weaknesses: The subcutaneous route of 

administration of a single dose was a weakness as were 

suboptimal sample sizes. Western blot analysis of 

cortactin was inappropriately presented as a function of 

the control value with no variability in the control 

sample. There were no apparent differences in levels of 

protein expression between various estrogenic agents/ 

treatments. No adverse outcomes of the changes in 

cortactin were explored. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate and not useful for the 

evaluation process. 

4.2.2.3 Other mammals: Moon et al. (2001), supported by Korea University Medical Science Research 

Center and the Korean Ministry of Education, examined 

the effects of bisphenol A exposure on penile function in 

rabbits. [No information was provided on feed or 

caging and bedding materials.] Male, 8–12 week-old 

New Zealand white rabbits were i.p. injected with corn 

oil vehicle or 150 mg/kg bw bisphenol A [purity not 

reported], every other day for 12 days to a cumulative 

dose of 900 mg/kg bw [75 mg/kg bw/day]. Rabbits were 

killed at 4 weeks (n 5 15/group) and 8 weeks (n 5 15/ 

group) following bisphenol A treatment. In 5 rabbits/ 

group, the penis was removed and fixed in 10% neutral 

buffered formalin for histological examination. In 10 

rabbits/group, the corpora cavernosa were removed 

from the penis, and in vitro responses to norepinephrine, 

acetylcholine, sodium nitroprusside, and L-arginine were 

studied. Data were analyzed by Student t-test. Treatment 

with bisphenol A significantly suppressed contraction of 

corpora cavernosa in response to norepinephrine and 

relaxation in response to acetylcholine, sodium nitroprusside, and L-arginine at both stages of evaluation. 

Histopathological observations in the bisphenol Atreated rabbits but not control rabbits at both ages 

included intracavernosal fibrosis in conjunction with 

decreased sinusoidal spaces. Compared to rabbits in the 

control group, both age groups of rabbits exposed to 

bisphenol A had significantly increased trabecular 

smooth muscle content (73.3–83.2 vs. 33.2% in controls) 

and a non-significant difference in thickness of tunica 

albuginea (0.93–1.12 mm vs. 0.32–0.43 mm in controls). 

The study authors concluded that bisphenol A may 

affect erectile responses by inducing histological alterations in the penis. 

Strengths/Weaknesses: There is no evidence that 

bisphenol A has any effect on the ability to attain an 

erection resulting in copulation in mice or rats. The 

lack of a plausible rationale is a weakness. This 

study does not have a concurrent control (e.g., 17bestradiol) to ascertain if the observed effects 

are the result of estrogenic responses in the penis. The 

route of administration and use of a single dose are 

weaknesses. 

Utility (Adequacy) for CERHR Evaluation Process: 

Due to the weakness identified above and the nature of 

the endpoints examined, this study is inadequate and of 

no utility for human risk assessment. 

Nieminen et al. (2002a), support not indicated, 

examined the effects of bisphenol A [purity not 

indicated] exposure on hormone levels in the European 

polecat (Mustela putorius). There were no significant 

effects on plasma levels of testosterone, estradiol, FSH, or 

thyroid hormones. Details of this study are discussed in 

Section 4.2.1.3. 

This study provides evidence that the bisphenol A 

administered to polecats increases GST and UDPGT 

activity. Since these findings were dose-related it appears 

that in the polecat bisphenol A increases Phase 2 

metabolism but has minimal effects on hormone levels. 

Due to the limited number of animals and the absence of 

a dose–response relationship, the hormonal changes in 

this study are difficult to interpret. 

Strengths/Weaknesses: Strengths include the use of a 

non-rodent species and multiple doses. Weaknesses 

include small sample sizes and the limited nature of 

reproductive endpoints. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate and not useful for the 

evaluation process. 

Nieminen et al. (2002b), support not indicated, 

examined the effects of bisphenol A exposure on 

endocrine endpoints in field voles (Microtus agrestis). 

Animals were housed in plastic cages with wood 

shavings and fed R36 diet (Lactamin, Sweden). Sexually 

mature field voles were randomly assigned to groups 

that received bisphenol A [purity not reported] in 

propylene glycol by s.c. injection for 4 days. Doses of 

bisphenol A (numbers of males in each group) were 0 

(n 5 6), 10 (n 5 4), 50 (n 5 6), and 250 (n 5 7) mg/kg bw/ 

day. Animals were killed the day following the last dose. 

Body and liver weights were measured. Blood was 

drawn for measurement of sex steroids, thyroxine, 

and weight regulating hormone levels in plasma using 

RIA or immunoradiometry methods. The activities of 

EROD, UDPGT, and GST were measured in hepatic 

and renal microsomes using appropriate substrates. 

Statistical analyses included ANOVA, post-hoc Duncan 

test, Student t-test, Kolmogorov–Smirnov test, Levene 

test, Mann–Whitney U test, w2 test, and Spearman 

correlation. [Results for females are discussed in 

Section 4.2.1.3.] 

Mortality was significantly increased by bisphenol A 

treatment, with incidences of 18, 36, and 20% in the lowto high-dose groups. No mortality was observed in the 

control group. Bisphenol A treatment did not significantly affect body, liver, or testis weight. Plasma 

testosterone levels increased with dose, and statistical 

significance was attained in high-dose males and 

females. Pooled (male1female) LH levels were not 

significantly altered by treatment. Liver EROD activity 

[apparently combined for males and females] was 

significantly decreased at the mid and high-dose and 

liver GST activities [not clear if for males or females or 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 353 

both] was significantly decreased at the highest dose 

level. There were no other significant effects on microsomal enzymes examined. The study authors concluded 

that wild mammals such as field voles could be more 

susceptible to bisphenol A-induced toxicity than laboratory rodents. 

Strengths/Weaknesses: Strengths include the use of a 

non-rodent species and multiple doses. Weaknesses 

include small sample sizes and limited nature of 

reproductive endpoints as well as the use of the 

subcutaneous route of administration. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is inadequate for the evaluation. 

4.2.2.4 Fish and invertebrates:: Although studies in 

fish and invertebrates may be important for understanding mechanisms of action and environmental 

impact, the Panel views these studies as not useful for 

the evaluation process. 

Shioda and Wakabayashi (2000), supported by the 

Japanese Ministry of Education, examined the effects of 

bisphenol A exposure on reproductive capability of male 

medaka (Oryzias latipes). Adult male medaka were 

housed for 2 weeks in glass beakers containing distilled 

water and bisphenol A [purity not indicated] at 0, 0.3, 1, 

3, or 10 mM [0, 0.07, 0.23, 0.69, or 2.3 mg/L]. [The number 

of male fish treated was not reported. Though not 

specifically stated, it was suggested that fish in the 

negative control group were exposed to the acetone 

vehicle.] Following exposure, each male was housed 

with two females in beakers containing distilled water. 

The numbers of eggs spawned, fertilized, and hatched 

were determined. Statistical analyses included F test 

followed by t-test or Welch test. Exposure to bisphenol A 

10 mM [2.3 mg/L] significantly reduced the number of 

eggs produced and hatched compared to the negative 

control group. Additional compounds were also examined, and it was reported that eggs and hatchings were 

significantly reduced following exposure to 17b-estradiol 

(Z 3 nM), but not nonylphenol or diethylhexyl phthalate. 

The study authors concluded that the reproductive 

effects induced by bisphenol A in this study occurred 

at a higher concentration than results observed in a yeast 

estrogen screen. 

Strengths/Weaknesses: This study appears to have 

been well conducted study and suggests that bisphenol 

A 2.3 mg/L in water decreases the number of medaka 

eggs produced and hatched. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study was not considered useful for the evaluation 

process. 

Kinnberg and Toft (2003), supported by the Danish 

Environmental Research Programme, examined the 

effects of bisphenol A exposure on the reproductive 

system of male guppies (Poecilia reticulata). Thirty 

sexually mature male guppies/group were exposed for 

up to 30 days to bisphenol A [purity not indicated] at 

nominal concentrations of 0 (acetone vehicle) 5, 50, 500, 

or 5000 mg/L. Levels of bisphenol A in water were 

verified. Exposure to the 5000 mg/L concentration was 

stopped after 21 days because of a high mortality rate. All 

fish in the high-dose group and 6 fish/group in the lower 

dose groups were killed and fixed in neutral buffered 

formalin. Histopathological examination was conducted 

in whole fish. The mortality rate in the 5000 mg/L group 

was 77%, but no increase in mortality was observed in the 

lower concentration groups. Testes of fish from the highdose group contained spermatozeugmata (bundles of 

spermatozoa with heads pointing outward and tails in 

the center) in ducts, and the authors stated the effect 

indicated blocked spermatogonial mitosis. [No information was provided on incidence or severity of testicular 

lesions, and it does not appear that statistical analyses 

were conducted.] Additional compounds were also 

tested, and it was indicated that effects induced by 

flutamide, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene, 

and 4-tert-octylphenol were similar to those observed 

with bisphenol A exposure. In contrast, exposure to 0.03 

and 0.1 mg/L 17b-estradiol resulted in hypertrophy of 

Sertoli cells and efferent duct cells. The study authors 

concluded that a high bisphenol A concentration induced 

adverse effects on testicular structure. 

Strengths/Weaknesses: This study appears to have 

been well conducted. The metabolism of bisphenol A in 

fish is unknown. It appears the bisphenol A does not 

exhibit the typical 17b-estradiol-like effect on the testis. 

Findings occurred at high relative exposures. There was 

no apparent low-dose effect. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study was not considered useful for the evaluation 

process. 

Oehlmann et al. (2000), supported by the Berlin 

Federal Environmental Agency, reported the effects of 

bisphenol A on reproductive organs in the freshwater 

ramshorn snail (Marisa cornuarietis) and the marine dog 

whelk (Nucella lapillus). Details of this study are 

discussed in Section 4.2.1.4, and most of the findings 

pertained to female snails. Adult ramshorn snails did not 

show abnormalities of male sexual organs or gonads after 

exposure to bisphenol A [purity not indicated] concentrations up to 100 mg/L for 5 months or after exposure for 

the first year of life. In the dog whelk, a 1-month 

exposure to 1, 25, or 100 mg/L bisphenol A significantly 

decreased the proportion of males with sperm in the 

seminal vesicles compared to the vehicle-exposed control. The length of the penis and prostate gland were also 

reduced by all concentrations of bisphenol A in this 

animal. The authors concluded that bisphenol A toxicity 

occurs in invertebrates at environmentally relevant 

concentrations. 

Strengths/Weaknesses: The study appears to have 

been well conducted and suggests that bisphenol A has 

an effect on the dog whelk. The potential stability/ 

biotransformation was discussed in the introduction but 

not determined during the exposure period. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study was not considered useful for the evaluation 

process. 

4.2.2.5 In vitro: While cell culture studies can provide 

useful insights into cellular and subcellular mechanisms, 

most of these studies are considered of no utility for the 

evaluation process. The study by Akingbemi et al. (2004) 

should nevertheless be considered for mechanistic value, 

and is considered adequate but of limited utility by the 

Panel for the evaluation process. 

Nikula et al. (1999), support not indicated, examined 

the in vitro effects of bisphenol A on steroidogenesis in 

mouse Leydig tumor cell cultures. Octyl phenols were 

also examined in this study, but results will not be 

discussed. In the first experiment, cells were incubated 

for 48 hr in media containing bisphenol A [purity not 

Birth Defects Research (Part B) 83:157–395, 2008
354 CHAPIN ET AL. 

indicated] at 0 (ethanol vehicle) or 10 -7–10 -4 M [0.023– 

23 lg/L] or estradiol at 10 -8 M [culture ware type not 

indicated]. Production of cyclic adenosine monophosphate (cAMP) and progesterone was measured following the incubation period and at 1 and 3 hr following a 

challenge with 10 ng/mL hCG. In additional experiments, the cells were exposed to bisphenol A at 0 or 10 -6 

M [0.23 lg/L] or 17b-estradiol or diethylstilbestrol at 10 -8 

M. Production of cAMP and progesterone was measured 

following the incubation period and at 1 and/or 3 hr 

following challenge with hCG, forskolin, cholera toxin, or 

8-bromo-cAMP. An additional study measured binding 

of 125I-hCG to the LH receptor following a 48-hr exposure 

to bisphenol A at 0 or 10 -6 M [0.23 lg/L]. Each 

experiment contained 5–8 replicates, and results from 3 

independent experiments were pooled. Data were 

analyzed by ANOVA followed by Fisher test. 

Bisphenol A had no effect on basal cAMP or 

progesterone production. At 3 hr following the hCG 

challenge, the increase in cAMP production was attenuated following previous exposure to bisphenol A at 

concentrations Z10 -7 M [0.023 lg/L] and increase in 

progesterone production was reduced at bisphenol A 

concentrations Z10 -6 M [0.23 lg/L]. At 3 hr following 

challenge, 10 -6 M [0.23 lg/L] bisphenol A decreased 

hCG-induced cAMP production but had no effect on 

forskolin- or cholera toxin-induced cAMP production. 

Following 3-hr challenges, hCG-induced progesterone 

production was reduced following exposure to 10 -6 M 

[0.23 lg/L] bisphenol A, but there were no effects on 

forskolin-, cholera toxin-, or 8-bromo-cAMP-induced 

progesterone production. Generally, 17b-estradiol and 

diethylstilbestrol attenuated hCG-, forskolin, and 8­

bromo-cAMP-induced progesterone production. Bisphenol A exposure had no effect on binding of 125I-hCG to 

the LH receptor. The study authors concluded that 

bisphenol A appears to inhibit cAMP formation and 

steroidogenesis in rat Leydig tumor cells by preventing 

coupling between the LH receptor and adenylate cyclase. 

Because no inhibition of cAMP production was observed 

following incubation of cells with 17b-estradiol, the 

study authors concluded that the effects of bisphenol A 

may not be estrogen-related. 

Strengths/Weaknesses: This appears to be a well 

conducted in vitro study. Stimulation occurred in the 

absence of steroid-rich fetal bovine serum. There was no 

mention of whether phenol red-free media were used. 

Cell viability does not appear to have been determined. 

Because this study used an in vitro system, the effects of 

metabolism were limited. Nonetheless, this study provides compelling evidence that the actions of bisphenol 

A may be non-estrogen mediated. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study was not considered useful for the evaluation 

process. 

Murono et al. (2001), from the Centers for Disease 

Control and Prevention, examined the effects of bisphenol A exposure on steroidogenesis in cultured rat 

Leydig cells. Leydig cell cultures were prepared from 

testes of 55–65-day-old Sprague–Dawley rats (n 5 8–10). 

Cells were incubated in 0 or 1–1000 nM [0.23–230 lg/L] 

bisphenol A [purity not indicated] in DMSO vehicle, 

with and without 10 mL U/mL hCG for 24 hr [culture 

ware not indicated]. Following the incubation period, 

testosterone level was measured by RIA and 125I-hCG 

binding to LH receptors was assessed. Media containing 

hydroxycholesterol was then added to the cultures, and 

testosterone production following a 4-hr incubation 

period was measured. The effects of 17b-estradiol and 

4-tert-octylphenol were also examined, but will not be 

discussed. Cell viability was evaluated by trypan blue 

exclusion and found to be unaffected at the bisphenol A 

concentrations used in this study. Three experiments 

with 4 samples/experiment were conducted. Data were 

analyzed by ANOVA and Student-Newman–Keuls test. 

Bisphenol A had no effect on basal or hCG-induced 

testosterone production or hCG binding to LH receptors. 

[Data were not shown by study authors.] Conversion of 

hydroxycholesterol to testosterone was also unaffected 

by exposure of Leydig cells to bisphenol A. No effect on 

testosterone production was observed following exposure of cells to 17b-estradiol. The study authors noted the 

similarity of effect between bisphenol A and 17bestradiol, which differed from the modest effects 

observed with 4-tert-octylphenol exposure. 

Strengths/Weaknesses: This study appears to have 

been well conducted. Phenol red-free media were used 

and cell viability after treatment was assessed. There was 

likely limited metabolism of bisphenol A, and the activity 

of metabolites cannot be assessed. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study was not considered useful for the evaluation 

process. 

Akingbemi et al. (2004), supported by NIEHS, U.S. 

EPA, NICHHD, and NIH, conducted in vitro studies to 

examine the effects of bisphenol A exposure on Leydig 

cell cultures. In vivo studies were also conducted and are 

described in Section 3 because exposures were commenced in immature animals. In a series of studies, 

testosterone production by Leydig cells was assessed 

following incubation of cells with various doses of 

bisphenol A or bisphenol A in combination with other 

compounds. Leydig cells were obtained from 90-day-old 

rats. In a dose–response study, testosterone and 17bestradiol levels were measured in Leydig cells that were 

incubated with bisphenol A [purity not indicated] at 0 

(ethanol vehicle), 0.01, 0.1, 1, 10, 100, or 1000 nM [0, 

0.0023, 0.023, 0.23, 2.3, 23, and 230 mg/L] bisphenol A for 

18 hr [culture ware not indicated]. To determine if 

bisphenol A induces estrogenic effects on Leydig cells, 

testosterone production was also measured in cells 

incubated with diethylstilbestrol or 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane, a metabolite of methoxychlor, at the same concentrations as bisphenol A. In 

mechanistic studies, Leydig cells were incubated with 

0.01 nM [0.0023 mg/L] bisphenol A, with and without the 

addition of LH or the antiestrogenic compound ICI 

182,780. Endpoints assessed included testosterone and 

17b-estradiol production and expression of mRNA for 

steroidogenic metabolizing enzymes, ER, and steroidogenic acute regulatory protein, a substance that transports the cholesterol used in testosterone synthesis. 

Levels of hormones in media were measured using RIA 

methods, and mRNA expression was evaluated using 

RT-PCR techniques. Statistical analyses included ANOVA and the Duncan multiple range test. 

In the concentration–response study, production of 

testosterone by Leydig cells was decreased following 

exposure to bisphenol A at 0.01 nM [0.0023 lg/L] but not 

at higher doses. Diethylstilbestrol reduced testosterone 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 355 

production at all dose levels, and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane reduced testosterone production at concentrations Z100 nM. Some statistically 

significant effects were observed in the mechanistic 

studies in which cells were exposed to 0.01 nM bisphenol 

A. In one study, LH-stimulated but not basal testosterone 

production was reduced by bisphenol A exposure. A 

second study demonstrated a decrease in basal testosterone production following bisphenol A exposure, but no 

decrease in testosterone level was observed following 

incubation of cells with bisphenol A in combination with 

ICI 182,270. 17b-Estradiol production was decreased in 

cells exposed to bisphenol A. Changes in mRNA 

expression following bisphenol A exposure included 

reduced expression of mRNA for the steroidogenic 

enzymes P45017b-hydroxylase and aromatase. ERb was 

not detected in Leydig cells, and expression of ERb

mRNA was not affected. The study authors concluded 

that environmentally relevant concentrations of bisphenol A act directly on Leydig cells to inhibit steroidogenesis, presumably via the ER. 

Strengths/Weaknesses: This study appears to have 

been very well-conducted. The study used a wide dose 

range and showed decreased testosterone production in 

in vitro Leydig cell cultures at low (0.1 nM) but not at 

higher concentrations. The response of multiple endpoints provides compelling evidence of a biological 

effect at 0.01 nM. An explanation for the selective effect 

of bisphenol A at this single low concentration (0.1 nM) 

was not provided, nor was the dose range of this effect 

explored. 

Utility (Adequacy) for CERHR Evaluation Process:. 

This study is adequate but of limited utility for the 

evaluation process. 

Song et al. (2002), supported by the Hormone 

Research Center and the Korean Andrological Society, 

examined the role of bisphenol A in inducing expression 

of orphan nuclear receptor Nur77, a receptor that plays 

an important role in the regulation of LH-induced 

steroidogenesis in Leydig cells. Methods used in this 

study are described in conjunction with the results. [It 

does not appear that statistical analyses were conducted in this study.] Following treatment of the mouse 

Leydig cell line K28 with bisphenol A [purity not 

indicated] atZ0.01 mM, expression of Nur77 mRNA was 

increased in a dose-related manner, with saturation of 

expression observed at 1 mM [0.23 mg/L] [culture ware 

not indicated]. In a time-response study with 1 mM 

[0.23 mg/L] bisphenol A, maximal expression of Nur77

mRNA was observed at 30 min following treatment, 

basal levels of expression were observed from 2–12 hr 

following treatment, and expression was again increased 

at 24 hr following treatment. When K28 cells were 

pretreated with the protein kinase inhibitor H89 or the 

mitogen-activated protein kinase (MAPK) inhibitor 

PD98059, induction of Nur77 mRNA by bisphenol A 

was reduced by 40–45%. Induction of c-fos and c-jun

mRNA occurred concurrently with induction of Nur77

mRNA. Bisphenol A-induced increases in Nur77 promotor activity were greater following transfection of cells 

with Nur77 promoter reporter and c-jun but not with cfos. Possible activation of MAPK by bisphenol A was 

examined using an immunoblot method with an antibody specific for phosphorylated MAPK. Phosphorylation of MAPK reached a maximum level at 10 min 

following bisphenol A treatment. No changes in bisphenol A-induced induction of Nur77 were observed 

following pretreatment with a protein kinase C inhibitor 

or P13 K inhibitor. The study authors stated that together 

these results suggest possible involvement of the protein 

kinase A and MAPK pathways in bisphenol A-induced 

induction of Nur77. 

In K28 cells transfected with Nur77 promoter or 

monomer binding site-luciferase reporters, gene promoter activities and transactivation were increased following treatment with Z0.1 mM [0.023 mg/L] bisphenol A, 

thus suggesting similar responses between promotor 

activity and mRNA induction. In a yeast assay, bisphenol 

A had no effect on interactions between Nur77 and its 

corepressor, silencing mediator of retinoid and thyroid 

receptor. 

Exposure of K28 cells to 1 mM [0.23 mg/L] bisphenol A 

resulted in increased progesterone production, which 

was inhibited 25% by the overexpression of dominant 

negative Nur77, which reduces the transactivation 

activity of Nur77. Expression of mRNA for steroidogenic 

enzymes was investigated and it was found that bisphenol A treatment increased expression of steroidogenic acute regulatory mRNA, cholesterol side-chain 

cleavage enzyme, and 3b-hydroxysteroid dehydrogenase. Effects of bisphenol A on expression of mRNA for 

Nur77 and steroidogenesis enzymes was tested in 

prepubertal mice (18 days old). Injection of 5 mice/ 

group with 125 mg/kg bw/day bisphenol A resulted in 

increased expression of Nur77 mRNA and testosterone 

levels in mouse testis from 1–6 hr following exposure. 

[Very few details were provided for the in vivo 

experiment.] The study authors concluded that the 

results of these studies indicate that bisphenol A induces 

Nur77 gene expression and alters steroidogenesis in 

Leydig cells, indicating a possible novel mechanism of 

toxicity. 

Strengths/Weaknesses: This study appears to have 

been well conducted and links in vitro bisphenol A 

administration to dose-related (classic, not inverted) 

activation of Nur77 and subsequent downstream signal 

transducing proteins. Various confirmatory experiments 

supported this relationship. These data strongly suggest 

that bisphenol A (40.1 mM) activates Nur77. The toxicological implications of these findings were not 

addressed. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study was not considered useful for the evaluation 

process. 

Hughes et al. (2000), supported by the Medical 

Research Council, the British Heart Fund, and the 

European Chemical Industry Council, examined the 

effects of bisphenol A on rat testicular calcium pumps. 

Other phenolic compounds were examined, some in 

greater detail than bisphenol A, but this discussion is 

limited to bisphenol A. Studies were conducted to 

determine the effects of bisphenol A exposure on calcium 

ATPase pump activity, calcium uptake in testicular 

microsomes, calcium levels in the TM4 Sertoli cell line, 

and TM4 cell viability [culture ware not indicated]. In 

the cell-viability study, cells were exposed to bisphenol A 

[purity not indicated] at 0, 100, 300, or 600 mM [0, 23, 68, 

or 137 mg/L] for 16 hr. In each study, 2–12 samples/group 

were analyzed. [For most studies, very few details were 

provided about procedures such as exposure 

Birth Defects Research (Part B) 83:157–395, 2008
356 CHAPIN ET AL. 

concentrations used and time that cells were incubated. 

There was no discussion of statistical procedures, and it 

was not clear if statistical analyses were conducted for 

some endpoints.] 

Bisphenol A inhibited calcium ATPase activity in rat 

testis microsomes. Mean7SEM median inhibitory concentration (IC50) values were reported at 0.4070.15 mM 

[91734 lg/L] for inhibition of calcium ATPase activity 

and 2.571.0 mM [5717228 mg/L] for calcium uptake. 

Exposure to 200 mM [47 mg/L] bisphenol A increased 

intracellular calcium levels in TM4 cells. A viability study 

was conducted to determine if increased intracellular 

calcium levels resulted in cell death. Bisphenol A 

exposure resulted in reduced TM4 cell viability (percent 

viability compared to control cells was 93, 64, and 17% at 

concentrations of 100, 300, and 600 mM). The study 

authors concluded that these results provide evidence 

that environmental estrogens may induce toxicity in male 

reproductive development by disrupting calcium 

homeostasis. 

Strengths/Weaknesses: This interesting mechanistic 

study examined the role of bisphenol A in modulating 

intracellular calcium levels. It is difficult to interpret the 

relationship between microsomal and intact cell effects of 

bisphenol A given the large difference in concentrations 

needed to produce an effect. Moreover, it is not clear if 

bisphenol A caused cytotoxicity by a calcium-dependent 

or non-calcium-mediated process. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study was not considered useful for the evaluation 

process. 

Tabuchi et al. (2002), supported by the Japanese 

Ministry of Education, Culture, Sports, Science, and 

Technology and Takeda Science Foundation, examined 

the effects of bisphenol A exposure on viability and gene 

expression in TTE3 cells, a mouse Sertoli cell line. The 

cells were incubated for 24 hr in media containing 0 or 

24–400 mM [5.5–91 mg/L] bisphenol A (99.7% purity) in a 

DMSO vehicle [culture ware not indicated]. Cell 

viability was determined, and gene expression changes 

were examined using microarray and PCR techniques. 

Data were analyzed by Dunnett multiple conversion test 

or Student t-test. Compared to values in control cells, 

bisphenol A exposure reduced cell viability by 25% at 

100 mM [23 mg/L], 33% at 200 mM [46 mg/L], and 96% at 

400 mM [91 mg/L]. Based on the results of the cell-viability 

studies, a bisphenol A concentration of 200 mM [46 mg/L] 

was selected for the gene expression studies. Of 1081 

genes examined by microarray, mRNA was downregulated in 3 cases and upregulated in 10 cases. Six genes 

were selected for evaluation of mRNA expression by 

PCR, and of those genes, 1 was downregulated (ERa) and 

5 were upregulated (iNOS, chop-10, odc, BipGRP78, and 

osip). The study authors concluded that microarray 

analysis is a useful tool for investing molecular mechanisms of bisphenol A-induced toxicity in testicular cells. 

Strengths/Weaknesses: This interesting mechanistic 

study appears to have been well conducted, but it is 

unclear from the data if bisphenol A-related changes in 

chop-10 are a primary (or secondary) effect or are the 

result of cytotoxicity. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is not useful in the evaluation. 

Tabuchi and Kondo (2003), supported by Japanese 

Ministry of Education, Culture, Sports, Science, and 

Technology, Takeda Science Foundation, and Toyama 

Daiichi Bank Foundation, conducted a series of experiments to examine the effects of in vitro bisphenol A 

exposure on gene expression in mouse Sertoli cells. The 

experiments used TTE3 cells, an immortalized Sertoli cell 

line established from transgenic mice expressing temperature-sensitive simian virus large T-antigen. Cells 

were exposed to bisphenol A (99.7% purity) in a DMSO 

vehicle [culture ware not discussed]. The majority of 

experiments were repeated 2–4 times, and data were 

analyzed by Student t-test. [Statistical significance was 

not reported in the results section of the study.] 

Before conducting gene expression studies, cells were 

exposed to 25–400 mM [5.7–91 mg/L] bisphenol A for 3– 

24 hr, and viability was determined using a tetrazolium 

compound. Cell viability was reduced at bisphenol A 

concentrationsZ200 mM [46 mg/L], and reductions in 

viability were increased with longer durations of 

exposure. Intracellular calcium levels were measured 

using a fluorescence imaging technique over a 15-min 

period in cells exposed to 0–400 mM [0–91 mg/L] bisphenol A, and a dose-related increase in calcium influx 

was observed at Z100 mM [23 mg/L]. Based on results for 

cell viability and calcium influx studies, a concentration 

of 200 mM [46 mg/L] was selected for the gene-expression 

experiments. 

Using a PCR technique, it was determined that 

expression of mRNA for transferrin was decreased and 

glucose-regulated protein mRNA was increased by 

bisphenol A exposure of up to 24 hr. Observations of 

increased intracellular calcium concentration and upregulated glucose-regulated protein mRNA expression led 

the study authors to conclude that bisphenol A stresses 

the endoplasmic reticulum. Gene expression was analyzed by a cDNA microarray technique after exposure for 

3, 6, 12, and 24 hr, and it was determined that 31 of 865 

genes examined were upregulated by exposure to 

bisphenol A; no downregulation of genes was observed. 

The greatest change in gene expression was observed for 

chop-10, a stress-response gene. Upregulation of 4 genes, 

c-myc, fra-2, odc, and chop-10, were confirmed by 

quantitative PCR. Chop-10 was determined to be the 

most responsive gene. To determine if chop-10 was 

required for development of endoplasmic reticulum 

stress and cell injury, a stably transfected cell line 

expressing chop-10 antisense RNA (chopR14) was developed. Mock cells were used as negative controls in 

studies where cells were exposed to 200 mM [46 mg/L] 

bisphenol A for up to 24 hr. Production of chop-10 

protein, as determined by Western blot analysis, was 

reduced in the chopR14 cells compared to the mock cells 

following exposure to bisphenol A. In contrast to the 

mock cells, no reductions in cell viability or transferrin 

mRNA expression were observed in the chopR14 cells 

following bisphenol A exposure. There were no changes 

in glucose-regulated protein mRNA expression in 

chopR14 versus mock cells. The study authors postulated 

that bisphenol A may disrupt the male reproductive 

system by altering calcium homeostasis in Sertoli cell 

endoplasmic reticulum without interacting with the ER 

and that genes such as chop-10 may be involved in the 

process. 

Strengths/Weaknesses: This mechanistic study appears to have been well conducted, but it is unclear 

from the data if bisphenol A-related changes in chop-10

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 357 

are a primary (or secondary) effect or are the result of 

cytotoxicity. Calcium levels were also affected and 

collectively these changes may be the result of apoptosis 

initiated by some other mechanism. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study was not considered useful for the evaluation 

process. 

Tabuchi et al. (2006), supported in part by the Japanese 

Ministry of Education, Culture, Sports, Science, and 

Technology, examined the effects of bisphenol A on gene 

expression in mouse Sertoli cell cultures. TTE3 cells were 

incubated in media containing bisphenol A [purity not 

reported] at 0 (DMSO vehicle) or 200 mM [46 mg/L] for up 

to 12 hr [culture ware type not discussed]. Cells were 

examined for viability using dye exclusion assays and for 

apoptosis by formation of DNA ladders. RNA was 

extracted from cells, and gene expression was determined by PCR and microarray analyses. Data were 

analyzed by Student t-test. Cell viability was decreased 

in a time-related manner between 3–12 hr of bisphenol A 

exposure, but there was no evidence of apoptosis. PCR 

analysis indicated that bisphenol A exposure significantly and time-dependently increased mRNA transcripts for 2 endoplasmic reticulum stress markers, 

hspa5 and ddit3. Microarray analysis demonstrated that 

661 sets of genes were downregulated and 604 sets of 

genes were upregulated 42-fold following bisphenol A 

exposure. Pathway analysis of decreased gene clusters 

revealed 2 significant genetic networks associated with 

the cell cycle or cell growth and proliferation. In 

increased gene clusters, two genetic networks were 

associated with cell death, DNA replication, recombination and repair, or injuries and abnormalities. The study 

authors concluded that the genes, genetic clusters, and 

genetic networks identified in this study are likely 

involved in Sertoli cell injury following bisphenol A 

exposure. 

Strengths/Weaknesses: State-of-the-art technology 

was used in this study to examine gene expression 

changes after in vitro bisphenol A exposure of a Sertoli 

cell line. Only one dose level was examined. The use of 

hormone rich fetal bovine serum in the media may be a 

confounder. The absence of DNA laddering is not 

conclusive evidence of the absence of apoptosis (e.g., 

adherent cells undergoing apoptosis often are released 

into the culture media). Moreover, it is not surprising that 

given this ‘‘high’’ bisphenol A concentration, ‘‘novel’’ 

and likely non-specific gene changes were noted. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study was not considered useful for the evaluation 

process. 

4.2.3 Male and female. 

4.2.3.1 Rat: Two unpublished studies performed by 

the International Research and Development Corporation for General Electric (General Electric, 1976, 1978) 

provided some information on reproductive toxicity in 

rats orally exposed to bisphenol A. The studies are 

described in detail in Section 3.2.3.1. There was no effect 

on fertility in male and female rats given feed containing 

up to 9000 ppm bisphenol A (B650 mg/kg bw/day in 

males and 950 mg/kg bw/day in females) for an 

unspecified period before mating (General Electric, 

1976). A second study reported no effects on estrus 

cyclicity or gestation length [data not shown by study 

authors] or male or female fertility in rats given feed 

containing bisphenol A at up to 1000 ppm (B60 mg/kg 

bw/day in males and 100 mg/kg bw/day in females) for 

B70 days before mating (General Electric, 1978). 

Ema et al. (2001), supported by the Japanese Ministry 

of Health and Welfare, conducted a multigeneration 

reproductive toxicity study of bisphenol A in CD rats. 

Animals were housed in suspended stainless steel cages 

at the beginning of the study. From GD 17, wood chips 

were used as bedding. Rats were fed CRF-1 chow 

(Oriental Yeast Co). In the study that was conducted 

according to GLP, F0 male rats and female rats with 4–5­

day estrous cycles were randomly assigned to groups of 

25/sex. Five-week-old males and 10-week-old females 

were gavaged with 0 (distilled water vehicle), 0.0002, 

0.002, 0.020, or 0.200 mg/kg bw/day bisphenol A (99.9% 

purity). Males were dosed for 10 weeks before mating and 

during the mating period, which lasted up to 2 weeks. 

Females were dosed from 2 weeks before mating, and 

during the mating, gestation, and lactation periods. Doses 

were based on results of studies by Nagel et al. (1997) and 

vom Saal et al. (1998). Stability and concentration of dosing 

solutions were verified. Dams delivered and nursed their 

pups. At weaning on PND 22 (day of birth defined as 

PND 0), 1 or 2 F1 weanlings/litter/sex (25/sex/group) 

were selected to continue in the study. Dosing of F1 

animals began on PND 23 and continued for 10 weeks 

before mating and through the mating period, which 

lasted up to 3 weeks. Dosing was continued through the 

gestation and lactation periods. Twenty-five F2 weanlings/sex/group were selected on PND 22. Beginning on 

PND 22, male F2 rats were dosed for 4 weeks and females 

were dosed for 11 weeks before being killed. 

Endpoints examined in adult rats included clinical 

signs, body weight, and food intake. Fertility, copulation, 

and gestational indices were examined in mating rats. 

Vaginal smears were evaluated for 2 weeks before mating 

in F0 and F1 females and at 9–11 weeks of age in F2 

females. Dams were killed and necropsied following 

weaning of their pups, and uterine implantation sites 

were examined. Males were killed following mating. 

Organs were weighed and histopathology examinations 

were conducted in control and high-dose animals. Sperm 

endpoints were measured in F0 and F1 adult males. 

Serum hormone levels were measured in 6 adult F0 and 

F1 males and proestrous females. At birth, pups were 

counted, sexed, and examined for viability and external 

malformations. On PND 4, litters were culled to 4 male 

and 4 female pups. At weaning, 1 male and female F1 

and F2 weanling was killed for organ weight measurement; histopathology exams were conducted in seminal 

vesicles and coagulating glands of F2 weanlings. Survival 

and growth were monitored during the postnatal period. 

Pups were examined for developmental landmarks and 

attainment of vaginal opening or preputial separation. 

Anogenital distance in pups was examined at numerous 

time points during the lactation period and through 

adulthood. Behavioral testing was conducted at 5–7 

weeks of age. The litter was considered the experimental 

unit in data obtained before weaning. Statistical analyses 

included Bartlett test for homogeneity of variance, 

ANOVA, and/or Dunnett multiple comparison, Kruskal–Wallis, Mann–Whitney U, w2

, or Fisher exact tests. 

In F0 and F1 adult animals, there were no treatmentrelated effects on clinical signs, body weight gain, or death. 

The only significant reproductive effects reported in adult 

Birth Defects Research (Part B) 83:157–395, 2008
358 CHAPIN ET AL. 

animals were non-dose-related decreases in percentages of 

females with normal estrous cycles (76 vs. 96% in controls) 

and reduced gestation duration (by 0.5 days) in the F1 

group treated with 0.020 mg/kg bw/day. Bisphenol A did 

not significantly affect the precoital interval, copulation 

index, fertility index, gestation index, number of implantations, or delivery index. There were no adverse effects on 

sperm endpoints such as count, motility, or morphology in 

F0 or F1 males. A significant decrease in abnormal and 

tailless sperm was observed in F1 males of the 0.020 mg/kg 

bw/day group. There was no evidence of histopathological effects in reproductive organs of F0 animals that did not 

copulate or had totally resorbed litters or in F1 animals of 

the high-dose group. [Data were not shown by study 

authors.] In F0 females, there were significant decreases in 

serum LH concentrations at 0.0002, 0.002, and 0.020 mg/kg 

bw/day and in serum triiodothyronine levels at 0.200 mg/ 

kg bw/day. [Data were not shown by study authors.] 

Organ weight changes in F1 adult males included 

decreased absolute weights of lung at 0.0002 and 

0.200 mg/kg bw/day, kidney at 0.2 mg/kg bw/day, and 

testis at 0.020 mg/kg bw/day. Absolute ovarian weight 

was decreased in females of the 0.0002 mg/kg bw/day 

group. Seminal vesicle weight was decreased in F2 males 

of the 0.200 mg/kg bw/day group. [Data were not shown 

by study authors]. 

There were no significant effects on number of F1 or F2 

pups delivered, sex ratio, or pup survival during the 

lactation period. Body weights of F1 pups in the 

0.020 mg/kg bw/day group were significantly lower 

[by 6–7%] on PND 14 and 21. Testicular descent was 

delayed by 0.7 days in F2 offspring from the 0.020 and 

0.200 mg/kg bw/day groups. There were no significant 

effects on age of pinna detachment, incisor eruption, or 

eye opening. Some significant but non-dose-related 

effects on reflex development were observed. Day of 

mid-air righting reflex was accelerated by 1.2 days in F1 

males and 1.5 days in F1 females of the 0.020 mg/kg bw/ 

day group. In F2 males, negative geotaxis was delayed by 

0.8 days at 0.0002 mg/kg bw/day, 0.5 days at 0.002 mg/ 

kg bw/day, and 0.8 days at 0.020 mg/kg bw/day. 

Bisphenol A treatment did not significantly affect age 

of vaginal opening or preputial separation in F1 or F2 

offspring. Some sporadic and small (within 5% of control 

values) changes in anogenital distance were observed in 

F1 and F2 offspring. In F1 males, decreased anogenital 

distance was observed in the 0.0002 mg/kg bw/day 

group on PND 57 and in the 0.020 mg/kg bw/day group 

on PND 106, 113, and on the day of sacrifice. In F1 

females, anogenital distance was decreased in the 

0.200 mg/kg bw/day group on PND 4 and increased in 

the 0.002 and 0.020 mg/kg bw/day group on PND 7. 

Decreases in anogenital distance of F2 females were 

observed in the 0.020 mg/kg bw/day group on PND 64, 

71, 85, 92, and on the day of sacrifice and in the 

0.200 mg/kg bw/day group on PND 57, 64, and on the 

day of sacrifice. In F1 offspring, there were no significant 

effects on behavior, as determined by open-field testing 

and performance in a T-maze. [Data were not shown by 

study authors.] There was no evidence of histopathological effects in seminal vesicle or coagulating gland of F2 

pups from the high-dose group. [Data were not shown 

by study authors.] Organ weight changes in F1 male 

weanlings included decreased absolute lung weight at 

0.020 and 0.200 mg/kg bw/day group and decreased 

kidney weight at 0.020 mg/kg bw/day. In male F2 

weanlings, significant decreases were observed in absolute and relative seminal vesicle weight and absolute 

thyroid weight at 0.002 mg/kg bw/day, absolute lung 

weight at 0.020 mg/kg bw/day, and relative heart weight 

at 0.200 mg/kg bw/day; relative liver weight was 

significantly increased in F2 males of the 0.002 mg/kg 

bw/day group. The study authors concluded that oral 

administration of bisphenol A at 0.0002 to 0.200 mg/kg 

bw/day to 2 generations of rats did not cause changes in 

reproduction or development. 

[The NTP Statistics Subpanel (NTP, 2001) reviewed 

an unpublished study that appeared to be the same 

study later published as Ema et al. (2001). The subpanel 

noted that in general they agreed with the statistical 

methodology used in the study but stated that the 

Dunnett test does not require significance of ANOVA. 

It was noted that the anogenital distance findings were 

the most difficult to interpret. The Subpanel noted that 

many of the anogenital distance effects remained 

statistically significant when analyzed by ANCOVA, 

a method they considered superior to adjustment by 

body weight. The NTP Subpanel agreed with the 

author’s conclusion that effects on anogenital distance 

were not biologically significant. They noted an error 

in the unpublished study abstract that described 

increases in anogenital distance in F1 and F2 females 

in the 0.020 and 0.2 mg/kg bw/day groups when actually 

the effect should have been decreased anogenital 

distance. [It was not clear to CERHR if this error was 

carried forward to the published report.] 

Strengths Weaknesses: This well-designed comprehensive low-dose assessment of potential bisphenol A-related 

effects on multiple generations of rats examined a wide 

variety of hormonally sensitive endpoints. The study had 

appropriate power with an appropriate number of rats per 

group. Route of administration (oral) was appropriate. 

The concentrations of the dosing solutions were verified 

(both prior and after). It would have been helpful if a dose 

level that caused maternal toxicity was also used; 

however, given the objective of this study it is a minor 

point. This thorough multiple generation rat study is 

highly valuable for human risk assessment of low dose 

oral exposure to bisphenol A. This study indicates that the 

NOAEL for bisphenol A exceeds 0.2 mg/kg bw/day 

under the conditions of this study. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

Tyl et al. (2000a, 2002b), sponsored by The Society of 

the Plastics Industry, Inc., conducted a multigeneration 

study of bisphenol A in rats. In the study that was 

conducted according to GLP, Sprague–Dawley rats were 

fed Purina Certified Rodent Chow 5002. F0 rats (30/sex/ 

group) were exposed to bisphenol A (99.5% purity) in 

feed for 10 weeks before mating. [Age at start of 

exposure was not reported, but based on information 

provided in the discussion, it appears that the animals 

were adults at the start of exposure.] Vaginal smears 

were evaluated during the last 3 weeks of the prebreeding period. Exposure continued through a 2-week mating 

period. Males were exposed an additional 3 weeks 

following mating, and females were exposed through 

gestation and lactation. Concentrations of bisphenol A 

added to feed were 0, 0.015, 0.3, 4.5, 75, 750, or 7500 ppm. 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 359 

Target intakes were B0, 0.0009, 0.018, 0.27, 4.5, 45, and 

450 mg/kg bw/day in males and 0.001, 0.02, 0.30, 5, 50, 

and 500 mg/kg bw/day in females. Actual intakes were 

0.0007–0.003, 0.015–0.062, 0.22–0.73, 4.1–15.4, 37.6–167.2, 

and 434–1823 mg/kg bw/day. The study was designed to 

include low-dose exposures reported to increase prostate 

weights (Nagel et al., 1997; vom Saal and Sheehan, 1998) 

and maximally tolerated doses expected to result in 

toxicity. Concentration, stability, and homogeneity of 

bisphenol A in feed were verified. During the study, 

body weight and food intake were measured and 

animals were examined for clinical signs. F0 males were 

killed and necropsied following delivery of the F1 litter. 

Histopathological evaluation of organs was conducted in 

all control animals and 10 animals/bisphenol A dose 

group. Reproductive organs were weighed and sperm 

endpoints were evaluated. F0 females were killed and 

necropsied following weaning of their litters. Selected 

organs were weighed and ovarian primordial follicles 

were counted. 

On PND 4, F1 litters were culled to 10 pups, with equal 

numbers of each sex when possible. Endpoints examined 

in pups included growth and survival in the prenatal 

period and retained areolae or nipples on PND 11–13. At 

weaning on PND 21, 30 F1 offspring/sex/group were 

randomly selected and exposed to bisphenol A in the 

diet according to the same protocol as F0 rats. Those 

selected offspring were monitored for vaginal opening 

and preputial separation and later mated. Up to 3 F1 

weanlings/sex/litter were killed for organ weight 

measurement. Mating and evaluation of F1 offspring 

were conducted according to the same procedures 

described for F0 rats. The same procedures were repeated 

in F2 rats and F3 litters during the lactation period. 

Anogenital distance was measured in F2 and F3 rats at 

birth. Following weaning of F3 offspring, up to 3/sex/ 

litter were randomly selected for necropsy. Thirty/sex/ 

dose were selected for evaluation of vaginal patency, 

preputial separation, and estrous cyclicity. Bisphenol A 

exposure was continued in those offspring until they 

were killed B10 weeks following weaning. F3 offspring 

were not mated, but necropsy evaluations were conducted as described above for previous generations. 

Statistical analyses for quantitative continuous data 

included Bartlett test for homogeneity of variances, 

ANOVA, Dunnett, linear trend, Kruskal–Wallis, or 

Mann–Whitney U tests. Frequency data were analyzed 

by w2

, Fisher exact, and Cochran–Armitage tests. Covariance and correlations analyses were also conducted. 

Treatment-related systemic findings with available 

quantitative information in adult rats are summarized 

in Table 93. Body weights and body weight gain were 

consistently lower in F0, F1, F2, and F3 adult rats of the 

750 and 7500 ppm dose groups, including during gestation and lactation periods. Terminal body weight effects 

are summarized in Table 93. Terminal body weight was 

reduced in all generations at 7500 ppm and in F1 females 

and F1 and F2 males at 750 ppm. There were no consistent 

or clearly treatment-related effects on feed intake. No 

treatment-related clinical signs were reported. In the 

7500 ppm group, absolute weights of the liver in males 

and the kidney in both sexes were decreased across 

generations. Relative weights were either increased or 

did not attain statistical significance. [According to 

Table 2 of the study, absolute liver weights were also 

decreased in males of the 750 ppm group. The study 

authors also mentioned reductions in weights of 

adrenal glands, spleen, pituitary, and brain at the 

high-dose, but there were no data shown in the report 

for those endpoints.] Other changes in non-reproductive 

organ weight occurred sporadically at lower dose and 

were not dose-related or consistent across generations. 

Relative organ weight changes that consistently attained 

statistical significance at the highest dose are summarized in Table 93. Histopathological analyses revealed a 

higher incidence of mild renal tubular degeneration and 

chronic hepatic inflammation in F0, F1, and F2 but not F3 

females of the 7500 ppm group. 

Treatment-related effects on reproductive endpoints in 

adult animals are summarized in Table 93. In evaluating 

organ weights, the study authors only considered organ 

weight effects to be biologically significant if statistically 

significant results were obtained in the same direction for 

absolute and relative weights. Therefore, the study 

authors concluded that the only treatment-related organ 

weight effects were reduced absolute and relative ovary 

weights. [Numerous statistically significant effects on 

reproductive organ weights were reported in Table 2 of 

the study. Reductions in testes, epididymides, prostate, 

and seminal vesicle weights were observed in most 

generations of the 7500 ppm group. When adjusted for 

body weight, organ weights were either increased or 

did not differ significantly from controls.] Relative 

reproductive organ weight changes that consistently 

attained statistical significance at the highest dose are 

summarized in Table 93. The authors reported no effect 

on mating, fertility, pregnancy, or gestational indices. 

[With the exception of gestational length, data were not 

shown by study authors.] Precoital interval, postimplantation loss, estrous cyclicity, and reproductive 

organ histopathology were also unaffected by bisphenol 

A treatment. In the high-dose group, there was no 

adverse effect on paired ovarian primordial follicle 

counts but counts were significantly increased by 43% 

in the F0 generation. Implantation sites were decreased in 

F0, F1, and F2 dams of the 7500 ppm group. The only 

significant effects on sperm endpoints were decreased 

epididymal sperm concentration in F1 males and 

decreased daily sperm production in F3 males of the 

7500 ppm dose group. There were no effects on sperm 

morphology or motility. The study authors considered 

sperm to be unaffected by treatment. 

Treatment-related effects observed in developing rats 

are summarized in Table 94. The number of live pups/ 

litter was reduced in F1, F2, and F3 litters of the 7500 ppm 

group. Body weights of F1, F2, and F3 pups of the 

7500 mg/kg bw/day groups were lower during the 

lactation period. Some small (B5%) decreases in pup 

body weight during the lactation period at lower 

doses were apparently not considered treatment-related 

by study authors. Postnatal survival was unaffected by 

bisphenol A treatment. In male rats, there were no 

effects on anogenital distance or the presence of 

areolas or nipples. Anogenital distance was significantly 

increased in F2 females at all doses except 75 and 

7500 ppm; there was no affect on anogenital distance in 

F3 females. The study authors did not consider 

anogenital distance effects to be biologically or toxicologically significant. Vaginal patency was delayed in F1, F2, 

and F3 females, and the effect remained significant 

Birth Defects Research (Part B) 83:157–395, 2008
360 CHAPIN ET AL. 

following adjustment for body weight. Preputial separation was delayed in F1 males of the 750 and 7500 ppm 

groups, F2 males in the 0.3, 75, 750, and 7500 ppm groups, 

and F3 males of the 7500 ppm group. When adjusted for 

body weight, the effect remained significant in F1 males 

of the 750 and 7500 ppm groups and F2 and F3 males of 

the 7500 ppm group. The study authors stated that 

reduced body weights were the most likely cause of 

puberty delay in males and females. [In rats killed at 

weanling, absolute organ weights were said to be 

decreased at the high-dose but increased when adjusted for body weight. The specific organs affected 

were not reported and no data were presented. The 

exception was ovarian weights, which were reported to 

parallel effects observed in adult females with decreases in both absolute and relative weight at 

7500 ppm.] 

The study authors concluded that there was no 

evidence of low-dose bisphenol effects (1 mg to 5 mg/kg 

bw/day) at any stage of the life cycle. They identified 

NOAELs of 75 ppm (B5 mg/kg bw/day) for adult 

systemic toxicity and 750 ppm (B50 mg/kg bw/day) 

for offspring and reproductive effects. The study authors 

concluded that bisphenol A should not be considered a 

selective reproductive toxicant. 

[The NTP Statistics Subpanel (NTP, 2001) stated 

that the study by Tyl et al. (2000a) apparently lacked a 

check for outliers, but noted that the study was in draft 

form at the time of review. The NTP subpanel agreed 

with most author conclusions but disagreed with a 

conclusion that relative uterine weights were equivalent 

across all groups. The unnecessary use of ANOVA with 

Dunnett test was noted. Some possible outliers and 10­

fold errors in data points that could have affected 

conclusions were observed. Overall, the NTP Subpanel 

concluded that Tyl et al. (2000a) study was the most 

comprehensive of the studies reviewed. They stated that 

the statistical methods were well thought out and 

appropriate.] 

Strengths/Weaknesses: This assessment of potential 

bisphenol A-related effects on multiple generations of 

rats was well-designed and comprehensive. The large 

number of rats/group (30), the multiple endpoints 

examined, and the oral route of administration (diet) 

are strengths. The concentration of bisphenol A in the 

test diet was verified, and maternal and paternal toxicity 

was identified. This study explored a wide dose range 

and demonstrates an absence of adverse effects on 

reproductive function at very low bisphenol A dose 

levels. This study is highly valuable for human risk 

assessment for oral exposure to bisphenol A. 

This study identified a NOAEL of 75 ppm (for general 

toxicity) and 750 ppm (for reproductive toxicity). 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

4.2.3.2 Mouse: NTP (1984) and Morrissey et al. 

(1989) sponsored a continuous breeding study in CD-1 

mice exposed to bisphenol A through s.c. implants. Mice 

were fed Purina certified ground rodent chow (#5002) 

and housed in polypropylene or polycarbonate cages 

containing Ab-Sorb-Dri bedding. Silastic implants were 

used for s.c. dosing of mice with bisphenol A (B95% 

purity) in corn oil vehicle. Stability and weight of 

bisphenol A in pumps was verified. In the dose-range 

finding portion of the study (Task 1), 8 mice/sex/group 

(8 weeks old) received implants containing vehicle or 

bisphenol A. Dosages were estimated by determining the 

difference in bisphenol A weight at the start and 

end of the 14-day dosing period. It was estimated 

that mice received 0, 6.25, 12.5, 25, 50, or 100 mg 

bisphenol A. Endpoints examined included body weight 

changes, survival, and uterine weight. Blood was 

collected to determine plasma bisphenol A levels. 

Data were analyzed by ANOVA, Duncan Multiple 

Range Test, w2 test, and Fisher exact test. The goal of 

Task 2 was to determine a maximum tolerated dose that 

produced signs of toxicity but did not reduce 

body weight or increase lethality by 410% and to 

identify a low dose that did not result in toxicity. 

Concentrations of bisphenol A in plasma were below 

the detection limit (3 ng/mL) in the 6.25 mg group but 

were reported at 7.0–7.7 mg/L in the 12.5 mg group, 

8.4 mg/L in the 25 mg group, 13.1–18.5 mg/L in the 50 mg 

group, and 31.5–56.2 mg/L in the 100 mg group. In mice 

treated with bisphenol A, there were no increases in 

death or effects on body weight gain. The study authors 

noted that reproductive tract weight in the high-dose 

group was greater [by 52%] than in the control group but 

statistical significance was not achieved because of high 

variability. 

In the continuous breeding portion of the study (Task 

2), mice were 11 weeks old at the start of dosing. Forty 

mice/sex/group received implants containing the vehicle and 20/sex/dose received implants containing bisphenol A at 25, 50, or 100 mg. Over a dosing period of 18 

weeks, it was estimated that animals in each treatment 

group received 11.65, 20.05, and 38.60 mg bisphenol A. 

[Assuming body weights of B38 g, as indicated in the 

study report, doses would have been B306, 527, and 

1015 mg/kg bw over 18 weeks or 2.4, 4.2, and 8.1 mg/kg 

bw/day.] Mice were 11 weeks old at the start of dosing, 

which began during a 7-day premating period. The mice 

were then randomly paired with animals from the same 

dose group and housed together during a 98-day 

breeding period. Litters born during the breeding period 

were examined for viability, weighed, sexed, and 

discarded. Following the 98-day mating period, mice 

were separated for 21 days to allow for the birth of the 

last litter. Dosing was continued throughout the breeding 

and separation periods. However, implants were often 

expelled through cutaneous lesions or the incision site. 

When animals expelled their implant, a new one was 

inserted but pregnant mice were allowed to complete 

their pregnancy before insertion of the new implant. 

Therefore dosing was not uniform. Endpoints examined 

in adult mice included body weight, number of litters/ 

pair, and fertility. Following delivery of the final litter, 

parental animals were killed and animals in the 0 and 

100 mg group were necropsied. Liver, brain, and reproductive organs were weighed. Data were analyzed by w2 

test, Fisher exact test, Kruskal–Wallis test, Jonckheere 

test, and Mann–Whitney U test. 

With the exception of cutaneous lesions at the 

implantation site, there were no clinical signs of toxicity. 

In parental mice, there were no effects on body weight, 

mortality, fertility, or number of litters born. There were 

no changes in weights of organs including, liver brain, 

pituitary, the female reproductive tract, testis, 

epididymis, prostate, or seminal vesicles. Statistically 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 361 

Table 93
 

Treatment-Related Effects in Adult Rats Fed Bisphenol A Through Diet in a Multigeneration
 

Reproductive Toxicity Studya
 

Dose, ppm diet [mg/kg bw/dayb] 

0.015 0.3 4.5 75 750 7500 

Endpoint [0.0095] [0.019] [0.285] [4.75] [47.5] [475] BMD10 BMDL10 BMD1SD BMDL1SD 

Terminal body weight 

F0 males 2 2 2 2 2 k 22% 3554 [225] 3137 [199] 3133 [198] 2701 [171] 

F1 males 2 2 2 2 k 6% k 26% 2811 [178] 2548 [161] 2443 [155] 2153 [136] 

F2 malesc 2 2 2 2 k 12% k 29% 733 [46] 554 [35] 648 [41] 484 [31] 

F3 malesc 2 2 2 2 2 k 26% 1456 [92] 913 [58] 1260 [80] 786 [50] 

F0 females 2 2 2 2 2 k 13% 5722 [362] 4753 [301] 4741 [300] 3876 [245] 

F1 females 2 2 2 2 k 6% k 16% 4600 [291] 3950 [250] 3730 [236] 3142 [199] 

F2 femalesc 2 2 2 2 2 k 14% 3863 [245] 1576 [100] 3115 [197] 1291 [82] 

F3 females 2 2 2 2 2 k 20% 3664 [232] 3194 [202] 3456 [219] 2949 [187] 

Relative paired kidney weight 

F0 males 2 2 2 2 2 m 14% 5903 [374] 4555 [288] 6536 [414] 5035 [319] 

F1 males 2 2 k 5% 2 2 m 10% 5729 [363] 4662 [295] 5053 [320] 4088 [259] 

F2 males 2 2 2 2 m 5% m 18% 4524 [287] 3893 [247] 3471 [220] 2950 [187] 

F3 males 2 2 2 2 2 m 16% 6986 [442] 4319 [274] 6720 [426] 3403 [216] 

F0 females 2 2 2 2 2 m 7% 8008 [507] 7521 [476] 7712 [488] 6578 [417] 

F2 females 2 2 2 2 2 m 6% 7930 [502] 7515 [476] 7621 [483] 6247 [396] 

Relative paired testis weight 

F0 males 2 2 2 2 2 m 27% 2924 [185] 2567 [163] 2998 [190] 2596 [164] 

F1 males 2 2 2 2 2 m 18% 3287 [208] 2763 [175] 4106 [260] 3428 [217] 

F2 males 2 2 2 2 2 m 24% 3086 [195] 2874 [182] 3245 [206] 2779 [176] 

F3 males 2 2 2 2 2 m 19% 4329 [274] 2593 [164] 5010 [317] 3298 [209] 

Relative paired epididymis weight 

F0 males 2 2 2 2 2 m 19% 3804 [241] 3072 [195] 5044 [319] 4068 [258] 

F1 males 2 2 2 2 2 m 19% 2963 [188] 2566 [163] 3255 [206] 2786 [17] 

F2 malesc 2 2 2 2 m 8% m 24% 884 [56] 596 [38] 951 [60] 641 [41] 

F3 males 2 2 2 2 2 m 22% 3449 [218] 2516 [159] 4117 [261] 3095 [196] 

Relative liver weight 

F0 females 2 2 2 2 2 m 11% 7663 [485] 5848 [370] 7965 [504] 7439 [471] 

F2 females m 2 2 2 2 m 19% 6912 [438] 3650 [231] 7454 [472] 5533 [350] 

Relative paired ovary weight] 

F0 females 2 2 2 2 2 k 19% 4103 [260] 3149 [199] 7126 [451] 5387 [341] 

F1 females 2 2 2 2 2 k 15% 5754 [364] 3964 [251] 10,237 [648] 6966 [441] 

F2 females k 15% 2 k 15% k 11% 2 k 24% 7053 [447] 3520 [223] 7646 [484] 6360 [403] 

No. with renal tubule degeneration 

F0 females 0/12 0/12 0/12 0/14 0/12 4/13 6491 [411] 3848 [244] 

F1 females 0/10 0/10 0/10 0/10 0/10 8/11 5498 [348] 2470 [156] 

F2 females 0/11 0/10 0/12 0/11 0/12 7/13 5884 [373] 3018 [191] 

No. females with chronic liver inflammation 

F0 females 0/12 1/12 0/12 0/14 1/12 3/13 4867 [308] 3214 [204] 

F1 females 0/10 0/10 3/10 1/10 1/10 3/11 

F2 females 1/11 0/10 2/12 2/11 2/12 5/13 3029 [192] 1856 [118] 

No. of implantation sites 

F0 dams 2 2 2 2 2 k16% 4088 [259] 3021 [191] 8020 [508] 5832 [369] 

F1 damsc 2 2 2 2 2 k26% 6120 [388] 2383 [151] 7000 [443] 4713 [298] 

F2 dams 2 k8% 2 2 2 k18% 4917 [311] 3597 [228] 7679 [486] 5631 [357] 

Epididymal sperm 2 2 2 2 2 k18% 5012 [317] 3407 [216] 11,050 [700] 7407 [469] 

concentration, F1 

Daily sperm production, F3 2 2 2 2 2 k19% 7399 [469] 4025 [255] 8279 [524] 7596 [481] 

a

Tyl et al. (2002b).
 

b

Based on target doses provided by the study authors and expressed as an average of the dose for males and females.
 

c

Benchmark dose values were estimated using a polynomial model.
 

m,k Statistically significant increase, decrease, 2 no statistically significant effect.
 

significant effects observed in pups included increased that further studies using a better route of exposure are 

numbers of live male and total pups and increased needed for bisphenol A. 

adjusted (for litter size) pup weight in the mid-dose Strengths/Weaknesses: This study appears to have 

group. Unadjusted and adjusted male and female pup been well conducted. When compared to studies that 

weights were significantly increased at the high-dose. used the oral route of exposure, this study provides 

The study authors noted that the effects observed in this evidence that the manifestation of maternal toxicity is 

study were random and most likely due to chance. They dependent on the route of administration and that routeconcluded that bisphenol A did not induce adverse dependent metabolism may be important for toxicity. 

effects on fertility in male or female mice. It was noted However, the administration of bisphenol A via silastic 

Birth Defects Research (Part B) 83:157–395, 2008
362 CHAPIN ET AL. 

Table 94 

Treatment-Related Effects in Developing Rats in a Multigeneration Reproductive Toxicity Study of Bisphenol Aa 

Endpoint 

0.015 

[0.0095] 

Dose, ppm diet [mg/kg bw/dayb

] 

0.3 

[0.019] 

4.5 

[0.285] 

75 

[4.75] 

750 7500 

[47.5] [475] BMD10 BMDL10 BMD1SD BMDL1SD 

Live pups/litter 

F1 2 2 2

F2 2 2 2

F3 2 k 11% 2

Pup body weight 

F1, PND 4 2 2 2

F1, PND 7 2 2 2

F2, PND 7 2 2 2

F3, PND 7 2 2 2

F1, PND 14 2 2 2

F2, PND 14 2 2 2

F3, PND 14 2 2 2

2c F1. PND 21 2 2

2c F2, PND 21 2 2

2c F3, PND 21 2 2

Anogenital distance, F2 females m 3% m 3% m 3% 

Age of vaginal opening adjusted for body weight 

F1 2 2 2

F2 2 2 2

F3 2 2 2

Age of preputial separation adjusted for body weight 

F1 2 2 2

F2 2 2 2

F3 2 2 2

2

2

2

2

2

2

2

2

2

2

2c 

2c 

2c 

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2c 

2c 

2c 

m4% 

2

2

2

m 1.7 days 

2

2

k 20% 

k 26% 

k 26% 

k 11% 

k 23% 

k 15% 

k 13% 

k 27% 

k 20% 

k 20% 

k 27% 

k 20% 

k 19% 

2

m 3.6 days 

m 4 days 

m 3.2 days 

m 4.9 days 

m 7.4 days 

m 4 days 

4232 

6661 

3733 

6412 

3432 

5179 

4976 

2890 

3840 

3704 

3284 

4253 

3972 

6225 

6381 

7444 

7350 

4740 

8637 

[268] 

[422] 

[236] 

[406] 

[217] 

[328] 

[315] 

[183] 

[243] 

[235] 

[208] 

[269] 

[252] 

[394] 

[404] 

[471] 

[466] 

[300] 

[547] 

3033 

2405 

2742 

4473 

2891 

4059 

3854 

2570 

3302 

3224 

2621 

3566 

3423 

5422 

5307 

6325 

6485 

4025 

7466 

[192] 

[152] 

[174] 

[283] 

[183] 

[257] 

[244] 

[163] 

[209] 

[204] 

[166] 

[226] 

[217] 

[343] 

[336] 

[401] 

[411] 

[255] 

[473] 

8823 

7241 

5943 

8860 

4179 

6023 

6474 

2789 

3579 

3323 

3523 

4219 

3575 

3248 

4367 

6249 

2974 

3809 

3503 

[559] 

[459] 

[376] 

[561] 

[265] 

[381] 

[410] 

[177] 

[227] 

[210] 

[223] 

[267] 

[226] 

[206] 

[277] 

[396] 

[188] 

[241] 

[222] 

6225 

4645 

4518 

6317 

3448 

4653 

4940 

2415 

3013 

2827 

2763 

3473 

3016 

2786 

3600 

3198 

2580 

3201 

2984 

[394] 

[294] 

[286] 

[400] 

[218] 

[295] 

[313] 

[153] 

[191] 

[179] 

[175]

[220]

[191]

[176] 

[228] 

[203] 

[163] 

[203] 

[189] 

a

Tyl et al. (2002b).
 

b

Based on target doses provided by the study authors and expressed as an average of the dose for males and females.
 

c

A significant (B5%) decrease in pup body weights observed only in F1 and/or F2 litters was apparently not considered treatment-


related by study authors.
 

m,k Statistically significant increase, decrease, 2 no statistically significant effect.
 

implants makes the extrapolation for human risk assessment difficult in the absence of an improved pharmacokinetic understanding. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate but of limited utility for the 

evaluation process. 

NTP (1985a) and Morrissey et al. (1989) sponsored a 

continuous breeding study in CD-1 mice exposed to 

bisphenol A (98% purity). Additional information on 

ovarian follicle counts in F0 and F1 females was 

published in a report by Bolon et al. (1997). In this study, 

mice were fed NIH-07 open formula rodent chow and 

housed in polypropylene or polycarbonate cages containing Ab-Sorb-Dri litter. The laboratory at which the study 

was conducted was stated to be in full compliance with 

GLP regulations. In the preliminary study (Task 1), 8 

mice/sex/group (8 weeks old) were fed diet containing 

bisphenol A at 0, 0.3125, 0.625, 1.25, 2.5, or 5.0% for 14 

days. By assuming that a 40-g mouse ingests 7 g feed/ 

day, the study authors estimated bisphenol intake at 0, 

437.5, 875.0, 1750.0, 4375.0, 8750.0 mg/kg bw/day. The 

aim of the preliminary study was to determine a 

maximum tolerated dose that induced significant toxicity 

but resulted in Z90% survival and r10% decrease in 

weight gain. Statistical analyses included ANOVA, and 

w2 test. Lethality was significantly increased in the highdose group. Body weight gain was depressed in groups 

exposed to Z1.25% bisphenol A. Clinical signs of toxicity 

were observed in the 2.5 and 5.0% dose groups and 

included dehydration, dyspnea, lethargy, tremors, ptosis, 

piloerection, and diarrhea. 

In the reproduction and fertility study (Task 2), 11­

week-old mice were randomly assigned to treatment 

groups according to body weight. The mice were fed 

diets containing 0, 0.25, 0.5, or 1.0% bisphenol A. The 

NTP stated that a 40-g mouse consuming 7 g of feed/day 

would be exposed to bisphenol A at 437.5, 875, and 

1750 mg/kg bw/day. [Based on body weight and feed 

intake values reported for males at B3 week intervals, 

CERHR estimated mean bisphenol A intake at B365, 

740, and 1630 mg/kg bw/day. Feed intakes were 

reported only at Week 1 and 18 for females, and Week 

18 most likely represented the lactation period. For 

Week 1, bisphenol A intake by females was estimated 

at 410, 890, and 1750 mg/kg bw/day. At Week 18, 

bisphenol A intake by females was estimated at 1090, 

1785, and 3660 mg/kg bw/day.] There were 40 mice/sex 

in the vehicle control group and 20/sex in each bisphenol 

A group. Exposure to bisphenol A began during a 7-day 

premating period. Following the premating period, 

males and females from the same treatment group were 

randomly paired and housed together for 98 days and 

following the mating period, each male and female was 

housed separately for 21 days. Bisphenol A dosing was 

continued throughout the mating and separation period. 

Concentration and stability of bisphenol A in feed were 

verified. During the 98-day cohabitation period, pups 

born were counted, sexed, and weighed. All litters 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 363 

Table 95 

Effects Observed in Adult Mice Dosed With Bisphenol A in a Continuous Breeding Studya 

Dose, % in diet [mg/kg bw/day] 

Endpoint 

0.25 [437.5] 0.5 [875] 1.0 [1750] BMD10 BMDL10 BMD1SD BMDL1SD 

F0 males and females 

Litters/pair 2 k 5% k 9% 1.0 [1750] 0.74 [1295] 0.96 [1680] 0.66 [1155] 

Postpartum dam weightb 2 2 k 6–9% 1.0 [1750] 0.83 [1452] 0.87 [1522] 0.66 [1155] 

Necropsy dam weight No data No data k 4% 

Percent motile sperm No data No data k39% 

Relative organ weight, malesc 

Liver No data No data m 29% 

Kidney/adrenal No data No data m 16% 

Seminal vesicle No data No data k 19% 

Relative organ weight, femalesc 

Liver No data No data m 27% 

Kidney/adrenal No data No data m 10% 

Liver lesions, males and femalesd No data No data me 

Kidney lesions, males and femalesd No data No data me 

F1 males and females 

Relative organ weight, malesc 

Liver m 7% m 7% m 29% 0.62 [1085] 0.42 [735] 0.59 [1032] 0.39 [682] 

Kidney/adrenalf m 16% m 20% m 20% 0.18 [315] 0.14 [245] 0.15 [262] 0.12 [210] 

Left testis/epididymisf 2 k 10% k 9% 0.64 [1120] 0.32 [560] 0.53 [928] 0.27 [472] 

Right testisg 2 k 13% 2

Right epididymisf k 11% k 16% k 18% 0.24 [420] 0.15 [262] 0.46 [805] 0.25 [438] 

Seminal vesicle k 11% 2 k 28% 0.40 [700] 0.29 [508] 0.66 [1155] 0.47 [822] 

Relative organ weight, femalesc 

Liver m 6% m 13% m 20% 0.49 [858] 0.38 [665] 0.45 [788] 0.35 [612] 

Kidney/adrenalg m 13% m 15% m 13% 

Percent motile spermg 2 k 31% 2

Liver lesions, malesd me me me 

Liver lesions, femalesd 2 me me 

Kidney lesions, males and females me me me 

a

NTP (1985a).
 

b

Values were reported following the birth of 5 litters, the benchmark doses are for values reported following the birth of the fifth litter
 

because the greatest magnitude of effect was observed at that time point.
 

c

Relative organ weights were adjusted for body weight; when absolute and relative organ weights changed in the same direction, only
 

the relative organ weights were listed in this table.
 

d

See text for a description of the types of lesions observed.
 

e

It does not appear that statistical analyses were conducted for histopathology data, but incidence was increased compared to controls.
 

f

Benchmark doses were estimated using a polynomial model.
 

g

Benchmark doses were not estimated for endpoints without dose-response relationships.
 

m,k Statistically significant increase, decrease compared to controls; 2 no statistically significant effects compared to controls.
 

Table 96
 

Effects in Immature F1 Mice in a Continuous Breeding Study With Bisphenol A
 

Dose, % in diet [mg/kg bw/day] 

Endpoint 0.25 [437.5] 0.5 [875] 1.0 [1750] BMD10 BMDL10 BMD1SD BMDL1SD 

Live pups/litter 

Proportion pups born alive 

Live birth weightb 

Mortality by PND 21c 

2

2

2

2

k 20% 

2

m5% 

2

k 48% 

k 4% 

m 6% 

m to 37.5% 

0.30 [525] 

3.0 [5250] 

0.43 [752] 

0.48 [840] 

0.20 [350] 

0.79 [1382] 

0.40 [700] 

0.43 [752] 

0.34 [595] 

0.30 [525] 

a

NTP (1985a).
 

b

Hill model used for benchmark dose calculations.
 

c

Control mortality was 6.3%. Mortality was reported on a per pup basis, which limits the utility of the benchmark dose model.
 

m,k Statistically significant increase, decrease compared to controls; 2 no statistically significant effects compared to controls.
 

excluding the last one born were killed on the day of parental rats included the number of litters born and 

birth so that animals could continue mating. The last fertility. Statistical analyses included Kruskal–Wallis 

litter was raised by the dam and weaned on PND 21 (day ANOVA on ranks, Mann–Whitney U test, w2 test, oneof birth not defined). Birth weight and weight gain were way ANOVA, arc sine square-root transformation, and 

recorded in the last litter. Reproductive endpoints in Duncan multiple range test. 

Birth Defects Research (Part B) 83:157–395, 2008
364 CHAPIN ET AL. 

Table 97 

Effects Observed in Mice Fed Bisphenol A-Containing Feed for One Generationa 

Endpoint 0.5 (840–1055)b 

Dose, % in diet (mg/kg bw/day) 

1 (1669–1988)b BMD10 BMDL10 BMD1SD BMDL1SD 

F0 females body weights and feed intake 

GD 17 body weightc,d 

PND 0 body weightd 

GD 0–17 body weight changec,d 

Study day 0–7 feed intake 

GD 14–17 feed intake (g/day) 

GD 0–17% food efficiency e Relative (to body weights) organ weights in F0

Liver, male 

Liver, female 

Kidney, female 

Clinical chemistry effects in F0 females, not examined in males 

Blood urea nitrogen 

Sodium 

Potassium 

Chloride 

Histopathology in F0 females (not examined in males)f 

Renal tubule epithelium degeneration (control: 0/20) 

Renal tubule epithelium necrosis (control: 0/20) 

Renal tubule regeneration (control: 2/20) 

Centrilobular hepatocyte hypertrophy (control 0/20) 

Diffuse hepatocyte hypertrophy (control 0/20) 

Reproductive/developmental effects 

Gestational length 

No. of live pups 

Total no. of pups 

Female pup body weight 

k 8% 

2

k 16% 

m11% 

2

k 16% 

m 22% 

m 27% 

m 8% 

2

k 9% 

k 18% 

k 8% 

9/20 

6/20 

12/20 

2/20 

6/20 

m 2% 

2

2

k0.6%g 

k 11% 

k 7% 

k 19% 

2

k 13% 

k 16% 

m 24% 

m 29% 

m 24% 

m 43% 

2

2

2

9/20 

8/20 

20/ 20 

11/20 

6/20 

m 2% 

k 15% 

k 15% 

k4%g 

1292 

2130 

472 

1454 

706 

615 

973 

628 

663 

223 

902 

1116 

1116 

2281 

646 

1675 

283 

898 

561 

484 

529 

266 

480 

151 

612 

727 

727 

1728 

742 

1813 

701 

1840 

705 

746 

1309 

1925 

1925 

2332 

404 

1193 

387 

1172 

555 

586 

863 

1189 

1189 

1733 

a

Tyl et al. (2002a).
 

b

Bisphenol A intakes included values estimated for males and females during prebreeding or gestation; intake values for the appropriate
 

sex were used in benchmark dose analyses; intakes during gestation were used for females.
 

c

The effect was reported at earlier time period but is shown here only for the latest or longest time period evaluated.
 

d

Benchmark doses were estimated using the polynomial model.
 

e

Only effects on relative organ weights were shown.
 

f

Histopathology data were not statistically analyzed.
 

g

By trend test.
 

m,k Statistically significant increase, decrease compared to controls; 2 no statistically significant effect.
 

In the cross-over trial (Task 3), B20 males and females 

from the high-dose group were randomly paired with 

control mice for 7 days in order to determine the affected 

sex. Twenty control males and females were also paired. 

The animals were not exposed to bisphenol A during the 

1-week mating period, but in animals from the high-dose 

group, dosing with bisphenol A was continued for 21 

days on separation of the mating pairs. Vaginal smears 

were obtained from females that did not mate or did not 

appear to be pregnant. Fertility and offspring survival 

were determined. Parental mice from the control (n 5 38/ 

sex) and high-dose groups (n 5 19/sex) were necropsied 

within a week following completion of the cross-over 

trial. Body, liver, kidney, and reproductive organ weights 

were obtained, and sperm count, morphology, and 

motility were determined. Testes, ovaries, and oviducts 

were fixed in Bouin solution and prostate, seminal 

vesicles/coagulating glands, uterus, liver, and kidney 

were fixed in 10% neutral buffered formalin for 

histopathological evaluation. 

In Task 4 of the study, 20 F1 mice/sex/group (at least 

2/sex from 10 randomly selected litters/group) were 

mated within dose groups for 7 days and examined for 

reproductive function. Because fewer F1 mice in highdose group were available as a result of increased 

mortality, only 11 mice/sex were mated. The animals 

continued to receive the same diet given to their parents. 

Vaginal smears were obtained from females that did not 

mate or did not appear to become pregnant. One litter/ 

pair was examined for sex, body weight, and viability. 

The parental F1 animals from all dose group were killed 

and examined as described for Task 3 of the study. 

Treatment-related effects observed in adult rats are 

summarized in Table 95, and effects occurring in 

immature rats are summarized in Table 96. Bisphenol A 

treatment had no effect on mating or fertility index in F0 

or F1 mice. Postpartum body weights were reduced in F0 

dams of the high-dose group. In F0 mice, the number of 

litters produced/pair and numbers of live F1 pups/litter 

were reduced at the mid- and high-dose level. A decrease 

in the proportion of pups born alive occurred in F0 mice 

of the high-dose group. No effects were observed on sex 

ratios of F1 or F2 pups. Weights of live F1 pups were 

increased at the mid and high-dose. There were no 

significant effects when pup weights were adjusted for 

total numbers of live and dead pups in the litter. 

Birth Defects Research (Part B) 83:157–395, 2008
98

T ough Diet in a Multigeneration Reproductive Toxicity Study hr

base on target intakes provided by study authors] d 

30 [5] 300 [50] 3500 [600] BMD10 BMDL10 BMD1SD BMDL1SD 

BISPHENOL A 

2 2 m2.2–fold 249 [42.2] 150 [25.4] 4258 [722] 2941 [498] 2 2 k10% 3455 [586] 2388 [405] 3503 [594] 2608 [442] 2 2 m17% 2189 [371] 1820 [308] 2021 [343] 1668 [283] 2 m5% m22% 1662 [282] 1425 [242] 1637 [277] 1389 [235] 2 2 m23% 1584 [268] 1383 [234] 1685 [286] 1405 [238] 2 2 m17% 2524 [428] 1595 [270] 3014 [511] 2155 [365] 2 2 m10% 3424 [580] 2438 [413] 3551 [602] 3024 [513] 2 m8% m20% 1861 [315] 1536 [260] 2100 [356] 1723 [292] m11% m10% m21% 2079 [352] 1913 [324] 862 [146] 773 [131] 2 2 m27% 1501 [254] 1229 [208] 1978 [335] 1610 [273] 2 2 m13% 3568 [605] 2504 [424] 4326 [733] 3041 [515] 2 2 m8% 3629 [615] 2976 [504] 3702 [627] 3393 [575] 2 m9% m19% 1899 [322] 1548 [262] 2249 [381] 1825 [309] m13% m10% m22% 2074 [352] 1650 [280] 2547 [432] 2020 [342] 2 m11% m28% 1466 [248] 1205 [204] 1937 [328] 1582 [268] 2 2 m11% 3746 [635] 2550 [432] 4773 [809] 3258 [552] 2 2 m10% 3413 [578] 2087 [554] 3627 [615] 3182 [539] 2 2 m16% 2678 [454] 1934 [328] 3476 [589] 2512 [426] 2 2 m9% 2678 [454] 1934 [328] 3476 [589] 2512 [426] 0/10 4/10 10/10 122 [20.7] 70 [11.8] 2/10 1/10 6/10 879 [149] 578 [98.0] 2/10 5/10 7/10 656 [111] 442 [74.9] 0/10 1/10 6/10 1348 [228] 947 [161] 0/10 3/11 7/10 962 [163] 679 [115] 2/10 1/10 4/10 1556 [264] 750 [127] 2/10 0/10 4/10 1418 [240] 838 [142] 2/10 0/10 3/10 1991 [337] 992 [168] 1/10 1/11 4/11 1646 [279] 847 [144] 

1/12 3/14 7/17 1328 [225] 833 [141] 2/10 2/11 7/15 1193 [202] 708 [120] 

2 2 k15% 3343 [567] 1884 [319] 3581 [607] 3241 [549] 2 2 m2% 21,351 [3619] 3770 [639] 6749 [1144] 3536 [599] 2 2 m2% 17,820 [3020] 3784 [641] 4552 [772] 3134 [531] 

365 to controls.
 

aT ble 

Treatment-Related Effects in Adult Mice Fed Bisphenol A 

Birth D

Dose, ppm diet [mg/kg bw/day 

efects

Endpoint 0.018 [0.003] 0.18 [0.03] 1.8 [0.3] 

Resea

Body weight gain during lactation, F0 2

2

2

Terminal body weight F1 retained males 

2

2

2

Relative liver to body weight

F0 males 

2

2

2

F1 parental males 

2

2

2

F1 retained males 

2

2

2

F0 females 

2

2

2

F1 females 

2

2

2

Relative right kidney to body weight

F0 males 

2

2

2

F1 parental males 

2 2 2

F1 retained males 

2

2

2

F0 females 

2

2

2

F1 females 

2

2

2

Relative left kidney to body weight

F0 males 

2

2

2

F1 parental males 

2 2 2

F1 retained males 

2

2

2

F0 females 

2

2

2

Relative pituitary to body weight

a F1 parental males

2

2

2

F1 retained malesa 2

2

2

a Relative brain to body weight F1 retained males

2

2

2

Hepatocyte centrilobular hypertrophy incidence (control incidence in parentheses)

F0 males (6/56) 1/10 2/10 2/10 

F1 parental males (7/55) 0/10 0/10 4/10 F1 retained males (4/50) 1/10 3/10 2/10 F0 females (1/56) 0/10 0/10 0/10 F1 females (2/55) 0/10 0/10 0/10 

Renal nephropathy incidence (control incidence in parentheses)

F0 males (12/56) 0/10 3/10 2/10 

F1 parental males (6/55) 2/10 0/10 1/10 F1 retained males (8/50) 1/10 0/10 0/10 F1 females (10/55) 1/10 3/10 0/10 

Paraovarian cysts (control incidence in parentheses)

F0 (9/56) 1/11 2/12 1/11 

F1 (14/55) 1/11 1/11 1/10 

a Epididymal sperm concentration, F0 2

2

2

Gestational length

F0 2

2

2

F1 2

2

2

rch (Part B) 83:157–395, 2008 

aNot considered a treatment-related effect by study authors.


m,k Statistically significant increase, decrease; 

2 no statistically significant effect compared 
366 CHAPIN ET AL. Table 99 Treatment- or Dose-Related Effects in Developing Mice in a Multigeneration Reproductive Toxicity Study With Bisphenol A Dose, ppm diet [mg/kg bw/day based on target intakes provided by study authors] a End point 0.018 [0.003] 0.18 [0.03] 1.8 [0.3] 30 [5] 300 [50] 3500 [600] BMD10 BMDL10 BMD1SD BMDL1SD Body weight F1, PND 7 2 2 2 2 2 k13% 3304 [560] 1849 [313] 3433 [582] 2403 [407] F1, PND 14 2 2 2 2 2 k11% 3453 [585] 2256 [382] 3639 [617] 2988 [506] F1, PND 21 2 2 2 2 2 k17% 3236 [548] 1577 [267] 3421 [580] 2342 [370] F1 male, PND 21 necropsy 2 2 2 2 2 k12% 3325 [564] 1845 [313] 3776 [640] 3536 [599] F1 female, PND 21 necropsy 2 2 2 2 2 k18% 2284 [387] 1501 [254] 4577 [776] 3529 [598] Lactational survival indices (control index, %, in parentheses) F2 PND 21 survival (100%)c 2 2 2 2 k to 86.6% 2 F2 Lactational index (97.2%)c 2 2 2 2 k to 86.6% 2 Relative thymus to body 2 2 2 2 m 13%b m 10%b weight, F1 male, PND 21b

Relative spleen to body weight

F1 male, PND 21 2 k 12% 2 2 2 k 30% 3123 [529] 1074 [182] 3538 [600] 3148 [534] 

F2 male, PND 21 2 2 2 2 2 k 20% 2148 [364] 1425 [242] 7013 [1189] 3560 [603] 

F1 female, PND 21 2 2 2 2 2 k 23% 3168 [537] 647 [110] 4571 [775] 3677 [623] F2 female, PND 21 2 2 2 2 2 k 21% 1787 [303] 1311 [222] 5022 [851] 3517 [596] 

Relative paired testes weight to

body or brain weight

F1, PND 21 (body weight) 2 2 2 2 2 k 8% 3578 [606] 2720 [461] 3861 [654] 3550 [602] 

F2, PND 21 (brain weight) 2 2 2 2 2 k 11% 3316 [562] 2003 [339] 5342 [905] 3571 [605] Relative paired epididymides 2 m 18% 2 2 2 2 b to body weight, F1

Relative brain to body weight 2 2 2 2 2 m 17%b 2219 [376] 1415 [240] 3576 [606] 2825 [479] 

F1 female, PND 21b

Relative left kidney to body 2 2 2 2 2 m 6% 6664 [1129] 3540 [600] 8501 [1441] 3589 [608] 

weight, F2 male, PND 21 

Relative seminal vesicles with 2 2 2 2 k 15% k 16% 2389 [405] 1315 [223] 11,294 [1914] 3631 [615] 

b coagulating gland to brain weight, F2 

Uterus with cervix and vagina 2 2 2 2 k16% 2

weight relative to bodyweight, F2 PND 21b b Relative paired ovary weights, F1 2 2 m 2 2 2

Birth Defects Research (Part B) 83:157–395, 2008 
BIS 367 

6/24 10/20 13/20 732 [124] 546 [92.5] 1/24 2/20 9/23 1442 [244] 1050 [178] 

4/26 8/16 6/22 1966 [333] 1182 [200] 

0/24 0/21 3/21b 3/51 2/45 5/43 3485 [591] 2398 [406] 

0/51 5/49 20/57 1670 [283] 1377 [233] 

5/78 7/50 12/600 2694 [462] 1755 [301] 

2 k4% k5% 8099 [1373] 3582 [607] 10,436 [1769] 3632 [616] 2 2 m2 days 4450 [754] 3397 [576] 3252 [551] 2445 [414] 

2 2 m1.8 days 4288 [727] 3375 [572] 2897 [491] 2145 [364] 

2 2 k22% 3076 [521] 1281 [217] 3294 [558] 1972 [334] 

2 2 k2.4 3501 [593] 2953 [501] 3404 [577] 2419 [410] 

PHENOL A live animals before or on PND 21 considered the litter as the statistical unit, but statistical analyses


Hepatic cytoplasm alteration

(control incidence in parentheses)

F1 males (6/44) 1/26 0/17 1/22 

F2 males (6/54) 1/25 1/25 1/25 F1 females (2/46) 1/27 2/21 3/24 

Unilateral hydronephrosis, 0/26 0/17 0/21 

F1 males (0/44)b

Seminiferous tubule hypoplasia

(control incidence in parentheses)

F1 (1/96) 0/54 0/37 1/45 

F2 (5/114) 1/53 2/61 2/55 Undescended testis, F1 PND 21 5/79 5/54 10/70 

(control 11/135) 

Anogenital distance adjusted for 2 2 2

body weight F1 male, PND 21b 

Age of preputial separation

(adjusted per body weight)

F1 parental males 2 2 2

F1 retained males 2 2 k0.6 days Body weight on day of 2 2 2

vaginal opening in F1

Age of vaginal opening 2 2 2 b adjusted for PND 21 body weight

Birth Defects Research (Part B) 83:157–395, 2008 

aBased on numbers of animals listed in data tables, it appears that statistical analyses in 

conducted at PND 21 necropsy considered the individual pup as the statistical unit.
 bNot considered treatment related by study authors.


cEffect was not discussed by study authors but it is unlikely to be related to treatment.
 
368 CHAPIN ET AL. 

Therefore the NTP concluded that the increased pup 

weights resulted from the smaller litter size. Body 

weights were evaluated through PND 21 in F1 pups, 

and no effects were found on pup body weight gain 

during the lactation period. Mortality in F1 offspring 

during the postnatal period was increased in the highdose group. 

The cross-over test revealed no effect on mating or 

fertility in either males or females exposed to bisphenol 

A. Postpartum body weight was not affected in the 

treated females. The number of live pups/litter was 

significantly reduced [by 26%] in the group containing 

treated males and [by 51%] in the group containing 

treated females. Live pup weight was increased in the 

group containing treated females, but there was no 

significant effect following adjustment for litter size. 

There were no effects on the proportion of pups born 

alive or on sex ratio. 

In sperm analyses conducted in high-dose F0 males 

and all dose groups of F1 males, sperm motility was 

reduced in high-dose F0 males and mid-dose F1 males. 

There were no effects on sperm count or morphology in 

either generation. Effects were observed on organ 

weights, which were examined in F0 adults of the highdose group and F1 animals from each treatment group. 

Effects on absolute reproductive organ weights of F1 

mice included decreased right epididymis weight at all 

doses, decreased left testis/epididymis weight at the mid 

and high-dose, and decreased seminal vesicle weight at 

the high-dose. Significant effects on relative organ 

weights adjusted for body weight in F1 rats included 

decreased right epididymis weight at all doses, decreased seminal vesicle weight at the low and high-dose, 

and decreased relative left testis and epididymis weight 

at the mid and high-dose. Reproductive organ weight 

effects observed in high-dose F0 males included decreased absolute and relative seminal vesicle weight. 

There were no effects on prostate weight. No effects were 

reported for estrous cyclicity of F0 females. There were no 

gross or histopathological alterations in F0 or F1 

reproductive organs including testis, epididymis, prostate, seminal vesicles, ovary, vagina, and uterus. Effects 

observed in high-dose F0 animals were also summarized 

in a report by Morrissey et al. (1988). 

Effects were observed on non-reproductive organ 

weights, which were examined in F0 adults of the highdose group and F1 animals from each treatment group. In 

the F1 mice, dose-related effects on absolute organ 

weights included increased kidney/adrenal weight at 

all doses in both sexes and increased liver weight in midand high-dose females and high-dose males. Significant 

effects on relative organ weight adjusted for body weight 

in F1 rats included increased liver and kidney/adrenal 

weights at all doses in both sexes. Organ weight effects 

observed in high-dose F0 males included increased 

absolute and relative liver and kidney/adrenal weight. 

In F0 female rats of the high-dose group, absolute and 

relative liver weight and relative kidney weights were 

increased. Body weights of high-dose F0 females were 

reduced at necropsy. Histopathology was examined in F0 

rats of the high-dose group and F1 rats from all dose 

groups. Treatment-related hepatic lesions observed in 

both generations included multifocal necrosis, multinucleated giant hepatocytes in males and females, and 

centrilobular hepatocytomegaly in males. Multifocal 

mineralization of liver cells was also observed in F1 

females of the high-dose group. Hepatic lesions were 

observed at all dose levels for F1 males and in F1 females 

of the mid- and high-dose group. Treatment-related renal 

lesions were observed in both generations and described 

as tubular cell nuclear variability, increased severity of 

spontaneous tubular interstitial lesions, cortical tubular 

dilatation, mineralization of renal cells, and micro-calculi 

in tubular epithelium that sometimes occurred with 

effaced tubular epithelium, tubular regeneration, and/or 

dilated tubules containing casts. [It appears that the 

incidence of renal lesions was increased at all doses in 

F1 rats.] Renal lesions were stated to generally be more 

prominent in females than males. The study authors 

concluded that exposure of mice to bisphenol A resulted 

in toxicity to the reproductive system, kidney, and liver. 

The possibility was noted that some or all effects on 

reproductive performance may have been secondary to 

the generalized toxicity of bisphenol A. 

This study demonstrates changes in F1 male absolute 

reproductive weights (seminal vesicle with coagulating 

gland as well as epididymis; the testis and prostate 

appear not to have been appreciably affected). This study 

also suggested that reproductive toxicity and general 

toxicity occurred at similar dose levels. Bisphenol Amediated general toxicity may have contributed to the 

observed female fertility effect, because this effect was 

noted with dosed females cohabiting with non-dosed 

males. In the male, however, the effect on motility is 

likely bisphenol A-related, resulting in the observed 

fertility deficits. 

In Task 2, a clear effect on fertility was found with a 

NOAEL of 0.25% bisphenol A in the diet. 

Strengths/Weaknesses: This comprehensive toxicology study was well-conducted. General toxicity was 

clearly demonstrated at all F1 dose levels, and histopathological findings appear to be a sensitive indicator of 

effect. As a limitation of this design, because bisphenol A 

was in the diet, exposure to bisphenol A did not occur 

during cohabitation; therefore, direct exposure to bisphenol A was minimal or nonexistent during sperm 

maturation, capacitation and ovulation. 

Utility (Adequacy) for CERHR Evaluation Process: 

These data are adequate and of high utility for the 

evaluation process. 

Tyl et al. (2002a), sponsored by the Society of the 

Plastics Industry, conducted a one-generation reproductive toxicity study in mice. The study was conducted to 

verify the findings of reduced pup numbers at birth in a 

continuous breeding study conducted by the NTP (NTP, 

1985a). GLP guidelines were applied in the conduct of 

the study. CD-1 mice were fed Purina Certified Rodent 

Diet Meal and housed in polycarbonate cages containing 

Sani-chip bedding. Mice were stratified according to 

body weight and randomly assigned to treatment 

groups. Starting at 9 weeks of age, 20 mice/sex/group 

were given feed containing bisphenol A (99.36% purity) 

0, 5000, or 10,000 ppm. Males and females were fed the 

bisphenol A-containing diets during a 2-week prebreeding period and a 1-week mating period. The day 

of vaginal plug detection was defined as GD 0. 

Exposures in females continued through the gestation 

period of B19 days. The study authors reported bisphenol A intakes of 0, 840, and 1669 mg/kg bw/day in 

males during the prebreeding period; 0, 1055, and 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 369 

1988 mg/kg bw/day in females during the prebreeding 

period, and 0, 870, and 1716 mg/kg bw/day in females 

during the gestation period. [Intake values were 

obtained from the Results section and study summary 

tables. They differed from values reported in text Table 

C, which were assumed to be in error.] Homogeneity 

and stability of bisphenol A in feed were verified. 

Parameters evaluated during the study included clinical 

signs, body weight, and feed intake. Reproductive 

endpoints evaluated included implantation loss and 

indices of mating, fertility, pregnancy, and gestation. F0 

Males were killed at the end of the breeding period; liver 

and kidney were weighed. At birth, pups were counted, 

sexed, weighed, and evaluated for viability and external 

alterations. F0 females and F1 pups were killed on the day 

of parturition (PND 0). Dams were assessed for clinical 

chemistry parameters of liver and kidney function; 

corpora lutea and implantation sites; uterus, ovary, 

kidney, and liver weight; and liver and kidney histopathology. The male, female, pregnant female, or the 

litter were considered statistical units. Statistical analyses 

included ANOVA, Levene test, GLM procedure, Dunnett 

test, w2 test, Cochran–Armitage test, and Fisher exact 

probability test. 

Treatment-related effects in F0 animals are summarized in Table 97. There were no treatment-related changes 

in clinical signs, body weight gain, feed intake, or food 

efficiency in males or in females during the prebreeding 

period. A transient increase in food intake occurring in 

females of the low-dose group on study stays 0–7 did not 

appear to be treatment-related. Gestational body weight 

gain was decreased in the high-dose group, beginning on 

GD 7 and in the low dose group beginning on GD 10. 

Body weights of live F0 females were significantly lower 

in the high-dose group on PND 0, but no significant 

differences were observed during necropsy conducted 

later in the day. A significant decrease in feed intake was 

reported for the high-dose group on GD 14–17, only 

when the values were expressed as g/day. [The results 

section indicated that food efficiency during gestation 

was not significantly affected, but a downward trend 

was observed. Table 10 of the study reported a 

significant decrease in food efficiency.] Significant 

necropsy findings observed in males included increased 

absolute and relative liver weight at both doses and 

increased absolute paired kidney weight at the low dose. 

Absolute and relative liver and paired kidney weight 

were increased significantly in females from both dose 

groups. Histopathological observations in females included dose-related increases in incidence and severity 

of hepatocyte hypertrophy and increased kidney lesions 

(renal tubular epithelial necrosis, degeneration, and 

regeneration) in both dose groups. Significant clinical 

chemistry findings in females included increased blood 

urea nitrogen in the high-dose group and decreased 

sodium, potassium, and chloride levels in the low-dose 

group. 

Treatment-related reproductive or developmental effects are summarized in Table 97. No significant effects 

were observed for mating, fertility, or pregnancy indices; 

time to insemination; numbers of ovarian lutea or 

implantation sites; or implantation loss. Gestation duration was extended by B10 hr in both dose groups; the 

study authors stated that the biological significance of 

the finding is not known. Total and live pup numbers 

were decreased in the high-dose group. No significant 

effects on pup weight were observed but a downward 

trend was statistically identified for female pup weight. 

The study authors concluded that their study confirmed the NTP (1985a) finding of reduced litter size in 

mice fed 10,000 ppm bisphenol A in feed. The NTP 

finding of decreased litter size at 5000 ppm bisphenol A 

was not confirmed in this study, likely due, according to 

the authors, to the shorter exposure duration in the 

current study than in the NTP study. The study authors 

concluded that the litter size decreases in their study 

were likely caused by the compromised status of dams. 

Strengths/Weaknesses: Strengths of this report include 

the comprehensive design with the assessment of multiple relevant endpoints. There were adequate numbers of 

animals, the doses and stability of the compound were 

verified, and the oral route of exposure was used. 

Weaknesses include the limited number of doses examined and the relatively high-doses studied. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and useful for the evaluation 

process. 

Tyl et al. (2006), sponsored by the American Plastics 

Council, conducted a two-generation study of bisphenol 

A in mice. The study was conducted accorded to GLP. 

CD-1 mice were received in two cohorts approximately 2 

weeks apart and data from the two cohorts were 

combined. Mice were fed Purina Certified Ground 

Rodent Diet No. 5002. The supplier provided information 

about phytoestrogen content of feed (177–213 ppm 

genistein, 173–181 ppm daidzein, and 39–55 ppm glycitein). Mice were housed in polypropylene cages with 

Sani-Chip bedding. Assignment of F0 animals to groups 

involved randomization stratified by weight. F0 and F1 

mice (28 sex/group/generation) were fed diets containing bisphenol A (99.70–99.76% purity) at 0.018, 0.18, 1.8, 

30, 300, or 3500 ppm. Target intakes were 0.003, 0.03, 0.3, 

5, 50, or 600 mg/kg bw/day, respectively. Based on 

measured feed intake, the study authors estimated 

bisphenol A intake in males at 0.0024–0.0038, 0.024– 

0.037, 0.24–0.37, 3.98–6.13, 39.1–60.8, or 529–782 mg/kg 

bw/day. Bisphenol A intakes (in mg/kg bw/day) by 

females were estimated at 0.0030–0.0041, 0.030–0.042, 

0.32–0.43, 5.12–7.12, 54.2–67.8, 653–910 during the premating period; 0.0027–0.0029, 0.027–0.028, 0.28–0.29, 

4.65–4.80, 47.0–48.6, 552–598 during the gestation period; 

and 0.0063–0.0087, 0.062–0.091, 0.61–0.89, 10.4–15.1, 

103.2–146.4, 1264–1667 during the lactation period. In 

each generation, there were 2 vehicle control groups with 

28 mice/sex/group. A positive control group was given 

feed containing 17b-estradiol at 0.5 ppm (target intake of 

0.08 mg/kg bw/day). Estimated intakes for 17b-estradiol 

(in mg/kg bw/day) were 0.074–0.104 in males, 0.093– 

0.12 in females during the pre-mating period, 0.08–0.081 

in females during the gestation period, and 0.160–0.25 in 

females during the lactation period. Dose selections were 

based on observations from several studies. [The Expert 

Panel notes that a separate 2-generation study was used 

to characterize the dose–response relationship for 17bestradiol.] Homogeneity, stability, and concentration of 

bisphenol A in feed were verified. Exposure of F0 mice 

began at B6 weeks of age. Exposure of F1 animals began at 

weaning, although it was noted that pups began eating the 

dosed feed in the late lactation period. F0 and F1 mice were 

fed the bisphenol A-containing diets for a minimum of 8 

Birth Defects Research (Part B) 83:157–395, 2008
370 CHAPIN ET AL. 

weeks before mating and during a 2-week mating period. 

Exposures of males continued through the gestation period 

of the litters they sired. Exposures of females continued 

through the gestation and lactation period. During the 

study, adult animals were monitored for clinical signs of 

toxicity, body weight, and food intake. 

Estrous cycles were evaluated in F0 and F1 females 

during the last 3 weeks of the pre-breeding exposure 

period. Day of vaginal plug was defined as GD 0 and day 

of birth was considered PND 0. F1 and F2 pups were 

counted, sexed, weighed, and assessed for viability and 

physical abnormalities at birth and throughout the 

lactation period. Anogenital distance was measured in 

F1 and F2 pups at birth and on PND 21. On PND 4, F1 and 

F2 litters were standardized to 10 pups, with equal 

numbers per sex when possible. Pups removed on PND 4 

were killed and examined for visceral alterations, with a 

focus on the reproductive system. The remaining pups 

were maintained and weaned on PND 21. At weaning, 28 

F1 pups/sex/group (1 per sex per litter) were randomly 

selected for mating and those animals were referred to as 

parental mice. An additional F1 male/litter was selected 

for a 3-month exposure (referred to as retained males). 

Two F1 pups/sex/litter were selected for gross necropsy 

and organ weight measurement at weaning. Histopathological examination of reproductive organs was conducted in one PND 21 pup/sex/litter. Histopathological 

evaluation of reproductive and systemic organs were 

conducted in the second F1 pup from each group at 

weaning. All F2 pups were killed at weaning and organ 

weights were measured. Vaginal opening and preputial 

separation were monitored in parental and retained F1 

mice. Parental F0 and F1 males were killed following 

delivery of the litters they sired. Retained F1 males were 

killed at the same time as the parental F1 males. Parental 

F0 and F1 females were killed after their pups were 

weaned. Organs, including those of the reproductive 

system, were weighed in adult F0 and F1 animals. 

Histopathological evaluations were conducted in all 

animals from the vehicle control group, in 10 F0 and F1 

parental animals from each treatment group, in all F1 

retained males, and 10 animals from the 17b-estradiol 

positive control group. Histopathological evaluation of 

reproductive organs was also conducted in animals with 

suspected reduced fertility. Testes were preserved in 

Bouin fixative. Daily sperm production, efficiency of 

daily sperm production, and epididymal sperm count, 

motility, and morphology, were evaluated in F0 and F1 

males. Data from the 2 control groups were analyzed 

separately and then pooled for statistical analysis of 

treatment groups. Statistical analyses included ANOVA, 

Levene test, robust regression methods, Wald w2 test, ttest, Dunnett test, Fisher exact probability test, and 

ANCOVA. 

Treatment- or dose-related results and observations in 

reproductive organs of adult animals are summarized in 

Table 98. There were no consistent effects on body weight 

or body weight gain in F0 males. Body weight gain 

during lactation was increased in F0 females from the 

3500 ppm group. During the premating period, body 

weights were decreased by r10% in F1 parental animals 

from the 3500 ppm group (study days 0, 7, 49, and 56 in 

males and study 0 in females). In retained F1 males from 

the 3500 ppm group, body weights were decreased at 

most time periods between study days 7 and 84 and at 

necropsy. No consistent or dose-related changes in feed 

intake or efficiency were observed throughout the study 

in F0 or F1 animals. There were no clinical signs of 

toxicity or treatment-related deaths in F0 or F1 males or 

females. Increases in absolute and relative to body or 

brain weights of kidney and liver were consistently 

observed in F0 and F1 adults. Significant and dose-related 

organ weight changes relative to body weight are 

summarized in Table 98. Other effects on organ weight 

(e.g., seminal vesicles, epididymides, coagulating glands, 

and pituitary) were not considered to be treatmentrelated by study authors due to factors such as lack of a 

dose–response relationship, no consistency between 

absolute and relative weights, no histopathology, or no 

consistency across generations. Absolute and relative 

prostate weights were unaffected by bisphenol A 

exposure. There were no treatment-related gross systemic findings in F0 or F1 adults. Incidence of minimal to 

mild hepatocyte centrilobular hypertrophy was increased in both generations at 300 and/or 3500 ppm 

(Table 98). Renal nephropathy incidence was increased in 

F0 males and in F1 males and females of the 3500 ppm 

group. [It did not appear that histopathological data 

were statistically analyzed.] 

Treatment- or dose-related reproductive effects in 

adult animals are summarized in Table 98. Bisphenol A 

exposure had no effect on numbers of implantation sites 

or resorptions or on mating, fertility, or gestational 

indices in F0 or F1 mice. Gestational length was increased 

in F0 and F1 females from the 3500 ppm group; the study 

authors stated the effect was of unknown biological 

significance. Epididymal sperm concentration was decreased in F0 males of the 3500 ppm group but no effect 

was observed in F1 parental or retained males. There was 

no effect on daily sperm production, efficiency of daily 

sperm production, or sperm motility or morphology in 

either generation. The study authors did not consider the 

decrease in sperm concentration in F0 animals to be 

treatment-related based on lack of consistency between 

generations, no effect on any other andrological endpoint, and no effect on fertility. Estrous cyclicity and 

numbers of ovarian primordial follicle counts were not 

affected by bisphenol A exposure in F0 or F1 females. The 

only gross observation in reproductive organs was a 

slightly increased incidence of gross ovarian cysts in F0 

females from the 3500 ppm group. The incidence of 

paraovarian cysts was increased in F0 and F1 females 

from the 3500 ppm group. [It did not appear that 

histopathological data were statistically analyzed.] 

Significant findings in developing mice are summarized in Table 99. Live F1 and F2 pups and litters at birth, 

sex ratio, and survival during the lactation period were 

not affected and there were no clinical or gross signs of 

toxicity in F1 or F2 offspring. A non-dose-related decrease 

in PND 21 survival index and lactational index (pups 

surviving on PND 21/PND 4) was described in F2 pups 

of the 300 ppm group. [The biological significance of 

the effect was not discussed by the study authors, but 

because the effect was not dose-related it is unlikely to 

be of biological significance.] In F1 pups from the 

3500 ppm group, body weights were reduced during 

PND 7, 14, and 21 in F1 females and both sexes combined 

and on PND 7 and 21 in F1 males. Body weight results for 

both sexes combined are summarized in Table 99. An 

increase in male pup body weight observed on PND 7 in 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 371 

the 1.8 ppm group was not considered to be treatmentrelated by the study authors because no dose–response 

relationship was observed. There was no effect on 

anogenital distance in F1 or F2 males or females on 

PND 0. Anogenital distance was also unaffected in F2 

males and F1 and F2 females on PND 21. Anogenital 

distance adjusted for body weight was reduced in F1 

males from the 300 and 3500 ppm groups on PND 21. 

Based on the lack of effect on anogenital distance at birth 

and inconsistencies between generations, the study 

authors did not consider the decreases in anogenital 

distance in F1 males to be treatment-related. An increase 

in anogenital distance in F2 females from the 0.018 ppm 

group on PND 0 was not considered to be treatmentrelated by the study authors. Preputial separation 

(absolute age and adjusted for body weight on day of 

acquisition) was delayed in parental and retained F1 

males of the 3500 ppm group. When adjusted for PND 30 

body weight, preputial separation was delayed in 

retained but not parental F1 males from the 3500 ppm 

group. Data for preputial separation adjusted for body 

weight on day of acquisition are shown in Table 99. Body 

weights on day of vaginal opening were lower in F1 

females from the 3500 ppm group. Day of vaginal 

opening was accelerated in the 3500 ppm group if 

adjusted for PND 21 body weight, but not body weight 

on the day of acquisition. Due to the lack of effect when 

adjusted for body weight on day of acquisition, the study 

authors did not consider effects on vaginal opening to be 

treatment-related. 

Shown in Table 99 are significant organ weight effects 

relative to body weight. Dose-related organ weight 

changes in F1 weanlings that were considered to be 

treatment-related by study authors included decreased 

absolute and relative (to body or brain weight) spleen 

and paired testes weights at 3500 ppm. Treatment-related 

absolute organ weight changes in F2 weanlings included 

decreased weights of spleen, paired testes, and seminal 

vesicles with coagulating glands in the 3500 ppm group. 

Changes in organ weights relative to body weight in F2 

weanlings included decreased spleen weight in males 

and females and increased relative left kidney weight in 

3500 ppm males. Treatment-related changes in organ 

weight relative to brain weight in F2 weanlings were 

decreased spleen weight in both sexes and decreased 

paired testes weight at 3500 ppm and seminal vesicles 

with coagulating glands at 300 and 3500 ppm. Other 

organ weight effects (e.g., affecting epididymides, 

thymus, brain, ovaries, and/or uterus with cervix and 

vagina weights) were not considered to be dose-related 

due to lack of dose–response relationships or no 

consistent effects across generations. Included in Table 99 

are significant organ weight effects relative to body 

weight. Significant organ weight effects relative to brain 

weight were included in Table 99 when the organ weight 

effect was significant only when normalized for brain 

weight. The study authors reported no gross findings in 

F1 or F2 weanlings. The incidence of undescended 

bilateral testes was increased in F1 and F2 weanling 

males of the 3500 ppm group. The incidence of hepatic 

cytoplasm alteration (clear hepatocellular cytoplasm, 

slightly more basophilic cytoplasm, and/or minute 

vacuoles) was apparently increased in F1 males from 

the 300 and 3500 ppm groups and F1 females and F2 

males from the 3500 ppm group. The incidence of 

seminiferous tubule hypoplasia was increased in F1 and 

F2 weanlings from the 3500 ppm group. [Another 

histopathological finding that appeared to be possibly 

increased in weanlings from the 3500 ppm group was 

unilateral hydronephrosis in F1 males. It did not appear 

that histopathological data were statistically analyzed.] 

Effects of 17b-estradiol in males were delayed preputial 

separation, reduced anogenital distance at weaning but not 

at birth, decreased weights of testes, epididymides, and 

seminal vesicles with coagulating gland, and increased 

incidence of seminiferous tubule hypoplasia and undescended testis. Effects of 17b-estradiol in female mice were 

accelerated vaginal patency, increased uterus with cervix 

and vagina weight, fluid filled/enlarged uterus, enlarged/ 

thickened vagina, increased vaginal epithelial keratinization, and prolonged gestation. Reproductive effects in the 

17b-estradiol group included decreased fertility, increased 

stillbirth, reduced live pups per litter, and increased dead 

pups. 

The study authors identified bisphenol A NOELs of 

30 ppm (B5 mg/kg bw/day) for systemic effects, 

300 ppm (B50 mg/kg bw/day) for developmental toxicity, and 300 ppm (B50 mg/kg bw/day) for reproductive 

toxicity. 

Strengths/Weaknesses: Strengths include the large 

number and range of doses examined, the rigor with 

which the study was performed, the large sample size in 

each group, the number of additional animals per litter 

that were retained and examined, the use of a concurrent 

estrogenic positive control group, and the thoroughness 

of the histologic evaluation. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study is adequate and of high utility for the 

evaluation process. 

4.2.3.3 Fish and invertebrates: Although studies in 

fish and invertebrates may be important for understanding mechanisms of action and environmental 

impact, the Panel views these studies as not useful for 

the evaluation process. 

Kwak et al. (2001), supported by the Korean Ministry 

of the Environment, exposed adult male swordtail fish 

(Xiphophorus helleri) to bisphenol A 0, 0.4, 2, or 10 ppm 

[mg/L] for 72 hr (n 5 20 fish/group). [No information on 

purity or culture ware was provided.] [Nonylphenol 

was also studied but will not be discussed here.] At the 

end of the exposure period, the fish were killed and 

livers were removed for measurement of vitellogenin. 

Testes of 10 fish/group were processed for flow 

cytometry by preparation of single cell suspensions 

stained with annexin V-fluorescein isothiocyanate and 

propidium iodide to detect necrosis and apoptosis. 

TUNEL staining was used to confirm apoptosis in testis 

sections. In a second experiment, juvenile male fish (30 

days old) were exposed to bisphenol A in water at 0, 0.2, 

2 and 20 ppb [lg/L] for 60 days, after which body length 

and sword length were measured. [The sword is a 

portion of the caudal fin that elongates as a secondary 

sex characteristic.] Statistical analysis used ANOVA 

followed by least significant difference test. Hepatic 

vitellogenin was increased by bisphenol A [data were 

not shown]. Apoptosis was increased in testes from fish 

exposed to bisphenol A at 10 ppm [mg/L] by TUNEL 

assay. [Flow cytometry was said to be more sensitive, 

but data did not appear to have been statistically 

analyzed.] Sword growth was decreased by bisphenol A 

Birth Defects Research (Part B) 83:157–395, 2008
372 CHAPIN ET AL. 

Table 100 

Summary of Serum Hormone Changes in Human Studies 

Study members Hormone effects Other Reference 

High utility 

Urine in male workers 

42 exposed 

42 non-exposed 

Limited utility 

Serum samples from: 

14 healthy women 

11 healthy men 

16 women with PCOS 

Serum samples from: 

26 healthy women 

19 women with PCOS 

28 women with other 

conditions 

Serum samples from women: 

11 controls 

10 simple hyperplasia (HP)
 

9 complex hyperplasia (HP)
 

7 endometrial cancer (EC)
 

k FSH (exp median 5.3 mIU/ml vs. 7.6 in 

controls) 

No difference LH, free testosterone 

m total testosterone (r 5 0.595, all subjects) 

m free testosterone (r 5 0.609, all subjects) 

No difference LH 

m total testosterone (r 5 0.391) 

m free testosterone (r 5 0.504) 

m androstenedione (r 5 0.684) 

m dehydroepiandrosterone 

sulfate (DHEAS) (r 5 0.514) 

No difference LH 

BPA exposure 

Exposed men: 

1.06 mmol/mol creatinine 

[0.043 mg/kg bw) 

Non-exposed men: 

0.52 mmol/mol 

creatinine [0.02 mg/kg bw) 

dec BPA in complex 

HP and EC patients
 

compared to controls
 

Hanaoka et al. (2002) 

Takeuchi and 

Tsutsumi (2002) 

Takeuchi et al. (2004a) 

Hiroi et al. (2004) 

exposure in a concentration-dependent manner, with 

statistically significant decreases from control at 2 and 

20 ppb [lg/L]. The authors concluded that bisphenol 

A at 20 ppb decreases sword growth and that reproductive impairment occurs in a concentration-dependent 

manner. 

Strengths/Weaknesses: This study of bisphenol A is 

consistent with previous reports on the effects of 

estrogenic compounds in fish (vitellogenin production 

and changes secondary sex characteristics). It is unclear 

exactly how these fish were maintained before exposure 

and during the long-term exposure. Bisphenol A concentrations in the test waters were not determined and 

only 3 concentrations of bisphenol A were used. 

Utility (Adequacy) for CERHR Evaluation Process: Of 

note is the classic dose response obtained in this 

apparently sensitive model. Given the absence of 

confirmation of exposure conditions and that this is a 

fish species immersed in the test agent, this study is not 

useful in the evaluation. 

Sohoni et al. (2001), supported by the Society of the 

Plastics Industry, exposed adult (122-day-old) fathead 

minnows (Pimephales promelas) to bisphenol A in water at 

0, 1, 16, 160, and 640 mg/L (n 5 60/group) [No information on purity or culture ware was provided]. Actual 

concentrations were 70–96% of nominal concentrations. 

After 42 days of exposure, 15 fish/group were killed for 

evaluation of somatic growth, relative gonad weight, 

plasma vitellogenin, and histologic assessment 

of the testis. Eight breeding pairs/group were segregated 

for continued exposure for 123 days. Eggs were 

removed and counted daily. On 2 occasions, eggs were 

continued in the same bisphenol A concentration 

as their parents and the percent hatching was assessed 

4 days after fertilization. The remaining adult fish were 

killed after 71 days of exposure for evaluation of somatic 

growth, relative gonad weight, and histologic 

assessment of the gonad. Data were analyzed using 2­

way ANOVA and Dunnett test or Kruskal–Wallis and 

Dunn multiple method test. Linear regression was used 

to evaluate the relationship between bisphenol A 

concentration and growth. There were no significant 

long-term effects of treatment on growth of female fish, 

but male fish showed a inverse relationship between 

bisphenol A concentration and growth with 

significant decrements in length and weight on pairwise comparison at bisphenol A concentrations of 640 

and 1280 mg/L. Relative gonad weight was also decreased in males and females at these bisphenol A 

concentrations. Plasma vitellogenin was increased in 

females beginning at bisphenol A concentrations of 

640 mg/L and in males beginning at 160 mg/L. A delay 

in spermatogenesis was suggested by an increase in 

spermatogonia or spermatocytes and a decrease in 

spermatozoa in testes beginning at a bisphenol A 

concentration of 16 mg/L. There were no intersex gonads 

and no treatment-related changes in ovarian histopathology. The number of eggs spawned per female was lower 

in the control than the treatment groups and attributed 

by the authors to an unexplained problem in one of the 

control tanks. The 1280 mg/L bisphenol A concentration 

resulted in failure of 7 out of 8 females to produce any 

eggs. Hatching was impaired in eggs exposed to bisphenol A concentrations of 640 and 1280 mg/L. The 

authors noted that the bisphenol A concentrations 

resulting in impairment of somatic growth and 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 373 

reproductive success were only 7-fold lower than the 96­

hr median lethal concentration, and concluded that the 

reproductive effects may have been the result of 

sublethal generalized toxicity rather than effects 

mediated through the endocrine axis. 

Strengths/Weaknesses: This study was well-conducted with multiple dose levels and concentrations in 

the test water were confirmed. ‘‘General toxicity’’ was 

identified and good histology was used. The conclusions 

regarding weak estrogenic activity were appropriate at 

160 mg/L and higher. Other effects were likely due to 

general toxicity. A classic dose response was noted. 

Utility (Adequacy) for CERHR Evaluation Process: 

Fish are apparently a sensitive model for assessment of 

responses to weak estrogenic compounds. Given that this 

study evaluated a fish species, it is not useful in the 

evaluation. 

Kang et al. (2002), supported by the Japanese Ministry 

of the Environment, exposed adult (4-month-old) breeding pairs of medaka (Oryzias latipes) to bisphenol A 

(499% purity) in the water at 0, 1000, or 4000 mg/L for 3 

weeks [culture ware not discussed]. Bisphenol A 

concentrations during the exposure period were 78– 

86% of nominal concentrations. Thirty-two pairs of fish 

had been selected for exposure during an acclimatization 

period based on their capacity to spawn daily, with the 

production of Z15 eggs/day and 90% fertility. During 

the exposure period, eggs were collected daily and 

assessed for fertility. Fertilized eggs collected on the last 

3 days of the exposure period were permitted to develop 

in untreated water, and 60 larvae/group were grown for 

60 days after hatching to assess normalcy of development. The parent fish were killed at the end of the 

treatment period for evaluation of external sex characteristics and for histologic assessment of the gonads. 

Hepatic vitellogenin was also assessed. Statistical comparisons of egg number were made using ANCOVA with 

female body weight as a covariate. Fertility, growth 

endpoints, and hepatic vitellogenin data were analyzed 

with ANOVA or Kruskal–Wallis test with post-hoc 

Dunnett or Mann–Whitney U test. There were no 

treatment effects on egg number, fertility, mortality, 

relative gonad weight, or relative liver weight in the 

adult fish. Ovarian tissue was found in the testis in some 

males in all bisphenol A-treated groups, although normal 

testicular tissue with apparently normal spermatogenesis 

was also found. Hepatic vitellogenin was increased in 

male fish in the high-dose group to control female levels. 

There were no treatment-related alterations in hepatic 

vitellogenin in female fish. Offspring at 60 days of age 

did not demonstrate treatment-related alterations in 

survival, growth, or secondary sex characteristics. The 

sex ratio was not significantly different in offspring of 

parents exposed to bisphenol A, although the authors 

noted that the low-dose group had a numerical deficit of 

males (41% males compared to 50% in the controls). The 

authors concluded that although bisphenol A increased 

hepatic vitellogenin in males and produced an intersex 

gonad, there were no adverse effects on reproductive 

capacity or the normalcy of offspring. 

Strengths/Weaknesses: This appears to have been a 

well conducted study. The bisphenol A findings are 

consistent with the work of others, using sensitive 

endpoints in fish such as vitellogenin production. Given 

the nature of the intersex gonad observation, it should be 

considered as adverse even though the severity was not 

sufficient to induce decreases in reproductive capacity 

under the conditions tested. 

Utility (Adequacy) for CERHR Evaluation Process: 

This study indicates that bisphenol A is able to induce 

vitellogenin in male fish and intersex gonads. This study 

exhibited classic dose responses in the affected endpoints. Because this study was conducted in fish, it is not 

useful in the evaluation. 

Lahnsteiner et al. (2005), supported by the Austrian 

Federal Ministry of Agriculture, Forestry, Environment, 

and Water Management, examined the effects of bisphenol A exposure on reproduction of male and female 

brown trout (Salmo trutta f. fario). Fish were caught and 

acclimated for 2 weeks before starting the study. Ten 

males/group and 6 females/group were exposed in a 

flow-through system to bisphenol A at 0 (DMSO vehicle), 

1.75, 2.4, or 5.00 mg/L beginning in the late prespawning 

period and continuing through the remainder of the 

spawning season [No information on purity or culture 

ware was provided]. The bisphenol A concentrations 

selected were said to occur in the Austrian water system. 

Endpoints examined included time point of spawning, 

sperm count and motility, ability of sperm to fertilize 

eggs from non-treated females, and numbers and 

viability of eggs produced by treated females. Statistical 

analyses included ANOVA and Tukey b post-hoc test. 

Throughout the entire spawning period, only 1 male in 

the high bisphenol A dose group produced semen and it 

was of low quality as indicated by significantly reduced 

sperm density, motility rate, swimming velocity, and 

fertility. In the low- and mid-dose groups, sperm density 

was significantly reduced in the early spawning period 

but was not affected in the mid or end part of the 

spawning period. Additional significant effects observed 

in the low-dose group included decreased sperm motility 

in the early spawning period, reduced swimming 

velocity in the early and middle spawning period, and 

increased circular motion and decreased linear motion in 

the middle of the spawning period. In the mid-dose 

group, sperm motility and swimming velocity were 

significantly decreased in the early and mid-spawning 

period, and a significant increase in circular motion and 

a decrease in linear motion occurred in the mid and late 

part of the spawning period. The study authors 

interpreted the sperm effects as representing a 4-week 

delay in spawning. Fertility of males in the low- and middose group was not affected by bisphenol A treatment. 

In females, no eggs were produced by fish in the highdose group. In all other dose groups, there were no 

significant effects on egg volume, viability, mass, mass 

increase during hardening, or on numbers of eggs 

produced by females. However, ovulation was delayed 

by 2 weeks in the low-dose group and by 3 weeks in the 

mid-dose group. The study authors concluded that 

exposure of trout to bisphenol A resulted in negative 

effects on semen and egg quality. 

Strengths/Weaknesses: In this study of fish, alterations in sperm motility were observed consistent with 

those observed in mice. Fertility effects in the female 

were also similar to those observed in other species. 

Weaknesses include a failure to determine the actual 

bisphenol A concentrations in the test system, the narrow 

dose range examined (1.75 to 5 mg/L), and the small 

number of fish/dose level assessed. 

Birth Defects Research (Part B) 83:157–395, 2008
374 

Table 101


ox Studies (Single and Multiple Dose Levels)


Bisphenol A dose level (mg/kg bw/day)

BMD10 BMDL10 BMD1SD BMDL1SD Reference 

CHAPIN ET AL. Single dose Della Seta et al. study (2005) 00 Data Yamasaki et al. presentation (2002a) does not permit modeling 

Ashby et al.

(2003)

Kim et al. (2002b) Nagao et al.

(2002) 

No dose Takahashi and

response Oishi (2001)

Data Yamasaki et al.

presentation (2002a) does notpermitmodeling 

Data

presentationdoes notpermitmodeling 

Summary of High Utility Reproductive T icity 

Model and treatment (doses in

mg/kg bw/day) Endpoint NOAEL LOAEL 

Female

Sprague–Dawley rat (fed kDuration of licking/ 0.04 (single

during pregnancy and grooming pups dose) 

lactation) CD rat (gavage, 40, 200, or Altered estrous cycle Unclear /6 r1000

600/1000 x 28 days) 

Male

Sprague–Dawley rat No effect on daily sperm 200 4200 

(gavaged, 0.020, 0.200, 2, 20, production, sperm count oror 200 x 6 days) reproductive organ weight F344 rats (drinking water No adverse effects reported 11.846 -with 0.011, 0.116, 1.094 or11.846 x 13 weeks)
 

C57BL/6N mouse (gavaged
 No effect on reproductive Z0.200 -

with 0.002, 0.020, or
 organ weight or epididymal0.200 x 6 days)
 sperm count F344 rat (diet 235, 466, 950)
 Histologic alterations in testis o235 235

CD rat gavaged with 40, 200, k Relative ventral prostate 200 000 600/1or 600/1000 x 28 days weight 

m Relative testis weight 200 000 600/1

Birth Defects Research (Part B) 83:157–395, 2008 
BISPHENOL A 375 aDose levels expressed as a mean of the estimated male-female target dose levels.
 bBenchmark doses are shown for the generation with the lowest values.
 m,k Statistically significant increase, decrease compared to controls; 2 no statistically significant effects compared to controls.
 

irth Defects Research (Part B) 83:157–395, 2008 Multigeneration CD rat (gavaged with No significant or dose-related Z0.200 - Ema et al. (2001) 0.0002, 0.002, 0.020, or 0.200 reproductive effects before mating and x 2 generations) Sprague–Dawley rat k F1 epididymal sperm 47.5a 475 317 216 700 469 Tyl et al. (2000b, (dietary with B0.0009, concentration 2002b) 0.018, 0.27, 4.5, 45, or 450 (male) and B0.001, 0.02, 0.3, 5, 50, or 500 (female) x 3 filial generations) k F3 daily sperm production 47.5 475 469 255 524 481 k Live pups/litterb 47.5 475 236 174 376 286 k Pup body weightb 47.5 475 183 163 177 153 Advanced vaginal openingb 47.5 475 394 343 206 176 Advanced F1 preputial 4.75 47.5 466 411 188 163 separation CD-1 mouse [F0 diet with k No. of live pups 840/1055 1669/1988 1116 727 1925 1189 Tyl et al. (2002a) 

B840 or 1669 (male) andB1055 or 1988 (female)] 

k Female pup body weight 2281 1728 2332 1733 

(trend test) 

CD-1 mouse (diet with k F0 epididymal sperm 50 600 567 319 607 549 Tyl et al. (2006) 

B0.003, 0.03, 0.3, 5, 50, or concentration 600 from 6 weeks of age x 2 filial generations) 

m Gestation lengthb 50 600 3619 639 1144 599 

k Relative testis weightb 50 600 562 339 905 605 k Seminal vesicle weight 30 50 405 223 1914 615 

relative to brain weight, F2 

Reproductive assessment by continuous breeding

CD-1 mouse (diet with k Litters/breeding pair 437.5 875 1750 1295 1680 1155 NTP (1985a) 

B437.5, 875, or 1750 over 14­week continuous breedingperiod) 

B
376 Table 102
 o Studies (Single and Multiple Dose Levels)


Bisphenol A dose level (mg/kg bw/day)

LOAEL BMD10 BMDL10 BMD1SD BMDL1SD Reference 

CHAPIN ET AL. 

B40 (single Single dose Funabashi et al.

dose) study (2001, 2004b) 

0.4 Data	 presentation

does notpermitmodeling

Funabashi

et al. (2003)

250 Goloubkova

et al. (2000) 

11 (low dose) 128 128 250 4 5 Park et al. (2004) 

0.05 (low

dose) 

0.5 0.5 only 

0.002 No dose Razzoli et al.

response (2005) 

20 (single Single dose Spencer et al.

dose) study (2002) 

0.0023 mg/L No dose Akingbemi et al.

response (2004)

0.005 Al-Hiyasat et al.

(2002) 

0.025 0.025 

0.0002 0.056	 0.021 0.014 0.0087 Chitra et al.

(2003a) 

Summary of Limited Utility Reproductive T xicity 

Model & treatment (doses in

mg/kg bw/day) Endpoint NOAEL 

Female

Wistar rat (ovariectomized; Altered progesterone receptor mRNA in

s.c. dosed with 

B40 x 1
 different brain regions 

day)


Wistar rat (ovariectomized;
 m Progesterone receptor in brain regions 0.04 

s.c. dosed with 

B0.004,


0.04, 0.4, or 4 s.c., single
dose)


Wistar rat (ovariectomized; k Body weight and body weight gain 

s.c. dosed with 11, 78, 128,or 250 x 7 days) 

m Blotted uterine weight (compared to

ovariectomized controls) 

m Pituitary weight (compared to

ovariectomized controls) 

m Serum prolactin (compared to

ovariectomized controls) 

m Prolactin immunopositive cells in anterior

pituitary

ICR mice, i.p. every 3 days No effect on body weight, uterine or ovarian 5 

over 2 wks histology, or hematological endpoints 

k Blood urea nitrogen k Ovarian weight (right) k Ovarian weight (left) 

Mongolian gerbil, fed 0.002 mSocial sniffing 

o0.002 

or 0.02 from Days 1–21 ofcohabitation

Sprague–Dawley m Uterine wet weight and protein content on

(pseudopregnant; s.c.) Days 1–4 with 

B60% k on Days 5–8 

Male

Long–Evans rat Leydig k Testosterone production; various effects on

cells (cell culture) mechanistic endpoints (e.g., LH-stimulated

and basal testosterone production, mRNAexpression) 

Swiss mouse, gavaged k Body weight	 

with 0.005, 0.025, and0.1 x 30 days 

m Relative testis weightb 0.005 

k Seminal vesicle weightb 0.005 

Wistar rat, gavage for 45 k Relative testis weight 

days with 0.0002, 0.002, or0.02 

Birth Defects Research (Part B) 83:157–395, 2008 
BISPHENOL A 377 

0.0002 0.011 0.0082 0.0069 0.0050 0.0002 0.014 0.0083 0.015 0.0089 5.0 Park et al. (2004) 5.0

0.020 No dose Sakaue et al.

response (2001)

0.020 Data presentation

does notpermitmodeling.

Sakaue et al.

(2001) 

Single dose Takahashi and

study Oishi (2003) 

B200 (single Single dose Takahashi and

dose) study Oishi (2003) 

B400 (single Single dose Takahashi and

dose) study Oishi (2003) 

Single dose Takahashi and

study Oishi (2003) 

20 7 5 9 6 Takahashi and

Oishi (2003)

20 3 2 16 9 235 124 86 171 114 

NTP (1984);

Morrisseyet al., (1989) 

sign icant effects compared to controls.
 if

k Relative epididymis weight k Relative ventral prostate weight 

ICR mice, i.p. every 3 days k Sperm concentration 0.5

over 2 weeks 

m Sperm abnormalities 0.5

Sprague–Dawley rat, k Daily sperm production (absolute and per g o0.020

gavaged with 0.020, 0.200, testis) 2, 20, or 200 x 6 days 

Sprague–Dawley rat, k Daily sperm production (absolute and per g 0.002

gavaged with 0.000002, testis)0.00002, 0.0002, 0.002,0.020, 0.200, or 2 x 6 days 

Wistar or Holtzman SD rat No effect on reproductive organ

(diet) histopathology, daily sperm production,

epididymal sperm reserves, or serumtestosterone

Wistar rat (s.c.) k Terminal body weight, absolute and relative

reproductive organ weight; altered testicularhistopathology

CD-1 (ICR) mouse (diet) m Absolute testis weight, kabsolute

epididymis weight; no effect on testishistopathology, epididymal sperm reserves,daily sperm production, or serumtestosterone

C57BL/6CrSlc mouse No effect on reproductive organ weights; no B400 (single

(diet) effect on testis histopathology, epididymal dose) 

sperm reserves, daily sperm production, orserum testosterone 

Wistar rat, 2 or 20 i.p. 4 k ventral prostate weight 2

days/week x 1 month 

k Serum testosterone 2

k Preputial gland relative weight o235 

Reproductive assessment

by continuous breeding

CD-1 mouse, B2.4, 4.2, or No adverse effects on fertility r8.1 

8.1 over 18-weekcontinuous breedingperiod, s.c. implant 

Birth Defects Research (Part B) 83:157–395, 2008 

a ose levels expressed as a mean of the estimated male-female target dose levels.
 Db enchmark doses are shown for the generation with the lowest values.
 B

m,k Statistically significant increase, decrease compared to controls; 2 no statistically 
378 CHAPIN ET AL. 

Table 103 

Summary of Blood LH and Testosterone Changes in Experimental Animal Studies 

Endpoints/protocol LH effectsa Testosterone effectsa Reference 

High Utility 

Experimental animal studies 

with oral exposure 

Adult male and female rats 2 at 40–1000 mg/kg bw/day 2 at 40–1000 mg/kg bw/day Yamasaki et al. (2002a) 

gavaged for 28 days 

4-week-old male rats fed Not examined 2 at 235–950 mg/kg bw/day Takahashi and Oishi (2001, 

bisphenol A in diet for 44 or or 200 mg/kg bw/day 2003) 

60 days 

Multiple generation gavage k in F0 adult females at 0.0002, 2 Ema et al. (2001) 

dosing study in rats 0.002, and 0.020 mg/kg bw/ 

day but not at high dose 

(0.2 mg/kg bw/day); not 

considered treatmentrelated. 

Experimental animal studies 

with parenteral exposure 

Female lambs i.m. injected at 4– 2 on blood levels during Not examined Evans et al. (2004) 

11 weeks of age; ovariectomy treatment; k pulsatile 

at 9 weeks of age secretion after treatment with 

3.5 mg/kg bw biweekly 

Limited utility 

Experimental animal studies 

with oral exposure 

Male rats gavaged from PND k at 0.0024 mg/kg bw/day but k at 0.0024 mg/kg bw/day but Akingbemi et al. (2004) 

21–35 2 at higher doses (0.010– 2 at higher doses (0.010– 

200 mg/kg bw/day) 200 mg/kg bw/day) 

Male rats gavaged from PND m at 0.0024 mg/kg bw/day 2 at 0.0024 mg/kg bw/day Akingbemi et al. (2004) 

21–90 

4-week-old mice fed bisphenol Not examined 2 at 400 mg/kg bw/day Takahashi and Oishi (2003) 

A through diet for 2 months 

Experimental animal studies 

with parenteral exposure 

4-week-old male rats s.c. dosed Not examined 2 at 200 mg/kg bw Takahashi and Oishi (2003) 

on 4 days/week for 1 month 

4-week-old male rats i.p. Not examined k at 20 mg/kg bw Takahashi and Oishi (2003) 

injected for 1 month 

a

Unless otherwise stated, animals were examined immediately after the treatment period.
 

m,k Statistically significant increase/decrease compared to controls; 2 no statistically significant effects compared to controls.
 

Utility (Adequacy) for CERHR Evaluation Process: This 

study suggests that fish are sensitive to bisphenol Ainduced abnormalities in reproductive endpoints. Because this study was conducted in fish, it is not useful in 

the evaluation. 

Ortiz-Zarragoitia and Cajaraville (2006), supported by 

the European Commission, examined the effects of 

bisphenol A exposure on the reproductive and digestive 

systems of adult blue mussels. For a period of 3 weeks, 

mussels were exposed to bisphenol A in acetone vehicle 

at 0 or 50 ppb [lg/L] [no information on purity or 

culture ware was provided]. Additional compounds 

were also tested but will not be discussed. Ten mussels/ 

sex/group were examined at the end of the exposure 

period. The digestive gland was examined for volume of 

peroxisomes and peroxisomal proliferation. Gonads were 

histologically evaluated and assessed for alkali-labile 

phosphate level, a vitellogenin-like protein that is a 

possible biomarker of endocrine disruption. Statistical 

analyses included ANOVA followed by Duncan post-hoc 

test, Kruskall–Wallis, and Mann–Whitney U test. 

Bisphenol A had no effect on gonadal development, 

gonadal alkali-labile phosphate levels, or digestive gland 

peroxisomal proliferation or peroxisomal volume. However, observations of follicular brown cell aggregates and 

gonadal hemocyte infiltration in 35% of male and female 

mussels indicated severe gamete resorption. 

Strengths/Weaknesses: This study evaluated bisphenol A-induced alterations in several reproductive endpoints in adult mussels. Severe gamete resorption was 

observed. Weaknesses include the failure to confirm 

bisphenol A concentrations in the test water and the use 

of only 1 concentration. 

Utility (Adequacy) for CERHR Evaluation Process: 

Because this study was conducted in the mussel, it is not 

useful in the evaluation. 

4.3 Utility of Reproductive Toxicity Data 

4.3.1 Human. One high utility study of 42 men 

occupationally exposed to bisphenol A diglycidyl ether 

and 42 unexposed men evaluated the relationship 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 379 

Table 104 

Estimates of U.S. General Population Intake of Bisphenol A 

Exposure source Population BPA mg/kg bw/day Notes Source 

Formula Infant 

Breast milk Infant 

Food Infant (0–4 months old) 

Infant (6–12 months old) 

Child (4–6 years old) 

Adult 

Aggregate Child (1.5–5 years old) 

Estimates based on urinary metabolites 

Aggregate Child 

Adult 

0.001 

0.001 

0.0016 

0.0008–0.00165 

0.0012 

0.00037 (canned food) 

0.00048 (canned 

food 1 wine) 

0.00004-0.00007 

0.00007 

0.000026 

Assumes 4.5 kg bw, 700 ml 

formula at 6 mg/L 

BPA (U.S. canned formula max) 

Assumes 4.5 kg bw, 700 ml at 6.3 mg/L 

(U.S. breast milk max) 

European Commission 

European Commission 

European Commission 

Max 5 0.00007–0.00157 

Assumes 50% absorption 

U.S. 6–8-year-old girls 

(max 5 0.00217) 

U.S. population 95th 

percentile 0.0.00159 

Expert Panel 

Expert Panel 

Table 11 Table 14 

Table 11 Table 14 

Table 11 Table 14 

Wilson et al. 

(2003, 2006) 

Table 15 

Table 15 

between urinary levels of bisphenol A and plasma LH, 

FSH, and free testosterone, found reduced FSH levels 

among the exposed men. No fertility endpoints were 

evaluated. Three studies were considered to have low 

utility in the evaluation process due to limitations in 

design and analysis but suggest directions for future 

research. Two of these studies measured serum bisphenol 

A in healthy women, women with polycystic ovary 

syndrome and healthy men and evaluated correlations 

with serum gonadotropins, prolactin, testosterone, and 

other androgens. No fertility endpoints were included in 

these studies. The third study of 37 women found 

significantly lower bisphenol A concentrations among 

women with endometrial cancer and complex endometrial hyperplasia compared to healthy women and 

women with simple hyperplasia. 

4.3.2 Experimental animal. Female reproductive 

toxicity testing using multiple dose levels has been 

evaluated in 2 rat, 1 mouse, and 1 gerbil study. Endpoints 

affected in these studies included brain progesterone 

receptor, estrous cyclicity, resorptions, and social sniffing. Male reproductive toxicity testing using multiple 

dose levels has been evaluated in 7 rat and 2 mouse 

studies. Affected endpoints in males included reproductive organ weight and histology, serum testosterone, 

daily sperm production, sperm motility, sperm concentration, percent pregnant females after mating, and 

females with resorptions after mating. There are 4 

multigeneration tests, 2 in rats and 2 in mice, involving 

gavage or dietary treatments with bisphenol A with dose 

levels as low as 0.0009 mg/kg bw/day. There are also 2 

reproductive assessments by continuous breeding, 1 of 

which involved subcutaneous implants for bisphenol A 

delivery and 1 of which used dietary administration in 

which the lowest dose level was B437.5 mg/kg bw/day. 

4.4 Summary of Reproductive Toxicity Data 

The hypothesis has been advanced that the Charles 

River Sprague-Dawley (SD) rat is insensitive to estrogens 

and other EDCs and therefore it should not be used for 

developmental EDC studies and the studies of the effects 

of BPA that used this strain should be discounted. In 

order to address this important issue Expert Panel 

members reviewed the literature on estrogen-sensitivity 

among rat strains and the following is a summary of our 

findings. 

Different strains of rats show clear, robust reproducible differences in responses to potent estrogens and 

antiandrogens. Several traits have been shown to be 

estrogen sensitive in rats including prolactin regulation 

in the pituitary, thymic involution, uterine pyometra, and 

liver carcinogenesis to name a few. It is evident that the 

SD rat and other rat strains are less sensitive to the effects 

of estrogens than the F344 rat. However, for some traits, 

the reverse is true. In addition, while the SD was less 

sensitive than the F344 to estrogen, the reverse was true 

for sensitivity to tamoxifen. 

The sensitivity to estrogens has been mapped to 

specific chromosomes for several traits. In no case has 

it been demonstrated that the SD strain is completely 

insensitive to any known estrogen. It is evident that 

different traits map to different chromosomes and the 

degree of estrogen sensitivity varies from tissue to tissue, 

likely depending on the tissue-specific gene regulated by 

ER on the chromosome. 

Therefore, one cannot conclude that the SD is 

insensitive to estrogens and the results of BPA studies 

with BPA should be ignored. In fact, there are several 

studies reporting low dose effects that used the SD rat. A 

comparison of the uterotrophic data from the OECD 

study with EE, BPA, and other estrogens does not 

indicate that the SD rat is less sensitive to any estrogen 

versus the Wistar. In this study, oral EE at 1 mg/kg/day 

for 3 days stimulated uterine weight whereas 0.3 mg/kg/ 

day was uterotrophic when administered s.c. In addition, 

in the pubertal female rat assay, EE, the antiestrogen 

tamoxifen and the estrogenic pesticide methoxychlor 

produced equivalent responses in the Long–Evans and 

SD female rats. 

Although some have hypothesized that the Crl: CD 

(SD) rat is more insensitive to estrogens than SD rats 

Birth Defects Research (Part B) 83:157–395, 2008
380 CHAPIN ET AL. 

from other suppliers, there are no data supporting this 

assertion. 

4.4.1 Human. Human reproductive studies are 

summarized in Table 100. A study of 42 men occupationally exposed to an epoxy hardening agent containing 

bisphenol A diglycidyl ether found higher urinary 

bisphenol A concentrations, corrected for creatinine, 

than were found in 42 men who worked in the same 

factory but did not have known exposure to the 

hardening agent (Hanaoka et al., 2002). Differences were 

not detected between the worker groups in plasma 

testosterone or LH, but plasma FSH was significantly 

lower in exposed workers [BPA: 0.043 mg/kg bw] than in 

workers not exposed to the hardening agent [BPA: 

0.021 mg/kg bw]. A significant correlation was noted 

between total urinary bisphenol A concentration and 

decreased FSH when adjusted for age and alcohol intake 

(r 5 0.23, P 5 0.045). 

Two studies from Takeuchi and Tsutsumi (2002) and 

Takeuchi et al. (2004a) suggested a relationship between 

serum bisphenol A concentration and serum testosterone 

(total and free). The first study (Takeuchi and Tsutsumi, 

2002) included women with and without polycystic 

ovary syndrome (POS), and healthy men. Statistically 

significant positive correlations were observed for 

women with and without POS (0.559 for total testosterone and 0.598 for free testosterone, Po0.01), and with all 

participants (0.595 and 0.609, respectively, Po0.001). The 

second study (Takeuchi et al., 2004a) reported only 

cycling women with and without obesity and women 

with POC, with and without obesity, hyperprolactinemia 

and hypothalamic amenorrhea. Statistically significant 

positive correlations were found for bisphenol A and 

total testosterone (r 5 0.391, Po0.001), free testosterone 

(r 5 0.504, Po0.001), androstenedione (r 5 0.684, 

Po0.001), and dehydroepiandrosterone sulfate (DHEAS, 

r 5 0.514, Po0.001). Although these studies used ELISA, 

which may overestimate bisphenol A compared to 

HPLC, significant correlations between bisphenol A 

levels and higher serum testosterone levels were found. 

The authors speculated that androgens either may affect 

bisphenol A metabolism or the reverse. 

A study of 37 women found differences in bisphenol A 

concentrations by health status. Significantly lower mean 

bisphenol A concentrations were found among women 

with endometrial cancer (1.4 ng/ml, n 5 7) and complex 

endometrial hyperplasia (1.4 ng/ml, n 5 9) compared to 

healthy women (2.5 ng/ml, n 5 11) and women with 

simple hyperplasia (2.9 ng/ml, n 5 10) (Hiroi, 2004). 

4.4.2 Experimental Animal. 

Reproductive toxicity studies of high and limited 

utility are summarized in Table 101 and Table 102 

respectively.(single and multiple dose level studies in 

the same utility category are combined within a table). 

Based on reproductive studies using a single dose level, 

the lowest dose level at which an effect was seen in these 

studies was 0.04 mg/kg/day fed to female rats during 

pregnancy and lactation and resulting in a decreased 

duration of licking/grooming pups (Della Seta et al., 

2005). This study of neural and behavioral effects is 

shown here for convenience but has been included with 

other studies focused on these endpoints for further 

discussion in Section 3. 

For high utility female reproductive studies using 

multiple doses, the lowest effect level, for altered estrous 

cycle, was Z600 mg/kg bw/day by gavage in rat for 28 

days (Yamasaki et al., 2002a). For high utility male 

reproductive studies, the lowest effect level, for histologic alterations in the testis, was 235 mg/kg bw/day by 

gavage in rat for 28 days (Takahashi and Oishi, 2001). The 

value of the histologic observations may be limited due 

to the fixation and embedding techniques employed, 

raising some concern over the validity of this endpoint. 

The reproductive assessments by continuous breeding 

included a study using very high-dose levels (NTP, 

1985a), and this study is not the most informative for 

reproductive risk assessment. In a multigeneration study, 

CD rats did not show statistically significant or doserelated reproductive effects over 2 generations with 

bisphenol A gavage doses of 0.0002, 0.002, 0.020, or 

0.200 mg/kg bw/day (Ema et al., 2001). In Sprague– 

Dawley rats treated for 3 generations, adverse reproductive effects consisted of decreased F1 epididymal sperm 

concentration, decreased F3 daily sperm production, 

decreased live pups/litter, decreased pup body weight, 

and delayed vaginal opening at an average dose level of 

475 mg/kg bw/day. Delayed preputial separation was 

seen in F1 and F2 males at an average dose level of 

47.5 mg/kg bw/day (Tyl et al., 2000a, 2002b). In CD-1 

mice given bisphenol A for 2 generations in the diet at 

dose levels as low as B0.003 mg/kg bw/day, the most 

sensitive effect was a reduction in F2 seminal vesicle 

weight relative to brain weight at 50 mg/kg bw/day. 

Effects on F0 epididymal sperm concentration, gestation 

length, and relative testis weight occurred at 600 mg/kg/ 

day, the next highest dose level (Tyl et al., 2006). 

A summary of LH and testosterone effects observed in 

bisphenol A-exposed experimental animals and in humans are included in Table 103. 

Data sufficiency statement for human data: In 

summary, there are insufficient data to evaluate whether 

bisphenol A causes male or female reproductive toxicity 

in humans. However, several studies collectively suggest 

hormonal effects, including one study of exposed male 

workers likely to have multiple routes of exposure 

including inhalation (Hanaoka et al., 2002). 

Data sufficiency statement for animal data: In 

summary, the experimental animal literature was assessed for its utility (high utility, limited utility, or no 

utility) based on the criteria established by this Expert 

Panel, including an evaluation of experimental design 

and statistical procedures. Studies with high and limited 

utility were further grouped according to female and 

male reproductive toxicity, their use of single or multiple 

dose levels, a multigenerational exposure paradigm, and 

the measurement of various hormonal endpoints. Greater weight was given to studies using the oral route of 

exposure, because of evidence that oral exposure 

predominates in humans and that target tissue exposure 

to parent compound (bisphenol A) is very low after oral 

exposure and first-pass metabolism as compared to 

subcutaneous or other routes of exposure. 

There is sufficient evidence in rats and mice 

that bisphenol A causes female reproductive toxicity, 

characterized as delayed vaginal opening with subchronic or chronic oral exposure NOAELs of 47.5 mg/kg bw/ 

day and a LOAEL of 475 mg/kg bw/day (Tyl et al., 

2002b). 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 381 

There is sufficient evidence in rats and mice that 

bisphenol A causes male reproductive toxicity, 

characterized as delayed preputial separation, with 

subchronic or chronic oral NOAEL of 4.75 mg/kg bw/ 

day and a LOAEL of 47.5 mg/kg bw/day (Tyl et al., 

2002b). 

There is inconsistent evidence in rats and mice that 

bisphenol A alters testosterone and gonadotropin levels 

in males after oral postnatal exposure. 

There is inconsistent evidence in male and female mice 

that bisphenol A produces aneugenic effects in germ 

cells after exposure. 

5.0 SUMMARIES, CONCLUSIONS, AND 

CRITICAL DATA NEEDS 

5.1 Developmental Toxicity 

No data on the effects of human developmental 

exposure to bisphenol A are available. There is a large 

literature describing studies in rodents and some work in 

other species. A large experimental animal literature was 

reviewed, assessed for its utility, and weighed based on 

the criteria established by this Panel. 

From the rodent studies we can conclude that bisphenol A: 

*	 Does not cause malformations or birth defects in 

rats or mice at levels up to the highest doses 

evaluated: 640 mg/kg/day (rats) and 1250 mg/kg/ 

day (mice). 

*	 Does not alter male or female fertility after gestational 

exposure up to doses of 450 mg/kg bw/day in the rat 

and 600 mg/kg bw/day in the mouse (highest dose 

levels evaluated). 

*	 Does not permanently affect prostate weight at doses 

up to 475 mg/kg/day in adult rats or 600 mg/kg/day 

in mice. 

*	 Does not cause prostate cancer in rats or mice after adult 

exposure at up to 148 or 600 mg/kg/day, respectively. 

*	 Does change the age of puberty in male or female rats 

at high doses (ca. 475 mg/kg/day). 

Rodent studies suggest that bisphenol A: 

*	 Causes neural and behavioral alterations related to 

disruptions in normal sex differences in rats and mice. 

(0.01–0.2 mg/kg/day). 

The data on bisphenol A are insufficient to reach a 

firm conclusion about: 

*	 A change in the onset of puberty in male rats or mice 

at doses up to 475–600 mg/kg/day. 

*	 An acceleration in the age of onset of puberty at a low 

dose in female mice at 0.0024 mg/kg/day, the only 

dose tested. 

*	 Whether Bisphenol A predisposes rats toward 

prostate cancer or mice toward urinary tract 

deformations. 

5.2 Reproductive Toxicity 

There are insufficient data to evaluate whether bisphenol A causes male or female reproductive toxicity in 

humans. A large experimental animal literature was 

reviewed, assessed for its utility, and weighted based on 

the criteria established by this expert panel, including an 

evaluation of experimental design and statistical procedures. These animal data are assumed relevant for the 

assessment of human hazard. 

Female effects: There is sufficient evidence in rats and 

mice that bisphenol A causes female reproductive 

toxicity with subchronic or chronic oral exposures with 

a NOAEL of 47.5 mg/kg bw/day and a LOAEL of 

Z475 mg/kg bw/day. 

Male effects: There is sufficient evidence in rats and 

mice that bisphenol A causes male reproductive toxicity 

with subchronic or chronic oral exposures with a NOAEL 

of 4.75 mg/kg bw/day and a LOAEL of Z47.5 mg/kg 

bw/day. 

5.3 Human Exposures 

Bisphenol A is FDA-approved for use in polycarbonate 

and epoxy resins that are used in consumer products 

such as food containers (e.g., milk, water, and infant 

bottles) food can linings (Staples et al., 1998; SRI, 2004) 

and in dental materials (FDA, 2006). Resins, polycarbonate plastics, and other products manufactured from 

bisphenol A can contain trace amounts of residual 

monomer and additional monomer may be generated 

during breakdown of the polymer (European-Union, 

2003). 

Environmental Exposures: Bisphenol A emitted 

from manufacturing operations is unlikely to be present 

in the atmosphere in high concentrations. However, it 

was found in 31–44% of outdoor air samples with 

concentrations of oLOD (0.9) to 51.5 ng/m3 (Wilson 

et al., 2006). Indoor air samples found concentrations 

r29 ng/m3 (Rudel et al., 2001, 2003; Wilson et al., 2003). 

Limited U.S. surface water sampling found bisphenol A 

in 0–41% of samples ranging from o0.1 to 12 ug/L 

(Kolpin et al., 2002; Boyd et al., 2003). Twenty-five to 

100% of indoor dust samples contained bisphenol A with 

concentrations of odetectable to 17.6 mg/g (Rudel et al., 

2001, 2003; Wilson et al., 2003, 2006). 

Exposures Through Food: The highest potential for 

human exposure to bisphenol A is through products that 

directly contact food such as food and beverage containers with internal epoxy resin coatings and through the 

use of polycarbonate tableware and bottles, such as those 

used to feed infants (European-Union, 2003). Studies 

examining the extraction of bisphenol A from polycarbonate infant bottles in the U.S. found concentrations 

o5 mg/L. Canned infant formulas in the U.S. had a 

maximum levels of 13 mg/L in the concentrate that 

produced a maximum of 6.6 mg/L when mixed with 

water (FDA, 1996; Biles et al., 1997a). Breast milk studies 

in the U.S. have found up to 6.3 mg/L free bisphenol A in 

samples (Ye et al., 2006). Measured bisphenol A 

concentrations in canned foods in the U.S are o39 mg/ 

kg (FDA, 1996; Wilson et al., 2006). Limited drinking 

water sampling in the U.S. indicates that bisphenol A 

concentrations were all below the limit of detection 

(o0.1 ng/L) (Boyd et al., 2003). 

Biological Measures of Bisphenol A in Humans: The panel finds the greatest utility in studies of 

biological samples that use sensitive and specific 

analytical methods (LC-MS or GC-MS) and report quality 

control measures for sample handling and analysis. The 

panel further focused on biological monitoring done in 

Birth Defects Research (Part B) 83:157–395, 2008
382 CHAPIN ET AL. 

U.S. populations. In the U.S, adult urine concentrations 

of free bisphenol A are o0.6 mg/L and total bisphenol A 

concentrations are o19.8 mg/L (Calafat et al., 2005; Liu 

et al., 2005; Ye et al., 2005). The 95th percentile total 

bisphenol A concentration for 394 adult volunteers 

(males and females; 20–59 years old) from the NHANES 

III survey was 5.18 mg/L (Calafat et al., 2005). Girls age 

6–9 in the U.S. have concentrations of total bisphenol A 

o54.3 mg/L, with median concentrations ranging from 

1.8–2.4 mg/L (Liu et al., 2005; Wolff et al., 2006). No U.S. 

studies have examined blood or semen concentrations of 

bisphenol A. Amniotic fluid total bisphenol A concentrations in the U.S are o1.96 mg/L. Dental sealant exposure 

to bisphenol A occurs primarily with use of the dental 

sealant bisphenol A dimethylacylate. This exposure is 

considered an acute and infrequent event with little 

relevance to estimating general population exposures. 

Bisphenol A Intake Estimates: The panel found that 

previous oral intake estimates for infants fed formula 

and breast milk did not use levels reported for the U.S. 

population, so the panel estimated intake based on 

typically-used parameters. The panel found the food 

intake estimates made by the European Commission 

(2002) used concentrations of bisphenol A comparable to 

U.S. food concentrations in their intake estimates, so have 

included these estimates as well (Table 104). Estimates 

from duplicate diets in U.S. children (Wilson et al., 2003, 

2006) found lower bisphenol A concentrations in foods 

than those estimated by the European Commission, 

therefore the aggregate estimates of intake by Wilson 

et al. were somewhat lower than those estimated by the 

European Commission. However, the aggregate intake 

estimates by Wilson et al. (2003, 2006) are in line with the 

estimates based on urinary metabolite measurements for 

children described above. 

Estimates of intake based on occupational air concentrations of bisphenol A from U.S. powder paint workers 

suggest exposures up to 100 ug/kg bw/day (USEPA, 

1988). Estimates of intake based on urinary metabolite 

levels among Japanese workers spraying epoxy coatings 

resulted in a mean estimate of exposure of 0.043 mg/kg 

bw/day (o0.002 pg to 0.45 mg/kg bw/day) (Hanaoka 

et al., 2002). 

5.4 Overall Conclusions 

The panel spent a considerable amount of time 

attempting to interpret and understand the inconsistent 

findings reported in the ‘‘low dose’’ literature for 

bisphenol A. Conducting low dose studies can be 

challenging because the effects may be subtle and small 

in magnitude and therefore more difficult to statistically 

distinguish from background variability. The inherent 

challenge of conducting these types of studies may be 

exacerbated with bisphenol A because the endpoints of 

concern are endocrine-mediated and potentially impacted by factors that include phytoestrogen content of 

the animal feed, extent of bisphenol A exposure from 

caging or water bottles, and the alleged sensitivity of the 

animal model to estrogens. The Panel believed that highdose studies are less susceptible to these types of 

influences because the toxicologic response should be 

more robust and less variable. While the Panel did not 

necessarily expect a specific effect to display a monotonic 

dose response (e.g., consistently increasing organ size), 

many members of the panel expected the high-dose 

studies with bisphenol A to detect some manifestation of 

toxicity (e.g., altered weight, histopathology) in tissues 

reported to be affected at low doses even if the 

study could not replicate the reported low dose effect. 

There are several large, robust, well designed studies 

with multiple dose groups using several strains of rats 

and mice and none of these detected any adverse 

reproductive effects at low to moderate dosage levels 

of BPA administered via the relevant route of human 

exposures. Further, none of these studies detected 

changes in prostate weight, age at puberty (rat), 

pathology or tumors in any tissue, or reproductive tract 

malformations. For this reason, Panel members gave 

more weight to studies that evaluated both low- and 

high-doses of bisphenol A compared to low-dose-only 

studies in cases where the target tissues were comparably 

assessed. 

Every chemical that produces low dose cellular and 

molecular alterations of endocrine function also produces 

a cascade of effects increasing in severity resulting in 

clearly adverse alterations at higher doses, albeit the 

effects can be different from those seen at low doses. With 

these endocrine disrupters, but not BPA, the low dose 

effects are often causally linked to the high-dose adverse 

effects of the chemical. This is true for androgens like 

testosterone and trenbolone, estrogens like DES, 17bestradiol and ethinyl estradiol, xenoestrogens like methoxychlor and genistein, and antiandrogens like vinclozolin, for example. Hence, the failure of BPA to produce 

reproducible adverse effects via a relevant route of 

exposure, coupled with the lack of robustness of the 

many of the low dose studies (sample size, dose range, 

statistical analyses and experimental design, GLP) and the 

inability to reproduce many of these effects of any adverse 

effect strains the credibility of some of these study results. 

They need to be replicated using appropriate routes of 

exposures, adequate experimental designs and statistical 

analyses and linked to higher dose adverse effects if they 

are to elevate our concerns about the effects of BPA on 

human health. The lack of reproducibility of the low dose 

effects, the absence of toxicity in those low-dose-affected 

tissues at high-doses, and the uncertain adversity of the 

reported effects led the panel to express ‘‘minimal’’ 

concern for reproductive effects. 

In contrast, the literature on bisphenol A effects on 

neural and behavioral response is more consistent with 

respect to the number of ‘‘positive’’ studies although it 

should be noted that the high-dose studies that proved to 

be the most useful for evaluating reproductive effects did 

not adequately assess neural and behavioral responses. 

In addition, even though different investigators assessed 

different neural and behavioral endpoints, the Panel 

concluded that the overall findings suggest that bisphenol A may be associated with neural changes in the 

brain and behavioral alterations related to sexual 

dimorphism in rodents. For this reason, the Panel 

expressed ‘‘some’’ concern for these effects even though 

it is not clear the reported effects constitute an adverse 

toxicological response. 

Concerns are expressed relative to current estimates of 

general population exposure levels in the U.S. 

1.	 For pregnant women and fetuses, the Expert Panel has 

different levels of concern for the different 

Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A 383 

developmental endpoints that may be susceptible to 

bisphenol A disruption, as follows: 

*	 For neural and behavioral effects, the Expert Panel 

has some concern; 

*	 For prostate effects, the Expert Panel has minimal 

concern; 

*	 For the potential effect of accelerated puberty, the 

Expert Panel has minimal concern; and 

*	 For birth defects and malformations, the Expert 

Panel has negligible concern. 

2.	 For infants and children, the Expert Panel has the 

following levels of concern for biological processes 

that might be altered by Bisphenol A, as follows: 

*	 Some concern for neural and behavioral effects; 

and 

*	 Minimal concern for the effect of accelerated 

puberty. 

3.	 For adults, the Expert Panel has negligible concern for 

adverse reproductive effects following exposures in 

the general population to Bisphenol A. For highly 

exposed subgroups, such as occupationally exposed 

populations, the level of concern is elevated to 

minimal. 

5.5 Critical Data Needs 

1.	 Neural and behavioral endpoints. A concerted 

effort is needed to better understand the effects of 

gestational and lactational exposure to bisphenol A on 

maternal behavior and offspring brain structure 

and behavior. This effort should include 

molecular and cellular studies to ascertain the 

sensitivity of the developing brain to bisphenol Ainduced structural and biochemical alterations. The 

association between bisphenol A and neural and 

behavioral endpoints should also be examined in 

longitudinal studies of pregnancy and child development in humans. 

2.	 Human exposure assessment. Additional data are 

needed to clarify bisphenol A exposures and internal 

dosimetry in the general population, newborns, and 

occupationally-exposed individuals. Available data 

demonstrate that a large fraction of children and 

adults have detectable levels of bisphenol A metabolites in their urine. What are needed are duplicate diet 

studies to identify in detail the sources and routes of 

exposure of bisphenol A. For example, while research 

suggests diet is the major source of BPA for U.S. 

infants and young children, the detailed analysis of 

BPA levels has primarily focused on polycarbonate 

baby bottle leachates and canned food. The contributions of non-canned food and drinking water 

routes of exposure for U.S. youth and adults not 

occupationally-exposed to BPA remain unknown and 

in need of further study. Levels of BPA in residential 

drinking water wells and community water sources 

have not been systematically studied. Also unknown 

is the impact of landfill leachates on levels of 

bisphenol A in U.S. drinking well waters and whether 

chlorinated congeners of bisphenol A are found in 

U.S. municipal water supplies. Finally, more measurement are needed of free and total bisphenol A, its 

glucuronide conjugate, and other metabolite 

concentrations from maternal, fetal, and neonatal 

tissues or fluids (i.e., placenta, amniotic fluid, breast 

milk, urine, serum). These data would provide insight 

into the roles of metabolism and exposure route on 

internal dose. 

3. Human studies relating adult exposure to reproduction and development, including effects on hormone 

levels. 

4.	 Physiologically-based pharmacokinetic (PBPK) models.

PBPK models are needed to facilitate the interpretation and applicability of animal studies, including 

rodents and nonhuman primates, for human risk 

assessment. 

5.	 Effects on prostate and mammary gland development.

Additional data are needed to understand the 

susceptibility to disruption of prostate and 

mammary gland development in humans and 

animals by bisphenol A exposure. Laboratory animal 

studies should initially focus on the oral route of 

exposure and should be informed by any new 

knowledge about human exposure and human 

internal dosimetry. A particular data need is an 

improved understanding of the biology of PIN 

(prostatic intraepithelial neoplasia) in animal 

models and its relationship to prostate cancer. 

Similarly, bisphenol A-induced alterations in mammary gland development and their potential relationship to mammary cancer should be investigated 

across a broad range of internal concentrations and 

external doses. 

6.	 Altered puberty. The robustness and biologic basis for 

altered puberty following bisphenol A exposure 

should be evaluated in mouse, rat, and gerbil. In 

laboratory animals, this evaluation should be performed following combined gestational and lactational exposure, and following pubertal exposure 

alone, and should include an assessment of any 

changes in hormonal responsivity at later ages, and 

all related to internal and tissue concentrations of 

bisphenol A. In addition, longitudinal cohort studies 

examining the potential modulation by bisphenol A 

of the onset, progression, and control of puberty in 

humans should be performed. 

7.	 Biological mechanism for low-dose-only effects. Most 

useful would be data that provided a biologicallyplausible explanation for effects that appear at low 

doses but not higher doses. This might involve the 

membrane-bound estrogen receptor and its possible 

activation by bisphenol A. 

8. More work directed toward urinary tract morphological and histologic changes after developmental 

exposure would be helpful to determine the robustness and relevance of the limited report of these 

effects in one study. 

9.	 Inter-laboratory replication of studies. Inter-laboratory 

replication of critical findings is a sine qua non for 

enhancing confidence in experimental results. Such 

studies should be supported by funding agencies, 

and should be facilitated by the open sharing of 

experimental details and approaches. The future 

reproducibility should also be considered by investigators as they design their studies. 

Birth Defects Research (Part B) 83:157–395, 2008
384 CHAPIN ET AL. 

10.	 Critical design components for all future research on BPA:

a.	 Appropriate experimental design and statistical analysis, especially accounting for litter effects; 

b. Appropriate	 route (oral) of exposure. Studies with 

non-oral route of administration should include 

internal dose measurements of free BPA; 

c.	 Multiple dose groups ranging from low to high; 

d. Linkage of effects to adverse effects; and 

e.	 Relevant endpoints, with biologically plausible outcomes especially for estrogen-mediated effects on 

reproduction and behavior. 

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III –.1

Appendix III

Appendix III. Public Comments and Peer Review ReporT

Public comments received during the NTP-CERHR evaluation of bisphenol A and the peer review report

for the draft NTP Brief on Bisphenol A are available on the CERHR website at http://cerhr.niehs.nih.gov/

chemicals/bisphenol/bisphenol.html.