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EFSA Journal 2010; 8(9):1829

SCIENTIFIC OPINION

Scientific Opinion on Bisphenol A: evaluation of a study investigating its


neurodevelopmental toxicity, review of recent scientific literature on its
toxicity and advice on the Danish risk assessment of Bisphenol A1
EFSA Panel on food contact materials, enzymes, flavourings
and processing aids (CEF)2, 3

European Food Safety Authority (EFSA), Parma, Italy

ABSTRACT
Bisphenol A (BPA) is used in the manufacture of plastics, to produce reusable drinking bottles, infant feeding
bottles and other food storage containers. EFSA was asked to evaluate a dietary developmental neurotoxicity
study in rats (Stump, 2009) and recent scientific literature (2007-2010) in terms of relevance for the risk
assessment of BPA. The impact of these studies on the current Tolerable Daily Intake (TDI) of 0.05 mg BPA/kg
body weight (b.w.)/day as set by EFSA in 2006 was assessed. Advice on the Danish risk assessment underlying
the Danish ban of BPA in food contact materials for infants aged 0-3 years is included. Overall, based on this
comprehensive evaluation of recent toxicity data, the Panel on food contact materials, enzymes, flavourings and
processing aids (CEF) concluded that no new study could be identified, which would call for a revision of the
current TDI. This TDI is based on the No-Observed-Adverse-Effect-Level (NOAEL) of 5 mg/kg b.w./day from a
multi-generation reproductive toxicity study in rats, and the application of an uncertainty factor of 100. This
factor is regarded as conservative based on all information on BPA toxicokinetics. The Panel noted that some
studies conducted on developing animals have suggested other BPA-related effects of possible toxicological
relevance, in particular biochemical changes in brain, immune-modulatory effects and enhanced susceptibility to
breast tumours. These studies had several shortcomings. At present the relevance of these findings for human
health cannot be assessed. Should any new relevant data become available in the future, the Panel will reconsider
this opinion. A minority opinion is expressed by a Panel member and presented in an Annex to this opinion.

© European Food Safety Authority, 2010

KEY WORDS
Bisphenol A, BPA, CAS No. 00080-05-7, developmental toxicity, neurobehaviour, low dose effects.

1
On request from the European Commission, Questions No EFSA-Q-2009-00864, EFSA-Q-2010-01023 and EFSA-Q-
2010-00709, adopted on 23rd September 2010.
2
Panel members: Arturo Anadón, Mona-Lise Binderup, Wilfried Bursch, Laurence Castle, Riccardo Crebelli, Karl-Heinz
Engel, Roland Franz, Nathalie Gontard, Thomas Haertlé, Trine Husøy, Klaus-Dieter Jany, Catherine Leclercq, Jean-Claude
Lhuguenot, Wim Mennes, Maria Rosaria Milana, Karla Pfaff, Kettil Svensson, Fidel Toldrá, Rosemary Waring, Detlef
Wölfle. Part of this Opinion is not shared by a member of the Panel, Catherine Leclercq, who expressed a Minority
Opinion related to parts II and IV (Overall conclusions) of this opinion. Correspondence: CEF-unit@efsa.europa.eu
3
Acknowledgement: The Panel wishes to thank the members of the Working Groups on Bisphenol A: David Bell (resigned
on 15 January 2010), Wilfried Bursch, Berit Granum, Ulla Hass (her participation was limited to the evaluation of the
Stump study until 26 March 2010), Edel Holene, Trine Husøy, Alberto Mantovani, Wim Mennes, Unni Cecilie Nygaard,
Terry Parker, Ulrike Reuter, Emanuela Testai, Rosemary Waring, Detlef Wölfle for the preparatory work on this scientific
opinion and EFSA staff: Anna F. Castoldi, Saghir Bashir, Laura Ciccolallo, Cristina Croera, Tomas Oberg, Roberta Pinalli
Anne Theobald and Didier Verloo for the support provided to this scientific opinion.

Suggested citation: EFSA Panel on food contact materials, enzymes, flavourings and processing aids (CEF). Scientific
Opinion on Bisphenol A: evaluation of a study investigating its neurodevelopmental toxicity, review of recent scientific
literature on its toxicity and advice on the Danish risk assessment of Bisphenol A. EFSA Journal 2010;8(9):1829. [110 pp.]
doi:10.2903/j.efsa.2010.1829. Available online: www.efsa.europa.eu/efsajournal.htm

© European Food Safety Authority, 2010 1


Bisphenol A

SUMMARY
Bisphenol A [2,2-bis(4-hydroxphenyl)propane, CAS Number 80-05-7] (BPA) is used as a monomer in
the manufacture of polycarbonates and epoxy resins, as an antioxidant in PVC plastics and as an
inhibitor of end polymerisation in PVC. Polycarbonates are used in food contact plastics such as
reusable beverage bottles, infant feeding bottles, tableware (plates and mugs) and storage containers,
whereas epoxy resins are used in protective linings for food and beverage cans and vats.

Small amounts of BPA can potentially leach out from food containers into foodstuffs and beverages
and therefore be ingested. BPA is permitted for use in food contact plastics in the European Union
with a specific migration limit of 0.6 mg/kg food4.

In 2006, EFSA set the TDI for BPA at 0.05 mg BPA/kg body weight (b.w.)/day. This is based on the
No-Observed-Adverse-Effect-Level (NOAEL) of 5 mg/kg b.w./day that has been identified in two
multi-generation reproductive toxicity studies in rodents, where the critical effects were changes in
body and organ weights in adult and offspring rats and liver effects in adult mice, respectively (EFSA,
2006). In 2008, EFSA reaffirmed this TDI, concluding that age-dependent toxicokinetics differences
of BPA in animals and humans would have no implication for the default uncertainty factor (UF) of
100 and in turn for the TDI.

The present opinion follows the requests of the European Commission (EC) to the Panel on food
contact materials, enzymes, flavourings and processing aids (CEF) (I) to evaluate a dietary
developmental neurotoxicity study of BPA in rats (Stump, 2009); (II) recent scientific literature (2007-
2010) in terms of relevance for the risk assessment of BPA and impact on the current tolerable daily
intake (TDI) of 0.05 mg BPA/kg body weight (b.w.)/day; (III) to provide advice on the Danish risk
assessment underlying the Danish ban of BPA in food contact materials for infants aged 0-3 years.

In order to provide a global view on the risk assessment of Bisphenol A, the CEF Panel decided to
address the three mandates as given above in a single opinion and postponed the adoption of this
comprehensive document to 23rd September 2010.

The three different questions raised by the Commission are dealt with in three different parts (PARTS
I to III) of the opinion. PART IV presents an overview of the conclusions from PARTS I to III,
together with an overall conclusion.

PART I

The GLP compliant study by Stump (2009) was performed according to OECD guideline 426 to
address any uncertainty regarding potential neurodevelopmental effects of BPA. BPA was
administered daily in the diet at concentrations of 0, 0.15, 1.5, 75, 750, and 2250 mg/kg feed to female
Sprague-Dawley rats from gestational day (GD) 0 to postnatal day (PND) 21. The relative estimated
intakes (in mg/kg b.w./day) were 0, 0.01, 0.12, 5.85, 56.4 and 164 during gestation and 0, 0.03, 0.25,
13.1, 129 and 410 during lactation. The CEF Panel considers this treatment schedule as relevant to
human exposure in utero and via either breastfeeding or infant bottle feeding (in this study the
estimated exposure of rat pups to BPA is ca. 30 times higher than that of bottle-fed infants). Dams
were evaluated for general signs of toxicity and offspring were evaluated for general toxicity including
developmental landmarks and for neurological effects, including behaviour and brain histopathology.

For systemic toxicity (i.e. decreased body weight and/or body weight gain), a NOAEL of 5.85 mg/kg
b.w./day during gestation was identified for both mothers and offspring.

Male and female pups were tested for developmental landmarks and for neurological effects, including
behaviour and brain histopathology.

4
Commission Directive 2002/72/EC of 6 August 2002 relating to plastic materials and articles intending to come into contact
with foodstuffs
EFSA Journal 2010; 8(9):1829 2
Bisphenol A

On PND 11 2 and 4 pups from the 750 and 2250 mg/kg feed groups, respectively, underwent popcorn
seizures and convulsions. This effect was not reproducible in any other published study/report
including a follow-up study by Stump (2009). The Panel concluded that the current NOAEL for BPA
(5 mg/kg b.w./day) would be sufficiently low to exclude any concern for this effect.

The study by Stump covers motor activity, learning and memory (spatial behaviour), auditory startle
response, brain histopathology and morphology. The study does not cover some specific aspects of
learning and memory (i.e. avoidance learning, schedule-controlled behaviour, and impulsiveness),
anxiety-related behaviour or sexual dimorphic behaviour, but this does not invalidate the study. No
treatment-related changes were observed in motor activity tests, auditory startle response or brain
histopathology and morphology.

According to the statistical analysis by the study authors, in the Biel maze swimming test for learning
and memory on PND 62 the male offspring from the 0.15 mg/kg feed exposure group showed an
increase in the number of errors in Path A trials 1-4, which could be interpreted as a delay in learning.
The effect reached statistical significance when compared to concurrent controls. No such an effect
was observed in pups studied on PND 22 or in any other exposure group on PND 62. When the
swimming trials were conducted in the reverse path (i.e. the path B) no delay in learning was observed
and also in the repeat Path A trials 11 and 12, no effect was observed in any exposure group, including
the 0.15 mg/kg feed group. Therefore, the authors concluded that there were no changes in learning
and memory.

The Panel noted that in the Biel Maze test only the error counts were reported as a measure of learning
and memory. The animals should have also been evaluated for effects on “time-to-escape” and
checked for long term memory effects. EFSA’s Assessment and Methodology Unit (AMU) applied a
more appropriate statistical evaluation to the Biel Maze data. It was realised that the data suffer from
censoring5. Based on the re-analysis the Panel considered that no conclusion can be drawn from this
study on the effect of BPA on learning and memory behaviour due to large variability in the data.

Based on the body weight effects on dams and offspring and also taking into account the occurrence of
seizures and convulsions in the two highest dose groups, which were not observed at the lower dose
levels, the study supports the NOAEL which was derived from multigeneration studies in the past (5
mg/kg b.w./day), leading to a TDI of 0.05 mg/kg b.w./day. However, the Panel considered that this
test on learning and memory was inconclusive and is of limited value in the risk assessment of BPA.

Overall, based on the body weight effects on dams and offspring and taking into account the
occurrence of seizures and convulsions only at the two highest doses, the study supports the previously
identified NOAEL of 5 mg/kg b.w./day. However, the Panel considered that this study was
inconclusive and is of limited value in the ongoing risk assessment of BPA.

PART II

The CEF Panel has reviewed toxicological data published between 2007 and July 2010 mainly
focusing on toxicokinetic, human and animal toxicity studies. For risk assessment purposes only
studies complying with these inclusion criteria were considered: full research papers in peer-reviewed
journals available in the public domains and reporting original data; all human studies (except for
purely biomonitoring studies). For the in vivo animal toxicity studies the focus was on low dose oral
studies employing several test doses including at least one <5 mg/kg b.w./day and involving
developmental exposure.

5
In statistical terminology censoring occurs when the value of an observation is only partially known. In the Biel water maze
test the number of errors made by the rats that did not complete the maze within the 3 minute-limit that was used, was
recorded and the time to escape was recorded as 180 seconds. These experiments are “right censored”, meaning that the rats
could have escaped and made more errors, had they been given sufficient time. Therefore the time to escape and the number
of errors recorded will have been underestimated.
EFSA Journal 2010; 8(9):1829 3
Bisphenol A

These studies were further assessed with respect to quality criteria (sufficient sample size, adequacy
of control procedures, inclusion of positive controls when applicable, assessment of correlation
between morphological and functional changes, and consideration of litter or dam as the appropriate
statistical unit) in order to assess the validity and/or applicability of the individual findings to human
risk assessment.

Studies on toxicokinetics of BPA have demonstrated a significantly lower internal exposure after oral
intake as compared to parenteral exposure. This confirms that toxicity studies with oral administration
have higher relevance for human risk assessment of BPA in food than studies with parenteral
administration. In addition, new findings in non human primates (both adults and newborns) further
strengthen the view that BPA is eliminated faster in humans than in rodents. This fast BPA elimination
in primates results in substantially lower internal exposure to free BPA in humans as compared to
rodents. Even human premature infants can metabolise and excrete BPA efficiently (via
glucuronidation and sulfation), as supported by recent human data and data in young monkeys. The
use of the standard uncertainty factor (UF) of 10 to take into account interspecies differences is
therefore considered quite conservative.

In relation to in utero exposure, studies on transplacental transport of BPA and BPA-glucuronide in


humans and rodents indicated that although transfer may occur, foetal free BPA levels are highly
limited by the efflux pump P-glycoprotein, and placental BPA glucuronidation may also take place.
The Panel noted that exposure to total BPA (the major constituent being the glucuronide) through
lactation is limited to a very low fraction. Therefore, in utero exposure and exposure through lactation
appear to be limited.

The Panel recognises that inter-individual differences occur in expression of the isoenzymes
responsible for the detoxification of BPA. However, even in persons with low expression of these
enzymes, the metabolising capacity is still sufficient to eliminate free BPA from blood at the low
levels of BPA resulting from consumer exposure. Dietary exposures of adults and infants aged 3-6
months were estimated to be up to 1.5 and 13 µg/kg body weight per day, respectively (EFSA, 2006),
based on conservative estimates of food consumption and migration from food contact materials.
These exposures are not anticipated to surpass the metabolic capacity for BPA in adults or infants.

Recent epidemiological studies have suggested some statistically significant associations of BPA
exposure (urine concentrations) and health effects (coronary heart disease, reproductive disorders) in
adults and behavioural changes in young girls. The Panel noted that cross sectional epidemiological
studies such as these can demonstrate statistical associations between BPA exposure and the presence
(e.g. coronary heart disease) or absence (e.g. cancer, asthma) of health outcomes, but the inherent
design of cross sectional studies does not allow establishment of a causal relationship between BPA
exposure and health effects (e.g. chronic diseases). In addition, the Panel has identified some
limitations in these studies, which raise further questions as to the significance of the reported
findings. Therefore, the Panel could not draw any relevant conclusion for risk assessment from these
studies.

The animal studies on developmental and reproductive toxicology reporting effects at doses lower
than 5 mg/kg b.w./day have severe shortcomings and were considered to be invalid. The Panel
considers that the valid studies do not raise concern regarding reproductive and developmental toxicity
of BPA at doses lower than 5 mg/kg b.w./day.

Potentially significant biochemical changes, e.g. altered receptor expression in different brain regions
(see section 5.3), have been reported. However, in the absence of a correlation with a functional
adverse effect, the relevance of these observations for human health cannot be assessed. The impact of
BPA on development of sexually dimorphic behaviour was addressed in the study by Ryan et al.
(2010a), who observed a male-like reduced saccharin preference and inhibition of lordosis behaviour
in female rat offspring from oestrogen-treated but not from BPA-treated dams. In the study reported
by Stump et al. (2009) (See Part I) the effects of BPA on learning and memory behaviour were

EFSA Journal 2010; 8(9):1829 4


Bisphenol A

inconclusive due to large variability in the data. Other recent studies have methodological
shortcomings. The Panel does not consider the currently available data as convincing evidence of
neurobehavioural toxicity of BPA.

The study by Jenkins et al. (2009) is the first oral study on a possible BPA-induced enhancement of
sensitivity of the mammary gland to carcinogen-induced breast tumour formation in rat offspring
following lactational BPA exposure of pups. Using the same model of dimethylbenzanthracene
(DMBA)-induced mammary carcinogenesis but in utero BPA exposure, Betancourt et al. (2010b) also
reported an enhancement of susceptibility for mammary gland carcinogenesis. In consideration of the
shortcomings in the design of both studies, in particular the uncertainty regarding the lactational as
well as in utero exposure of the offspring to BPA, and of the limitations in reporting the Panel
concluded that these results cannot be taken into consideration for derivation of a TDI. However, the
Panel noted that at the highest dose level studied there is a shift of the ratio between cell proliferation
and apoptosis in favour of cell multiplication in the mammary gland. In view of the mechanistic data
obtained upon in utero exposure in other studies (see section 5.3) and the implications of an increased
cell proliferation/apoptosis ratio in carcinogenesis, the effects reported by Jenkins and Betancourt
deserve further consideration.

Modulation of immune system-related parameters is also an emerging field also in BPA research.
Several studies have reported changes in cytokines, changes in T-cell populations and other aspects of
immune modulation. However, the studies were all suffering from shortcomings in experimental
design and reporting. Therefore, at the moment, these studies cannot be taken into consideration for
derivation of a TDI.

In vitro- and in vivo-studies (not compliant with the selection criteria in section 3) on receptors,
hormones, immune system, cell proliferation, apoptosis, proteomic, genomic and epigenetic changes
have been presented to compile recent data on potentially relevant endocrine mechanisms of action of
BPA. High doses of BPA (>5 mg/kg b.w./day) may have biochemical and molecular effects consistent
with those observed with other oestrogenic substances. Effects have been claimed to occur at low
levels of BPA exposure, which could be independent of the classical hormone receptors. BPA has only
weak binding affinities to these receptors, but these effects may alternatively be induced by cell
membrane-triggered signalling pathways via protein kinases. However, in the absence of clear dose
response curves and due to the shortcomings in experimental design, a conclusion cannot be reached
on the implications of the observed biochemical and molecular changes or establish whether they have
any impact on human health. Because of the lack of a common clearly defined mode of action of BPA
at low doses, the toxicological relevance of the BPA effects described cannot be evaluated and the
results cannot be taken into consideration for derivation of a TDI. While low dose effects of BPA are
reported for some biochemical changes the Panel is not aware of any clearly reproducible adverse
effect expressed specifically at low BPA doses only.

EFSA has established an internal task force to initiate the development of a common strategy towards
endocrine active substances. The Panel is aware of EFSA’s ongoing work to monitor trends and
developments in the assessment of health risks of endocrine active substances.

PART III

This part deals with the EFSA advice on the Danish risk assessment of BPA. The conclusion of the
DTU Food Institute is based upon three major lines of arguments: (i) a degree of uncertainty with
regard to the effects on learning ability, since in the study by Stump et al (2009) impaired learning
ability was found in male offspring with low dosage of BPA; (ii) doubts on the monotonic (“normal”)
dose-response for BPA; (iii) some endpoints which have not been considered, namely certain aspects
of learning and memory (avoidance learning, schedule-controlled behaviour, impulsiveness), anxiety-
related behaviour and gender-specific (i.e., sexually dimorphic) behaviour.

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Bisphenol A

With respect to the learning and memory endpoint of the Stump study, as examined in the Biel Maze
test, the Panel concluded that the influence of BPA on learning and memory behaviour cannot be
evaluated. Regarding this endpoint, the study is inconclusive and cannot be used for the risk
assessment of BPA.

It has been argued that BPA may show a non-monotonic dose-response curve. Low dose effects of
BPA have been reported, which might be independent of the effect on the classical hormone receptors
(See Part II). However, most of these studies have several shortcomings such as lack of dose responses
and limitations in experimental design. The Panel is not aware of any clearly reproducible adverse
effect expressed specifically at low BPA doses only.

Altogether, the Panel concluded that the study by Stump et al. (2009) cannot be used for the risk
assessment of BPA, because of large variability in the data. Therefore, the study is inconclusive and
cannot impact on the risk assessment of BPA. Methodological shortcomings also apply to a number of
studies addressing other neurobehavioural endpoints (e.g. learning and memory behaviour, anxiety-
related behaviour and gender-specific behaviour), which were considered invalid or inadequate for
risk assessment purposes. The Panel does not consider the currently available data sufficiently
indicative of neurobehavioural toxicity as an endpoint of concern for BPA.

Overall, based on the comprehensive evaluation of recent human and animal toxicity data, the Panel
concluded that no new study could be identified, which would call for a revision of the current TDI of
0.05 mg/kg b.w./day. This TDI is based on the NOAEL of 5 mg/kg b.w./day from a multi-generation
reproductive toxicity study in rats, and the application of an uncertainty factor of 100, which is
regarded as conservative based on all information on BPA toxicokinetics.

The Panel noted that some studies conducted on developing animals have suggested other BPA-related
effects of possible toxicological relevance, in particular biochemical changes in brain, immune-
modulatory effects and enhanced susceptibility to breast tumours. These studies had many
shortcomings. At present the relevance of these findings for human health cannot be assessed, though
should any new relevant data become available in the future, the Panel will reconsider the current
opinion.

A minority opinion expressed by a Panel member is presented in an Annex to this opinion.

EFSA Journal 2010; 8(9):1829 6


Bisphenol A

TABLE OF CONTENTS
Abstract .................................................................................................................................................... 1 
Summary .................................................................................................................................................. 2 
Table of contents ...................................................................................................................................... 7 
Background as provided by the European Commission........................................................................... 9 
Terms of reference as provided by the European Commission ................................................................ 9 
Interpretation of the terms of reference by the EFSA ............................................................................ 10 
Part I – Evaluation of the dietary developmental neurotoxicity study of Bisphenol A in rats by Stump
(2009) ........................................................................................................................................ 11 
Part I - Assessment ................................................................................................................................. 11 
1.  Introduction ............................................................................................................................... 11 
1.1.  Previous risk assessments ..................................................................................................... 12 
1.2.  Discussion of five developmental neurotoxicity studies, highlighted in previous evaluations . 15 
2.  Assessment of a newly available developmental neurotoxicity study in rats............................ 17 
2.1.  Summary of the study as reported by Stump (2009)............................................................. 17 
2.2.  The Panel’s comments to the study design and results ......................................................... 19 
2.2.1.  F0 data (Maternal generation) .......................................................................................... 19 
2.2.1.1.  Detailed Clinical Observations ................................................................................ 20 
2.2.2.  F1 data (Litter) .................................................................................................................. 20 
2.2.2.1.  Postnatal survival, growth and developmental landmarks ....................................... 20 
2.2.2.2.  Detailed Clinical Observations (Subset A) .............................................................. 20 
2.2.2.3.  Motor Activity (Subset A) ....................................................................................... 21 
2.2.2.4.  Auditory Startle Test (Subset A).............................................................................. 21 
2.2.2.5.  Biel Maze Swimming Trials (Subsets A and B) ...................................................... 21 
2.2.2.6.  Macroscopic Examinations ...................................................................................... 25 
2.2.2.7.  Neuropathology (Subsets A and C) ......................................................................... 25 
2.3.  Discussion of the study outcome .......................................................................................... 26 
Part I - Conclusions ................................................................................................................................ 27 
Part I - Documentation provided to EFSA ............................................................................................. 29 
Part I - References .................................................................................................................................. 30 
Part I - Abbreviations ............................................................................................................................. 33 
Part I - Appendices ................................................................................................................................. 34 
Part II - Review of recent scientific literature on the toxicity of Bisphenol A ....................................... 38 
Part II - Assessment................................................................................................................................ 38 
3.  Introduction ............................................................................................................................... 38 
4.  Toxicokinetics ........................................................................................................................... 40 
4.1.  New pharmacokinetic studies in rats and monkeys (Doerge et al. 2010a, 2010b) ............... 41 
4.2.  The enzymes involved in BPA biotransformation ................................................................ 43 
4.3.  In utero exposure and kinetics .............................................................................................. 44 
4.4.  Neonatal exposure and kinetics............................................................................................. 47 
4.5.  Human physiologically based pharmacokinetic (PBPK) modelling ..................................... 49 
4.6.  BPA repeated exposure ......................................................................................................... 51 
4.7.  Summary and conclusions on toxicokinetics ........................................................................ 52 
5.  Toxicity ..................................................................................................................................... 52 
5.1.  Human studies ....................................................................................................................... 52 
5.1.1.  Braun et al. (2009) ............................................................................................................ 52 
5.1.2.  Melzer et al. (2010) .......................................................................................................... 54 
5.1.3.  Other human studies ......................................................................................................... 55 
5.1.4.  General comments on human studies ............................................................................... 59 
5.1.4.1.  Analytical aspects .................................................................................................... 59 
5.1.4.2.  Comment on cross-sectional studies ........................................................................ 60 
5.1.5.  Conclusions on human studies ......................................................................................... 61 
5.2.  Animal toxicity studies ......................................................................................................... 61 
5.2.1.  Reproductive and developmental toxicity ........................................................................ 62 

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Bisphenol A

5.2.1.1.  Developmental studies by Howdeshell at al. (2008) and Ryan et al. (2010a) ......... 63 
5.2.1.2.  Other developmental toxicity studies....................................................................... 64 
5.2.2.  Conclusions on animal toxicity studies ............................................................................ 70 
5.2.2.1.  Studies on developmental and reproductive toxicity ............................................... 70 
5.2.2.2.  Developmental neurotoxicity and neurodevelopmental studies .............................. 70 
5.2.2.3.  Cell proliferation and apoptosis related to enhancement of tumourigenesis ........... 70 
5.2.2.4.  Studies on immunotoxicity ...................................................................................... 70 
5.3.  Other information on the endocrine-mediated action of BPA .............................................. 70 
5.3.1.  Effects on receptors and hormone homeostasis ................................................................ 71 
5.3.2.  Effects on signal transfer and gene expression ................................................................. 73 
5.3.3.  Effects on the immune system .......................................................................................... 74 
5.3.4.  Cytogenetic and epigenetic effects, cell proliferation and apoptosis ................................ 74 
5.3.5.  Conclusions on the endocrine-mediated action of BPA ................................................... 76 
Part II - Conclusions ............................................................................................................................... 76 
5.4.  Conclusions on toxicokinetics .............................................................................................. 77 
5.5.  Conclusions on human studies .............................................................................................. 77 
5.6.  Conclusions on studies in animals ........................................................................................ 78 
5.6.1.  Studies on developmental and reproductive toxicity ........................................................ 78 
5.6.2.  Developmental neurotoxicity and neurodevelopmental studies ....................................... 78 
5.6.3.  Cell proliferation and apoptosis related to enhancement of tumourigenesis .................... 78 
5.6.4.  Studies on immunotoxicity ............................................................................................... 78 
5.7.  Conclusions on endocrine-mediated action of BPA ............................................................. 79 
Part II - References ................................................................................................................................. 80 
Part II - Abbreviations ............................................................................................................................ 94 
Part III - Advice on the Danish risk assessment of Bisphenol A ........................................................... 96 
Part III - Assessment .............................................................................................................................. 96 
6.  Introduction ............................................................................................................................... 96 
7.  Conclusions of the Danish risk assessment ............................................................................... 96 
8.  Discussion of the Panel on the Danish risk assessment ............................................................ 97 
Part III - Conclusions ............................................................................................................................. 98 
Part III - References ............................................................................................................................... 99 
Part III - Abbreviations......................................................................................................................... 100 
Part IV - Overall Panel conclusions ..................................................................................................... 101 
9.  Background ............................................................................................................................. 101 
10.  Conclusions from PART I: Evaluation of the dietary developmental neurotoxicity study of
Bisphenol A in rats .................................................................................................................. 102 
11.  Conclusions from PART II: Review of recent scientific literature on the toxicity of Bisphenol
A .............................................................................................................................................. 103 
11.1.  Conclusions on toxicokinetics ............................................................................................ 104 
11.2.  Conclusions on human studies ............................................................................................ 104 
11.3.  Conclusions on studies in animals ...................................................................................... 105 
11.3.1.  Studies on developmental and reproductive toxicity ...................................................... 105 
11.3.2.  Developmental neurotoxicity and neurodevelopmental studies ..................................... 105 
11.3.3.  Cell proliferation and apoptosis related to enhancement of tumourigenesis .................. 105 
11.3.4.  Studies on immunotoxicity ............................................................................................. 105 
11.4.  Conclusions on endocrine-mediated action of BPA ........................................................... 105 
12.  Conclusions from PART III: Advice on the Danish risk assessment of Bisphenol A ............ 106 
13.  Overall conclusion................................................................................................................... 107 
Part IV - References ............................................................................................................................. 108 
Part IV - Abbreviations ........................................................................................................................ 110 

EFSA Journal 2010; 8(9):1829 8


Bisphenol A

BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION


The substance 2,2-bis(4-hydroxyphenyl)propane, CAS Number 80-05-7, more commonly known as
bisphenol A (BPA), is used as a monomer in the manufacture of polycarbonates and epoxy resins, as
an antioxidant in PVC plastics and as an inhibitor of end polymerisation in PVC. Polycarbonates are
used in food contact materials such as reusable beverage bottles, infant feeding bottles, tableware
(plates and mugs) and storage containers. Epoxy resins are used in protective linings for food and
beverage cans and vats.

BPA was first evaluated in 1984 by the Scientific Committee on Food (SCF, 1986) for use in plastic
materials and articles intended to come into contact with foodstuffs. At that time, the Committee
allocated a Tolerable Daily Intake (TDI) of 0.05 mg/kg b.w.. This was based on 90-day and long-term
oral studies in rats and mice, in which BPA was given via the diet. These showed an overall no-
observed-adverse-effect level (NOAEL) of 25 mg/kg b.w./day for effects on body weight, taken from
the 90-day rat study, to which an uncertainty factor of 500 was applied because of the incomplete
database.

The substance was re-evaluated in 2002 by the SCF in the light of new information published up to
2001. The Committee allocated a temporary TDI (t-TDI) of 0.01 mg/kg b.w.. This was based on 90-
day and comprehensive three-generation studies in the rat, in which BPA was given via the diet. These
showed an overall no-observed-adverse-effect level (NOAEL) of 5 mg/kg b.w./day for effects on body
weight, taken from the comprehensive three-generation study in the rat, to which an uncertainty factor
of 500 was applied because of the incomplete database. The SCF recommended that the t-TDI be
reviewed once significant new data were available.

The substance was re-evaluated in 2006 by EFSA in light of the new information published up to
2005. EFSA has, in its opinion of 26 November 2006, established for BPA (BPA) a tolerable daily
intake (TDI) of 0.05 milligram per kilogram (mg/kg) body weight based on the no adverse effect level
of 5 mg/kg body weight in rodent studies.

A new opinion on the toxicokinetics of Bisphenol was adopted by EFSA on 9 July 2008. In this
opinion, the AFC Panel considered that its previous risk assessment based on the overall NOAEL for
effects in rats and using a default uncertainty factor of 100 can be regarded as conservative for
humans. The Panel concluded that the differences in age-dependent toxicokinetics of BPA in animals
and humans would have no implication for the assessment of BPA carried out by EFSA in 2006.

Health Canada in its risk assessment issued on 18 April 2008 concluded on neurobehavioural toxicity
that even though the weight of evidence supporting neurobehavioural effects in rodents at exposure
below the established NOAEL for reproductive/developmental toxicity is limited, the outcome of the
existing studies warrant the application of precaution for sensitive life stages such as pregnant
women/foetus and infants. Based on this evaluation the government of Canada proposed legislation
banning the use of polycarbonate in baby feeding bottles.

In 2008 the Polycarbonate/BPA Global Group of the American Chemistry Council commissioned a
neurobehavioural study to address the concerns raised by the Canadian Government. The results of
this study have now been transmitted to the European Commission by Plastics Europe in the context of
Article 11(5) of Regulation (EC) No 1935/2004.

TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION


In October 2009 the Commission asked the European Food Safety Authority (EFSA) to:

- Assess the relevance of the dietary developmental neurotoxicity study of Bisphenol A in rats
and its implication for hazard and risk assessment of Bisphenol A.

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Bisphenol A

- Update, if necessary, the currently applicable tolerable daily intake for Bisphenol A by May
2010.

In relation to the above request, the European Commission clarified in a letter dated 3 February 2010
that EFSA should address any new scientific evidence that may affect the conclusions of the
previously adopted opinions on BPA by May 2010.

On 30 March 2010, EFSA received an additional request from the European Commission to advise by
15 April 2010 on the Danish risk assessment underlying the Danish ban, on 26 March 2010, of
Bisphenol A in food contact materials for infants aged 0-3 years.

INTERPRETATION OF THE TERMS OF REFERENCE BY THE EFSA


In order to provide a global view on the risk assessment of Bisphenol A, the CEF Panel decided to
address the three mandates received from the European Commission in a single opinion and postponed
the adoption of this comprehensive document to September 2010.

The present opinion consists of four distinct parts:

PART I: Evaluation of the dietary developmental neurotoxicity study of Bisphenol A in rats

PART II: Review of recent scientific literature on the toxicity of Bisphenol A

PART III: Advice on the Danish risk assessment underlying the Danish ban of Bisphenol A in food
contact materials for infants aged 0-3 years.

PART IV: Overall conclusions

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Bisphenol A

PART I – EVALUATION OF THE DIETARY DEVELOPMENTAL NEUROTOXICITY STUDY OF


BISPHENOL A IN RATS BY STUMP (2009)

PART I - ASSESSMENT

1. Introduction
Bisphenol A (BPA; 2,2-bis(4-hydroxyphenyl)propane, CAS Number 80-05-7) is primarily used as a
monomer in the manufacture of polycarbonate (PC) and epoxy resins. Regarding food contact
applications, polycarbonate is used to make food containers such as reusable beverage bottles, infant
feeding bottles, tableware (plates and mugs), microwave ovenware and storage containers including
reservoirs for water dispensers. Epoxy resins are used to make protective coatings and linings for food
and beverage cans and vats. BPA is permitted for use in food contact materials in the European Union
(EU) with a specific migration limit of 0.6 mg/kg food, under Commission Directive 2002/72/EC of 6
August 2002 relating to plastic materials and articles intended to come into contact with foodstuffs. It
is also permitted for food contact use in other countries such as the USA and Japan. Another field of
potential use of BPA is in water distribution networks for human consumption. BPA-based epoxy-
phenolic resins are widely used as a surface-coating on residential drinking water storage tanks (Bae et
al., 2002). For use of BPA in materials in contact with drinking water no legislation at the EU level is
available. Individual Member States may have their own national legislation in force.

BPA is also used in a variety of non-food applications: epoxy resin based paints, PVC medical
devices, wood fillers, adhesives, surface coatings, printing inks, carbonless and thermal paper, flame
retardants, tyre and brake fluid manufacture, resin-based composites and sealants used in dentistry
(EFSA, 2006). PC finds wide application in construction materials, light fittings and other non food
consumer products.

As a result of food contact uses, consumers may be exposed to BPA via their diet. Small amounts of
BPA can potentially leach out from food containers into foodstuffs and beverages and can therefore be
ingested (EFSA, 2006; EC, 2003; 2008). Based on calculations with a EU environmental exposure and
risk assessment model (EUSES), for the general population the main route of human exposure to BPA
is the oral route (EC, 2003; 2008).

There is a concern that BPA has potential endocrine-disrupting properties, which may adversely
impact physical, neurological and behavioural development. BPA is considered as a weak oestrogen-
like agent (NTP-CERHR, 2008), and a multitude of biological effects has been postulated for BPA
(see also Part II of this Opinion). Toxicokinetic data indicate that BPA is rapidly absorbed from the
gastrointestinal tract, and that formation of BPA-glucuronide is the major pathway of BPA
biotransformation in primates and in rats. Conjugation of BPA is considered a deactivation reaction
since BPA-conjugates are devoid of endocrine activity. Major differences in disposition of BPA-
glucuronide have been reported in primates, including humans, and rodents due to different pathways
of elimination from the liver. In humans, 80% of the ingested BPA is excreted in the urine within 5
hours. In rodents, enterohepatic circulation of BPA takes place and this causes a slower clearance of
BPA from the organism than in humans (the half-life in rodents is 19-78 hrs; EFSA, 2006).

The toxicology of BPA has been extensively investigated by industry, government and academic
research groups in short and long term animal tests, including several reproductive toxicity studies,
multi-generation exposure studies and a cancer bioassay. The acute toxicity of BPA is low through all
exposure routes relevant to humans (EC, 2003). Repeated oral toxicity studies have indicated the
intestine, liver, kidney and reproductive system as the possible target organs/systems for toxicity in
laboratory animals at doses ranging from 25 to ≥500 mg/kg b.w./day (EFSA, 2006; NTP-CERHR,
2007). BPA was shown not to be genotoxic in bacteria and in mammalian cells, or tumourigenic in
mice or rats (NTP, 1982) exposed for life to high doses of BPA by dietary administration (EC, 2003;
EFSA, 2006).

EFSA Journal 2010; 8(9):1829 11


Bisphenol A

There is evidence that prenatal and/or lactational exposure to “high” BPA doses (i.e., those causing
maternal toxicity) can adversely affect the development of laboratory animals, reducing survival, birth
weight, and growth of offspring early in life, and modify the onset of puberty in males and females
(EFSA, 2006; NTP-CERHR, 2008). Other studies in rodents reported that also “low” BPA doses (i.e.,
those that are more relevant to human environmental exposure) administered during development can
produce a number of effects including neural and behavioural alterations in rats and mice. However,
the validity of these studies as well as the implications of these “low” dose behavioural for human
health is heavily debated (EFSA, 2006; NTP-CERHR, 2008).

1.1. Previous risk assessments


In recent years, several international agencies and bodies have comprehensively reviewed the
available toxicological studies on BPA (AIST, 2005; SCF; 2002; EC, 2003; 2008; EFSA, 2006; 2008;
Health Canada, 2008; NTP-CERHR, 2007; 2008, AFSSA, 2010a, U.S. FDA, 2010a).The current
opinion was partly presented as a draft at a meeting in Parma with experts from Member States on 26
March 2010. It was also communicated to the U.S. Federal Drug Administration (FDA), the Food
Standards Australia New Zealand (FSANZ), the New Zealand Food Safety Authority (NZFSA), the
World Health Organisation (WHO) and Health Canada.

In 1984, the Scientific Committee on Food (SCF, 1986) evaluated BPA for use in plastic materials and
articles intended to come into contact with foodstuffs. The Committee set a Tolerable Daily Intake
(TDI) of 0.05 mg/kg b.w.. This was based on 90-day and long-term dietary studies in rats and mice6.
An overall no-observed-adverse-effect level (NOAEL) of 25 mg/kg b.w./day was identified for effects
on body weight (90-day rat study), to which an uncertainty factor (UF) of 500 was applied because of
the incomplete database.

In 2002, BPA was re-evaluated by the SCF. The SCF considered the overall oral NOAEL for BPA to
be 5 mg/kg b.w./day, based on the results of a comprehensive three-generation study in the rat,
showing significant reductions in adult body weight and pup body and organ weights (Tyl et al.,
2002). A temporary TDI of 0.01 mg BPA/kg b.w./day was derived by applying an UF of 500 (100 for
inter- and intra-species differences, and 5 for uncertainties in the database) to the NOAEL of 5 mg/kg
b.w./day.

In 2003, the European Union published a comprehensive Risk Assessment Report (EU-RAR) for BPA
from all sources of exposure in the context of Council Regulation (EEC) No. 793/93 on the evaluation
and control of existing substances. The key health effects of BPA through different exposure routes
were considered to be eye and respiratory tract irritation, skin sensitisation, repeated dose toxicity to
the respiratory tract, effects on the liver and reproductive toxicity (effects on fertility and on
development). With respect to human health risks, a need for further research was identified, to
resolve the uncertainties surrounding the potential for BPA to produce adverse effects on neurological
and neurobehavioural development at low doses (EC, 2003). Several studies addressing these
endpoints are discussed in section 1.2.

In 2005, the Japanese National Institute of Advanced Industrial Science and Technology (AIST)
published a comprehensive human health risk assessment report based on a thorough review of the
toxicological profile of BPA combined with estimates of human exposure. The key toxicological
endpoints for human health risks posed by BPA were reduction in body weight gain, effects on the
liver and reproductive toxicity. For all three endpoints the NOAEL or the calculated Benchmark Dose
Lower Limit (BMDL) were in the range of 5-50 mg/kg b.w./day. Comparison of the most realistic
exposure estimates with the NOAELs and BMDL led to Margins of Exposure (MOEs) in the range of
85,000-1,800,000. The MOEs were also sufficiently large (>1,000) for adults and children when more

6
SCF (1986) refers to CIVO (currently TNO-voeding, Zeist, The Netherlands) report number R 6229, november, 1979. This
report was not available to the CEF Panel.
EFSA Journal 2010; 8(9):1829 12
Bisphenol A

conservative, worst-case exposure estimates were used for the comparison. AIST concluded that the
current BPA exposure levels were unlikely to pose unacceptable risks to human health.

In 2006, on a request from the European Commission, EFSA re-assessed BPA for use in food contact
materials, focusing on its effects on reproduction and the endocrine system. The Scientific Panel on
Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (AFC) confirmed
the validity of the overall NOAEL of 5 mg BPA/kg b.w./day from a comprehensive three generation
study in the rat (Tyl et al., 2002), taken as the key study by the SCF in 2002. This NOAEL was further
supported by the results of a newer two-generation reproductive toxicity study in mice, which also
provided a NOAEL of 5 mg/kg b.w./day for liver effects (Tyl et al., 2007; same study as Tyl et al.,
2006, cited in EFSA, 2006). Because the database concerning reproduction and development had been
considerably strengthened since the 2002 SCF report, the Panel considered that the additional UF of 5
was no longer needed. Thus, the AFC Panel set a full TDI for BPA at 0.05 mg BPA/kg b.w., by
applying a default UF of 100 to the overall NOAEL of 5 mg/kg b.w./day. This revised UF was
considered to be conservative in view of the well-described species differences in toxicokinetics,
showing a low systemic concentration of free BPA in humans compared with rats. The Panel found
that intakes of BPA through food and drink were well below the TDI, even for infants and children
(EFSA, 2006).

In April 2008, the EU-RAR (EC, 2008) was updated after submission and evaluation of the two-
generation reproductive study in mice by Tyl et al. (2007) along with the new data on human exposure
and effects of BPA that had become available since the original risk assessment report was completed
(EC, 2003). The Rapporteur (UK) came to the conclusion that there was no need for further
information and/or testing and for risk reduction measures beyond those which were already being
applied. However, Denmark, Sweden and Norway considered that the results of four
neurodevelopmental studies (Adriani et al., 2003; Carr et al., 2003; Negishi et al., 2004; Ryan and
Vandenbergh, 2006) warranted further consideration (EC, 2008). These studies will be addressed in
more detail in part I, section 1.2. In June 2008, the Norwegian Scientific Committee for Food Safety
(VKM) issued an opinion regarding the assessment of the same four studies (Adriani et al., 2003; Carr
et al., 2003; Negishi et al., 2004; Ryan and Vandenbergh, 2006) as highlighted in the EU-RAR (EC,
2008), on developmental neurotoxicity of BPA following low dose exposure. The VKM concluded
that these studies did not provide sufficient evidence for setting a robust lower NOAEL for BPA than
that previously reported by EFSA, i.e. 5 mg/kg b.w./day. In order to eliminate any uncertainty
regarding potential developmental effects of BPA at low doses, the VKM recommended that a GLP
compliant study should be carried out according to OECD guideline 426, using a broad concentration
range from the very low doses up to those with known maternal effects. The new study evaluated in
Part I of the current opinion is in compliance with this recommendation.

In its opinion of July 2008, EFSA further assessed the possible age-dependent toxicokinetics of BPA
in animals and humans and their implications for hazard and risk assessment of BPA in food, taking
into account the most recent information and data available. The AFC Panel considered the exposure
of a human foetus to free BPA as negligible, due to the increased maternal capacity for conjugation of
BPA with glucuronide during pregnancy, whereas the foetal rat would be exposed to free BPA from
the maternal circulation (due to decreased glucuronidation capacity). The AFC Panel also concluded
that human neonates would have sufficient capacity to biotransform BPA to hormonally inactive
conjugates at the BPA exposure levels reported in the EFSA opinion of 2006 and the EU-RAR (EC,
2003; 2008). Because of these metabolic differences, exposure to free BPA, and therefore toxicity,
would be greater in rats of any age than in humans on an equivalent dose basis. The Panel concluded
that the differences in age-dependent toxicokinetics of BPA in animals and humans would have no
implication for its earlier risk assessment and reaffirmed the TDI value of 0.05 mg/kg b.w./day
(EFSA, 2008) and the conservative nature of the uncertainty factor used (100).

In August 2008, the Health Canada’s Food Directorate reported in its assessment of BPA from food
packaging applications that, based on the overall weight of evidence, the current dietary exposure to

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Bisphenol A

BPA through food packaging uses was not expected to pose a health risk to the general population,
including newborns and young children (Health Canada, 2008). Health Canada did not revise the
provisional tolerable daily intake (pTDI) for BPA of 0.025 mg/kg b.w./day which had been set by
them in 1996 based on the lowest NOEL for general toxicity parameters of 25 mg/kg b.w./day
observed in a 90-day study in rats (NTP, 1982). However, Health Canada recommended that the
general principle of ALARA (as low as reasonably achievable) should be applied to limit BPA
exposure from food packaging applications of newborns and infants. This advice was based on the
results of a number of neurodevelopmental and behavioural studies in experimental animals, which
suggest a heightened sensitivity during stages of development in rodents. At the same time, these
studies presented interpretational uncertainties related to methodological concerns, raising questions as
to the actual significance of these findings in the assessment of the potential risk of BPA to human
health. Health Canada concluded that although highly uncertain, these data sets suggest the need for
more focused attention on products consumed by newborns and infants. In 2009, the Health Canada’s
Food Directorate issued a statement (Health Canada, 2009) endorsing the same conclusions on the
safety of BPA first drawn by them in 2008. Nevertheless, due to uncertainties in the
neurodevelopmental database the Government of Canada reiterated the need to limit BPA exposure of
infants and young children from food packaging applications, i.e. from pre-packaged infant formula
products.
In September 2008, the U.S. NTP-CERHR (National Toxicology Program - Center for the Evaluation
of Risks to Human Reproduction) published a monograph on the potential human reproductive and
developmental effects of BPA. This included the NTP Brief and the Expert Panel Report that had been
published the year before (NTP-CERHR, 2007). The NTP expressed some concern (“Some” concern
is the midpoint on a five-level scale, ranging from “negligible” to “serious”) for effects on the brain,
behaviour, and prostate gland in foetuses, infants, and children at current human exposures to BPA.
The NTP had minimal concern for effects on the mammary gland and for an earlier age for puberty for
females, in foetuses, infants, and children at current human exposure levels to BPA. NTP expressed
negligible concern that exposure of pregnant women to BPA will result in fetal or neonatal mortality,
birth defects, or reduced birth weight and growth in their offspring. NTP also expressed negligible
concern that exposure to BPA will cause reproductive effects in non-occupationally exposed adults
and minimal concern for workers exposed to higher levels in occupational settings.

On 15 October 2009, EFSA received a request from the European Commission to assess the relevance
of a new study on possible neurodevelopmental effects of BPA (Stump, 2009) and, if necessary, to
update the existing TDI accordingly. The study in question was commissioned by the American
Chemistry Council to address concerns raised by the Canadian government. The study was conducted
according to OECD guideline 426 as recommended by the VKM in 2008. This study is discussed in
depth in Part I, section 2.

After the release of the previous EFSA opinions on BPA by the former Panel on additives,
flavourings, processing aids and materials in contact with food (AFC Panel) and the Panel on food
contact materials, enzymes, flavourings and processing aids (CEF Panel), several hundreds of new
papers on BPA were published, which required consideration. An update review on these new studies
is presented in Part II of this Opinion.

During the preparation of this opinion, the U.S. FDA (2010b), the Danish DTU Food (2010), the
French Food Safety Agency (AFSSA, 2010a, b) and the German Institute for Risk Assessment (BfR,
2010) published updates on BPA, including comments on the study by Stump (2009). The Danish risk
assessment is discussed in Part III of this opinion, as requested in the terms of reference of this
opinion.

In January 2010, the U.S. FDA (2010b) stated that standard toxicity studies had thus far supported the
safety of low level exposure of humans to BPA. However, FDA had some concern for human
development in terms of potential neurobehavioural and reproductive effects that had been reported in
some novel approach-based studies testing for subtle effects. At the same time, FDA emphasised

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Bisphenol A

substantial uncertainties on the overall interpretation and implications of these data for human health
and informed that new in-depth research was being performed to answer key questions about the risk
assessment of BPA.

The AFSSA (February and May 2010) examined the Stump (2009) study as well and did not point out
any neurotoxic effect on the litters at the low dose levels. However, AFFSA identified some concern
for convulsions at the two highest dose levels. This effect will be addressed in the discussion on this
study in Part I, section 2, as well as in Part II.

1.2. Discussion of five developmental neurotoxicity studies, highlighted in previous


evaluations
Previous evaluations have addressed developmental neurotoxicity as an issue for discussion. The
discussion has focussed on four studies (Adriani et al., 2003; Carr et al., 2003; Negishi et al., 2004;
Ryan and Vandenbergh, 2006), which according to three Nordic countries, should be considered a
reason for concern as mentioned in a minority opinion in the EU-RAR (EC, 2008). The following
paragraphs present a discussion of EFSA and EU evaluations with focus on these four studies.
Broadly, the studies indicated that BPA at low doses might affect anxiety-related behaviour in female
offspring but not in males (Adriani et al., 2003 and Ryan and Vandenbergh, 2006). In addition,
maternal BPA exposure during gestation and lactation might cause behavioural alterations consistent
with decreased impulsiveness/facilitated learning (schedule-controlled test) or learning deficits (active
avoidance test) in male offspring (Adriani et al., 2003 and Negishi et al., 2004). In the EFSA (2006)
opinion, it was also mentioned that in a fifth study (Kubo et al., 2003) changes in the size of the locus
coeruleus in the brain stem were reported, which preliminary result needed further support. This study
is also discussed in the paragraphs below. A table summarising the data and the experimental details
from these five studies is given in Part I, Appendix A.

Two of the studies (Adriani et al., 2003 and Negishi et al., 2004) have already been reviewed by the
AFC Panel of EFSA (EFSA, 2006). The AFC Panel stated that “there were no consistent treatment-
related effects in the behavioural endpoints and apparently contradictory observations were
published”. The AFC Panel indicated limitations in the various studies considered, such as lack of
assessment of dose-response in several studies, partial lack of information on blinding of the
investigators with respect to the treatments of the animals, and insufficient information on food
consumption. The AFC Panel did not specifically address the quality of the studies by Adriani and
Negishi, but the overall conclusion was that the neurobehavioural database revealed that there were no
consistent adverse effects of perinatal exposures to doses of BPA below 50 mg/kg b.w./day. The CEF
Panel noted that in the study by Adriani et al. (2003) only one dose level of BPA was administered.
From the study description, although not clearly stated, it can be inferred that the BPA dose level was
40 μg/kg b.w./day. Oestrous cyclicity of females was not monitored and this may have influenced the
outcome of the study. Also in the study by Negishi et al. (2004) only one dose level (100 μg/kg
b.w./day) of BPA was administered.

The EFSA (2006) opinion did not address the study in rats by Carr et al. (2003; see also Part I,
Appendix A). In this study only the offspring of an unspecified number of untreated dams was
exposed to BPA via oral gavage dosing (100 or 250 μg/kg b.w./day) during the lactation period. The
effect of BPA on learning and memory was studied using a Morris water maze. Statistical significance
between controls and BPA-exposed groups for acquisition of Maze performance (i.e. learning) was not
reached. In the high dose group a statistically significant reduced retention (i.e. memory) was observed
in females but not in the males. These changes were not observed with the oestrogenic reference
compound, oestradiol-17β. The authors extensively discussed if oestradiol-17β was a suitable positive
control since the responses with this substance were different than expected from theoretical
considerations. Following parturition, male and female rat pups within the same litter were assigned to
the various treatment groups such that in each litter there was a member of each treatment group for
each sex. Since the animals were dosed at a very young age, the pups had to be returned to their dam
after dosing. This results in the presence of pups from more than one exposure group in each nest, with
EFSA Journal 2010; 8(9):1829 15
Bisphenol A

a possibility of cross-exposure between BPA-treated, oestradiol-17β-treated or control pups. Also,


from the study description it is not clear if only one or more than one littermate per treatment group
could have been present. Further limitations are the unspecified number of litters, the limited number
of exposure levels tested, the limited degree of changes in the parameters studied and the lack of a
mechanistic background. Because of these shortcomings, the Panel concludes that this study should be
considered of limited value for risk assessment purposes.

Another study carried out in C56/Bl-6 mice, which was not addressed in the previous assessment of
BPA (EFSA, 2006) is the paper by Ryan and Vandenbergh (2006; see also Part I, Appendix A). From
the study report it is not clear if the offspring were also dosed directly, or whether only the dams were
treated. No effects were observed in anogenital distance in either sex, but in the females an earlier
onset of puberty (cornified cells in vaginal smears) was observed with the high BPA dose (2 and 200
μg/kg b.w./day) and with ethynyloestradiol (EE; oestrogenic reference compound). No statistically
significant decrease was observed in the time spent in open arms of an elevated plus Maze (i.e.
increased anxiety) for any of the BPA treatment groups, whereas a significant decrease was observed
with the oestrogenic reference compound. The mice exposed to the highest dose of BPA and to EE
spent significantly less time in the light part of a light/dark preference test. This would also indicate
increased anxiety. With EE but not with BPA, changes were observed in two tests for learning and
short-term spatial memory. The Panel notes that since the behavioural tests were performed in
ovariectomised animals, the relevance of the findings for humans is highly questionable. In addition,
the wide dose-interval and the number of dose groups limit the relevance of the findings on anxiety
behaviour for human risk assessment.

These four studies (Adriani et al., 2003; Carr et al., 2003; Negishi et al., 2004; Ryan and Vandenbergh,
2006) were considered in the EU-RAR (EC, 2008), together with numerous other developmental
neurotoxicity studies. The results of these four studies were discussed in an extensive “weight-of-
evidence” approach. The EU-RAR concluded that the limited reporting for many studies made it
difficult to fully understand how the studies were performed and statistically evaluated. As noted in
the EU-RAR (EC, 2008), for most of the end-points studied (i.e. open field behaviour, anxiety and
related behaviour, learning and memory, social and sexual behaviour and amphetamine or morphine
responses, receptor expression in brain, or brain morphology/chemistry) contradictory results were
obtained. However, a consistent absence of any BPA-related effect on morphology of the sexually
dimorphic nucleus of the pre-optic area (SDN-POA) was observed in the three studies in which this
endpoint was addressed.

Notably, in the EU-RAR (EC, 2008) three Nordic countries expressed a minority opinion on these four
studies (Adriani et al., 2003; Carr et al., 2003; Negishi et al., 2004; Ryan and Vandenbergh, 2006)
which they considered sufficiently reliable for regulatory use. The minority opinion stated that “The
reliability of these studies is judged to be adequate because the behavioural testing has been
conducted according to acceptable methods, the group sizes are quite close or equal to those
recommended in the OECD TG 426, and the litter has been used as the statistical unit. The effects
found in these studies indicate that there is a possible risk for developmental neurotoxicity of BPA at
very low exposure levels (0.1-0.25 mg/kg/d). These effects cannot be dismissed based on the other
unreliable studies in the DNT database. These countries would therefore prefer one of two possible
conclusions: 1) the available, but limited data are used for the risk assessment or 2) there is a need for
further information (the countries certainly evaluate the database as sufficient to justify a concern
warranting further investigation of developmental neurotoxicity), similarly to the proposed conclusion
in the final expert panel report on the reproductive and developmental toxicity of BPA performed by
NTP, US in November 2007.”

In the EFSA 2006 evaluation, the AFC Panel also noted a reported influence of low doses of BPA
(100 or 1000 μg/L in drinking water) on the sex difference in morphometric measurements of the
locus coeruleus (LC) in a study by Kubo et al. (2003; see also Part I, Appendix A). Dams were
exposed during pregnancy and lactation up to weaning. Two oestrogenic reference compounds were
also included. Treatment with BPA resulted in an inverted sex difference in LC size, caused by a
EFSA Journal 2010; 8(9):1829 16
Bisphenol A

decrease in female LC size and an increase in male LC size. In addition to the effect on LC size, a
reduced difference in behaviour in an open field test was observed between male and female offspring,
in connection to exposure to BPA. This was mainly due to a “demasculinisation” of male behaviour
rather than a “defeminisation” of females. A statistically significant difference in male open field
behaviour with the control males was only observed for pups from the low dose group, and the effects
were not consistent with those observed for the two oestrogenic reference compounds. No effects were
observed in the size of the sexually dimorphic nucleus of the preoptic area (SDN-POA), or on male or
female gonads and sex hormone levels. In low-dose male offspring a reduced intromission rate was
observed but other parameters of sexual behaviour were not affected in either sex. The AFC Panel
considered the effect on the LC “as a preliminary finding that needs to be repeated in a larger study,
with the litter as the experimental unit, blinded evaluation and comparison to historical control data”.
The CEF Panel noted that the effect of BPA on LC size was inversely related to dose (i.e. the effects
were slightly larger at the lower dose level as compared to the higher dose level). In addition, statistics
were based on the pup as a statistical unit rather than the litter and from the study description and the
animal number it could be inferred that the various treatment groups comprised more than one pup per
litter, with unequal numbers of pups per litter in each group. The EU-RAR concluded that the effects
on LC volume could be due to chance rather than to BPA exposure.

Conclusion

The CEF Panel noted that the studies by Adriani et al. (2003) and Negishi et al. (2004) were already
reviewed by the EFSA’s AFC Panel in 2006, who did not consider that these studies would raise a
concern regarding the safety of BPA. In the view of the CEF Panel there is no reason to revise this
conclusion. In fact, the studies by Carr et al. (2003) and Ryan and Vandenbergh (2006) are not
considered sufficiently robust to have an impact on the risk assessment of BPA, considering the fact
that these two studies also have several limitations and their findings are not supported or are even
contradicted by other studies. Also the study by Kubo et al. (2003) which was already evaluated by the
AFC Panel in 2006 has too severe limitations to impact on the risk assessment for BPA.

2. Assessment of a newly available developmental neurotoxicity study in rats

2.1. Summary of the study as reported by Stump (2009)


The text shown under this heading is taken directly from the Stump (2009) study report. It does not
necessarily reflect the opinion of the Panel.

The study aimed at determining the potential of BPA to induce functional and/or morphological effects
to the nervous system of rat offspring following exposure of the mother during pregnancy and
lactation. The study was conducted in accordance with OECD Test Guideline 426 and U.S. EPA
OPPTS Guideline 870.6300 and was in conformance with Good Laboratory Practice Regulations (40
CFR Part 792) and the OECD Principles of Good Laboratory Practice [C(97) 186/Final]. As
specified in the guidelines, positive control data, using reference chemicals to establish the reliability
and sensitivity of the test methods at the appropriate offspring ages used for neurotoxicity endpoints
were developed by WIL Research Laboratories, LLC.

The test substance, bisphenol A (BPA), was given at concentrations of 0 (control), 0.15, 1.5, 75, 750,
and 2250 parts per million (mg/kg feed) on a continuous basis in the diet to six groups of 24 bred
female Crl:CD(SD) rats (F0 dams) daily from gestation day 0 through lactation day 21. The mean test
substance consumption in the 0, 0.15, 1.5, 75, 750, and 2250 mg/kg feed groups was 0, 0.01, 0.12,
5.85, 56.4, and 164 mg/kg b.w./day, respectively, during gestation and 0, 0.03, 0.25, 13.1, 129, and
410 mg/kg/day, respectively, during lactation. Clinical observations, body weights, and food
consumption were recorded at appropriate intervals. In addition, detailed clinical observations
(DCO) were conducted outside of the home cage on all dams in each group on gestation days 10 and
15 and on lactation days 10 and 21. All females were allowed to deliver and rear their offspring (F1
generation) to lactation day 21, at which time the dams were euthanized and necropsied. The liver and
kidneys were weighed and examined microscopically for all F0 females surviving to the scheduled
EFSA Journal 2010; 8(9):1829 17
Bisphenol A

necropsies. Clinical observations, body weights, and sex were recorded for the F1 pups at appropriate
intervals. Surface righting response was evaluated for all available F1 pups beginning on postnatal
day (PND) 2. On PND 4, litters were culled to eight pups/litter (four pups/sex/litter, when possible).

Following culling, a subset (Subset A) consisting of one pup/sex/litter was assigned to detailed clinical
observations (PND 4, 11, 21, 35, 45, and 60), auditory startle response (PND 20 and 60), motor
activity (PND 13, 17, 21, and 61), and learning and memory using the Biel maze (PND 62). This same
subset of animals was selected for brain weight measurements on PND 72; of these, one pup/litter (10
pups/sex) from all groups was selected for neuropathological and morphometric evaluations on PND
72. A second subset (Subset B) consisting of one pup/sex/litter was selected for learning and memory
using the Biel maze on PND 22. A third subset (Subset C) consisting of one pup/sex/litter was selected
for brain weight evaluations on PND 21; of these, one pup/litter (10 pups/sex) from all groups was
selected for neuropathological and morphometric evaluations on PND 21. Developmental landmarks
(balanopreputial separation or vaginal patency) were evaluated for all F1 animals in Subset A. All F1
animals not selected for behavioural evaluations (Subset D) were euthanized and necropsied on PND
21.

F0 maternal survival was unaffected by test diet exposure. No remarkable clinical findings were noted
at the daily examinations and no test substance-related changes in detailed clinical observations were
noted at any F0 exposure level. Maternal toxicity was expressed at 750 and 2250 mg/kg feed in the F0
generation by lower mean body weight gains during gestation (gestation days 0-20) of 9.5% and
22.4%, respectively, with an associated reduction in mean food consumption from the onset of test
substance exposure through gestation days 10 and 20, respectively. Mean body weight gains and food
consumption in the 750 and 2250 mg/kg feed groups were similar to the control group throughout
lactation. Mean body weights, body weight gains, and food consumption in the 0.15, 1.5, and 75 mg/kg
feed groups were unaffected by test substance exposure throughout gestation and lactation. No
additional evidence of systemic toxicity was observed in F0 females, including liver weights, kidney
weights, and microscopic changes. There were no effects on reproductive performance. With the
exception of reduced mean pup body weights at exposure levels of 750 and 2250 mg/kg feed, there
were no effects on litter or offspring developmental parameters. Reductions in mean male and female
pup body weight gains occurred during PND 7-14. This resulted in lower mean male and female pup
body weights during PND 14-17 in the 750 mg/kg feed group (up to 5.6% lower) and PND 11-21 in
the 2250 mg/kg feed group (up to 8.3% lower) relative to the control group. There were no test
substance-related effects on post-weaning body weight or the onset of sexual maturation as measured
by the age of attainment of vaginal opening or preputial separation. For F1 males and females, there
were no test substance-related effects on DCO, motor activity, auditory startle, learning and memory,
macroscopic examinations and measurements, neuropathology, or brain morphometry at any dietary
concentration at any age. At the PND 11 DCO, two pups in the 750 mg/kg feed group and four pups in
the 2250 mg/kg feed group exhibited clonic movements that were recorded as convulsions and/or
popcorn seizures. These observations were not reproduced in a follow-up study (Stump, 2009)
conducted under conditions designed to replicate, as closely as possible, this original developmental
neurotoxicity study through PND 11. A single dietary concentration (2250 mg/kg feed) was used in the
follow-up study, with DCO conducted on twice the number of pups per litter (from 27 litters). The
initial observations were not considered to be test substance-related because they were not noted at
any other age, and they were not reproduced in the follow-up study. In addition, no other
neurobehavioral or neuropathological parameters were altered in any animals, including those that
exhibited clonic movements on PND 11.

In conclusion, there were no test substance-related effects observed in this study at dietary
concentrations of 75 mg/kg feed or lower (0.15 and 1.5 mg/kg feed). Reductions in mean body weight
and body weight gain were observed in dams and offspring at dietary concentrations of 750 and 2250
mg/kg feed. There was no evidence of non-monotonic exposure-response curves for any parameter.
Therefore, the no-observed-adverse-effect level (NOAEL) for systemic toxicity of BPA was 75 mg/kg
feed (5.85 mg/kg/day and 13.1 mg/kg/day during gestation and lactation, respectively).

EFSA Journal 2010; 8(9):1829 18


Bisphenol A

There was no evidence of developmental neurotoxicity at any dietary concentration. Thus, the NOAEL
for developmental neurotoxicity of BPA was 2250 mg/kg feed (164 mg/kg/day and 410 mg/kg/day
during gestation and lactation, respectively), the highest dietary concentration tested. Based on the
conditions of this study, there was no evidence that BPA is a developmental neurotoxicant in rats.

2.2. The Panel’s comments to the study design and results


The testing was performed according to OECD test guideline 426. In agreement with previous
evaluations (EFSA, 2006; NTP-CERHR, 2008), the most appropriate route of dosing was determined
to be via the diet, since this takes account of the first-pass elimination of BPA seen in typical human
dietary exposure. The dose selection was consistent with previous studies on BPA (i.e. the multi-
generation studies in rats; Tyl et al., 2007). The top dose gave adverse effects in the maternal animals
(see section 2.2.1), indicating that adequate levels of exposure to the test compound have been
obtained. The concentration of the test compound in the diet, and diet consumption, were determined
throughout the study and this represents an important control for ensuring that dosing levels were
appropriate. The number of dosing levels (five) and use of a concurrent control group were adequate,
and the number of dams per group (23-24) met the stipulated minimum requirement (20). Dietary
phytooestrogen (sum of aglycon units of daidzin, daidzein, genistin, genistein, glycitin, glycitein and
coumestrol) was measured and the level in all batches of feed was 0.03% (300 μg/g).

The adequacy of the rat strain for the purpose of the study is substantiated by evidence showing that
Sprague-Dawley rats are as responsive to oestrogens as other rat strains (Diel et al., 2004). However, a
mechanism of action of BPA on animal behaviour through interaction with oestrogen receptors on
animal behaviour has not yet been unequivocally demonstrated.

In the study by Stump (2009) an oestrogenic reference compound was not included. Therefore,
sensitivity of the test parameters to oestrogenic substances has not been demonstrated. It is not a
general requirement in OECD guideline 426 to include a concurrent reference compound in the study.
Non-inclusion of such a reference compound does not invalidate the study: U.S. FDA (2010a) and
AFSSA (2010a) have expressed similar views in their 2010 opinions.

The Panel noted that the exposure of the pups in the study may reflect exposure of breast-fed infants,
but it is also reflective of the exposure of bottle-fed children. This is based on the estimation of
transfer of BPA via milk, based on maternal exposure in the Stump study and data on transfer taken
from the study by Snyder et al. (2000; see Part II). A dam in the Stump high dose group receives 2250
ppm via the feed, which according to the study is equal to 410 mg/kg b.w./day during the lactation
period, equivalent to 120 mg/animal/day, since the maternal body weight at day 14 of lactation was
300 g. Snyder et al. reported a transfer of BPA to milk of 0.003% of the dose (mg/dam)/g of milk at
one hour after gavage dosing of dams with 100 mg/kg b.w.. This declined to 0.0008% of the dose
(mg/dam)/g of milk at 24 hours after dosing. From these data a total 24 hr exposure of 3.15 µg
BPA/pup, or 0.3% of the dose (as mg/dam)/kg pup weight could be estimated. Assuming equal
transfer for the pups in the high dose group of the Stump study, these pups could have been exposed to
ca. 350 µg BPA/kg b.w.). The conservative estimate of exposure of bottle-fed infants in the 2006
opinion (ca. 12 µg/kg b.w./day) is ca. 30 times less than the exposure of the high dose pups in the
Stump study. It could be argued that, in contrast to infant formulae, in rat milk the BPA is conjugated
and therefore inactive, but due to glucuronidase activity in milk and GI tract this conjugate will be
hydrolysed to give the free substance again (Snyder et al., 2000).

2.2.1. F0 data (Maternal generation)


Animals on all dose groups were mated successfully, with no evidence for a chemical-related effect on
conception. One animal died during parturition, and there were no other clinical observations worthy
of note. During pregnancy, animals in the top two dose levels showed a reduction in food intake in the
initial three days of pregnancy, coupled to a reduction in body weight gain during the first week of
gestation. The two highest dose groups maintained this weight difference with control animals
throughout gestation, and it was maintained throughout lactation in the high dose group. There were

EFSA Journal 2010; 8(9):1829 19


Bisphenol A

no dose-dependent effects of BPA on gestation length. The parental females showed no dose-
dependent effects of BPA on liver and kidney weight or pathology, in a limited necropsy on lactation
day 21. The mean number of pups born, litter size, percentage of males per litter at birth, and postnatal
survival between birth and weaning were unaffected at all exposure levels.

2.2.1.1. Detailed Clinical Observations


Clinical observations were performed twice during gestation and lactation.

Some animals with red deposits around the nose were observed in controls and some of the dose
groups; there was no dose-effect relationship. No other significant findings were observed for F0
females.

2.2.2. F1 data (Litter)


The evaluation of the offspring was carried out according to the following set up (Table 1).

Table 1: Scheme of the study design for the evaluation of the offspring exposed to BPA

Sub Test Postnatal day


set procedure 4 11 13 17 20 21 22 35 45 60 61 62 72
A1 Clinical X X X X X X
observation
Auditory startle X
response
Motor activity X X X X
Biel maze X4
Brain weight X
2
Neuropatholo- X
gical evaluation
3
Sexual
maturation
B1 Biel maze X4
C1 Brain weight X
2
Neuro- X
pathological
evaluation
1
one pup/sex/litter, 2 one pup/litter (10 pups/sex) from all groups, 3 balanopreputial separation or vaginal patency investigated
daily from about day 30 onwards. 4 Not the same animals tested on PND 22 and 62.

2.2.2.1. Postnatal survival, growth and developmental landmarks


Male and female weight gain of the pups was significantly reduced between PND 7-14 in the top two
dose groups, resulting in significantly lower body weight compared with controls at weaning. There
was no statistically significant effect of treatment on body weight at lower dose levels (75 mg/kg feed
and below). Developmental landmarks (surface righting response, balanopreputial separation and
vaginal patency) and onset of puberty were unaffected by any treatment. There were no differences in
morbidity between treatment groups, and body weight gain showed no difference related to treatment
group after weaning.

There were no historical data provided for surface righting response. However, the comparison
between the concurrent control and treated rats did not show any deviation.

2.2.2.2. Detailed Clinical Observations (Subset A)


No BPA-induced effects were noted at detailed clinical observations on PND 4, 21, 35, 45, and 60. On
PND 11 only some irregular jerking movements of limbs, head, and/or body and/or jumping with all
four feet in the air (recorded as convulsions and popcorn seizures, respectively) were observed in two

EFSA Journal 2010; 8(9):1829 20


Bisphenol A

animals from the 750 mg/kg feed group and four animals from the 2250 mg/kg feed group. No other
clinical observations were observed in PND 11 animals. The popcorn seizures seen as a non-
statistically significant result on PND 11 in some animals did not correlate with any histopathology. In
the study report it was mentioned that such convulsions or seizures are also observed in historical
control group, but at much lower incidence than in the two groups in which they were seen in the BPA
study. For this reason a follow-up study was conducted (0 and 2250 mg/kg feed, n= 15 and 30 F0
females, respectively) but in this follow-up study no such seizures or convulsions were observed. It is
unclear whether these observations are related to the administration of BPA.

2.2.2.3. Motor Activity (Subset A)


Motor activity, as measured by total activity counts in F1 animals was unaffected by BPA exposure at
0.15, 1.5, 75, 750 and 2250 mg/kg feed when evaluated on PND 13, 17, 21, and 61. The pattern of
activity obtained from the six intervals evaluated (0-10, 11-20, 21-30, 31-40, 41-50, and 51-60
minutes) and the overall 60-minute test session values were comparable to the concurrent control
group. There were no statistically significant differences from the control group when the data were
evaluated by repeated measures analysis. No remarkable shifts in the pattern of habituation occurred in
any of the groups exposed to the test diet when the F1 animals were evaluated on PND 13, 17, 21, and
61.

Sensitivity of the test was demonstrated in separate experiments with positive control substances: to
detect increases in motor activity nicotine and amphetamine were used; to detect decreases in motor
activity haloperidol was used. A high variability of the data was observed in the motor activity
assessments. There were no statistically significant dose-dependent or sex-dependent effects; factors
included in the statistical analysis were treatment group, sex, and time.

Motor activity is generally sexually dimorphic in sexually mature animals, with females exhibiting
higher activity levels than males. This also seems to be the case in the controls of the two positive
control studies. However, the historical control data showed high variability and this would make it
difficult to find statistical significant differences between sexes and between control and treated
groups.

The two positive control studies report separate results for total activity count (combination of gross
and fine motor movement) and ambulatory count (gross motor movement). However, for the study on
BPA, only total activity counts have been given but no rationale is given for omitting ambulatory
count for BPA. Since the two counts provide different types of activity data, the Panel cannot evaluate
the effects of BPA on gross motor movements.

2.2.2.4. Auditory Startle Test (Subset A)


The auditory startle response habituation paradigm was conducted as a longitudinal assessment with
selected F1 animals evaluated on PND 20 and PND 60. Test substance exposure at 0.15, 1.5, 75, 750,
and 2250 mg/kg feed had no significant effect on auditory startle response amplitude on PND 20 or
60. No effects were noted in the pattern of the habituation over the entire 50-trial test session at either
age.

Sensitivity of the test was demonstrated in separate experiments with positive control compounds.
There were no significant differences between the two sexes similar to what has been reported for the
historical controls.

2.2.2.5. Biel Maze Swimming Trials (Subsets A and B)


The Biel water maze testing paradigm is a modified T-maze that examines visual spatial maze
performance associated with cognitive function. Animals are placed in the maze and are required to
traverse the maze and escape by locating a submerged platform.

EFSA Journal 2010; 8(9):1829 21


Bisphenol A

Beginning on PND 22 and PND 62, respectively, swimming ability and learning and memory were
assessed for one rat/sex/litter. In accordance with the guideline, different animals were used for testing
on PND 22 and 62 (see the study design in Table 1, above). Each testing sequence consisted of three
phases that were conducted over seven consecutive days:

• Phase 1 = day 1: test for swimming ability and motivation to escape from the maze.
• Phase 2 = days 2-6: evaluated sequential learning; measure of learning and shorter-term memory
ƒ Path A: two 180-sec trials per day for two days (trial 1 to 4; totally 4 trials).
ƒ Path B (reverse of path A): two 180-sec trials per day for three consecutive days (trial 5 to
10; totally 6 trials)
• Phase 3 = on day 7: memory task (repetition of path A): two 180-sec trials (trial 11 to 12; totally 2
trials); measure of long-term memory of path A

• Biel maze data were evaluated as the numbers of errors committed during Phases 2 and 3 (i.e.
sequential learning and memory). Time to escape (latency) was recorded to verify that the animals
were removed within the specified time. Data were analysed by RANOVA fitting linear mixed
effects models that included fixed effects for treatment, sex and trial (and their corresponding
interactions). Potential variability resulting from the fact that the animals were received by the
testing laboratory in two separate shipments was also taken into account. Random effects for litter
were included in the PND 22 model but not in the PND 62 one and for this no rationale was
provided. The statistical analysis by Stump et al. was only carried out on the numbers of error
data, but not on the time-to-escape data.

• Sensitivity of the test was demonstrated in separate experiments with positive control substances.
To detect decreases in learning and memory scopolamine was used. A study with methimazole
showed increased latency (i.e. the time the animals needed to reach the end of the swimming
trajectory) in the absence of effects on the number of errors and swimming ability on PND 62. No
concurrent reference compound was used to demonstrate the sensitivity of the test to detect effects
on learning and memory. However, it should be noted that no generally accepted reference
compounds are available for this purpose.

• According to the study authors, in the Biel maze swimming test for learning and memory, the
females in the 0.15 and 2250 mg/kg feed groups showed an improved performance (i.e. they
committed less errors than the controls) on PND 22 in Path B (trials 5-10). However, the numbers
of errors committed was within the historical control range, and the number of errors committed
by the concurrent controls was higher than that for the historical controls. Therefore, the improved
performance of the females in the 0.15 and 2250 mg/kg feed groups could partially be attributed to
a less than normal performance of the concurrent controls as compared to the historical controls.
The study authors concluded that no changes were observed in the path A trials (1-4 and 11-12) on
PND 22.

• According to the analysis by the study authors, on PND 62 the male offspring from the 0.15 mg/kg
feed exposure group showed an increase in the number of errors in Path A trials 1-4, which could
be interpreted as a delay in learning, because the difference was mainly caused by an increased
number of errors during trials 3 and 4. The effect reached statistical significance when compared
to concurrent controls. The study authors argued that no such an effect was observed in pups
studied on PND 22 or in any other exposure group on PND 62. When the swimming trials were
conducted in the reverse path (i.e. the path B) no delay in learning was observed and also in the
repeat Path A trials 11 and 12, no effect was observed in any exposure group, including the 0.15
mg/kg feed group.

• Based on the reported data, the performance of the male offspring with respect to numbers of
errors and time-to-escape is shown in Figure 1, below (PND 62 animals only). From the general
shape of the learning curved, one could conclude on first glance that similar to what was claimed

EFSA Journal 2010; 8(9):1829 22


Bisphenol A

by the study authors, there is no dose-related response and the increased numbers of errors in the
0.15 mg/kg feed group is a chance finding. For the sake of clarity, no error bars are included.
However, the Panel noted that the horizontal axis is not a time axis and the time gap between the
trials for the individual animals is unknown, which impedes interpretation and comparison of
slopes.

Average number of errors, males biel maze pnd 62

35

30

number of errors 25 0
0,15
20
1,5
75
15
750
10 2250

0
1 2 3 4 5 6 7 8 9 10 11 12


trial nr

average time-to-escape (inc fails), males biel maze PND62

160

140

120
0
100 0,15
time (sec)

1,5
80
75
60 750
2250
40

20

0
1 2 3 4 5 6 7 8 9 10 11 12
trial nr

Figure 1: Performance in Biel maze test for learning and memory in male pups on PND 62
The two panels show learning curves for all treatment groups. Performance is given as average
number of errors and average time-to-escape. The data are not corrected for censoring (see text).

Apart from the error counts, the performance of the animals should have also been evaluated for
effects on “time-to-escape”, i.e. the time needed to reach the exit platform of the swimming maze.
Also, to check for long term memory effects, results in trials 11 and 12 should have been compared to
the results of trials 1 to 4. However, this comparison was not made by the study authors.

The Panel realised that the data suffer from censoring, as the animals were only allowed to fulfil the
Biel maze task for three minutes. In each trial where an animal did not reach the escape platform, it
was taken out of the maze, and the number of errors committed and a maximum time-to-escape of 180
seconds for that animal were recorded. In practice this means that Stump (2010) assumes that all rats
escaped from the maze leading to a biased estimate and under estimates for the number of errors. This
problem (i.e. censoring) has also been identified in the paper by Holson et al. (2007) according to
which Stump et al. have evaluated their data, but it was not accounted for in their analysis.

The consequence of this censoring for the PND 62 Biel maze trials is shown in Figure 2. The two
panels in this Figure show median number of errors and median time-to-escape and the variability of
the data. All data points in Panel 2B running against the 180 sec limit represent censored data.
Censoring can affect over 50% of the trials per group in trial number 5. Also in other less affected
trials censoring has occurred in up to 20 % of the animals per group. In Figure 2, the data were
combined for males and females (48 instead of 2 times 24 animals per group).

EFSA Journal 2010; 8(9):1829 23


Bisphenol A

Panel A: Number of errors

Panel B: Time-to-escape

Figure 2: Presentation of Biel maze trial data combined for male and female pups on PND 62

The graphs show medians (solid horizontal line and 25th and 75th percentiles for the number of errors
(panel A) or time-to-escape (panel B) per dose group. The dotted lines show the ranges of the data.
The empty circles are considered to be “outliers/extreme” values. Only data for the first trials (called
“seq” in the Figure) of each day of the Biel maze testing are presented. Dose groups: 1 = 0, 2 = 0.15, 3
= 1.5, 4 = 75, 5 = 750, and 6 = 2250 mg BPA /kg feed.

EFSA Journal 2010; 8(9):1829 24


Bisphenol A

EFSA’s Assessment and Methodology Unit (AMU) has submitted the Biel maze study data for both
PND 22 and PND 62 to a more appropriate statistical evaluation, taking into account the complexity of
the study design, covering the issue of data censoring and including an analysis of time-to-escape data.
Among others gender was included as a covariate in the analysis.

AMU results reveal an extreme high variability, not only in the study data for the PND 62 trials, but
also in the data for PND22. This variability might be due to other non-modelled or not-possible-to-
model aspects of the experimental design or execution of the experiment. This indicates that the Biel
maze test as performed by Stump et al. does not have the potential to demonstrate equivalence of BPA
compared to a control. Therefore this study should be considered as being inconclusive.

EFSA AMU will publish a full report on the statistical evaluation of the Biel maze data in this study
shortly after the adoption of this Opinion. For further details the reader is referred to this report.

2.2.2.6. Macroscopic Examinations


One female and three male pups in the control and 1.5 mg/kg feed groups, respectively, were found
dead or euthanized in extremis following the pre-weaning period. No remarkable gross findings were
noted for any of these animals at necropsy. There were no indications of substance-related
macroscopic effects in unscheduled deaths or in animals euthanized on PND 22 following learning
and memory testing.

2.2.2.7. Neuropathology (Subsets A and C)


Macroscopic Examinations - PND 21

One male and female per litter were killed for examination on PND 21. Fixation of tissues was carried
out by transcardiac perfusion, with postfixation in neutral buffered formalin for 36 hours. Brains were
weighed, and size and length recorded. There were no significant observations, beyond minor
alterations associated with perfusion artefacts. There were no significant effects on brain size or
measurements.

Qualitative Histopathology and Brain Morphometry- PND 21

Ten pups per sex per group were used to prepare brain and peripheral nervous system sections, and
their histopathology was examined. There were no significant findings associated with exposure to
BPA. Nests of embryonal cells were seen in five animals, but there was no dose-response relationship,
and this is known to be a spontaneous lesion in control animals. There were no significant effects on
morphometric measurements.

Macroscopic Examinations - PND 72

One male and female per litter were killed for examination on PND 72. Macroscopic examination was
carried out as described for PND 21rats. Brains were weighed, and size and length recorded. There
were no significant observations, beyond minor alterations associated with perfusion artefacts, and one
animal with hydrocephalus7. There were no significant effects on brain size or morphometric
measurements.

Qualitative Histopathology and Brain Morphometry - PND 72

Ten pups per sex per group, plus animals with clonic convulsions, were used to prepare brain and
peripheral nervous system sections, and their histopathology was examined. There were no significant

7
This animal (26492-03) was in dose group 0.15 mg/kg feed and in the Biel maze test group for PND 62. It did not perform
much worse or better than the rest of the group. Leaving it out would slightly improve the learning behaviour of the group
(not statistically analysed).
EFSA Journal 2010; 8(9):1829 25
Bisphenol A

findings with a dose-response relationship. Minor evidence of change in peripheral nerves was seen in
some animals, but this is a common endogenous lesion.

In summary, the study examined an adequate number of animals to draw conclusions on


neuropathology. The methodology was in general adequate for purpose.

2.3. Discussion of the study outcome


The new developmental neurotoxicity study on BPA by Stump (2009; published as a paper in 2010)
considered in this opinion is supplemental to the extensive database on BPA, in which a large number
of studies have addressed interactions of BPA with early brain development. The Stump (2009) study
has been performed according to OECD guideline 426. Exposure of animals to BPA was via the diet,
which would mimic the human oral route of exposure. The Panel noted that the exposure of the pups
in the study may reflect exposure of breast-fed infants. Since it can be estimated that the pups in the
2250 mg/kg fed exposure group may have been exposed to dose levels ca. 30 higher than can be
anticipated for bottle-fed children, the study is also relevant for bottle-fed infants. The dose selection
was consistent with previous studies on BPA and some adverse effects in the maternal animals were
observed at the highest dose levels, namely 750 and 2250 mg/kg feed (equivalent to 56.4 and 164
mg/kg b.w./day, respectively, during gestation). Exposure was adequately controlled in the diet and a
wide range of exposure levels was covered in sufficiently large groups of animals. Dietary phyto
oestrogen (sum of aglycon units of daidzin, daidzein, genistin, genistein, glycitin, glycitein and
coumestrol) was measured and the level in all batches of feed was 0.03% (300 μg/g). Although the
adequacy of the SD-rat strain for toxicity testing of oestrogenic substances has been challenged
(Myers et al., 2009), there is evidence showing that Sprague-Dawley rats are as responsive to
oestrogens as the other rat strains (Diel et al., 2004; Sharpe, 2010).

In the study by Stump (2009) an oestrogenic reference compound was not included. Therefore,
sensitivity of the test parameters to oestrogenic substances has not been demonstrated. Missing of such
a reference compound does not invalidate the study; the U.S. FDA (2010a) and AFSSA (2010a) have
expressed a similar view.

Maternal toxicity was monitored throughout the test. No effects on litter parameters were observed at
any dose, but the dams in the two top dose levels showed a reduction in food intake during the initial
three days of gestation, coupled to a reduction in body weight gain during the first week of gestation.
The animals did not recover from this throughout gestation and in the top dose animals this resulted in
a reduced body weight at the end of the exposure period. Based on this observation, a NOAEL of 75
mg/kg feed corresponding to 5.85 mg/kg b.w./day during gestation and 13.1 mg/kg b.w./day during
lactation could be derived for the parental animals.

General toxicity was monitored in the offspring with respect to survival, growth, development, onset
of puberty and clinical observations. The battery for behavioural neurotoxicity included the following
tests: motor activity, auditory startle reflex and Biel maze swimming test (learning and memory). In
addition, neurohistopathology and brain morphometry was carried out at weaning and on PND 72.

Concerning survival, growth, developmental landmarks, clinical observations and onset of puberty in
offspring, the only effects were reduced mean pup body weights at exposure levels of 750 and 2250
mg/kg feed and convulsions and “popcorn seizures” on PND 11 in 2 and 4 pups from the 750 and
2250 mg/kg feed groups, respectively. Since the latter effect was dose-related, the Panel is not
confident to disregard this observation. However, the Panel considered the toxicological significance
of this observation as being very limited because convulsions and seizures were not seen in any other
occasion during the study, and could not be reproduced in a follow-up study with animals exposed to 0
or 2250 mg/kg feed according to the same study design (Stump, 2009). Also, convulsions or “popcorn
seizures” or similar clonic movements have not been reported in any other study on BPA, including
the available reproductive toxicity and developmental neurotoxicity studies, as summarised in EFSA
previous evaluations, in the EU-RAR and its addendum or the NTP-CERHR Monograph (EFSA,
2006; EC, 2003, 2008; NTP-CERHR, 2008).
EFSA Journal 2010; 8(9):1829 26
Bisphenol A

No adverse effects were reported upon neuropathological or brain morphological examination of the
offspring on PND 21 or 72.

No remarkable observations were obtained from the tests for auditory startle response and for motor
activity. Control data in the BPA study, as well as the historical control data consistently showed no
gender related differences in auditory startle response. Also in literature auditory startle response is not
known as a sexually dimorphic behaviour (Meyer, 1998). With BPA no findings were reported in
either sex.

No sex- or treatment-related differences in motor activity were found. Motor activity is generally
sexually dimorphic in adult animals, with females exhibiting higher activity levels than males.
However, the concurrent and historical control data from the WIL Research Laboratories8 showed
high variability and this would make it difficult to find statistically significant differences between
sexes and between control and treated groups. For the study on BPA, only total activity counts have
been presented but no rationale has been given for omitting ambulatory count for BPA. The two
counts provide different types of activity data and therefore the effects of BPA on gross motor
movements cannot be evaluated.

From the graphical presentation of the learning curves, no effect of BPA seems to emerge. However,
these learning curves may suffer from censoring bias. Based on analysis of the Biel Maze data as
carried out by EFSA AMU, taking into account the problem of censoring, no conclusion can be drawn
on the effect of BPA on learning and memory behaviour. Also the finding of delayed initial learning in
PND62 males in group 0.15 mg/kg feed cannot be substantiated. The Panel has also evaluated Biel
maze performance in the same way for the PND22 pups obtaining the same result.

The Panel noted that the study by Stump does not cover some specific aspects of behaviour (i.e.
avoidance learning, schedule-controlled behaviour, and impulsiveness) and anxiety-related behaviour,
because tests addressing such behavioural endpoints were not included.

PART I - CONCLUSIONS
Following earlier evaluations of the impact of BPA on neurologic and behavioural developmental in
rodents indicating overall possible concern for these endpoints (for references see EC, 2003; 2008;
NTP-CERHR, 2008; VKM, 2008), an industrial consortium has submitted results from a
developmental neurotoxicity study with BPA, in accordance with OECD guideline 426 and in
compliance with GLP. On request of the European Commission to EFSA, the Panel has evaluated the
outcome of this study. In the new study (Stump, 2009), BPA was administered to pregnant rats via the
diet at concentration ranging from 0.15 to 2250 mg/kg feed equivalent to 0.01-164 mg/kg b.w./day
during gestation and to 0.03-410 mg/kg b.w./day during lactation. The treatment schedule was relevant
to human exposure in utero and to breastfeeding. Since it can be estimated that the pups in this study
were exposed up to ca. 30 times higher levels of BPA than bottle-fed infants, the BPA exposure and
the results from the study are also regarded as relevant to bottle-fed infants. Dams were evaluated for
general signs of toxicity, and offspring was evaluated for general toxicity including developmental
landmarks and for neurological effects, including behaviour and brain histopathology.

• General toxicity expressed as reductions in mean body weight and body weight gain in dams
and offspring was detected at levels of 750 and 2250 mg/kg feed, for which a No-Observed-
Adverse-Effect-Level (NOAEL) of 75 mg/kg feed (equal to 5.85 mg/kg b.w./day during
gestation) could be derived. This NOAEL is close to that of 5 mg/kg b.w./day identified by the
SCF (2002) for similar effects, and which is the basis for the current TDI of BPA. No effects
were observed on brain development as examined by histopathology.

8
WIL Research Laboratories, LLC is the contract research organization that performed the study
EFSA Journal 2010; 8(9):1829 27
Bisphenol A

• In a few pups of the 750 and 2250 mg/kg feed (equal to 56.4-164 mg/kg b.w./day during
gestation) dose groups, popcorn seizures and convulsions were observed on postnatal day
(PND) 11. The absence of seizures or convulsions in a follow-up study and in any study
reported in the EU-RAR (EC, 2003; 2008) or in other recent reviews (e.g. NTP-CERHR, 2008)
casts doubt on the relevance of this observation. The Panel noted that the current Tolerable
Daily Intake (TDI) for BPA (0.05 mg/kg b.w/day) would be sufficiently low to exclude a
possible concern for this effect, seen only at the two highest dose levels.

• The neurodevelopmental toxicity study by Stump (2009) covers motor activity, learning and
memory (spatial behaviour), auditory startle response, brain histopathology and morphology.
The study does not cover some specific aspects of learning and memory (i.e. avoidance
learning, schedule-controlled behaviour, and impulsiveness), anxiety-related behaviour or
sexual dimorphic behaviour, but this does not invalidate the study. No statistically significant
effects were observed in tests on motor activity or auditory startle or in brain histopathology and
morphology.

• According to the statistical analysis by the study authors, in the Biel maze swimming test for
learning and memory on PND 62 the male offspring from the 0.15 mg/kg feed exposure group
showed an increase in the number of errors in Path A trials 1-4, which could be interpreted as a
delay in learning, because the difference was mainly caused by an increased number of errors
during trials 3 and 4. The effect reached statistical significance when compared to concurrent
controls. No such an effect was observed in pups studied on PND 22 or in any other exposure
group on PND 62. When the swimming trials were conducted in the reverse path (i.e. the path
B) no delay in learning was observed and also in the repeat Path A trials 11 and 12, no effect
was observed in any exposure group, including the 0.15 mg/kg feed group. Therefore, the
authors concluded that there were no changes in learning and memory.

• In the Biel Maze test only the error counts were reported as a measure of learning and memory.
The animals should have also been evaluated for effects on “time-to-escape” and checked for
long term memory effects. However, this comparison was not made by the study authors.
EFSA’s Assessment and Methodology Unit (AMU) applied a more appropriate statistical
evaluation to the study data. It was realised that the data suffer from censoring9. Therefore,
based on the re analysis of the Biel Maze data the Panel considered that no conclusion can be
drawn from this study on the effect of BPA on learning and memory behaviour due to large
variability in the data.

• Based on the body weight effects on dams and offspring and also taking into account the
occurrence of seizures and convulsions in the two highest dose groups, which were not observed
at the lower dose levels, the study supports the NOAEL which was derived from
multigeneration studies in the past (5 mg/kg b.w./day), leading to a TDI of 0.05 mg/kg b.w./day.
However, the Panel considered that this test on learning and memory was inconclusive and is of
limited value in the risk assessment of BPA

The Panel is aware of ongoing studies by the U.S. Food and Drug Administration which will address
some neurodevelopmental endpoints.

9
In statistical terminology censoring occurs when the value of an observation is only partially known. In the Biel water maze
test the number of errors made by the rats that did not complete the maze within the 3 minute-limit that was used, was
recorded and the time to escape was recorded as 180 seconds. These experiments are “right censored”, meaning that the rats
could have escaped and made more errors, had they been given sufficient time. Therefore the time to escape and the number
of errors recorded will have been underestimated.
EFSA Journal 2010; 8(9):1829 28
Bisphenol A

PART I - DOCUMENTATION PROVIDED TO EFSA


Stump (2009) study report. A Dietary Developmental Neurotoxicity Study of Bisphenol A in rats.
October 2009. Submitted by Polycarbonate/BPA Global Group American Chemistry Council,
Arlington, VA, USA.

EFSA Journal 2010; 8(9):1829 29


Bisphenol A

PART I - REFERENCES
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AIST (National Institute of Advanced Industrial Science and Technology, Japan), 2005. Bisphenol A
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Bae B, Jeong JH and Lee S J, 2002. The quantification and characterization of endocrine disruptor
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BfR (Bundesinstitut für Risikobewertung), 2010. Bisphenol A: Studies by Stump et al. (2010) and
Ryan et al. (2010) provide no indications for adverse effects on neurological development and
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Carr R, Bertasi F, Betancourt A, Bowers S, Gandy BS, Ryan P and Willard S, 2003. Effect of neonatal
rat bisphenol A exposure on performance in the Morris water maze. Journal of Toxicology and
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Diel P, Schmidt S, Vollmer G, Janning P, Upmeier A, Michna H, Bolt HM, and Degen GH, 2004.
Comparative responses of three rat strains (DA/Han, Sprague-Dawley and Wistar) to treatment
with environmental estrogens. Archives of Toxicology 78, 183-193.
DTU Fødevareinstituttets, 2010. Evaluation by the DTU Food Institute of the industry’s new
developmental neurotoxicity study (DNT, OECD TG 426) of bisphenol A and the significance of
the study for the Food Institute’s assessment of the potential harmful effects of bisphenol A on the
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EC (European Commission), 2003. European Union Risk Assessment Report. Bisphenol A, CAS No:
80-05-7. Institute for Health and Consumer Protection, European Chemicals Bureau, European
Commission Joint Research Centre, 3rd Priority List, Luxembourg: Office for Official Publications
of the European Communities. Available from http://ecb.jrc.it/Documents/Existing-
Chemicals/RISK_ASSESSMENT/REPORT/bisphenolareport325.pdf
EC (European Commission), 2008. Updated Risk Assessment Report of 4'-Isopropylidenediphenol
(Bisphenol-A) (human health). Final approved version awaiting publication, April 2008. Available
from http://ecb.jrc.it/documents/Existing-
Chemicals/RISK_ASSESSMENT/ADDENDUM/bisphenola_add_325.pdf
EFSA (European Food Safety Authority), 2006. Opinion of the Scientific Panel on Food Additives,
Flavourings, Processing Aids and Materials in Contact with Food on a request from the
Commission related to 2,2-Bis(4-hydroxyphenyl)propane (Bisphenol A). The EFSA Journal 428,
1-75. Available from http://www.efsa.europa.eu/en/scdocs/scdoc/428.htm
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EFSA (European Food Safety Authority), 2008. Scientific Opinion of the Panel on Food additives,
Flavourings, Processing Aids and Materials in Contact with Food (AFC) on a request from the
Commission on the toxicokinetics of Bisphenol A . The EFSA Journal 759, 1-10. Available from
http://www.efsa.europa.eu/en/scdocs/scdoc/759.htm
Health Canada, 2008. Health Risk Assessment of Bisphenol A from Food Packaging Applications.
August 2008. Available from http://www.hc-sc.gc.ca/fn-an/alt_formats/hpfb-
dgpsa/pdf/securit/bpa_hra-ers-eng.pdf
Health Canada, 2009. Bisphenol A. Available from http://www.hc-sc.gc.ca/fn-
an/pubs/securit/bpa_survey-enquete-eng.php
Holson R, Freshwater L, Maurissen, PJ, Moser VC and Phang W, 2007. Statistical issues and
techniques appropriate for developmental neurotoxicity testing: a report from the ILSI Research
Foundation/Risk Science Institute expert working group on neurodevelopmental endpoints.
Neurotoxicology and teratology 30, 326-348.
Kubo K, Arai O, Omura M, Watanabe R, Ogata R, Aou A, 2003. Low dose effects of Bisphenol A on
sexual differentiation of the brain and behavior in rats. Neuroscience Research 45, 345-356.
Meyer J.S. (1998) Behavioural Assessment in developmental neurotoxicology. Approaches involving
unconditioned behaviours and pharmacologic challenges in rodents. In: Slikker W. and Chang
L.W. (eds) Handbook of developmental neurotoxicology. Academic Press, New York.
Myers JP, vom Saal FS, Akingbemi BT, Arizono K, Belcher S, Colborn T, Chahoud I, Crain DA,
Farabollini F, Guillette LJ Jr, Hassold, T, Ho S-m, Hunt PA, Iguchi T, Jobling S, Kanno J, Laufer
H, Marcus M, McLachlan JA, Nadal A, Oehlmann J, Olea N, Palanza P, Parmigiani S, Rubin BS,
Schoenfelder G, Sonnenschein C, Soto AM, Talsness CE, Taylor JA, Vandenberg LN,
Vandenbergh JG, Vogel S, Watson CS, Welshons WV and Zoeller RT, 2009. Why Public Health
Agencies Cannot Depend on Good Laboratory Practices as a Criterion for Selecting Data: The Case
of Bisphenol A. Environmental Health Perspectives 117, 309-315.
Negishi T, Kawasaki K, Suzaki S, Maeda H, Ishii Y, Kyuwa S, Kuroda Y and Yoshikawa Y, 2004.
Behavioral Alterations in Response to Fear-Provoking Stimuli and Tranylcypromine Induced by
Perinatal Exposure to Bisphenol A and Nonylphenol in Male Rats. Environmental Health
Perspectives 112, 1159-1164.
NTP (National Toxicology Program), 1982. Carcinogenesis Bioassay of Bisphenol A in F344 rats and
B6C3F1 mice - Feed Study, NTP Technical Report 215.
NTP-CERHR (National Toxicological Program-Center for the Evaluation of Risks to Human
Reproduction), 2007. NTP-CERHR Expert Panel Report on the reproductive and developmental
toxicity of bisphenol A. November 26, 2007. U.S. Department of Health and Humane Services.
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NTP-CERHR (National Toxicological Program-Center for the Evaluation of Risks to Human
Reproduction), 2008. NTP-CERHR Monograph on the Potential Human Reproductive and
Developmental Effects of Bisphenol A. September 2008. NIH Publication No. 08-5994. Available
from http://cerhr.niehs.nih.gov/chemicals/bisphenol/bisphenol.pdf
Ryan BC and Vandenbergh JG, 2006. Developmental exposure to environmental estrogens alters
anxiety and spatial memory in female mice. Hormones and Behavior 50, 85–93.
SCF (Scientific Committee on Food), 1986. EUR 10778 Report of the Scientific Committee for Food
(17th series). European Communities-Commission. Office for Official Publications of the European
Communities, Luxembourg. Catalogue no CD-NA-10778-EN-C Available through:
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SCF (Scientific Committee on Food), 2002. Opinion of the Scientific Committee on Food on
Bisphenol A. Expressed on 17 April 2002. Available from
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Sharpe RM, 2010. Bisphenol A exposure and sexual dysfunction in men: editorial commentary on the
article 'Occupational exposure to bisphenol-A (BPA) and the risk of self-reported male sexual
dysfunction' Li et al., 2010. Human Reproduction 25, 292-294.
Snyder RW, Maness SC, Gaido KW, Welsch F, Sumner S C and Fennell T R, 2000. Metabolism and
disposition of bisphenol A in female rats. Toxicology and Applied Pharmacology 168, 225-234.
Stump DG, Beck MJ, Radovsky A, Garman RH, Freshwater L, Sheets LP, Marty MS, Waechter JM,
Dimond SS, Van Miller JP, Shiotsuka RN, Beyer D, Chappelle AH and Hentges SG, 2010.
Developmental neurotoxicity study of dietary bisphenol A in Sprague-Dawley rats. Toxicological
Sciences 115, 167-182.
Tyl RW, Myer CB, Marr MC, Thomas BF, Keimowitz AR, Brine DR, Veselica MM, Fail PA, Chang
TY, Seely, JC, Joiner RL, Butala JH, Dimond SS, Cagen SZ, Shiotuka RN, Stropp GD and
Waechter JM, 2002. Three-Generation Reproductive Toxicity Study of Dietary Bisphenol A in CD
Sprague-Dawley Rats. Toxicological Sciences 68, 121-146.
Tyl RW, Myers CB, and Marr MC, 2006. Draft Final Report. Two-generation reproductive toxicity
evaluation of Bisphenol A (BPA; CAS No. 80-05-7) administered in the feed to CD-1® Swiss
mice (modified OECD 416). RTI International Center for Life Sciences and Toxicology, Research
Triangle Park, NC, USA.
Tyl RW, Myers CB, and Marr MC, 2007. Final Report. Two-generation reproductive toxicity
evaluation of Bisphenol A (BPA; CAS No. 80-05-7) administered in the feed to CD-1 Swiss mice
(modified OECD 416). RTI International Center for Life Sciences and Toxicology Volumes 1-8,
Research Triangle Park, NC, USA.
U.S. FDA (Food and Drug Administration), 2010a. Document 3 - Memorandum of 11/24/2009,
Bisphenol A, update regarding studies added to "Review of low dose assessment" [FDA-2010-N-
0100-0001]. Available from
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U.S. FDA (Food and Drug Administration), 2010b. Update on Bisphenol A for Use in Food Contact
Applications. Available from http://www.fda.gov/NewsEvents/PublicHealthFocus/ucm197739.htm
VKM (Norwegian Scientific Committee for Food Safety), 2008. Opinion of the Scientific Panel on
Food Additives, Flavourings, Processing Aids, Materials in Contact with Food and Cosmetics of
the Norwegian Scientific Committee for Food Safety. Assessment of four studies on developmental
neurotoxicity of bisphenol A.

EFSA Journal 2010; 8(9):1829 32


Bisphenol A

PART I - ABBREVIATIONS
AFC Scientific Panel on additives, flavourings, processing aids and materials in
contact with food
AFSSA French Food Safety Agency
AIST Japanese National Institute of Advanced Industrial Science and Technology
ALARA As Low As Reasonably Achievable
AMU Assessment and Methodology Unit of EFSA
BMDL Benchmark Dose Lower Limit
BPA Bisphenol A
b.w. Body weight
CAS Chemical abstracts service
CEF Scientific Panel on food contact materials, enzymes, flavourings and
processing aids
Da Dalton
DNT database Developmental neurotoxicity database
DTU Technical University of Denmark
EC European Commission
EE Ethynyloestradiol
EEC European Economic Community
EFSA European Food Safety Authority
EU European Union
EU-RAR European Union Risk Assessment Report
EUSES EU environmental exposure and risk assessment model
FCM Food Contact Material(s)
FSANZ Food Standards Australia New Zealand
GD Gestational day
GI Gastrointestinal
GLP Good Laboratory Practice
LOAEL Lowest-Observed-Adverse-Effect-Level
LC Locus coeruleus
MOE Margin of Exposure
MW Molecular weight
N or n Number of subjects/animals
NIEHS National Institute of Environmental Health Sciences
NOAEL No-Observed-Adverse-Effect-Level
NTP National Toxicology Program
OECD Organisation for Economic Co-operation and Development
PBPK Physiologically based pharmacokinetic
PC Polycarbonate
PND Postnatal day
ppm Parts per million
PVC Polyvinyl chloride
SDN-POA Sexually dimorphic nucleus of the preoptic area
SOPs Standard Operation Procedures
SCF Scientific Committee on Food
TDI Tolerable Daily Intake
t-TDI Temporary Tolerable Daily Intake
UF Uncertainty Factor
UK United Kingdom
U.S. EPA U.S. Environmental Protection Agency
U.S. FDA U.S. Federal and Drug Administration
U.S. NTP-CERHR National Toxicology Program - Center for the Evaluation of Risks to Human
Reproduction

EFSA Journal 2010; 8(9):1829 33


Bisphenol A

VKM Norwegian Scientific Committee for Food Safety


WHO World Health Organisation

PART I - APPENDICES

A. SHORT OVERVIEW OF CHARACTERISTICS AND RESULTS OF THE STUDIES EVALUATED IN


SECTION 1.2

EFSA Journal 2010; 8(9):1829 34


Bisphenol A

A. SHORT OVERVIEW OF CHARACTERISTICS AND RESULTS OF THE STUDIES EVALUATED IN SECTION 1.210

Ryan & Vandenbergh,


Parameters Adriani et al., 2003 Carr et al., 2003 Kubo et al., 2003 Negishi et al., 2004
2006

Species/strain/route Rat/SD/oral by Rat/Fisher/oral by gavage Rat/wistar/oral via drinking Rat/Fisher/oral by Mice/C57/BL-6/oral by


micropipette water gavage gavage
Dose groups 1) Control/arachis oil 1) Control /safflower oil 0.5 ml/ 1) control /0.01% ethanol 1) Control /corn oil 1) Control/corn oil 40
2) BPA in arachis oil at a kg b.w. 2) BPA 30 μg/kg b.w./d 2ml/kg b.w. μl/dose
concentration of 0.04 2) 17 beta-oestradiol E2 3) BPA 300 μg/kg b.w./d 2) BPA 0.10 mg/kg 2) BPA 0.002 mg/kg
11
mg/kg b.w. 3) BPA 0.10 mg/kg b.w./day 4) resveratrol 1500 μg/kg b.w./day b.w./day
4) BPA 0.25 mg/kg b.w./day b.w./d 3) NP 0.10 mg/kg 3) BPA 0.20 mg/kg
5) DES 6.5 μg/kg b.w./d b.w./day b.w./day
4) NP 10 mg/kg b.w./day 4) EE 5 μg/kg b.w./day

Dosing period GD 0 - PND 25 (mating PND 1 to PND 14 (directly to GD 0 – weaning GD 3 to PND 20 GD 3 to PND 21
to weaning) pups)
Number (N) dams 9 /group Not given 5 – 6/group 10-11/group Probably 14-16/group
Offspring exposed In utero and through Directly In utero and through In utero and through In utero and through
lactation lactation lactation lactation
Offspring tested 1 male and 1 female per All exposure groups within the Numbers vary with test; 1 male per litter used in 1 ovariectomizied
litter same litter. Totally: usually the numbers of pups all behavioural tests. female per litter
Totally: 9/sex /group 10/sex/group per test-group is larger than Totally: 7-10 Totally: 14-16
the number of litters, so that males/treatment group females/group
per test group more than
one littermate must have
been present.

10
BPA – bisphenol A, NP – nonylphenol, EE – ethinyloestradiol, TCY - Trans-2-phenylcyclopropyl-amine hydrochloride, GD – gestational day, PND – postnatal day, PNW – postnatal week.
AGD – anogenital distance; VO – vaginal opening. RVT - trans-resveratrol; DES – diethylstribestrol; b.w. – body weight. SDN-POA: sexually dimorphic nucleus of the preoptic area;
LC - locus coeruleus
11
It is not known whether the concentration given is per kg oil or per kg body weight of rats. The authors stated the administered dose was “within the range of human exposure”. Based on this,
it may be assumed that the dose was given as mg/kg b.w./day.

EFSA Journal 2010; 8(9):1829 35


Bisphenol A

Ryan & Vandenbergh,


Parameters Adriani et al., 2003 Carr et al., 2003 Kubo et al., 2003 Negishi et al., 2004
2006

Reproductive and Not performed Not performed Sexual development Body weight of dams At weaning: AGD, b.w.,
developmental (various PNDs): males: and pups litter size.
parameters 1/litter/group for b.w., Pathological exams of Assessment of puberty
and AGD dams and pups (n=4-7)
Females: 1/litter/group
for b.w., and AGD 24 -
31/group for VO and
b.w..
Sex organs and hormone
levels (PNW 12): 10-
15/sex/group
Sexual behaviour (PNW 11):
7-13/sex/group
Activity Not performed Not performed Open field behaviour: 20 - - Open field behaviour Not performed
24/sex/group (PNW 6) (PNW 8)
- Spontaneous motor
activity (PNW 12)
Brain morphology / Not performed Not performed 7-8 /sex/group; only for Not performed Not performed
histology SDN-POA and LC
Anxiety-related Novelty preference test Not performed Not performed Elevated plus-maze test Elevated plus-maze
behaviour (PND 35-45) (PNW 14) (PND 42)
Light/dark preference
chamber
Learning and memory Impulsive behaviour Spatial memory: Not performed Learning and memory: Short-time spatial
(PND > 70) : - Swim channel test (on PND - Passive avoidance test memory:
- Schedule-controlled 33) (PNW 13) - Radial-arm maze
test (nose poking holes, - Morris water Maze test (on - Active avoidance test - Barnes maze
increased delay in food PND 34) (PNW 15)
delivery)
Pharmacological Open field response to Not performed Not performed Open field response to Not performed
challenge amphetamine challenge TCY-challenge
(PND> 70) (Monamine disruption
test)

EFSA Journal 2010; 8(9):1829 36


Bisphenol A

Ryan & Vandenbergh,


Parameters Adriani et al., 2003 Carr et al., 2003 Kubo et al., 2003 Negishi et al., 2004
2006

Results for BPA BPA caused increased During acquisition, exposure to Control female pups more Perinatal exposure to Perinatal exposure to 5
according to authors neophobia in female rats E2 or BPA-low eliminated the active in the open field test BPA causes an adverse μg/kg b.w.
and reduced impulsive normal gender differences. with larger LC volume than behavioural effect when ethinyloestradiol or 200
behaviour in male rats This was due to worsen male males. BPA abolished sex the animals were forced μg/kg b.w. BPA
performance and not facilitated differences in open field to avoid fear-provoking accelerated onset of
female performance (unlike behaviour and inverted sex stimuli. puberty in female
what was expected). Exposure differences in LC volume. No offspring mice.
to BPA-high exaggerated the effect on SDN-POA and Ethinyloestradiol was
normal gender differences male or female reproductive found to masculinise
during acquisition, due to function. In males reduced behaviour in
worsen female performance. intromission rate but no ovariectomised mice in
Females exposed to E2 (and other parameters of sexual all behavioural assays
BPA) were expected to perform behaviour affected. DES used. BPA increased
as well as males and in such a inverted sex differences in anxious behaviour in a
way eliminate the normal LC volume and affected dose-dependent
gender differences because reproductive system (toxic to fashion, but had no
exposure to androgen-derived sex organs; defeminisation effect on short-time
oestrogens during brain of reproductive system and spatial memory.
development is supposed to sexual behaviour). RVT
masculine the female brain. abolished sex differences in
Also unlike what was expected, LC and defeminised the
exposure to BPA-high worsens sexual reproductive system.
the retention of spatial It demasculinised and
information in particular in defeminised sexual
females (statistically significant) behaviour. The effects of
but also in males (not BPA may vary from those of
statistically significant). RVT and DES.

EFSA Journal 2010; 8(9):1829 37


Bisphenol A

PART II - REVIEW OF RECENT SCIENTIFIC LITERATURE ON THE TOXICITY OF


BISPHENOL A

PART II - ASSESSMENT

3. Introduction
The no observed adverse effect level (NOAEL) of 5 mg/kg body weight (b.w.) per day
identified by the SCF in 2002 has provided the basis for setting the health based guidance
values for BPA both in Europe and the United States (EFSA, 2006; U.S. FDA, 2008). This
NOAEL has been identified in a multi-generation reproductive toxicity study, where the
critical effects were changes in body and organ weights in adult and offspring rats (Tyl et al.,
2002). In a subsequent multi-generation study in mice, an identical NOAEL was identified
based on liver effects in adult mice (Tyl et al., 2006).

Since then, there have been ongoing discussions on the reported low-dose effects of BPA,
particularly neurodevelopmental and behavioural effects in laboratory animals, and on the
immaturity of metabolic pathways in the foetus and neonate, both of which are important
issues for risk assessment.

In its opinion of 2006, the EFSA had reviewed a large number of literature publications,
addressing various aspects of the toxicity of BPA, including its fate in the body and the issue
of possible low-dose effects, i.e. at doses below 5 mg/kg b.w./day (EFSA, 2006). In its
evaluation, the EFSA expressed reservations both about the biological significance of the
reported observations and the robustness of the low-dose studies, ruling out their possible use
as pivotal studies for risk assessment. In 2008, the EFSA re-confirmed its previous
conclusions (EFSA, 2008a; 2008b).

Recently, the U.S. FDA has expressed some concern about the potential effects of BPA on the
brain, behaviour, and prostate gland in foetuses, infants, and young children, recognising at
the same time that the current literature could not yet be fully interpreted for biological or
experimental consistency or for relevance to human health (U.S. FDA, 2010a). In Europe, the
French Food Safety Agency (AFSSA, 2010) reviewed approximately 50 recent publications
on BPA. AFSSA interpreted some low dose effects (e.g. subtle modifications of neurological,
motor or sensorial functions, hormone balance or metabolism), as “warning signals” because
of their unknown significance in terms of health safety. Notably, publications classified by
AFSSA as “warning signals” included studies using exposure routes other than the oral. The
French Agency also recommended discussing the relevance of increasing the safety factor of
the Tolerable Daily Intake (TDI) for BPA and questioned the suitability of the TDI approach
for the risk assessment of low doses of endocrine disruptors. As of March 27, 2010 Denmark
temporarily banned BPA in materials in contact with food for children aged 0-3 years until
new studies document that low doses of BPA do not have an impact on development of the
nervous system or on behaviour. The scientific basis for this ban is a risk assessment report by
the Danish National Food Institute (DTU Food Institute, 2010) mainly focusing on the
developmental neurotoxicity study in rats by Stump (2009). According to the DTU report,
“The study does not provide any clear evidence that BPA has harmful effects on the types of
behaviour investigated in the study, but does give rise to a degree of uncertainty with regard
to the effects on learning ability, since impaired learning ability was found in male offspring
with a low dosage of BPA”.

In relation to the Commission request (Question No. EFSA-Q-2009-00864) to EFSA to


evaluate the impact of the rat developmental neurotoxicity study by Stump (2009) on the risk
assessment and on the current TDI of BPA, the EC further clarified (letter dated 3 February

EFSA Journal 2010; 8(9):1829 38


Bisphenol A

2010) that EFSA should address any new scientific evidence that may affect the conclusions
of the previously adopted opinions on BPA. Additionally, the Danish Minister for Food,
Agriculture and Fisheries sent two letters on 19 November 2009 and 10 February 2010,
respectively, encouraging EFSA to evaluate three studies on BPA, namely those of Ryan et al.
(2010a), Braun et al. (2009) and Melzer et al. (2010), as soon as possible.

Therefore, the CEF Panel undertook the task of reviewing new toxicological data that may
have an impact on the previous risk assessments of BPA (EFSA, 2006; 2008a; 2008b). In
particular, this review focuses on toxicokinetics, human studies and animal toxicity studies.
The overall scientific literature on BPA was searched for the time period 2007-July 2010,
using MEDLINE and ISI Web of knowledge, retrieving about 800 articles. A full list of the
retrieved articles will be made available by EFSA on request. The study design of all these
publications was examined in the first instance. Only studies complying with the inclusion
criteria indicated below were considered for risk assessment purposes, and were then
reviewed according to specific quality criteria (see section 5.2.) in order to assess the validity
and/or applicability of the individual findings to human risk assessment. In this review, the
studies by Ryan et al. (2010a), Braun et al. (2009) and Melzer et al. (2010) are addressed in
depth to meet the requests of the Danish Minister for Food, Agriculture and Fisheries.

Criteria for study inclusion for consideration in risk assessment

The criteria for study inclusion in this review were set as follows:

• Full research papers published in peer-reviewed journals available in public domains


since the EFSA 2006 opinion (2007 – July 2010)

• Original data (no reviews, discussions or others)

• Human studies12

• For the animal toxicity studies the focus was on studies having the following
experimental design:

a. Developmental exposure, i.e. pre-, peri-, and/or early post-natal exposure


b. oral route of exposure
c. several tested doses (plus a control) including at least one dose level lower than
the NOAEL of 5 mg/kg b.w./day (low-doses) from which the current TDI has
been derived.
Each of these design aspects is discussed in more detail as follows.

a. Developmental exposure

In view of the possible concern on children’s health associated with low dose exposure
to BPA, as expressed by the U.S. FDA, AFSSA and the Danish Veterinary and Food
Administration, the CEF Panel decided to review the studies where BPA was
administered at any stage during the perinatal period (in utero and/or early postnatal
development).

12
The Panel excluded from the selection purely biomonitoring studies, which mainly deal with exposure, and
therefore are not useful for TDI setting. The Panel noted that most biomonitoring data obtained in different
biological samples obtained so far have been tabled in a recent FDA report (U.S. FDA, 2010b).

EFSA Journal 2010; 8(9):1829 39


Bisphenol A

b. Oral route of exposure

The CEF Panel considers oral toxicity studies more appropriate for a quantitative risk
assessment than non-oral route studies for the following reasons: (i) oral intake is the
most relevant route of human exposure, occurring through migration of BPA from food
contact materials into food; (ii) other routes of exposure (e.g. intraperitoneal (i.p.),
intracranial (i.c.), subcutaneous (s.c.) injection or through implantation of s.c. pumps),
show kinetic differences with respect to oral uptake of BPA (i.e. bypass of intestinal
absorption and first-pass detoxifying metabolism), affecting the internal free BPA level
and dose-effect relationships as demonstrated in rats and non human primates
(Tominaga et al., 2006). In contrast to results reported by Taylor et al. (2008; discussed
by EFSA, 2008a) there is clear evidence that the serum levels of free BPA are higher
after parenteral rather than oral administration in rodents as well as in non human
primates (Tominaga et al., 2006; Doerge, 2010a,b; see section 4 “Toxicokinetics”).

The Panel has also considered several studies employing non-oral routes of exposure, in
order to properly characterize the potential toxicity endpoint and mode of action of
BPA. However, such studies were not used for risk assessment for the reasons indicated
above.

c. Dose-response analysis

A dose-response characterization is of paramount importance for quantitative risk


assessment. Dose dependency of an effect provides an increase in the level of
confidence in an endpoint. The issue of dose-response relationships is crucial to the
debate on endocrine active substances, for which non-monotonic (U-shaped or inverted
U-shaped) dose-response curves have been postulated (Melnick et al., 2002). U-shaped
dose-response curves could occur if a ligand activated two separate pathways with
differing threshold sensitivities, but which impinge on a similar downstream pathway.
To demonstrate U-shaped dose-responses in a robust way, it is necessary to have a wide
dose range and reasonably closely spaced dose intervals, usually of less than 10-fold
within the same study. The presence of a response at one dose level only is not
sufficient to demonstrate a causal relationship between the administration of a
substance and an observed change.

The current NOAEL for BPA is 5 mg/kg b.w./day. The Panel defined as “low dose”
studies those investigations that included at least one dose of BPA below 5 mg/kg
b.w./day. If studies employed only doses at or higher than this NOAEL, they were not
deemed relevant to low dose risk assessment.

Studies not compliant with the inclusion criteria

Several research studies not compliant with the inclusion criteria, but still useful for hazard
identification and for support of biological plausibility e.g. in vitro studies or non-oral in vivo
studies are also discussed in this opinion (see e.g. section 5.3 “Other information on the
endocrine-mediated action of BPA”).

4. Toxicokinetics
As already reported in the two previous opinions (EFSA, 2006; EFSA, 2008a), a marked
species difference exists in BPA disposition in humans and rodents. In humans, both low and
high single oral doses of BPA are well absorbed and undergo complete first-pass metabolism
in the liver to BPA-glucuronide as major metabolite, which is rapidly excreted in the urine,
with a half-life of less than 6 hours (Völkel et al., 2002, 2008; Tsukioka et al., 2004).

EFSA Journal 2010; 8(9):1829 40


Bisphenol A

Bisphenol A-sulphate has been reported as a minor urinary metabolite of BPA in humans (Ye
et al., 2005). In rats BPA is biotransformed by the same reactions (EFSA, 2006), but in this
species, BPA undergoes enterohepatic recirculation. Once excreted from the liver via bile into
the gastrointestinal tract as BPA conjugates, these conjugates are cleaved back to BPA and
the free BPA is reabsorbed. As a result BPA clearance takes longer in rodents than in humans,
the half-life in rodents being 19-78 hrs (EFSA, 2006). Free BPA is the toxicologically
relevant compound, since the conjugates do not retain the biological activity of the parent
compound (Snyder et al., 2000; Shimizu et al., 2002, Willhite et al., 2008). Therefore the
ability and level of conjugation is important for risk-assessment of BPA.

4.1. New pharmacokinetic studies in rats and monkeys (Doerge et al. 2010a, 2010b)
A very recent pharmacokinetic study by Doerge et al. (2010a) measured by LC/MS/MS serum
levels of free and conjugated deuterated BPA in neonatal, immature (PND 3, 10 and 21) and
adult Sprague-Dawley rats dosed via oral (gavage) and injection routes (i.v and s.c.). Animals
were given a single dose of 100 μg/kg b.w. BPA, which was demonstrated to be within the
linear range of pharmacokinetics, so that extrapolation to lower doses is feasible. The use of
labelled BPA (methyl-d6-BPA) avoided confounding background contamination (from
laboratory materials or other sources), which was reported by the authors to be as high as 2
ng/ml in buffer blanks. These findings confirm the importance of taking into account sample
contamination by BPA in the study design, and also strongly limit the value for risk
assessment of studies where this issue has not been properly taken into account.

In adult rats, direct comparisons of i.v. and oral routes of administration indicate: i) extensive
absorption from the gastrointestinal tract; ii) rapid elimination of free BPA from the
circulation (> 50% of circulating BPA was conjugated 5 min after i.v. injection); iii)
importance of first-pass conjugation in liver and gut after ingestion: indeed, at maximum
serum concentration (Cmax), the fraction present as conjugated BPA was substantially higher
following oral compared to i.v. administration (99.5% vs. 55%). Moreover, the higher volume
of distribution following i.v. vs. oral administration was due to higher distribution of free
BPA to tissues. In addition, the occurrence of enterohepatic recirculation of BPA in adult rats
is reflected by a secondary peak in the concentration of total BPA later in the rat serum
concentration-time profiles.

The Cmax of free BPA observed in individual rats ranged from 0.1-0.6 nM (average value of
0.39 ± 0.19 nM, corresponding to about 89 ng/L) so that internal exposures of adult rats to
free BPA are below 1% of the total (Cmax = 73 ± 29 nM, corresponding to 16.6 µg total
BPA/L).

A comparison of BPA pharmacokinetics in adult vs. neonatal rats was also performed.

Oral administration of BPA (100 μg/kg b.w.) to PND 3 pups produced higher Cmax in serum of
total (6-fold) and free (74-fold) BPA when compared to adults: the fraction present as
conjugates was 93.4% (PND 3) and rapidly increased with age up to 96.9 and 98.9% (PND 10
and 21, respectively), indicating a regular development of metabolic and excretory functions
toward the adulthood situation (99.5% BPA in conjugated form). The percentage of total BPA
present in neonatal blood (PND 3) as free BPA was very limited (1.4% of AUC, Cmax: 6.6%).

Administration of an identical dose (100 μg/kg b.w.) by s.c. injection to PND 3 rats produced
34-fold higher Cmax and 17-fold higher AUCs for free BPA compared to oral. The age-related
changes (described above) after oral administration were not observed after s.c. injection,
indicating that even in early postnatal pups, which possess lower conjugation activity, the first
pass effect is extremely relevant. This confirms that the effect due to the route of

EFSA Journal 2010; 8(9):1829 41


Bisphenol A

administration is very relevant, and highlights the limitations in interpreting toxicity data from
studies in which BPA is administered via routes other than the oral one, without any internal
dosimetry.

Assuming that the impact of repeated dosing would be more relevant for young animals, with
slower elimination, higher Cmax and lower conjugation capacity, based on the kinetic
parameters obtained in PND 3 pups after a single oral BPA dose, a simulation was carried out
to reflect the typical once-daily dosing used in many BPA toxicity studies (Doerge et al.,
2010a). Although the Panel noted that potential repeated exposure in infants is 6 times a day,
the estimate of the average free BPA concentration at the steady state was 2.3 nM, and no
significant increase in rat plasma or tissues is expected as a result of repeated dosing (ratio
free BPA Cmax vs. steady state level is 1.1 to 1.2.

With a similar study design, free deuterated BPA (100 μg/kg b.w.) was administered to adult
and neonatal rhesus monkeys orally (PND 5, 35 and 70) and i.v. (PND 77). Free and
conjugated BPA were measured in the blood (Doerge et al., 2010b).

In adult rhesus monkeys, results for the first sampling points show that the percentage of free
BPA was higher following parenteral (i.v) administration (29 ± 19% of total BPA at 5 min
post-injection) than after oral administration of the same dose (0.21 ± 0.14% of total BPA at
30 min post-gavage). This confirms that in monkeys the systemic availability of free BPA is
much lower after oral administration. Overall, these findings are in line with those in the rat
(Doerge et al., 2010a), although after i.v. exposure the percentage of free BPA was higher in
adult rats than in monkeys (52 ± 10% vs. 29 ± 19% at 5 min post-injection). Results are in
agreement with those obtained in a previous study comparing BPA toxicokinetics in rats and
cynomolgus monkeys after oral administration of 10 mg/kg b.w. BPA: the Cmax of BPA
metabolites in cynomolgus monkeys was around 60-fold greater than in rats, with a ratio for
metabolite/parent compound of about 30 and 350 in rats and monkeys, respectively
(Tominaga et al., 2006). Also the half-life of total BPA (3.5 h) was similar to those reported
in cynomologous monkeys (4.2 h; Tominaga et al., 2006), and humans (3.4 h, Völkel et al.,
2002), indicating that the monkey is a better model for human adults than are adult rats.

Overall, the results of the studies by Doerge et al. (2010a,b) indicate that following oral
administration of 100 μg/kg b.w.BPA, the degree of conjugation is similar in rats and
monkeys (>99%), and support that presystemic conjugation with glucuronide occurring in the
gut and in the liver after oral administration is a crucial factor in determining the internal dose
of free BPA in both species. In adult monkeys no evidence for enterohepatic recirculation was
observed, which resulted in a shorter elimination half-life in monkeys than in rats.

When BPA was administered to neonatal non-human primates orally (PND 5, 35 and 70) or
i.v. (PND 77), the toxicokinetic parameters were not statistically significantly different from
those in adults. Indeed, the percentages of AUCs0→∞ for free BPA vs. total BPA after i.v.
administration were 14 ± 4.0% (n=4) and 13 ± 5% (n=5) in adult and PND 77 animals,
respectively. After oral dosing, these percentages were 0.19 ± 0.18%, 0.14 ± 0.11, 0.16 ±
0.11, and 0.45 ± 0.37 in adult (n=4) and PND 5, 35 and 70 neonatal animals (n=5),
respectively. These data show that the degree of conjugation was not affected by
developmental age at the dose administered. By comparison, in rats the fraction present as
free BPA was clearly age-related, being higher in pups and decreasing with age. In both
monkeys and rats, the predominant conjugate was the glucuronide with the sulfate
representing <20% for monkeys and <5% for rats. The glucuronide/sulfate ratio was not
significantly affected by age from early perinatal to adulthood in either species. In conclusion,
although the level of variability was higher in young non human primates, the conjugation

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Bisphenol A

capacity for the tested BPA dose resulted in a free BPA fraction <1%, starting from PND 5,
with no significant age-related changes (Doerge, 2010b).

The Panel noted that following the same oral dose of 100 µg/kg b.w. BPA to adult rat and
monkeys, free BPA concentrations in both species was similarly low (<1%), the only notable
difference being the longer elimination half-time in rats, due to the enterohepatic re-
circulation in the rat. On the contrary, comparing newborn animals, PND3 rats have longer
elimination half-time and approximately 10 times higher plasma levels of free BPA than PND
5 monkeys, when treated with the same oral BPA dose (Doerge, 2010b). These data provide
evidence for a different developmental profile of hepatic and intestinal conjugation of BPA in
rats and monkeys, consistent with literature data describing a higher degree of immaturity of
rats at birth as compared to primates, in relation to UGT activity (Coughtrie et al, 1988;
Matsumoto et al, 2002).

To evaluate the impact of repeated dosing, the single dose (100 μg/kg b.w.) toxicokinetic
parameters derived for a representative PND 5 monkey were used to simulate the serum
concentration-time profile after repeated exposure with a 4 h frequency of dosing to mimick
the typical infant food and beverages consumption over a 24 h period (Doerge et al., 2010b).
The estimate of average free BPA concentration at the steady state was 0.86 nM, and no
significant increase in plasma or tissues is expected as a result of repeated dosing (ratio Cmax
vs. steady state level is 1.1).

The authors (Doerge et al., 2010b) used the toxicokinetic parameters as identified in the study
with non human primates with deuterated BPA to estimate exposure to BPA based on
reported measured concentrations of free BPA in human serum. They calculated that a
concentration of 3 µg free BPA/ml serum would correspond to an oral exposure to 1300 µg
BPA/kg b.w.. This value is far higher than any conservative estimate of potential exposure to
BPA (EFSA, 2006). It follows that studies reporting high levels of free BPA in serum, up to
20 µg/L, are very likely affected by sample contamination, representing a significant flaw of
many studies not using labelled BPA or not applying specific procedures to take background
contamination into account (see section 5.1.4.1).

4.2. The enzymes involved in BPA biotransformation


The enzymes which are involved in BPA conjugation are UDP-glucuronyl-transferases
(UGT) and sulfotransferases (SULT). In both monkeys and rats, the predominant pathway is
glucuronidation, with the sulfation reaction representing <20% for monkeys and <5% for rat.
The glucuronide/sulphate ratio was not age-dependent in both species (Doerge et al., 2010b).
Both enzyme families consist of different isoforms, which can have different affinities
towards BPA (expressed as Km) and efficiencies in metabolizing it (expressed as intrinsic
clearance =Vmax/Km) as well as different ontogenetic patterns. In addition they show genetic
polymorphisms. Information on which isoform is involved at dose levels relevant for human
exposure can be used as valuable input in PBPK modelling in order to identify whether
groups of individuals can be at higher risks due to the presence of allelic variants with altered
activity or a different age-related enzyme expression level.

Among the different recombinant human isoforms, UGT2B15 showed the highest activity
over the range of BPA concentrations (1-20 μM) tested. The biochemical parameters (Km and
Vmax) of UGT2B15 and of pooled human liver microsomes were of the same order of
magnitude. Clearly a role for UGT2B15 is identified (Hanioka et al., 2008). In addition, by
using a complementary panel of recombinant enzymes, UGT1A9 (which is expressed both in
the gastrointestinal tract and in the liver) was the most efficient one, and to a lesser extent also
2B7 and 1A8 (hepatic and intestinal enzyme, respectively) (Doerge et al 2010b).

EFSA Journal 2010; 8(9):1829 43


Bisphenol A

A single nucleotide polymorphism has been identified in the UGT2B15 gene, resulting in
either an aspartate (UGT2B15*1) or tyrosine (UGT2B15*2) at position 85 of the protein
(Levesque et al., 1997). The polymorphism can result in a modification of the activity for
different substrates. With a number of substrates (i.e. dihydrotestosterone, androstane-3-diol,
S-oxazepam, naringenin, eugenol, and 4-methylumbelliferone), the UGT2B15*1 allelic
variant has been demonstrated to have 2 to 5 fold higher rates of glucuronidation when
compared to UGT2B15*2 (Court et al., 2002; Court, 2010). Polymorphisms of UGT 1A9 and
2B7 have been also identified, whose functional consequence is unclear. However, the
coefficient of variation for UGT 2B15, 1A9 and 2B7 in a large human liver bank is 72, 55 and
45% (the lowest among different UGT isoforms), respectively. Therefore, although factors
influencing UGT-activity variability include genetic polymorphism, the impact of the
polymorphic allelic variants is expected to be limited (Court, 2010).

Due to the redundancy in UGTs for conjugation and the overlapping substrate specificity, it is
expected that a single polymorphism would not significantly affect the total BPA
glucuronidation capacity of individuals (Doerge et al., 2010b).

At present no specific information about ontogeny in humans is available for UGT2B15,


whereas some information is available on other UGT isoforms, such as UGT1A1, the activity
of which is low at birth, reaching levels comparable to adulthood within 3-6 months later (de
Wildt et al., 1999), or UGT2B7 whose activity in neonates at term is only 5% that of adults,
increases to 30% by 3 months of age, and reaches adult levels by 1 year of age (Edginton et
al., 2006). This pattern is the one which has been used in human PBPK models to account for
possible limited UGT activity for BPA conjugation during early life (Edginton and Ritter,
2009; Mielke and Gundert-Remy, 2009). Notably, most information on UGT ontogeny refers
to the liver, which is usually endowed with the highest glucuronidation activity. However, in
the foetus UGT immunoreactivity in liver and kidney tissue is considerably lower when
compared with the red blood cells. For this reason it has been hypothesised that circulating
UGTs may substantially contribute to detoxification of xenobiotics in the foetus (de Wildt et
al., 1999).

Regarding the role of SULT isoform(s) in the conjugation and deactivation of BPA, human
recombinant SULT1A1, which is involved in the conjugation reaction of other phenols
(Campbell et al., 1987), has been identified as the major isoform mediating BPA sulfation in
the human liver, although recombinant SULT2A1 and 1E1 showed also some activity
(Nishiyama et al., 2002). The human SULT1A1 gene has common single nucleotide
polymorphisms resulting in three allelic variants (Nagar et al., 2006; Ung and Nagar, 2007)
for which the differences in specific activity can be up to 10-fold (Nowell and Falany, 2006),
although they are not necessarily translated into the same degree of interindividual variability
in in vivo sulfation capacity. The differences due to polymorphism are expected to be covered
by the interindividual standard uncertainty factor.

For SULT enzymes no age-dependency has been described (Pacifici et al., 1993: Duanmu et
al., 2006) and consequently, in humans the sulfation activity is comparable at birth and in the
adult.

4.3. In utero exposure and kinetics


In utero exposure is directly dependent on the possibility of BPA transfer from the exposed
mother to the embryo/foetus. It is important to underline that during pregnancy in humans the
glucuronidation pathway is induced as compared to the activity in non-pregnant women
(Anderson, 2005; Hodge and Tracy, 2007). Higher glucuronidation activity would result in
higher metabolic clearance of BPA and a consequent reduction of free BPA plasma

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Bisphenol A

concentrations. In contrast, in rodents metabolite profiles in non-pregnant and pregnant


animals were similar, and therefore there would be a possible systemic availability of free
BPA for the foetus.

After oral administration of 10 mg/kg BPA to GD 16 rat dams, Domoradzki et al. (2003)
demonstrated that the concentration of BPA-glucuronide in the foetus is approximately 0.1%
compared to that in maternal plasma (0.013 vs. 1.7 µg equivalents/g plasma in foetus and
maternal plasma, respectively). Kurebayashi et al. (2005) also reported that a small amount of
radioactivity is transferred to the rat foetus, but only during late pregnancy (not before GD
18). No distinction could be made between free- or conjugated BPA, due to the low levels
observed.

Lee et al. (2008) have studied maternal and foetal exposure to BPA, measured as total (free +
conjugates) BPA concentration in maternal and the umbilical cord blood in 300 subjects in
Korea. From the available information it was not possible to associate maternal levels with
dietary BPA exposure (use of food in contact with BPA-containing material), and the
possibility for other sources of exposure is not excluded by the authors.

The study showed that 22% and 60% of the maternal and foetal samples, respectively, did not
contain any detectable level of total BPA (LOD: 0.625 μg/L). The maternal blood
concentrations were highly variable, with 77% of the samples having total BPA levels <10
µg/L and the remaining 23% spread over a wide range of concentrations (median 2.7 µg/L;
higher level 66.48 µg/L). The high variability shown by data on maternal exposure may be
due to the short half-life of BPA, which makes the time of sample collection (not under
control in the study) with respect to recent exposure particularly critical, and makes further
comparisons difficult.

The BPA-positive umbilical cord blood samples (40% of the total) showed a median
concentration of <0.625 µg/L. The highest reported value was below 10 µg/L.

Maternal and foetal blood total BPA concentrations showed some correlation, although levels
in the umbilical cord blood were always considerably lower, indicating limited placental
transfer. Since the study did not discriminate between free and conjugated BPA, it is not
possible to evaluate the form in which BPA was present and to know if placenta could
preferentially/selectively prevent diffusion of conjugated BPA, which is very likely to
represent the major BPA portion in maternal blood.

The concept of the placenta as an efficient barrier has been supported by a recent study on
placental transfer of BPA (Mørck et al., 2010). [14C]-ring-labelled BPA permeability was
tested in an in vitro model of human origin for the placental transport (BeWo cell monolayer)
and in term human placentae (ex vivo). The use of the radiolabelled BPA avoids the problem
of background contamination of samples; however, total BPA was detected by liquid
scintillation, without differentiating between free and conjugated BPA. The in vitro
permeability of total BPA in BeWo cells was higher in the foetal-to-maternal direction as
compared to the opposite. Inhibition of the P-glycoprotein (P-gp) reduces the foetal-to-
maternal BPA transport, indicating active efflux of BPA by the BeWo cells, resulting in the
majority of the added BPA remaining in the maternal compartment. Since the BPA
concentration tested was quite high (0.5 µM, corresponding to 114 µg/L), the possibility of P-
gp saturation (limiting the efflux action) cannot be excluded.

About 20% of the initially added free BPA (0.5 µM consisting of 10% [14C]-ring-labelled free
BPA and 90% of cold BPA) was transferred across the barrier in human term placentae ex
vivo, with 15% recovered in the perfused cotyledon and 12.7 ± 8.5% in the surrounding

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Bisphenol A

placental tissue (Mørck et al., 2010). However, the metabolism of BPA (which is expected to
occur in the placental tissue) was not investigated: therefore some of the radioactivity
detected in placental tissue or in the perfusate probably represents BPA-conjugates. In
addition, the efflux of BPA by P-gp in the placental tissue (evidenced with the cell line) was
not investigated ex-vivo and, since P-gp is expressed in human placenta, BPA efflux probably
occurs, reducing the transplacental transfer of BPA to the foetal circulation.

Another paper investigating the transfer of BPA across 7 human placentae by means of
placental perfusion, by using a 10 µg/L BPA solution (corresponding to 44 nM) reports that a
similar fraction (about 27%) was transferred to the foetal compartment (Balakrishnan et al.,
2010). Only less than 10% of the total BPA in the foetal compartment was present in the
conjugated form. The authors excluded sample contamination by free BPA, by using 2 control
placentae in which no BPA was found; furthermore, they excluded the occurrence of
deconjugation reaction during sample analytical processing, by adding β-oestradiol-17β-
glucuronide and β-oestradiol-3-sulfate as ‘reference’ conjugated compounds. However, the
relative stability of the ‘reference’ compounds when compared with BPA-conjugates was not
reported, so that results are not conclusive. The authors claim that BPA was transferred in the
free form, due to a postulated inability of human placenta to conjugate BPA. However, the
activity of UGT and SULT in the placentae was not determined. Indeed, a loss of
glucuronidation and sulfation capacity during the entire testing period (more than 4 hours
from delivery to transport sample collection) cannot be excluded. In the absence of these data
no direct conclusion can be drawn on this issue.

The barrier function of the human placenta is supported by data obtained in pregnant rats
(GD18), by using uterine perfusion with 2 μM BPA-glucuronide (Nishikawa et al., 2010),
corresponding to about 968 μg/L, a concentration much higher than realistic exposure levels.
After perfusion, almost all the BPA-glucuronide was detected in the maternal vein, but only
0.09% of total amount was detected in the foetus. Extremely small amounts of free BPA were
detected in the foetal tissues (about 6% of the amount of BPA-glucuronide found in the
foetus, that is 0.005% of the initial concentration of BPA in the uterine perfusate). The
authors attributed the occurrence of this fraction of free BPA in the foetus to the presence of
enzymatic deconjugation activity in foetal tissues. However, no specific measures were
adopted to avoid sample contamination with free BPA during analytical procedures, which
therefore cannot be excluded (see section 5.1.4.1).

The potential role of enzymatic deconjugation reactions (catalyzed by β-glucuronidases and


sulfatase), has also been postulated by Ginsberg and Rice (2009): this would be the cause of a
local activation of the conjugated form back to free BPA, initiating a conjugation–
deconjugation cycling. The authors considered BPA deconjugation as a potentially important
pharmacokinetic factor during the perinatal period, due to the presence of β-glucuronidase in
the placental and foetal tissues, with a rapid ontogeny during development (Lucier et al.,
1977). However, the experimental data available so far on in utero exposure do not seem to
support this hypothesis. Data in humans on the expression and activity of sulfatase are not
available. Maturation of sulfatase activity has been investigated in developing rats (Huang et
al., 1996). In hepatic microsomal preparations from rat foetuses the desulfation activity was
extremely low. The Panel noted that the profile of BPA elimination from plasma in monkeys
showed no evidence for any recycling due to deconjugation reactions in any organs both in
neonatal and adult animals. Based on this, the Panel considers that deconjugation is also not
likely to occur in humans and therefore a relevant production of free BPA from circulating
conjugates is not anticipated in humans.

In addition to the placenta, also the rat uterine epithelial cells could act as a barrier system.
Matsumoto et al (2007) after incubating a rat uterine sac in buffer solutions containing free

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BPA (25 μM, corresponding to more than 5 mg/L), demonstrated that most BPA perfused into
the uterus was glucuronidated in the epithelium, and then excreted into the serosal side
(maternal side), probably via active transporters. The almost complete BPA glucuronidation
suggests that the enzymes are not yet saturated also at the extremely high BPA concentration
tested.

Overall these findings indicate that the transplacental transfer of BPA and BPA-glucuronide
can occur but foetal exposure to free BPA is limited due to limited placental transfer of
(conjugated and) free BPA from mother-to-foetus, efficient foetal-to-mother transfer by the
efflux pump P-glycoprotein (which is known to be expressed and functional in human
placenta) and due to the expected BPA detoxification in placental tissue, also taking place in
the uterine epithelium. Although BPA placental detoxification has not been specifically
measured in the described studies, it is well recognized that UGT are present in the placenta
throughout gestation, with UGT2B family (UGT2B4, 2B7, 2B10, 2B11, 2B15 and 2B17)
protein expression and enzymatic activity toward different drugs detected in first trimester
and term human placentae (Syme et al., 2004; Collier et al, 2004). Also UGT1A mRNA and
protein expression have been detected in the first-trimester but not in term placenta (Syme et
al., 2004)

4.4. Neonatal exposure and kinetics


The urinary concentrations of 54 low-birth-weight premature infants (gestational age ≤ 44
weeks) undergoing intensive therapeutic medical interventions in two different neonatal
intensive care units in the US were investigated (Calafat et al., 2009). These premature infants
had median total urinary BPA concentrations (28.6 μg/L) markedly higher than the median
concentration (3.7 μg/L) among children 6-11 years old (Calafat et al., 2008). The repeated
urinary measurements from the same infants available for 13 individuals were highly
correlated. The median total BPA concentrations were inversely related to gestational age,
with median total BPA levels 10 times higher among infants 25-27 weeks of age when
compared with the older (28-34 weeks) infants, indicating that the observed differences in
BPA concentrations are likely to be attributable to the recent treatment given to the infants
after birth rather than to the in utero exposure. No correlation was evidenced between total
BPA levels and length of stay in intensive care units or feeding type. However, these are
preliminary results since information on gestational age, hospitalization period or feeding type
was incomplete for many infants and taken by observation, since the authors had no access to
medical records. Notably, BPA total concentrations among infants in one health care unit
were about 17 times higher than those among the infants hospitalized in the second one. In the
unit where infants experienced higher levels of BPA, intensity of use of plasticised PVC
products containing di(2-ethylhexyl) phthalate (DEHP) (low, medium and high exposure
group) was strongly associated with BPA total concentrations. The presence of BPA in some
samples of plasticised PVC was reported by Lopez-Cervantes and Paseiro-Losada (2003).
This suggests atypical exposure conditions and specific source(s) of exposure to BPA, not of
dietary origin. Considering that such medical devices could represent the exposure source, a
parenteral route could imply a higher fraction of circulating free BPA, due to the bypass of
presystemic intestinal and hepatic conjugation.

Notably, the study showed that > 90% of the BPA excreted in the urine by the premature
infants was in its conjugated (e.g. glucuronide, sulfate) form, clearly indicating that premature
infants are able to metabolize BPA, despite the very high exposure and the possible major
contribution of the parenteral source. The analyses were carried out about 5 years after the
sample collection, but since samples were stored at -40°C, de-conjugation during storage
should have been prevented: indeed, the recovery of 90% BPA as conjugates supports the
validity of the results. However, since the same samples after collection were used for another

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analysis possibly requiring repeated freezing and thawing, the partial hydrolysis of conjugates
(sulphates and/or glucuronides) cannot be ruled out, and free BPA data should be interpreted
with caution, as advised by the authors themselves. The fraction of free-BPA in the freshly-
collected urine samples may have been less than 10%. Overall, these findings confirm that
BPA is biotransformed efficiently by newborns (EFSA, 2008a).

The concentrations of free and total BPA were linear over the range of detected BPA levels,
suggesting that the enzyme(s) responsible for the conjugation of BPA are not saturated. Since
the urinary levels reported in the general adult U.S. population are 10-fold lower (1.2-2.6
μg/L; Calafat et al., 2005; 2008) than those measured in premature infants, saturation is not
expected also in adults. This is in agreement with the first order elimination of BPA described
by Völkel et al. (2002; 2008) even at extremely high BPA doses, as cited in previous EFSA
opinions.

The passage of free BPA into breast milk is an important parameter. Breast milk is the major
route through which breast fed infants can be exposed to environmental chemicals and indeed
this exposure pattern has been used in some recent toxicity studies on BPA with rodents.

The study of Snyder et al. (2000), which was considered by EFSA (2006) for other kinetic
aspects, provides detailed information about the passage of BPA into breast milk from
lactating rats receiving 100 mg/kg b.w. 14C-labelled BPA by gavage on day 14 post-delivery,
1 and 8 hours post-dosing. Minimal levels of radioactivity were found in milk (0.003% of the
administered dose (mg/dam)/g of milk at 1 hour; 0.002%/g of milk at 8 hrs and 0.0008%/g
after 24 hrs. The major radioactive peak observed in milk in females was identified by
retention time to be BPA-glucuronide. Free BPA was also detected in milk of 2/10 treated
females. Total exposure of the pups was estimated to be 3.15 µg equivalents/pup, or
approximately 0.3% of the dose (mg/dam)/kg pup weight.

A second group of animals experiencing the same treatment was returned to their pups
immediately following dosing (10 pups per dam). Groups of 4 pups per litter were killed at 2,
4 and 6 hours post-dosing of the dams: radioactivity in pup carcasses accounted for <0.01% of
the administered dose. Overall, the data from Snyder et al. (2000) provide evidence of very
limited transfer of BPA to pups via the breast milk. The detection of radiolabelled material
allows excluding any ‘environmental’ contamination of the samples.

Very recently, an additional paper addressed the issue of excretion of BPA into rat milk
(Okabayashi and Watanabe, 2010). Dams were orally administered BPA (100 mg/kg b.w., 20
fold higher than the NOAEL for the rat) 2, 4, 8, or 24 h prior to milking. Beside milk samples
from the treated dams, samples obtained from control dams, and dams which were provided
the drinking water in polycarbonate bottles or in glass bottles, were also analysed by LC/MS.
The detail provided in the paper was limited: the authors did not mention the time for
sampling of the samples from control animals, nor the detection of any BPA-conjugate. Data
are reported for milk samples obtained from dams dosed with 100 mg/kg b.w.. BPA
concentrations were higher in milk of dams treated 2 and 4 h prior to milking (462 ± 182 μg/L
and 138 ± 18.5 μg/L, respectively), then decreased at 8 and 24 h (80 ± 19.7 μg/L and 23.2 ±
5.1 μg/L, respectively). It is stated that BPA could not be detected in the milk samples
obtained from untreated rats with BPA or receiving drinking water stored in glass bottles.
Milk samples from rats receiving drinking water stored in polycarbonate bottles contained
BPA levels of 18.4 ± 5.0 μg/L, that is only one order of magnitude lower than the levels
measured in rats treated with 100 mg/kg b.w.. Considering that the BPA concentration in
water in polycarbonate bottles was indicated to be as low as 0.33 ± 0.15 μg/L, this finding
seems to be not fully consistent and limits the value of the described results.

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Kurebayashi et al. (2005; cited in the 2006 EFSA opinion) reported the recovery of BPA-
derived radioactivity in neonatal tissues (mostly located in the intestinal content) from two
pups 24 and 48 hr after oral administration of 14C-BPA (500 μg/kg b.w.) to a single lactating
rat on PND 11 (Kurebayashi et al., 2005). The results also indicate a transfer of BPA via milk
during lactation, with a peak concentration in milk 8 h after treatment of 3 lactating dams
(4.46 ng/ml). No indication about the number of pups in the litter or about the volume of
collected milk was given. The radioactivity was given as total BPA, without differentiating
between free BPA and its glucuronide.

The Panel underlined that the total daily yield of milk in rats is highly dependent on the PND
and on the litter size. A 10 pup litter at PND11 showed an average daily yield of 20 ml milk
(Russel, 1980), whereas a litter of one pup would yield about 1.25 ml. At the peak
concentration in milk obtained 8 h post treatment as described in the Kurebayashi study, the
transfer of total BPA to the pup via milk can be estimated to be in the range of 0.003-0.066%
of the administrered dose, depending on the litter size. The mean daily milk yield of animals
nursing one pup (considered as a single group) was only about 5% of the daily milk yield of
animals nursing ten pups (Russel, 1980). Therefore the transfer from lactating animals to
neonates and the BPA-derived radioactivity concentration in milk are strictly linked to the
study design and to the number of pups in the litter.

Yoo et al. (2002) also reported transfer of free BPA into rat milk following i.v. infusion of
doses from 0.47-1.88 mg/kg b.w. with a steady state milk/serum ratio of approximately 2.5.

The Panel noted that exposure to total BPA (the major constituent being the glucuronide)
through lactation is limited to a very low fraction (approximately 0.3%) of the administered
dose (mg/dam)/kg pup weight.

4.5. Human physiologically based pharmacokinetic (PBPK) modelling


In two recent studies (Edginton and Ritter, 2009; Mielke and Gundert-Remy, 2009),
physiologically based pharmacokinetic (PBPK) modelling was applied to estimate levels of
BPA in the blood in young children. Both studies used a similar approach, assessing the age
dependence of the toxicokinetics of BPA and its glucuronidated metabolite, by using a human
based model. In view of the metabolic differences among rodents and humans, this makes
these two studies more appropriate than a previous one using PBPK models, which were
extrapolated from a rat model (Teeguarden et al., 2005). The PBPK model by Kawamoto et
al. (2007) was specifically developed for pregnant mice. Good consistency between the model
simulations and the experimental data were observed for BPA and its metabolite
concentrations, as well as for total BPA concentrations in maternal blood and liver. This
model indicated that the enterohepatic recirculation is responsible for the time course in dams
and for the longer stay of BPA and its metabolites in the foetal compartment (Kawamoto et al.
2007). Since enterohepatic recirculation does not occur in humans, the applicability of such a
model to humans would not be appropriate.

Concerning human-based models, Edginton and Ritter used information gathered from
toxicokinetic studies in adults to build their PBPK model, which was further scaled to
children < 2 years of age, by replacing the age-dependent physiologic parameters relevant for
kinetics in newborn. For BPA metabolism, the age-dependent activity of the human UDPGT
was approximated from the UGT2B7 isoform, since information on the ontogeny of
UGT2B15 (the isoform indicated as the most active one) was currently not available (Court,
2010). As pointed out by the authors themselves, there is uncertainty when using enzyme
activity ontogeny data (Bjorkman, 2006) because a period of high interindividual variability is

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present between the postnatal onset and increase in expression of hepatic enzymes (Hines,
2008).

The average steady-state plasma concentration of total BPA in adults after a dietary exposure
of 1.5 μg/kg b.w./day (EFSA, 2006) was 0.58 μg/L, which is similar to the LOD of the
method used in Völkel et al. (2005), and therefore consistent with the finding described in that
paper; simulation of BPA glucuronide kinetics in adults and distribution volume were also
equivalent to experimental data (Völkel et al., 2002; 2005).

Edginton and Ritter reported average free BPA modelled plasma concentrations at steady
state in newborns and 3 months-old infants, which are, respectively, 11 and 2 times greater
than that in adults, when given 1 μg/kg b.w./day. By using the BPA exposure data for adults
and different age children from EFSA (2006) and from Casey et al. (1986) and Ye et al.
(2006) for breast-fed newborn exposure, free BPA average plasma concentration at steady
state in newborns and in breastfed 3 month-old infants were 1.8 and 0.26-fold that in adults
(0.004 μg/L). In formula-fed 3 and 6 month-old infants the modelled plasma concentrations
were approximately 5 times greater than those in adults.

Under the same age-specific EFSA exposure scenarios, the estimates of the urinary
concentrations in young children were higher than in adults, with values 25 times higher than
those described in the literature for adults (Lakind and Naiman, 2008), suggesting that the
exposure scenarios as described by EFSA (2006) can be considered as conservative.

In their model Mielke and Gundert-Remy included not only UDPGT-mediated BPA
conjugation, considering the age-dependent activity of the human UGT2B7, but they also
took the sulfation pathway into account. They assumed a SULT capacity in the adult to be
about 15% of that of glucuronidation and considered that SULT expression at birth is already
at the same level as in adults. Blood concentrations were simulated in humans of various ages
after intake of 0.05 mg/kg b.w./day, corresponding to the TDI (EFSA, 2006), with a dosing
schedule mimicking the age specific pattern of meals per day (i.e. considering that newborn
are usually fed more frequently than older children and of the adults). The PBPK model
estimated a 3 times greater blood free BPA concentration in newborns as compared with
adults after intake of 0.05 mg/kg b.w./day (0.44 μg/L vs. 0.13 μg/L).

The difference in the children/adult ratio between the two studies (about 3 in Mielke and
Gundert-Remy (2009) and in the range 2-11 in Edginton and Ritter (2009), depending on
different ages) may be explained with both the pattern of exposure and the consideration of
sulfation in BPA metabolism. The simulation by Mielke and Gundert-Remy suggests that the
well-expressed sulfation activity in the newborn can counteract at least partly the lower
glucuronidation activity in neonates, as already highlighted in the EFSA opinion of 2008.

Mielke and Gundert-Remy also calculated in the adult a steady-state concentration of free
BPA of 0.0014-0.0026 μg/L, resulting from a daily intake of 0.905 μg/kg b.w. (50 times lower
than the TDI). The value compares well with the steady state concentration of 0.004 µg free
BPA/L following exposure to 1 μg/kg b.w./day reported by Edington and Ritter (2009). The
derived estimated Cmax value was 2-3 orders of magnitude lower than mean measured values
reported by some authors (Schönfelder et al., 2002; Lee et al., 2008; Padmanabahn et al.,
2008). The discrepancy could be partially explained, since some assumptions used in the
model were affected by uncertainty. At the same time this underlines the need for cautious
interpretation of data on extremely high concentrations of free BPA, due to the possible
background contamination affecting the analytical detection (see 5.1.4.1).

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The Panel noted that both models assumed an intestinal absorption of 100%, irrespective of
age. However, the immature pancreatic and biliary secretion and the scant presence of
intestinal flora (which usually develop starting from the 8th postnatal month) significantly
limit the absorption of lipophilic compounds such as BPA in infants (Ginsberg et al., 2002).
The assumption of complete absorption is therefore conservative for infants.

The results from the human-based PBPK models underline the importance of taking into
account both pathways (i.e. glucuronidation and sulfation) for BPA metabolism at different
ages. Simulations taking into account both age-dependent metabolic differences and specific
pattern of exposure predict for newborns a 3-fold greater free BPA blood concentration as
compared with the adult (0.44 μg/L versus 0.13 μg/L) after intake of the same quantity of
BPA per kg b.w..

4.6. BPA repeated exposure


BPA is rapidly cleared following acute (single) doses; as a consequence, following dietary
exposure, total BPA blood concentrations should be directly related to contaminated food
consumption, and individuals with long fasting times before urine collection should have
substantially lower BPA urine levels than those with shorter fasting times. However, a recent
statistical analysis of a population study of 1469 adults (2003-2004 NHANES) indicate that
urinary BPA concentrations decline rapidly during the first 4.5- 8.5 hours of fasting, whereas
the elimination curve was flat from 8.5 to 24 hours (Stahlhut et al., 2009). These results are
consistent with a dietary exposure which is rapidly eliminated (with a half-life shorter than 5
hours), but also suggest that BPA exposure may also be related to non-food sources which
will not be affected by fasting. The highest total BPA urinary levels after fasting were 12–80
μg/L, measured in 7.5% of participants. The authors hypothesised that these findings could be
due to non food exposure, to accumulation in body tissue, or to both (Stahlhut et al., 2009). If
following repeated exposure BPA accumulation in slowly releasing tissues, such as fat,
occurs, then BPA concentrations in urine should increase with age. However, in the study
individuals in the range 60-85 years old show lower levels than younger people enrolled in
the study. Beside a non identified source of exposure, the authors listed among the limitations
of their study possible sample contamination or measurement error, self-reported fasting time,
non-use of complex survey variables, which cast some doubts on the reported results.

Other recent studies have reported on the presence of BPA and chlorinated derivatives of
BPA in adipose tissue of women (Fernandez et al., 2007). Twenty adipose tissue samples
were collected from 11 women (24-81 years old) in the course of surgical treatment in Spain.
Free BPA was found above the LOD in about half of the enrolled individuals, with mean ±
standard deviation equal to 3.16 ± 4.11 ng/g of adipose tissue, indicating a high degree of
variability in results (Fernandez et al., 2007). The most obese women in these series showed
BPA levels below the LOD. The authors reported a positive correlation with age, although the
the authors themselves underlined that the small sample size (11 women) strongly limited the
value of this association. Perhaps most importantly, possible contribution of free BPA from
medical devices related to surgical treatment cannot be ruled out.

The Panel noted that considering the toxicokinetics profile of BPA, any significant degree of
bioaccumulation is not expected.

Data on percutaneous absorption with human in vitro reconstituted skin, were obtained with
an extremely high dose of BPA (17.5 mM corresponding to 3.99 g/L, inconsistent with BPA
solubility) over a 48 h period (Mørck et al., 2010). However, the exaggerated BPA application
used does not allow any conclusion to be drawn from this study.

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4.7. Summary and conclusions on toxicokinetics


Studies on toxicokinetics of BPA have demonstrated a significantly lower internal exposure
after oral intake as compared to parenteral exposure. This confirms that toxicity studies with
oral administration have higher relevance for human risk assessment of BPA in food than
studies with parenteral administration. In addition, new findings in non human primates (both
adults and newborns) further strengthen the view that BPA is eliminated faster in humans than
in rodents. This fast BPA elimination in primates results in substantially lower internal
exposure to free BPA in humans as compared to rodents. Even human premature infants can
metabolise and excrete BPA efficiently (via glucuronidation and sulfation); this is supported
by recent human data and data in young monkeys. The use of the standard uncertainty factor
(UF) of 10 to take into account interspecies differences is therefore considered conservative.

In relation to in utero exposure, studies on transplacental transport of BPA and BPA-


glucuronide in humans and rodents indicated that although transfer may occur, foetal free
BPA levels are highly limited by the efflux pump P-glycoprotein, and placental BPA
glucuronidation may also take place. The Panel noted that exposure to total BPA (the major
constituent being the glucuronide) through lactation is limited to a very low fraction.
Therefore, in utero exposure and exposure through lactation appear to be limited.
The Panel recognises that inter-individual differences occur in expression of the isoenzymes
responsible for the detoxication of BPA. However, even in persons with low expression of
these enzymes, the metabolising capacity is still sufficient to eliminate free BPA from blood
at the low levels of BPA resulting from consumer exposure. Dietary exposures of adults and
infants aged 3-6 months were estimated to be up to 1.5 and 13 µg/kg body weight per day,
respectively (EFSA, 2006), based on conservative estimates of food consumption and
migration from food contact materials. These exposures are not anticipated to surpass the
metabolic capacity for BPA in adults or infants.

5. Toxicity

5.1. Human studies


This section addresses recent human epidemiological studies. The studies by Braun et al.
(2009) and Melzer et al. (2010) are discussed in detail in sections 5.1.1. and 5.1.2.,
respectively, according to the request of the Danish Minister for Food, Agriculture and
Fisheries.

Most of the human studies discussed in this section, including the Melzer et al. (2010) study,
are cross-sectional studies. In principle this design is not suitable to establish causal
relationship between exposure to a substance and health effects (Levin, 2006), as discussed in
section 5.1.4.2.

5.1.1. Braun et al. (2009)


Braun et al. (2009) investigated the possible association between prenatal BPA exposure and
behaviour in 2-year old children using data collected during an ongoing prospective birth
cohort study (HOME study). Two hundred forty-nine mothers and their children from
Cincinnati (USA) were included in the study. Total (free plus conjugated) BPA levels were
determined in maternal urine samples that were collected around the 16th and 26th week of
gestation and at birth. Median concentrations (not adjusted for creatinine) were 1.8 (16
weeks), 1.7 (26 weeks), and 1.3 (birth) ng/ml. These levels can be compared with the levels in
the general population reported in the study by Calafat et al. (2008), in which median
concentrations (not adjusted for creatinine) were 2.7 (2.4–3.0) ng/ml in the age group
corresponding to the selected women and 2.4 (2.2–2.8) ng/ml among females generally. In the

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Braun (2009) study, child behaviour was assessed at 2 years of age using the second edition
of the Behavioural Assessment System for Children (BASC-2). Composite scores were
computed for externalizing (hyperactivity and aggression scales), internalizing (depression,
anxiety, and somatisation scales) and Behaviour System Index (BSI) scales (overall level of
problem behaviours consisting of scales for aggression, hyperactivity, depression and
attention). The association between maternal total BPA concentrations and BASC-2 scores
was analyzed using linear regression. After adjustment for confounders (maternal age, race,
education, marital status, annual household income, child sex, maternal depression during
pregnancy and caregiving environment), log10-transformed mean maternal BPA levels were
associated with the children’s externalizing scores, but only among females. The analysis
according to the timing of exposure revealed that the positive association between BPA
concentrations and externalising scores in girls was present at 16 weeks of pregnancy, but not
at the two later time points. All the adjusted scores for any group were in the physiological
range (<50), i.e. well below the threshold for both clinical significance (>70) and score ranges
for “at risk” behaviour (60-69).

This is the first study examining the association of BPA prenatal exposure and behavioural
effects in young children. The strengths of the study include the availability of three different
sampling times for exposure assessment and of valid and reliable measurements of adaptive
and problem behaviours in children, the use of directed acyclic graphs to examine the role of
potential confounders in the observed exposure-behaviour association, and the performance of
a sensitivity analysis to evaluate possible misclassification of exposure.

The Panel noted that the HOME study was not designed to detect potential effects of BPA,
but rather to quantify the possible impact of environmental toxicants (in some instances with
cumulating properties) like lead, mercury, PCBs, pesticides, environmental tobacco smoke
and alcohol on child development (cognition, behaviour, growth, and hearing).

The limitations of the study by Braun et al. (2009) were pointed out by the authors themselves
and include possible exposure misspecification due to (i) intra-individual variability in urinary
BPA concentrations, (ii) influences of the glucose tolerance test (26th week of gestation)
and/or birthing process on maternal urinary BPA levels at sampling times, (iii) sample
contamination (iv) lack of BPA biomonitoring during the postnatal period. The demographic
profile of the analysed women, those for whom both urine samples and children’s behaviour
assessments were available, differed from that of the whole study population, potentially
resulting in selection bias. Behaviour was assessed at only one time point and this may not
adequately reflect the overall pattern during early childhood. Since all adjusted scores for any
group were in the physiological range the clinical significance of the association between
child behaviour and maternal BPA exposure may be questioned. The reported statistical
associations could potentially be due to unmeasured confounding, e.g. not adequately
assessed parental psychopathology, alcohol or drug consumption, maternal behaviour toward
the child, etc. Statistical limitations were identified in testing for a large number of
associations (despite of the consistency in associations between prenatal urinary BPA
concentrations and externalizing behaviours in girls) and in the lack of statistical power to
detect sex- and time- specific associations (also resulting in imprecise estimates).

In assessing the biological plausibility of the association of BPA exposure during pregnancy
(at 16 weeks) with changes in the externalizing behaviour in girls it should be noted that the
impact of (xeno-/phyto-)oestrogens on human brain development is still unclear (EFSA, 2006;
Longnecker, 2009). Extrapolations of experimental results with BPA from rodents to humans
are challenging due to differences in plasma levels of endogenous oestrogens (which are
considerably higher in primates) and the differential influence of sex hormones on the brain
development in both species. While in rodents the development of masculine behaviour is

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triggered by oestrogens, in primates it is induced directly by testosterone. To further clarify


the effect of BPA on brain development in primates, the interference of subcutaneous BPA
treatment (50 µg/kg b.w./day) with the synaptogenic effect of oestrogens was studied in
ovariectomized female monkeys (Leranth et al., 2008). The study results suggest that BPA
inhibits the oestradiol-induced spine synapse formation in the hippocampus and the prefrontal
cortex. However, Leranth et al. (2008) did not provide data on behavioural or cognitive
changes. In addition, the persistence of the BPA-mediated inhibition of oestrogen-induced
synaptogenesis and the underlying mechanism of action remains unclear. The Panel
considered also other experimental studies for adverse BPA effects on the behavioural
development in rodents and concluded that evidence from these studies and the study by
Leranth et al. (2008) is insufficient to support the biological plausibility of the effects
observed by Braun et al. In addition, the study by Leranth et al. was done in ovariectomised
animals and with s.c. exposure which further limits the relevance of the effects observed in
this study for humans.

In summary, the study by Braun et al. (2009) reports a positive statistical association between
maternal urinary BPA levels and externalizing behaviour among 2-year old girls, with
associations being statistically significant for urinary samples taken at 16 weeks of pregnancy,
but not with urinary samples taken at 26 weeks or at term. Considering the limitations of the
study the Panel concluded that the study results are not sufficiently robust to express concern
on neurobehavioural toxicity of BPA in humans. The Panel is aware that the authors of the
study are continuing to follow the children through 5 years of age to examine whether the
observed associations persist throughout early childhood. In the light of future findings the
Panel might wish to reconsider this issue for risk assessment.

5.1.2. Melzer et al. (2010)


This study follows a study by Lang et al. (2008; reviewed in EFSA, 2008b) that had reported
associations between raised urinary BPA concentrations and cardiovascular disease, diabetes
and enhanced liver enzymes in adult participants of the National Health and Nutrition
Examination Survey (NHANES) 2003/04. In this second study (Melzer et al., 2010) the
authors re-tested the originally identified associations using data from the US NHANES
2005/06 cross-sectional survey and estimated the strength of these associations in all the
available data (pooled data across the collection years NHANES 2003/04 and 2005/06).

The study sample included 1493 (2005/06 wave) and 1455 (2003/04 wave; Lang et al., 2008)
US adult subjects (aged 18–74 years). The 2005/06 analyses were based on an entirely new
population sample, providing an opportunity for independent replication of the earlier
findings. A spot urine sample was collected from each individual to assess total (free and
conjugated) urinary concentrations of BPA. The health outcomes were self reported diagnoses
of angina, heart attack or coronary heart disease, ‘cardiovascular disease’ (CVD) defined as a
combination of the three, ‘diabetes’ as a combination of self-reported diagnosed and
borderline diabetes (fasting blood glucose levels were not available for all participants and
were not used in the analyses) and serum liver enzyme (gamma-glutamyl transferase, lactate
dehydrogenase, alkaline phosphatase) levels.

The results showed that urinary BPA concentrations in the 2005/06 cohort were substantially
lower than in that of 2003/04, with a geometric mean value of 1.79 ng/ml and 2.49 ng/ml
respectively. As reported by the authors “Higher BPA concentrations were associated with
coronary heart disease in 2005/06 and in pooled data. Associations with diabetes did not reach
significance in 2005/06 (pooled estimates remained significant). There was no overall
association with gamma glutamyl transferase concentrations, but pooled associations with
alkaline phosphatase and lactate dehydrogenase remained significant”. The authors concluded

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that they had replicated the earlier findings of an association between higher urinary
concentrations of BPA and an increased prevalence of coronary heart disease.

The association between higher urinary concentrations of BPA and increased prevalence of
coronary heart disease was much weaker in the 2005/06 than in the 2003/04 NHANES
survey. Notably, in the study by Melzer et al. (2010) only ‘fully adjusted’ (age, gender,
ethnicity, urinary creatinine, education, income, body mass index (BMI), waist circumference,
and smoking status) models showed a statistically significant association with coronary heart
disease, whereas ‘crude’ (adjusted for age, gender, ethnicity and urinary creatinine) models
showed a statistically significant association with myocardial infarction. For comparison, in
the study by Lang et al. (2008) statistically significant associations were reported for the
overall diagnoses of CVD as well as for any of the diagnoses of its three individual
components (i.e. angina, coronary heart disease and heart attack). With respect to other
medical disorders, associations with diabetes or increase in liver enzyme levels that were seen
in the 2003/04 NHANES survey were not evident in the 2005/06 NHANES wave. The Panel
considered that the statistical associations reproduced in the pooled 2003/04 and 2005/06 data
analysis are not sufficient to rule out the contribution of unidentified confounders and chance
findings as a possible alternative explanation. The Panel compared the studies by Lang et al.
(2008) and Melzer et al. (2010) and considered that the overall results from the first NHANES
wave had not been confirmed but only partly supported by the second wave.

The strengths of the study include the availability of large-scale population-representative


epidemiological data on urinary BPA concentrations with sufficient statistical power to detect
low-dose effects, the use of a comprehensive quality control system to rule out sample
contamination during handling, storage and analysis, addressing potential bias due to co-
morbidity (minimized by age restriction, i.e. subjects aged less than 74 yrs).

There are also several limitations, some of which were pointed out by the authors themselves.
BPA measures in NHANES were based on single spot specimens. No information was
available on exposure prior to/during the development of diseases. Unmeasured confounding
may underlie the statistical association of urinary BPA levels and cardiovascular disease, the
latter resulting from a complex interaction of genetic, lifestyle and environmental factors.
Health outcome definitions were based on self-reporting, including diabetes diagnosis, for
which no laboratory test confirmation was available. The paper lacks information on how the
authors dealt with possible concurrent diseases (e.g. diabetes with CVD or any of the three
components) in order to minimize outcome misclassification.

In summary, the study by Melzer et al. (2010) is a sound cross-sectional study which only
partly reproduced findings from a previous study (Lang et al., 2008). The Panel concluded
that further (prospective and/or animal) studies would be needed to demonstrate the biological
plausibility of these findings and to explain the potential underlying mechanism of action.

5.1.3. Other human studies


Only a few studies investigating the association between BPA exposure and
reproductive/developmental disorders in human subjects have been published since 2007 (Itoh
et al., 2007; Padmanabhan et al., 2008; Wolff et al., 2008; Braun et al., 2009; Cobellis et al.,
2009; Yang et al., 2009; Li et al., 2010a, b; Meeker et al., 2010; Mendiola et al., 2010; Mok-
Lin et al., 2010).

Itoh et al. (2007) reported no association between urinary BPA concentrations and
endometriosis in a cross-sectional study of 140 infertile Japanese women. Negative results
were found in two other studies (Padmanabhan et al., 2008; Wolff et al., 2008) with respect to

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an association between maternal levels of BPA and birth outcomes such as gestational length
and infant size at birth. The above three studies (Itoh et al., 2007; Padmanabhan et al., 2008
and Wolff et al., 2008) were reviewed by the NTP-CERHR in 2008. In agreement with the
NTP, the Panel considers that drawing firm conclusions about a correlation between BPA and
potential reproductive or developmental effects from these studies in humans remains difficult
due to study-related factors such as small sample size, cross-sectional design, lack of large
variations in exposure, or lack of adjustment for potential confounders.

In the study by Cobellis et al. (2009) the concentrations of both BPA and 2,2-bis(4-
hydroxyphenyl)butane (BPB) were measured in the sera of 58 endometriotic women vs. 11
healthy controls, using a new method for the simultaneous determination of BPA and BPB in
serum for routine monitoring. Neither BPA nor BPB was found in any serum from healthy
women; in contrast, at least one of the bisphenols was detected in 63.8% of sera of patients
with endometriosis. No formal testing for an association was applied because of the small size
of the control group. The mean BPA concentration was described as being 2.91 (± 1.74)
ng/ml (± SD) but this value was based only on 15 sera, since just 30 endometriotic women had
detectable concentrations and among them only 50% had concentrations over 0.50 ng/ml.

The Panel agreed with the authors that further studies would be needed to confirm the
hypothetical association between BPA exposure and endometriosis.

The studies by Li et al. (2010a, b), Meeker et al. (2010) and Mendiola et al. (2010) have
addressed male reproductive toxicity in adult individuals.

The occupational study of 634 Chinese male workers involved in the manufacture of BPA and
of epoxy resins (Li et al., 2010a) showed that high exposure to BPA was associated with a
significant increase in occurrence of sexual dysfunction, e.g. quadruple risk of erectile
dysfunction, and 7-fold higher risk of ejaculation difficulty. Most likely, these workers were
occupationally exposed to BPA through multiple routes including inhalation. Their urinary
BPA levels were more than 50-fold higher than in the control group, the latter consisting of
Chinese men working in facilities where BPA was not present.

The authors recently published a second paper (Li et al. 2010b) in which they analysed a sub-
group of workers from the previous study for whom urinary samples were available; they
stated that the association between BPA exposure and male sexual dysfunction observed in
the previous study was confirmed.

The Panel considered the study design as having significant shortcomings. The high
complexity of the factors underlying possible changes in the test endpoint, i.e. sexual
performance, would call for a thorough consideration of a number of possible confounders,
which instead were not sufficiently addressed. There was no clinical evaluation of the subjects
(e.g. hormonal status, vascular problems, diabetes, etc.), including assessment of their
psychological status (e.g. depression). No data were available with respect to occupational
exposure to chemicals other than BPA. The exposure assessment to heavy-metals, pesticides,
organic solvents as well as the presence of chronic diseases was based on self-reports. As for
biological plausibility, the Panel considered that the low oestrogenic activity and weak anti-
androgenic potency of BPA do not provide a logical mechanistic explanation for the findings
of Li et al. Therefore the study by Li et al. is considered of no relevance for the assessment of
BPA-associated health effects in humans.

The study by Meeker et al. (2010) measured urinary BPA concentrations and serum thyroid
and reproductive hormone levels in 167 men recruited through an infertility clinic. In
multivariable regression models adjusted for potential confounders (specific gravity, age,

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BMI, current smoking status, and season and time of day of blood/urine sample collection)
BPA concentrations in spot urine samples collected on the same day as blood samples were
inversely associated with serum levels of inhibin B and oestradiol:testosterone ratio (E2:T)
and positively associated with follicle-stimulating hormone (FSH) and FSH:inhibin B ratio. In
a subset of 75 men for whom two/three repeated BPA urinary determinations were available
(within 3-75 days after blood withdrawal) the inverse relationship with E2:T ratio remained
consistent, whereas the associations involving FSH and inhibin B weakened.

The study limitations include (i) the cross-sectional design; (ii) the small sample size of the
subset with more than one urine sample available (less than 50% of subjects) (iii) exposure
measurement error due to high within-individual temporal variability; (iv) limited
generalisability (only men from an infertility clinic).

The study by Mendiola et al. (2010) investigated the relationships between environmental
BPA exposure (measured in urine) and reproductive parameters, including semen quality and
serum reproductive hormone levels, in prospectively recruited fertile men (n=375). These
men were partners of pregnant women who participated in the multicenter Study for Future
Families in four U.S. cities between 1999 and 2005. Information on demographics, recent
fever, history of sexually transmitted diseases, lifestyle factors (smoking, alcohol and caffeine
consumption) and diet was collected through a questionnaire.

Serum samples were analyzed for reproductive hormones, including follicle-stimulating


hormone, luteinizing hormone (LH), testosterone, inhibin B, oestradiol, and sex hormone-
binding globulin (SHBG), as well as the free androgen index (FAI). Semen analyses were
performed according to World Health Organization criteria. Pearson correlations were used
for unadjusted analyses, and multiple linear regression analyses were used to examine
associations controlling for age, body mass index, smoking, ethnicity, urinary creatinine
concentration, time of sample collection, and duration of abstinence.

After multivariate adjustment, no significant associations between any semen parameter and
urinary BPA concentration were observed. A significant inverse association was found
between urinary BPA concentration and FAI levels and the FAI/LH ratio, as well as a
significant positive association between BPA and SHBG.

It should be noted that the magnitude of the effect of BPA on FAI was small compared with,
for example, the diurnal variation in this parameter in healthy young men. Furthermore,
urinary concentrations of BPA in the subjects under study were only about half as high as
those reported in the national sample of U.S. men from NHANES 2003-2004.

The inverse associations between urinary BPA and FAI, E2, and thyroid-stimulating hormone
levels found in the Meeker et al. study (2010) on infertile men were observed only in the
subset who provided at least two urine samples (n = 75); when they analyzed the entire cohort
of men (n = 167, single urine sample), urinary BPA concentrations were inversely associated
only with serum inhibin B levels and positively associated with serum FSH levels.

The strengths of the Mendiola study include: quality controls carried out on urinary BPA
measurements; data analysis performed independently by two different analysists; sensitivity
analysis carried out excluding outliers (with extremely high concentrations of urinary BPA).
The study limitations include (i) the cross-sectional design; (ii) assessment of BPA exposure
by single urine sample (iii) assessment of hormone function by single serum sample; (vi)
likely exposure measurement error due to high within-individual temporal variability; (v)
limited external validity (only fertile men); (vi) small participation rate, potential selection

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bias and uncontrolled confounding; (vii) uncertain clinical significance of observed


associations.

In a prospective cohort (Mok-Lin et al., 2010) of 84 US women undergoing in vitro


fertilization (IVF, a total of 122 cycles), urinary BPA concentrations and ovarian response
were measured. The geometric mean of two specific-gravity (SG) adjusted urinary BPA
concentrations collected during each cycle was used as the cycle-specific BPA exposure level.
For each log unit increase in SG-BPA, there was an average decrease of 12% in the number of
oocytes retrieved and an average decrease of 213 pg ⁄ ml in peak oestradiol. Associations were
adjusted for age, BMI and day 3 FSH levels, a clinical measure of ovarian reserve.

Study subjects were female partners of couples seeking infertility evaluation and treatment.
Infertility diagnoses consisted of some female factors only in 29/84 patients, the rest being
male factors or unexplained causes. Notably, subjects underwent one out of three different
IVF treatments protocols. The small number of individuals studied limits the statistical power
and the ability to perform stratum-specific analyses within the infertility diagnoses and type
of treatment protocol, which are likely to be relevant confounders of the estimated
associations. Other study limitations mentioned by the authors include the number (only 2
samples) and timing (not preceding oocyte retrieval) of urinary collections, the limited
generalizability of findings to women who are not receiving infertility treatment and potential
confounding effects of many related factors. The Panel noted that there is not supporting
evidence from animal studies on the biological plausibility of the relationship between BPA
low-exposure and female fertility (e.g. Tyl et al., 2002, 2008, Ryan et al., 2010a). For these
reasons, the study of Mok-Lin et al. is considered of no relevance for the assessment of BPA-
associated health effects in humans.

In the Yang et al. study (2009) 152 Korean women, 70 breast cancer patients and 82 controls
(women not confirmed as having breast cancer after clinical assessment) were interviewed to
collect information on characteristics considered as potential breast cancer risk factors, such
as age, BMI and lifestyle and reproduction factors. Blood samples were collected (and kept
over 10 years in a freezer under controlled conditions) between 1994 and 1997 to characterize
women BPA exposure. There was no statistically significant difference in blood BPA levels
between breast cancer cases and controls (P = 0.42).

The Panel considers the study as having several shortcomings. The study design is not
sufficiently described and statistical power calculations are not reported; the statistical
methods applied are not adequate to the study design (case-control study); the questionnaire
used to collect information is not a standardized one. Overall an evaluation of the quality of
the study is not possible.

Moreover, the limitations of the cross-sectional design of the study are particularly relevant in
the case of breast cancer, for which latency between initial exposure to risk factors and first
clinical symptoms is expected to last years.

Therefore, from this study no conclusions can be drawn on the association between BPA
exposure and breast cancer.

The Panel considered the study by Ahrens et al. (2007) examining the association between
occupational exposure to endocrine disrupters and cancer of the extrahepatic biliary tract in
men. However, it noted that the investigation was focussed on compounds other than BPA,
e.g. polychlorinated biphenyls, with just 9 individuals exposed to BPA. Therefore the Panel
concluded that this study was not relevant to the specific purpose of this review.

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5.1.4. General comments on human studies

5.1.4.1. Analytical aspects


As pointed out in the study description, the quality of sample collection, storage and
processing as well as of the analytical methods used to detect BPA and its conjugates strongly
affect the robustness of some of the results obtained in both animal and human studies.

The rapid conjugation of BPA and its rapid plasma clearance indicate that spot analyses of
BPA (free plus conjugates) in blood are not reliable indicators of average exposure, since
blood BPA concentrations strongly depend on the time of blood sampling relative to BPA
exposure time.

The almost complete urinary excretion within 24 hr after exposure and the non-invasive
sampling procedure make urine the most appropriate biological matrix for estimating daily
exposure to BPA in humans. A 24 h pooled urine sample is preferable to determine average
daily exposure to BPA: indeed, single spot urine samples serve as a measure of very recent
BPA exposures, due to the short half-life of BPA and its dependence on the frequency of food
intake, time of sampling between food consumption and the last urination, and urine
production rate. However, in large cohorts the high number of spot urine samples collected
will average out variations in urinary concentrations of total BPA among individuals arising
from temporal factors within a day. It has been reported that mean values from studies
reporting BPA concentrations in spot urine samples with a larger number of participants
correlate well with those using cumulative excretion over 24 h (Völkel et al., 2008).

In order to have useful data for risk assessment purposes, both free BPA and its metabolites
should be measured, considering that most of BPA in human is present as conjugates. The
presence of free BPA in human specimens at levels higher than expected raises questions as
to the obtained results, underlining the need for inclusion of a careful quality control in the
analysis to avoid/minimize possible artefacts or contaminations.

Data on spontaneous hydrolysis of BPA conjugates, due to microbial contamination, during


short-term storage and processing of urine samples at room temperatures (Schöringhumer and
Cichna-Markl, 2007; Ye et al., 2007) indicate the instability of BPA conjugates as a possible
within-sample contamination. Therefore, urine (or any other biological sample) should
undergo rapid freezing immediately after collection. The possible degradation of conjugates
to products other than BPA (Waechter et al., 2007) could also result in an underestimation of
BPA levels.

An additional source of free BPA has been identified in the leakage of BPA from plastic
containers for their sampling and storage, as well as from plastic disposable laboratory
devices, sample workups and analysis (including HPLC capillary systems and solvents). The
background contamination from such sources has been quantified by Doerge et al. (2010a) to
be as high as 2 ng/ml in buffer blanks, and this can be responsible for the differences in free
BPA levels reported in many studies. Results obtained with the administration of 14C-labelled
or deuterated BPA are not affected by background contamination and are the most reliable for
risk assessment purposes and the studies by Doerge et al. (2010a,b) substantiate the need for a
rigorous quality control in biomonitoring studies.

For biomonitoring studies, independently of the detection method used, the use of a stably-
labelled BPA isotope as an internal standard is suggested as a means to determine the effect of
complex matrices of biological samples likely affecting recovery, and the stability of BPA
during analysis.

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Another source of differences among studies is the variety of analytical methods used to
detect BPA and its metabolites, especially at the low levels expected in biological samples.
The features of different methods have been recently summarized by the U.S. FDA (2010b):
among the methods for BPA analysis, the MS-based methods are considered the most
appropriate, whereas those based on immunochemical determinations (ELISA) are the least
reliable, due to low sensitivity, cross-reactivity with other phenols, and strong matrix effects.
As far as reporting is concerned, data on BPA in urine samples are either reported with or
without adjustment for urine dilution (based on e.g. urinary creatinine or specific gravity); for
statistical processing, samples with non-detectable BPA levels may be asssigned the LOD
value or a different arbitrary value, set by the authors. For this reason all data below the LOD
and LOQ, as well as some average (median) values should be interpreted with caution.

In addition, the variability of results obtained in biomonitoring studies also depends on human
inter-individual differences, which include age, diet, presence of pathologies (e.g. renal
failure, hepatic dysfunction), genetic factors (e.g. polymorphisms of UDPGT enzymes)

5.1.4.2. Comment on cross-sectional studies


In principle, cross sectional studies are suitable for the generation or testing of working
hypotheses for a chemical-induced effect for which there is sufficient evidence e.g. from
animal studies. Their design foresees the simultaneous measurement of exposure and outcome
(Levin, 2006), and therefore it is unsuitable to establish a causal relationship between
exposure and effect, especially in the case of chronic diseases developing over a long time
period, for which no information on past exposure to the substance is collected. Thus, cross
sectional studies as performed by Lang et al. (2008) and Melzer et al. (2010) can demonstrate
statistical associations between BPA exposure and the presence (e.g. coronary heart disease)
or absence (e.g. cancer, asthma) of health outcomes, but, by design, cannot establish a causal
relationship between BPA exposure and any induced adverse effect. The problems associated
with the above mentioned studies are also reflected in the different outcomes in the two waves
(2003/2004 vs. 2005/2006) concerning BPA exposure, inconsistent association with certain
diseases (e.g. diabetes, angina) or biochemical findings (liver enzyme abnormalities). In
addition, no supporting evidence from corresponding treatment- and dose-related BPA effects
have been observed in animal studies with oral administration. As demonstrated in the study
by Melzer et al. (2010) the selection of confounders can have a major impact on the observed
results. In this respect, the impact of unidentified confounders (e.g. diet, alcohol consumption)
on the outcome of the human BPA studies may also be relevant.

In the case of BPA various modes of actions are suggested and numerous effects have been
reported in animal experiments (see section below 5.2 and EFSA, 2006). Based on the
outcomes of studies in animals with low doses of BPA, some concern was expressed in a
previous evaluation of BPA by NTP (NTP-CERHR, 2008) regarding the possibilities of
effects of BPA on brain, behaviour and prostate gland in foetuses, infants and children.
However, these endpoints were not addressed by the human studies reviewed by the Panel,
with the exception of the Braun study (2009). In contrast, minimal and negligible concern for
reproductive toxicity was concluded by NTP-CERHR (2008) for workers and
environmentally exposed adult men, respectively. The studies from Meeker et al. (2010) and
Li et al. (2009) suggest an association between BPA exposure and fertility (associated with
changes in sex hormone ratio) or male sexual dysfunction. However, these associations are
currently not supported by a recent animal study (e.g. Howdeshell et al., 2008: section
5.2.1.1.) with low BPA exposure.

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5.1.5. Conclusions on human studies


Recent epidemiological studies have suggested some statistically significant associations of
BPA exposure (urine concentrations) and health effects (coronary heart disease, reproductive
disorders) in adults and behavioural changes in young girls. The Panel noted that cross
sectional epidemiological studies such as these can demonstrate statistical associations
between BPA exposure and the presence (e.g. coronary heart disease) or absence (e.g. cancer,
asthma) of health outcomes, but the inherent design of cross sectional studies does not allow
establishment of a causal relationship between BPA exposure and health effects (e.g. chronic
diseases). In addition, the Panel has identified some limitations in these studies, which raise
further questions as to the significance of the reported findings. Therefore, the Panel could not
draw any relevant conclusion for risk assessment from these studies.

5.2. Animal toxicity studies


As described in the introduction to Part II of this opinion, only in vivo toxicity studies
complying with certain inclusion criteria were considered for the purpose of this risk
assessment. Specifically, the CEF Panel focused on studies involving in utero and/or early
postnatal exposure to BPA, using an oral route for BPA administration and testing several
BPA doses (including at least one dose ≤5 mg/kg b.w. per day).

The Panel evaluated these studies according to established quality criteria in order to assess
the validity and/or applicability of the individual findings to human risk assessment.

Such quality criteria include a sufficient sample size, the adequacy of control procedures, the
inclusion of positive controls when applicable, the assessment of a correlation between
morphological and functional changes, and the consideration of litter or dam (and not of
individual pups) as the appropriate statistical unit. In addition, the same analytical
considerations applicable to human studies also apply to animal studies (see section 5.1.4.).

The Panel also acknowledged potential sources of experimental bias, which were taken into
account when the Panel evaluated the validity of the available new toxicological studies on
BPA. These comprise:

Strain sensitivity
Strain differences have been claimed as a major factor influencing the outcome of animal
studies: specifically, the Sprague-Dawley CD rat is reported to have a limited sensitivity to
exogenous oestrogens (Richter et al., 2007). However, there is other evidence showing that
Sprague-Dawley rats are as responsive to oestrogens as the other rat strains (Diel et al., 2004;
Moral et al., 2008; Sharpe, 2010). Similarly, the Long Evans rats used by Howdeshell et al.
(2008) and Ryan et al. (2010a) have been criticized as an insensitive animal model (vom Saal
et al., 2010). In the study by Howdeshell et al. (2008) the lowest effective oral dose of
ethinyloestradiol was 1.5 µg/kg b.w.. Notably, an oral dose of 5 µg/kg b.w. per day for a
reference oestrogenic compound (e.g. oestradiol) is regarded as appropriate to demonstrate
sensitivity of an animal model towards oestrogenic substances (Richter et al., 2007). As in the
study by Howdeshell et al. (2008) effects were observed at lower dose levels of
ethinyloestradiol, this would demonstrate the suitability of the Long Evans rat to study BPA-
oestrogenic effects.

Housing conditions
Animal housing in polycarbonate cages has been reported to be a potential source of BPA
exposure through contact or licking. Howdeshell et al. (2003) examined whether new and
used polycarbonate cages and polysulfone cages passively released BPA into water at a
neutral pH after 1 week contact at room temperature. The results indicate that polycarbonate

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and polysulfonate cages released a small amount of BPA when new (respectively 0.3 and 1.5
μg/L). Used polycarbonate cages released about 1000 times more than the new ones (310
μg/L compared with 0.3 μg/L). No BPA was reported to be released from polypropylene or
glass cages. The authors also reported that prepubertal female mice housed in used
polycarbonate cages had a mean uterine weight (calculated from more than 20 litters of at
least six 19-day females) 16% higher than females placed in polypropylene cages, but this
result was not statistically significant (Howdeshell et al., 2003).

Drinking bottles
The use of plastic drinking bottles may represent an additional source of exposure to BPA.
Therefore, the use of glass water bottles in such animal studies is highly recommended, but
the material from which drinking bottles were made is rarely reported.

Phytooestrogen-containing diet
The presence of phytooestrogens in commercial animal diets results primarily from the
inclusion of soy and alfa products in the laboratory diets and concentrations of
phytooestrogens can vary from batch-to-batch of standard laboratory diets (Heindel and vom
Saal, 2008). There is indication that high levels of phytooestrogens in laboratory diets affect
the results of experimental studies designed to identify endocrine disruption. For example,
high concentrations of phytooestrogens have been known to increase uterine weights in
rodents (Owens et al., 2003).

As reported in the OECD TG 440, “High levels of phytooestrogens and of metabolizable


energy in laboratory diets may also result in early puberty, if immature animals are used”.
Furthermore “As a guide, dietary levels of phytooestrogens should not exceed 350 μg of
genistein equivalents/gram of laboratory diet for immature female Sprague Dawley and
Wistar rats. Such diets should also be appropriate when testing in young adult ovariectomised
rats because food consumption on a body weight basis is less in young adult as compared to
immature animals. If adult ovariectomised mice or more phytooestrogen-sensitive rats are to
be used, proportional reduction in dietary phytooestrogen levels must be considered. In
addition, the differences in available metabolic energy from different diets may lead to time
shifts for the onset of puberty. Prior to the study, careful selection is required of a diet without
an elevated level of phytooestrogens or metabolizable energy that can confound the results”.

Bedding
Some bedding materials may contain naturally occurring oestrogenic or antioestrogenic
substances (e.g. corn cob appears to be antioestrogenic). Therefore, low level of of such
potentially interfering is recommended in bedding material. As with drinking water bottles, in
public literature the nature of the bedding is usually not specified.

5.2.1. Reproductive and developmental toxicity


The effect of prenatal and perinatal exposure to BPA on development has been investigated in
a large number of recent studies where many developmental toxicity endpoints were
evaluated including reproductive, neurological and behavioural parameters. In this section
only in vivo studies fully compliant with the selection criteria set in section 3 “Introduction”
(PART II) are addressed. To meet the request of the Danish Minister for Food, Agriculture
and Fisheries, in this section also the studies by Ryan et al. (2010a) and Howdeshell et al.
(2008) in female and male rats, respectively, of the same reproductive toxicity study are
addressed (see section 5.2.1.1).

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5.2.1.1. Developmental studies by Howdeshell at al. (2008) and Ryan et al. (2010a)
Effects on behavioural sexual differentiation, the age at puberty and the reproductive function
were examined in one study with male (reported in Howdeshell et al. 2008) and female
(reported in Ryan et al. 2010a) F1 offspring of Long-Evans rats following maternal oral
exposure to low levels of BPA in comparison to oestrogen throughout gestation and lactation.
All animals were housed individually in transparent polycarbonate cages with laboratory-
grade, heat-treated pine shavings. Pregnant and lactating dams were fed Purina Rat Chow
5008 ad libitum, and weanling and adult rats were fed Purina Rat Chow 5001 ad libitum.
Filtered municipal drinking water was provided through an automatic watering system.

Pregnant females (F0; two sets of experiments; n=13-29 dams/treatment group in the first set,
6-14 F0 dams/treatment group in the second set) were daily dosed via oral gavage with the
vehicle (corn oil), BPA (2, 20 or 200 µg/kg b.w./day) or ethinyloestradiol (EE: 0.05, 0.5, 1.5,
5, 15, or 50 μg/kg b.w./day) as positive control from GD 7 to PND18. The following
endpoints were evaluated in dams and/or their F1 male and female offspring: maternal
pregnancy data, neonatal and pup data (anogenital distance on PND 2, pup body weight), age
at puberty, and morphology of external genitalia. In addition, female F1 offspring were tested
for fecundity, saccharin preference, locomotor activity (Figure-8 maze) and lordosis
behaviour, as indices of sexual dimorphic behaviour. Male F1 offspring were tested for
areolae/nipple retention, reproductive organ weights and sperm numbers. BPA did not affect
any of the above parameters at any dose tested. EE reduced maternal body weight gain from
GD 7 to GD 20 (≥1.5 µg/kg b.w./day), reduced the number of live pups per litter on PND 2
(≥15 µg/kg b.w./day) and decreased implantations (≥50 µg/kg b.w./day). In female offspring,
EE increased anogenital distance and reduced pup body weight on PND 2 and PND 21 (50
μg/kg b.w./day), accelerated the age at vaginal opening, induced malformations of the
external genitalia and reduced F1 fecundity and F2 litter sizes (5 μg/kg b.w./day). F1 females
exposed to EE also displayed a reduced (male-like) saccharin preference (5 μg/kg b.w./day)
and absence of lordosis behaviour (15 μg/kg b.w./day), indications of defeminisation of the
CNS. None of the effects of EE displayed a non-monotonic dose-response pattern over a dose
range of four log units. In male offspring, EE reduced seminal vesicles and paired testis
weights (5 μg/kg b.w./day), reduced pup body weight on PND 2 and PND 150, reduced
ventral prostate, levator ani-bulbocavernous muscle and glans penis weights, and reduced the
number of epididymal sperm counts (50 μg/kg b.w./day).

The size of the experimental groups was adequate and the statistical analysis of the data was
appropriate. The inclusion of the oestrogenic reference compound EE at several doses
demonstrates the sensitivity of the test animals (≥1.5 µg/kg b.w./day) and of the methods to
detect oestrogen-related toxicity. However, the Panel noted that EE-induced behavioural
changes (e.g. suppression of lordosis) were observed in Wistar rats at a much lower dose, i.e.
0.4 µg/kg b.w./d, when given orally to dams during gestation (day 5-21) and lactation
(postnatal day 0-21) and to the offspring during postnatal day 21-32 (Della Seta et al., 2008).
The prolongation of the treatment time including direct EE administration to the offspring in
the latter study may contribute to the amplification of the estrogenic effect compared to the
Howdeshell study. The study weaknesses included the housing of animals in polycarbonate
cages (although the authors specified that these were clear and without evidence of significant
wear, thus suggesting low release of BPA (Howdeshell et al., 2003), the administration of
phytooestrogen-containing feed (though this problem was attenuated by the inclusion of
several doses of a positive reference oestrogen compound) and the absence of higher doses of
BPA to correlate the observations at lower dose levels studied with other more obvious toxic
effects. The sensitivity of the Long Evans rat strain to BPA has been previously demonstrated
(e.g. Adewale et al., 2009). The dosage levels of BPA used by Howdeshell et al. (2008) and
Ryan et al. (2010a) are in line with those used in ‘‘low-dose’’ BPA studies with rats, with the

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lowest dose (2 µg/kg b. w./day) being in the same range or below the estimated average
intake of the European population including infants, children and adults (1.5-13 µg/kg
b.w./day) (EFSA, 2006). In summary, in these studies the NOAEL for reproductive and/or
developmental endpoints were at least 200 μg BPA/kg b.w./day (highest dose) and no
LOAEL for these effects could be identified. Therefore, in principle this study demonstrates
the absence of any adverse effects of BPA up to 200 μg BPA/kg b.w./day on the endpoints
included in the study. This is, however, 25 times lower than the 5 mg/kg b.w./day NOAEL
used to derive the TDI.

Overall, the Panel considered the experimental design and the performance of the study as
valid. This study shows no effects on male offspring regarding anogenital distance, age at
puberty, morphology of external genitalia, areolae/nipple retention, reproductive organ
weights and sperm numbers (Howdeshell et al., 2008). Also in female offspring, no effects on
anogenital distance, age at puberty, morphology of external genitalia or fecundity were
reported (Ryan et al., 2010a). The latter study also addressed specifically the impact of BPA
on the sexually dimorphic behaviour of female rats, i.e. sweet preference and lordosis
behaviour, which were clearly affected by ethinyloestradiol treatment, but not by any BPA
dose. Therefore, the Panel concludes that the study results did not indicate any low-dose
effects of BPA on the development of sexually dimorphic behaviour in female rats.

5.2.1.2. Other developmental toxicity studies


Studies on fertility
Kobayashi et al. (2010) have assessed the effects of dietary exposure to low doses of BPA on
reproduction and development in two generations of C57BL/6J mice. Pregnant female mice
(F0; n = 14 – 16 per group) were fed a standard laboratory diet (CE-2, Clea Japan, Inc.,
Tokyo, Japan) containing BPA (0, 0.33, 3.3, or 33 mg/kg feed; equivalent to 0, 0.05, 0.5 or 5
mg/kg b.w./day ) from GD 6 through PND 22, and the weanlings (F1 and F2) from each F0
and F1 dam group, respectively, were also fed diets with the same concentrations of BPA ad
libitum until sacrifice. At week 13 after birth of the F1, 12 females per group were randomly
selected from the F1 generation. These were cohabited for 2 weeks with 6 randomly selected
males from the same group (1M+2F) to become parents of the F2.

There were no treatment-related changes in body weight, body weight gain, food
consumption, gestation length, or the number of live births on postnatal day 1 in F0 dams
between the control group and BPA groups. Sex ratio and viability were similar in all F1
pups.

No treatment-related changes were observed in body weight, food consumption,


developmental parameters, anogenital distance, or weight of any of the organs (liver, kidney,
heart, spleen, thymus, testis, ovary, or uterus) in 13-weeks old F1 and F2 adults in either sex
(n=12), although on occasional instances statistically significant deviations in organ or body
weights were recorded: in F1-females: a slight increase in mean liver weight in the low dose
group only; and in the F2 males: increases in mean body weight and mean heart weight also
in the low dose group only. These were considered chance findings.

For the evaluation of sperm counts and testes, epidydymus and sperm histology, the six F1
males used for breeding the F2 were supplemented with 6 virgin F1 males at week 15. In
these F1 males, the paired epididymis weight was slightly higher with 0.33 and 3.3 mg
BPA/kg feed, which was related to increased right but not left epidydymus weights in all
exposure groups, without a clear dose response relationship. There was no such increase in
epidydymus weights in the 15-weeks old F2 males (n=12). There were no treatment-related
effects of BPA on cauda epididymal sperm count or sperm motility in F1 or F2 males. These

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findings indicate that dietary exposure to BPA up to 33 mg/kg feed (equivalent to 5 mg/kg
b.w./day in adults) does not adversely affect reproduction or development as assessed in two
generations of mice.

The Panel noted the lack of information in the study report with respect to control for
environmental sources of contamination (animal housing conditions, phytooestrogen content
of the diet, type of drinking bottles and bedding) and unsufficient clarity as to whether the
analysis of the effects in the offspring was performed on a“per litter” or individual basis
despite the use of a “per litter” statistical analysis was stated by the authors themselves. Only
12 pups per sex per group were studied (i.e. 2-4 less than the number of dams/group in the
F0). The Panel noted that the F0 generation was not exposed prior to pregnancy. It is also
unclear if F1 animals were mated among littermates, which should not be the case. Mating
should have been on a 1M+1F basis, rather than 1M+2F.

The study by Kobayashi et al. (2010) does not indicate adverse effects on the reproductive
development in two generations of mice. However, the Panel observed that the study has
some shortcomings in its design and in the reporting.

Salian et al. (2009) performed a 3 generation-study assessing the effects of very low oral
doses of BPA on the fertility of male Holtzman rats. Eight pregnant rats per group were
gavaged with either BPA (1.2 or 2.4 µg/kg b.w./day), a vehicle control or diethylstilbestrol
(DES; 10 µg/kg b.w./day) from GD 12 to PND 21. Litters were culled to 4 to 5 male
offspring, weaned on PND 22, cohabited (n=24) on PND 75 with unexposed adult females
(n=48) to obtain F2 male generation; by the same procedure, F3 male generation was derived.
Adult F1 males were subjected to fertility assessment by mating with unexposed females. The
reproductive functions of the subsequent F2 and F3 litters were investigated in a similar
manner. Immunohistochemical localization of steroid receptors was carried out in the testes of
F1, F2 and F3 generation adult rats. A significant increase in post implantation loss and a
decrease in litter size and sperm count and motility were observed in the F1 male offspring. A
reduction in the testicular expression profile of steroid receptors was also observed. No dose
response relationships were reported by the authors for any of these effects. The authors
reported that these impairments in the fertility due to exposure to oral doses as low as 1-2
µg/kg b.w./day became more prominent in the F2 and F3 generations of males which had
never been directly exposed to the chemical.

The Panel indicates several limitations of this study. One limitation of the study is the small
number of treated dams in the first and most important phase of the study. Only 8 dams per
group were used and it may be plausible for a genetic defect in one pair to develop and
magnify as subsequent generations are examined. Very few details of the actual conduct of
the study are reported, so that it is impossible to track effects in individual litters and animals.
It is unclear how many males were used for mating (contradictory information in the text of
the experimental design - one male per each female, vs. Figure 1 - one male per two females),
what happened if mating failed to occur, e.g. whether a second male was used. Similarly, the
fertility testing of the males of the subsequent generations was not clearly defined. Forty-eight
offspring per dose group and generation were examined: it is unclear how "litter as a unit"
was defined. Surprisingly, the male and female rats at 9 weeks of age weighed about the same
(~250 g). Usually, males are significantly heavier than females at that age. The nature of the
'in house' prepared diet used is unclear (soy-free, in-house prepared rat pellets consisting of
crude protein, fiber and nitrogen-free extract), and perhaps impossible to replicate. Also the
statement about the quality check of the diet, i.e. by qualitative and quantitative proximal
analysis) is insufficiently explicative.

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With respect to the results the Panel noted the following: the number of resorptions in the
controls is unusually low with none in the F1 matings and only one single foetus in one
female in each of the F2 and F3 groups of litters (as assessed by the pre-implantational and
post-implantational percentages). This may indicate that the controls may be the unusual
groups and not the BPA-treated animals in which the pre-implantation and post-implantation
losses of about 1-2 implants per litter may be more usual. The epididymal sperm count
reduction and its variation were smaller than expected (the samples shown in the pictures
cannot be representative and show an artefact that is different from control). There was no
effect on testis weight in the BPA groups, and the lack of any dose response relationship in
other organ weights does not suggest a treatment-related effect. The lack of dose response
relationships in the androgen and oestrogen receptor studies again suggests random variations
rather than treatment-related effects.

Overall, the unusual nature of the reported effects at very low doses of BPA (Salian et al.
2009), coupled with incomplete reporting of results and the absence of dose response
relationships makes this study unsuitable for risk assessment of BPA.

Developmental neurotoxicity and neurobehavioural studies


Miyagawa et al. (2007) analysed the behaviour (anxiogenic-like effects, motor learning and
memory) and a hippocampal cholinergic marker in the adult (aged 7-11 weeks) male
offspring of C57BL/6J mice (≥10 dams per group) which had been exposed throughout
gestation and lactation to BPA via maternal diet (BPA doses of 0, 30 µg/kg diet and 2 g/kg
diet; assuming an intake of 3 g diet/day and a body weight of 0.02 kg, this results in 0, 4.5
µg/kg b.w./day, and 300 mg/kg b.w./day). No information was given on type of feed, caging
and bedding materials. The treatment did not result in any significant body weight loss. In
male offspring, no anxiogenic effects (as assessed by the light-dark paradigm and the elevated
plus-maze paradigm) were detected and motor learning (time spent on a rotating rod) was not
impaired. However, memory (passive avoidance test) was impaired both at the low and high
dose. Choline acetyltransferase-like immunoreactivity (a marker of acetylcholine production)
was significantly decreased (Low: 64.3 ± 5.6% of control mean, High: 50.0 ± 3.8% of control
mean) in the hippocampus. Since no dose-related impairment of memory and reduction in
acetylcholine production was observed (almost the same for low and high dose groups despite
a ca. 100,000-fold difference in the maternal intake of BPA) the causal relationship of this
effect to the BPA treatment remains questionable. Methodological shortcomings, with a study
design using only two very widely spaced doses of BPA, only one sex examined, and
unclarity with respect to the use of the litter as the statistical unit limit the validity of these
results for risk assessment.

Tian et al. (2010) treated 2 pregnant ICR mice per group by oral administration of BPA (0,
100, 500 µg/kg b.w./day) from GD 7 to PND 21. The 5 week old male and female offspring
(9 to 17 pups/litter; not further specified) were tested PND 22 to PND 36 for spontaneous
exploratory activity in the open field, anxiety-related behaviour in the elevated plus-maze test,
spontaneous alteration behaviour in the Y-maze test, novelty recognition in the novel object
test, ligand-binding studies for D1, D2 and dopamine transporters (involved in anxiety-related
behaviour) and NMDA receptors (contribute to memory and learning). There was no effect on
total locomotion in the open field, but central locomotion increased at 100 µg/kg b.w., but not
at 500 µg/kg b.w.. BPA increased time spent in the open arm (significant at 500 µg/kg b.w.)
of the elevated plus-maze but did not change total number of entries of the open arms. Both
BPA groups significantly decreased percentage of alteration behaviour in the Y-maze test.
BPA decreased novel object recognition at 100 µg/kg b.w. but not at 500 µg/kg b.w..
Receptor binding assays did not show an effect on D1 receptor, a significant increase in D2
receptor binding at 100 µg/kg b.w., an almost identical reduction in dopamine transporter
binding at 100 and 500 µg/kg b.w. and a dose-dependent reduction in NMDA receptor

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binding in frontal cortex and hippocampus with 100 and 500 µg/kg b.w.. The Panel
considered the study design by Tian et al. (2010) as not valid (insufficient number of dams
per group and inappropriate statistics not performed on a “per dam” basis).

Xu et al. (2010) gavaged groups of 8 to 9 pregnant Sprague-Dawley rats from GD 7 through


PND 21 with different doses of BPA (0.05, 0.5, 5, 50, 200 mg/kg b.w./day). At birth, pups
were culled to eight and pups were sacrificed to examine the hippocampal region on PND 4,
7, 14, 21, and 56 for expression of the N-methyl-D-aspartate receptor (NMDAR) subunits
NR1, NR2A and NR2B, of oestrogen receptor beta (ERβ), and of aromatase cytochrome P450
(P450arom) protein. All the determinations were done only in male pups. At the lower doses
of 0.05 to 50 mg/kg b.w./day, BPA concentration dependently inhibited the expression of
NMDAR subunits. At the highest dose (200 mg/kg b.w./day), the effects of BPA on these
subunits were different, with a stronger inhibition of NR1 expression and a slighter inhibition
of NR2A, NR2B expression when compared with those at the lower dosage of BPA.
According to the authors, these results indicate a change in the composition of NMDARs
thereby affecting function of the hippocampus such as synaptic plasticity, learning, and
memory. Perinatal exposure to BPA at doses ≥5 mg/kg b.w./day significantly inhibited the
expression of ERβ protein in a dose-dependent manner early after birth (on PND 4 and 7). At
later time points (from PND 14 to 56) the expression of this protein was significantly
decreased in comparison with control values only in the high dose group (200 mg/kg
b.w./day). Aromatase was dose-dependently (≥0.05 mg/kg b.w./day) increased during the first
1-3 postnatal weeks, with no effects being detectable on PND 56. The Panel considers that
biochemical changes are not sufficient to revise the TDI in the absence of a clear link to an
adverse outcome (i.e. a functional correlate). In addition, the Panel has identified
shortcomings mostly in study reporting that bring into doubt the validity of the study.
Specifically, there is no indication of the number and the identity of the animals used for
testing, and on whether the statistical analysis was performed on a “per litter” basis. In
addition, only one sex was tested and there was no reporting on the animal housing
conditions, the diet administered and the type of bottles used for drinking water.

Studies on metabolic effects


The effects of peri- and postnatal exposure to BPA on adipose tissue mass were investigated
by Miyawaki et al. (2007). Groups of 3 pregnant ICR mice were exposed to BPA in drinking
water (0, 1 or 10 µg/ml, resulting in 0, 0.26, 2.72 mg/kg b.w./day) from GD 10 to end of
lactation. Offspring were exposed up to PND 31 and groups of 16 to 25 offspring per sex and
dose group were evaluated. Body weights of female offspring were increased at the low and
high dose group, body weights of the males at the high dose group. Adipose tissue weight was
increased significantly in females at the low dose and in males at the high dose group. Serum
leptin was increased only in females of the low dose group. Total cholesterol was increased
only in females with the highest increase in the low dose group. Serum triacylglycerol and
non-esterified fatty acid levels were increased and serum glucose levels decreased only in
males of the low dose group. The low number of dams per group invalidates this study.

Studies on cell proliferation and apoptosis related to enhancement of tumourigenesis


To examine the effect of lactational exposure to BPA on dimethylbenzanthracene (DMBA)-
induced mammary cancer in female offspring, Jenkins et al. (2009) gavaged nursing Sprague-
Dawley rats with BPA (0, 25 or 250 µg/kg b.w./day) from lactation day 2 to 20. All female
offspring (5-8 per litter) and enough males were retained to yield 10 offspring/litter. Cell
proliferation and apoptosis were measured in the mammary gland of the female offspring (n =
5/group) at 21 days of age (at end of BPA treatment) and at 50 days of age (before DMBA
exposure). In the absence of genotoxicity of BPA (EFSA, 2006; EC, 2008), the rate of cell
proliferation and apoptosis are major determinants of cancer development, both of which are

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threshold phenomena (see also section 5.3). Increased cell proliferation and reduced apoptosis
in the mammary gland of female offspring were observed at the high dose group at 50 days of
age but not at 21 days of age. Consistent with increased proliferation and reduced apoptosis,
expression of the following proteins was increased in the high dose group: Akt and
phosphorylated Akt (pAkt; proteins linked with apoptosis), progesterone receptor (PR)-A,
steroid receptor activator (SRC) 1 to 3, and erbB3. The expression of oestrogen receptor
(ER)-α was slightly reduced. At 50 days of age, one female offspring from each litter of each
treatment group was given a single gavage of DMBA (30 mg/kg b.w.) which was expected to
result in a low number of mammary adenocarcinomas. In total, 32, 34, and 24 female
offspring in the control (no BPA during lactation), low and high BPA group, respectively,
received DMBA. Offspring were palpated twice weekly to monitor tumour development and
underwent necropsy at 12 month of age or when tumour burden exceeded 10% of body
weight. All tumours were prepared for pathological examination. BPA-treatment increased
the number of tumours (not further specified between adenoma and carcinoma) per animal
(2.84 ± 0.31, 3.82 ± 0.43, and 5.00 ± 0.88 for control, low and high BPA groups, respectively)
with the effect at the high dose group being statistically significant. The authors reported that
there was “no change in the carcinomas score”. Tumour latency was also reduced (65, 53,
56.5 days for control, low and high BPA groups, respectively) with statistically significance at
the high dose group.

The study, however, revealed limitations as follows:

(a) The toxicokinetic studies showed that only minimal fraction of BPA administered to dams
is transferred to breast milk (see section 4.4). Therefore, the exposure of the pups to BPA
under this condition is anticipated to be very low. Information on internal BPA levels would
improve the interpretation of the study results.

(b) The score of carcinoma formation which would indicate tumour progession is not
changed.

(c) When considering the results of the study on tumour latency, the measurement
uncertainties involved in the data collection (palpation) should be taken into account.

In conclusion, the shortcomings in the study design, in particular the uncertainty regarding the
exposure of the offspring to BPA, and the limitations in reporting preclude these results to be
used for risk assessment of BPA and the re-evaluation of the existing TDI. However, the
Panel noted that a dose-related response of BPA on cell proliferation and apoptosis in the
mammary gland was reported in the study and this deserves further considerations. This
mechanistic aspect is further discussed in section 5.3 under the subheading “Epigenetic
effects, cell proliferation and apoptosis”.

Betancourt et al. (2010b) also used the model of DMBA-induced mammary carcinogenesis,
but BPA was administered in utero by gavaging pregnant Sprague-Dawley rats with 0, 25 or
250 μg BPA/ kg b.w./day (GD 10-21).
Female offspring of BPA treated dams did not differ from controls with respect to body
weight development, vaginal opening and on PND 50, serum concentration of 17ß-estradiol,
progesterone as well as estrus cyclicity. On PND 50, expression of oestrogen receptor (ER)-α,
PR-A and bcl-2 was reduced. On PND 100, ER-α and bcl-2 were upregulated, PR-A was as
in controls. As to SRC-1,-2 and -3, only SRC-3 was increased on PND 50, but all members of
the SRC-family were up-regulated on PND 100. Cell proliferation (n=6/group) and apoptosis
(n =5/group) were measured in the mammary gland of the offspring of controls and high
BPA-dose group on PND100 (before DMBA treatment). Upon prenatal BPA exposure,
proliferation of epithelial cells was increased but apoptosis was not affected. Consistent with

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increased proliferation expression of the following proteins was increased in the high dose
group at PND100: EGFR, phosphorylated -IGF-1R, phosphorylated –c-Raf, phosphorylated
pERKs 1/2, phosphorylated ErbB2, phosphorylated Akt.
For tumourigenesis studies, one female offspring per litter was given a single gavage of 30
mg DMBA/kg b.w. on PND 50 (31, 29 and 33 rats in the control, low- and high-dose groups,
respectively) or on PND 100 (30 and 28 rats in the control, and high-dose groups,
respectively). Animals underwent necropsy at 12 months of age or when tumour burden
exceeded 10% of body weight. Offspring were palpated twice weekly to monitor tumour
development and underwent necropsy at 12 month of age or when tumour burden exceeded
10% of body weight. DMBA administration on PND50, prenatal BPA-treatment did not result
in increased the number of tumours per animal (2.94 ± 0.48, 2.38 ± 0.42, and 2.88 ± 0.4 for
control, low and high BPA groups, respectively). The tumour latency was not reduced (109 ±
11, 116 ± 14, 106 ± 14 days for control, low and high BPA groups, respectively). DMBA
administration on PND 100 caused a significant increase in tumour incidence (53 to 83%)
along with a non-significant increase in tumour multiplicity (1.96 ± 0.53 to 2.53 ± 0.55). The
latency period was reduced from 267 to 189.5 days. Finally, a significant greater proportion
of DMBA-induced tumours classified as grade II (Bloom-Richardson system; control: 3 of 13
tumours (23%); BPA high dose group: 9 of 20 tumours (45%)) was observed.
The authors concluded that the high BPA dose (250 μg BPA/kg b.w.) enhanced cell
proliferation in mammary glands of the offspring, associated with an increased cancer
susceptibility and shift of the window for susceptibility for DMBA-induced tumourigenesis in
rat mammary gland from PND50 to PND100. However, the study revealed similar
shortcomings in design and reporting as the study by Jenkins et al. (2009), i.e. internal BPA
doses were not measured, measurement uncertainties involved in tumour data collection by
palpation, time of necropsy of individual animals not exactly reported but given as “at 12
months of age or when tumour burden exceeded 10% of body weight”. The Panel concluded
that these data cannot be taken into consideration for derivation of a TDI for BPA.

Studies on immunologic effects


Yan et al. (2008) examined the response of mice to a bacterial pathogen after exposure to
BPA during development. Mice were housed in polymethylpentene cages, fed commercial
diet and drank water from glass bottles. Bedding was not described. Pregnant BALB/c mice
were exposed to BPA in drinking water at concentrations of 0, 1, 10, or 100 nM (equivalent to
0.03, 0.3 and 3 µg/kg b.w./day) for 2 weeks before mating and 1 week after mating. At 10
weeks of age, male offspring were given a subcutaneous injection of Leishmania (L.) major in
the hind footpad. The offspring of exposed mothers showed dose-dependent enhanced footpad
swelling at 6 and 8 weeks after infection with L. major antigen. Production of interleukin-4
(IL-4) and interferon-γ (IFN-γ) by splenocytes was significantly increased in offspring from
dams exposed to 10 and 100 nM BPA but not in those born to 1 nM BPA-treated females
compared with the nonexposed control mice. In addition, diminished occurrence of
CD4+CD25+ T cells was detected at the same dose levels, suggesting that the increased
cytokine production could be due to the decreased number of regulatory T cells. However, the
lack of inclusion in the experimental design of the appropriate marker (Foxp3) in addition to
that of CD4+ and CD25+ does not allow the conclusion that the measured cells were indeed
regulatory T cells. In addition, the study has some shortcomings (small experimental groups
of 3-4 animals and evaluations in males only, no indication of the number of exposed dams,
or whether animals in the tested groups were littermates).
In conclusion, due to the limited study reporting, no clear conclusions can be drawn from this
study and, therefore, this study cannot be taken into consideration for derivation of a TDI for
BPA.

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5.2.2. Conclusions on animal toxicity studies

5.2.2.1. Studies on developmental and reproductive toxicity


The studies reporting effects at doses lower than 5 mg/kg b.w./day including the study by
Salian et al. (2009) have severe shortcomings and were considered to be invalid. The Panel
considers that the valid studies do not raise concern regarding reproductive and
developmental toxicity of BPA at doses lower than 5 mg/kg b.w./day.

5.2.2.2. Developmental neurotoxicity and neurodevelopmental studies


Potentially significant biochemical changes, e.g. altered receptor expression in different brain
regions (see section 5.3), have been reported. However, in the absence of a correlation with a
functional adverse effect, the relevance of these observations for human health cannot be
assessed. The impact of BPA on development of sexually dimorphic behaviour was addressed
in the study by Ryan et al. (2010a), who observed a male-like reduced saccharin preference
and inhibition of lordosis behaviour in female rat offspring from oestrogen-treated but not
from BPA-treated dams. In the study reported by Stump et al. (2009) (See Part I) the effects
of BPA on learning and memory behaviour were inconclusive due to large variability in the
data. Other recent studies have methodological shortcomings. The Panel does not consider the
currently available data as convincing evidence of neurobehavioural toxicity of BPA.

5.2.2.3. Cell proliferation and apoptosis related to enhancement of tumourigenesis


The study by Jenkins et al. (2009) is the first oral study on a possible BPA-induced
enhancement of sensitivity of the mammary gland to carcinogen-induced breast tumour
formation in rat offspring following lactational BPA exposure of pups. Using the same model
of DMBA-induced mammary carcinogenesis but in utero BPA exposure, Betancourt et al.
(2010b) also reported an enhancement of susceptibility for mammary gland carcinogenesis. In
consideration of the shortcomings in the design of both studies, in particular the uncertainty
regarding the lactational as well as in utero exposure of the offspring to BPA, and of the
limitations in reporting the Panel concluded that these results cannot be taken into
consideration for derivation of a TDI for BPA. However, the Panel noted that at the highest
dose level studied there is a shift of the ratio between cell proliferation and apoptosis in
favour of cell multiplication in the mammary gland. In view of the mechanistical data
obtained upon in utero exposure in other studies (see section 5.3) and the implications of an
increased cell proliferation/apoptosis ratio in carcinogenesis, the effects reported by Jenkins
and Betancourt deserve further consideration.

5.2.2.4. Studies on immunotoxicity


Modulation of immune system-related parameters is an emerging field also in BPA research.
Several studies have reported changes in cytokines, changes in T-cell populations and other
aspects of immune modulation (see section 5.3). However, all the studies including that of
Yan et al. (2008) suffered from shortcomings in experimental design and reporting.
Therefore, at the moment, these studies cannot be taken into consideration for derivation of a
TDI.

5.3. Other information on the endocrine-mediated action of BPA


Endocrine disruption is a collection of modes of actions rather than a toxicological endpoint in
itself. BPA is one of the most extensively studied endocrine disrupting chemicals. It has been
subjected to a huge number of test systems in which a variety of parameters have emerged
that may be indicative of BPA-interactions with biological systems. Criteria for selection of
studies which can be used for risk assessment of BPA are described in section 3. The section

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5.3 deals with studies not compliant with these criteria. Nevertheless, these studies addressed
cellular and molecular events which could be informative and which could be used for
identification of potential hazards and for support of biological plausibility.

5.3.1. Effects on receptors and hormone homeostasis


Based on in vitro data BPA is confirmed to be a weak agonist at the oestrogen receptor (ER)
and an antagonist at the androgen receptor (Bonefeld-Jørgensen et al., 2007). A slightly to
markedly increased expression of oestrogen receptor 1α and of androgen receptor was
reported in prostate mesenchyme cells treated with nanomolar to micromolar concentrations
of BPA (Richter et al., 2007). Additionally, very sensitive alternative receptor pathways for
BPA are reported, e.g. activation of protein kinases via a membrane G-protein-coupled ER
(Bouskine et al., 2009) and high affinity binding of BPA to the oestrogen-related receptor-γ,
which is strongly expressed in fetal brain and in placenta (Okada et al., 2008; Takeda et al.,
2009).

In in vivo studies, oestrogen-like activity (reviewed in SCF, 2002) and inhibition of


oestrogenic activity by BPA (e.g., the prevention of the synaptogenic response to oestradiol in
hippocampus and prefrontal cortex of ovariectomized monkeys: Leranth et al., 2008) have
been reported. Subcutaneous administration of BPA in neonatal female rats resulted in
alterations in the hypothalamic-pituitary function, i.e. decreased gonadotropin-releasing
hormone-induced secretion of luteinizing hormone (Fernández et al., 2009). A differential
modulation of ERα protein expression in the preoptic area of prepubertal female rats was
reported for different doses of subcutaneously applied BPA: While a low dose of BPA (0.05
mg/kg b.w./day, postnatal day 1 to 7) increased the protein expression, a high BPA dose (20
mg/kg b.w./day) reduced the expression compared to controls (Monje et al., 2007). Both
postnatal doses of BPA were shown to decrease the ERα protein expression in hypothalamic
nuclei of adult female rats and partly changed their sexual behaviour, i.e. proceptive
behaviour (measured as hops and darts, but not ear wiggling or receptive behaviour, i.e.
lordosis) mediated by these hypothalamic regions (Monje et al., 2009). Similarly a recent
study with oral pre- and postnatal (until puberty) exposure of female Sprague-Dawley rats to
ethinyloestradiol at a low dose (0.004 µg/kg b.w./d) inhibited female proceptive behaviour,
i.e. solicitation, but not lordosis; in contrast a higher dose of ethinyloestradiol (0.4 µg/kg
b.w./day) markedly suppressed the oestrous cycle and lordosis (Della Seta et al., 2008).

Ceccarelli et al. (2007) investigated the effect of oral BPA exposure (only at a single daily
dose of 40 µg/kg b.w./day from PND 23 to PND 30) on brain development of Sprague-
Dawley rats. A ~2-fold increase of ERα levels in the ventromedial nucleus was found among
BPA-exposed females on PND 37. In the ethinyloestradiol group there were occasional
differences in ERα levels, which were not consistent with the effect seen in the BPA group.
Serum testosterone levels were reduced by ~30% in males exposed to BPA, but were
increased in males exposed to ethinyloestradiol. 17β-Oestradiol levels were not affected in the
BPA-treated group, but were increased in the ethinyloestradiol female group. Finally, some
differences in ERα were detected in one sexually dimorphic region of the hypothalamus in
female rats exposed to BPA as juveniles. Time-dependent up-regulation of ER α and ER β in
the dorsal raphe nucleus were reported in newborn male mice after prenatal oral BPA
treatment (2 µg/kg b.w./day on GD 11-17 to pregnant ICR mice) at 5 and 13 weeks but not at
9 weeks of age (Kawai et al., 2007). No statistically significant changes in serotonin,
serotonin transporter, and serum testosterone concentrations were observed in the latter study.

In addition to the interaction with sex hormone-dependent effects, BPA could also have an
impact on the regulation of brain organization via corticosterone-mediated actions: this was
studied by Poimenova et al. (2010) in perinatally treated Wistar rats (40 µg BPA/kg b.w./day

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was administered orally during pregnancy and lactation to four dams). Female offspring (at
PND 46) from the BPA-treated dams had significantly higher plasma corticosterone levels
while the hippocampal glucocorticoid receptor and minieralcorticoid receptor levels remained
unchanged. After performing a Y-maze experiment, corticosterone levels were increased in
BPA-treated offspring of both sexes. The decreased basal corticosterone levels in females
were associated with changes in the performance of the Y-maze (increased “anxiety-like”
behaviour and loss of exploration attitude). The limitations of the study included a low
number of treated animals and the use of one dose only.

Several in vitro studies suggest that BPA can also interact with other central nervous system
components such as rat dopamine transporters (Alyea and Watson, 2009), GABA receptors in
rat CA3 pyramidal neurons (Choi et al., 2007) and rat foetal hypothalamic cells (Yokosuka et
al., 2008).

Oral administration of BPA to pregnant Sprague-Dawley rats at 4 mg/kg alters dopamine


metabolism in the offspring (Honma et al., 2006) while subcutaneous prenatal and neonatal
exposure to a low dose of BPA (20 µg/kg b.w./day) alters the function of dopaminergic
receptors at the DL-striatum (Zhou et al., 2009). The in vivo results of Xu et al. (2010) show
an effect of BPA on NMDA-receptor expression (see 5.2). Recently, kisspeptin neuropeptides
have been described as key players in the neuroendocrine control of reproduction (Navarro et
al., 2009). Disruption of sex specific organization of hypothalamic kisspeptin was observed
after neonatal exposure of rats to BPA (50 mg/kg body weight, s.c.; Patisaul et al., 2009). This
dose level and the route of exposure preclude these findings from having an impact on the risk
assessment of BPA.

Similar considerations apply to the study of Nakagami et al. (2009) who applied BPA
subcutaneously to pregnant cynomolgus monkeys at doses of 10 µg/kg b.w./day via implanted
pumps. There was a high variability in the age of the dams (5 to 13 years of age) and it is not
stated whether it was the first pregnancy of each dam; therefore, variability in the behaviour
of the dams and their offspring can not be excluded. Moreover, the animals´ behaviour was
studied outside normal social environment which further raises questions on the validity of
the study.

BPA has the potential to modulate insulin secretion in mice as argued by Ropero et al. (2008)
who quoted data indicating that a single injection (no further details) of 10 μg/kg b.w. rapidly
increased plasma insulin and concomitantly decreased glycaemia. Perinatal exposure to BPA
(1 mg/L in drinking water from GD 6 to PND 21) in rats (Somm et al., 2009) was reported to
increase adipogenesis in female offspring at weaning. Some of BPAs’ effects may therefore
be due to interactions with peptide hormonal pathways as well as steroid metabolism and
function. Exposure to BPA at dose of 10 and 100 µg/kg b.w./day (s.c) aggravated insulin
resistance in mice during pregnancy and was associated with decreased glucose tolerance and
increased plasma insulin, triglyceride and leptin concentrations (Alonso-Magdalena et al.,
2010).

Ryan et al. (2010b) investigated the effect of gestational and lactational exposure to BPA or
DES on metabolic syndrome (obesity and diabetes) in CD-1 mice. Female mice were fed a
control diet, a diet containing 4 µg DES/kg diet (approx. 1 µg/kg b.w./day) or a diet
containing 1 µg BPA/kg diet (approx. 0.25 µg/kg b.w./day) during the whole pregnancy until
the end of lactation. The only effect was an increased pup body weight during lactation; there
were no indications of increased susceptibility to high-fat diet-induced obesity and glucose
intolerance in the mice when adult.

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The oestrogen related receptor-gamma is also expressed in adipose tissue in which BPA has
been shown to inhibit at nanomolar concentrations the release of adiponectin, an adipocyte-
specific hormone (Hugo et al., 2008). Micromolar BPA concentrations inhibited purified and
microsomal aromatase activities (Benachour et al., 2007) and potentially reduced oestrogen
synthesis by reducing aromatase activity in placental cells (Huang and Leung, 2009) or
aromatase expression in granulosa cells (Kwintkiewicz et al., 2010). This is in line with other
recent studies (Zhou et al., 2008; Huang and Leung, 2009). In contrast, a concentration-
dependent (0.1-10 nM) induction of aromatase expression by BPA was reported in rat
testicular Leydig cells, which was correlated with an up-regulation of cyclooxygenase-2 and a
reduction of testosterone synthesis (Kim et al., 2010). Other in vitro studies show also a weak
antagonism to thyroid hormone receptors with affinities several orders of magnitude below
that of triiodothyronine (Sun et al., 2009; Jung et al., 2007). Additionally BPA at micromolar
concentrations can interact with the function and protein binding of triiodothyronine (Okada
et al., 2007). Weak endocrine-disrupting effects involve interactions with growth hormone
expression (Dang et al., 2009).

5.3.2. Effects on signal transfer and gene expression


In addition to BPA´s interactions with hormone receptors the activation of protein kinase
pathways, e.g. via the extracellular signal-regulated kinase (ERK), was also reported in
association with genomic instability and neurotoxic effects (Kabil et al., 2008; Lee et al.,
2007; Lee et al., 2008). The rapid activation of cytoplasmic signal transduction pathways
linked to changes in calcium influx, adenylate cyclase and MAPK activation usually results in
changes in gene expression.

As an additional mechanism of action of BPA the induction of oxidative stress has been
described (Rashid et al., 2009): Wistar rats were treated for 6 months starting from weaning to
adult stage with BPA at relatively high concentrations in drinking water (1, 5 and 10 mg/L
corresponding to roughly 0.3, 1.5 and 3 mg /kg b.w./day). The mid and the high dose groups
showed significant increases in lipid peroxidation and reductions in glutathione (GSH) levels,
antioxidant enzymes in liver, kidney and in gonads from males and females.

The impact of BPA treatment on gene expression was studied in various cell types.
Concentration and time-dependent analyses of BPA exposure to human endometrial cell lines
showed that the majority of gene expression changes were induced by higher micromolar
concentrations of BPA (10-100 µM) (Naciff et al., 2010; Bredhult et al., 2009). However, at
lower BPA concentrations the limited gene expression changes did not correlate to the
oestrogenic effects of BPA (Naciff et al., 2010). Using different human cell lines treated with
various oestrogenic chemicals including BPA, cell type- and substance-specific patterns of
gene expression were observed (Inuoe et al., 2007). Dairkee et al. (2008) studied genomic
effects of BPA (0.1 µM) in cultures of nonmalignant breast epithelial cells from breast cancer
patients in the presence of 17 β-oestradiol and progesterone: BPA treatment resulted in
overexpression of genes facilitating cell cycle progression and multidrug resistance with
similarities to phenotypes of aggressive breast tumour cells. In Leydig tumour cells BPA (10
µM) treatment changed the expression profiles of several genes related to steroid/cholesterol
metabolism/transport and cell cycle regulation (Takamiya et al., 2007). The same BPA
concentration has been suggested to interfere with gene expression in the differentiation of
embryonic stem cells of monkeys (Yamamoto et al., 2007). A cell-specific effect of BPA at a
low concentration (1 nM) was reported in androgen receptor mutated prostate cancer cells:
BPA induced a pattern of gene expression partly overlapping with that of dihydrotestosterone,
specifically down-regulating oestrogen receptor β (Hess-Wilson et al., 2007).

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5.3.3. Effects on the immune system


Miao et al. (2008) studied the immunological effects resulting from a continuous prenatal and
postnatal (until PND 30) oral exposure to BPA (4, 40 and 400 mg/kg b.w./day; gavage) in
F344 rats. Although the results suggest that BPA affects immune-related responses, the
interpretation of the results in relation to immune disorders (dose-dependent downregulation
of IL-2, IL-12, IFN-γ and TNF-α in spleens; changes in ERα expression in mid- and high-
dose groups only; histological changes in spleens, liver and kidney in the high dose groups
only) is uncertain due to shortcomings both in the experimental design (insufficient number of
dams, i.e. 5/group, and not described exposure regimen) and lack of a link between the
observed molecular effect (mRNA expression levels of cytokines in the spleen) and a certain
adverse outcome (in the absence of evaluation of any other disease-related endpoints).

Midoro-Horiuti et al. (2010) investigated whether prenatal BPA exposure could affect
asthma-related endpoints in neonates via maternal loading in an ovalbumin (OVA)-induced
asthma model using BALB/c mice. The only significant finding was an increased IgE anti-
OVA level found in offspring of BPA-primed females. The Panel noted that this finding was
largely dependent on one animal, which the authors themselves called an “outlier”. Only one
dose level (ca. 2 mg /kg b.w./day) was tested. For all lung function and immunological tests,
more pups were used than that there were dams in the two parental BPA treatment groups.
That means that always littermates and non-littermates were mixed / combined to give the
four different treatment groups for the neonates. Also, there is no statement as to whether only
male or female pups were used. The description of statistics is rather limited (one-way
ANOVA, with T-test as follow up) and no information is given on the differentiation between
sexes.

A study in a mouse model by Oshima et al. (2007) gives some supportive qualitative evidence
that prenatal BPA exposure may partially interrupt the development of oral tolerance;
however only one dose of BPA was used for parts of the experiments.

Goto et al. (2007) studied antigen-specific responses of immune cells after oral administration
of BPA (1.5 to 1.8 mg/kg b.w./day) to T cell receptor transgenic mice. BPA induced complex
effects on the immune response mainly suppressing antigen-specific cytokine production of T
cells.

Guo et al. (2010) have performed in vitro experiments studying the effect of human
monocyte-derived dendritic cells (DC) exposed to BPA on the cytokine production and
expression of ER-α, ER-β and GPR30 in naïve human Th cells. This study demonstrated that
the presence of BPA during DC maturation influences the function of human DCs, thereby
polarizing the subsequent Th response. The authors suggest that DCs exposed to BPA can
provide one of the initial signals driving the development and perpetuation of Th2-dominated
immune responses in allergic reactions.

Yang et al. (2008) studied protein upregulation (including apo-A1, DPPIII and VAT1) in the
spleen and thymus of mice exposed to BPA in utero. Apo-A1 mRNA expression was also
increased. The authors considered that these three proteins could serve as biomarkers for BPA
developmental immunotoxicity.

5.3.4. Cytogenetic and epigenetic effects, cell proliferation and apoptosis


Naik and Vijayalaxmi (2009) reported that oral administration of BPA as single (10, 50, 100
mg/kg b.w.) or repeated doses (5x10 mg/kg b.w.) did not increase the incidence of structural
chromosomal aberrations or micronuclei in bone marrow of Swiss albino mice.
Administration of BPA, however, was associated with an increased incidence of achromatic

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lesions (gaps) which can not be considered as an evidence of a clastogenic potential in the
absence of a concurrent increase in structural chromosomal aberrations. Therefore, the
findings of this study do not change the previous conclusion (EFSA, 2006) that BPA has no
clastogenic potential in vivo. The authors also reported on an interference of BPA with the
spindle structure, which could be interpreted as an indication of aneuploidy. However,
considering the thresholded mechanism for aneuploidy induction, the large margin between
the doses tested negative in the micronucleus test and the TDI provides adequate reassurance
on the lack of aneugenic effects.

In addition Muhlhauser et al. (2009) presented data showing a borderline effect of BPA on
chromosome alignement or spindle abnormalities. The subtle effects of BPA on meiotic
spindle can be modulated by the amount of phyto oestrogens present in the diet. However, the
consequences of these cytological effects on chromosome segregation are unknown and
therefore these effects cannot be considered as markers of aneuploidy. Another paper by
Pacchierotti et al. (2008) showed that BPA can induce some cytological abnormalities in
oocytes, but that these do not result in increased aneuploid zygotes. In summary, these data
have no impact on the Panel´s previous conclusion on the lack of aneugenic activity of BPA
in mouse germ cells (EFSA, 2006).

BPA has recently been linked to transgenerational and developmental epigenetic changes in
rodents. BPA has been implicated in DNA hypomethylation resulting in aberrant expression
of key growth regulatory genes in rat prostate (Ho et al., 2006; Prins et al., 2008), mouse
forebrain (Yaoi et al., 2008) and mouse reproductive tract (Bromer et al., 2010). Maternal
BPA exposure of mice (50 mg/kg diet corresponding roughly to 10 mg/kg b.w./day) induced
alterations in the adult phenotype of the offspring by hypomethylation of the epigenome
(Dolinoy et al., 2007). Enhancer of Zeste Homolog 2 (EZH2), a histone methyltransferase has
been linked to breast cancer risk and epigenetic regulation of tumourigenesis. BPA elicited an
increase in (EZH2) expression in MCF-7 cells as well as in mammary glands of animals
exposed in utero (Doherty et al., 2010). The expression of the homeobox gene Hoxa10 in the
reproductive tract of mice, which controls uterine organogenesis, is increased upon BPA
exposure in utero; enhanced gene expression is associated with DNA-hypomethylation of
Hoxa10 promoter and intron (Bromer et al., 2010). Similarly, in utero and perinatal exposure
of mice to BPA was reported to elicit an endometriosis like-phenotype in female offspring,
associated with the expression of Hoxa10-protein. The authors suggest that endometriosis
may result from BPA-induced altered developmental programming (Signorile et al., 2010).

An oral study by Betancourt et al. (2010a) reports on proteomic changes in mammary glands
of Sprague Dawley rats treated with BPA in utero. The doses used in this study (25 or 250 μg
BPA/kg b.w. GD 10-21) did not appear to induce overt toxicity nor to affect sex hormone
regulation (17-β oestradiol and progesterone), but altered the expression of key proteins
(vimentin, SPARC, 14-3-3) steering cell proliferation and survival. In a subsequent
publication, Betancourt et al. (2010b; see section 5.2.1.2) supported these findings by
showing that - consistent with increased proliferation of epithelial cells of the mammary gland
in the high dose group - the levels of EGFR, phosphorylated IGF-1R, phosphorylated c-Raf,,
phosphorylated ERKs 1/2, phosphorylated ErbB2 and phosphorylated Akt were increased.
Studies using s.c. application of BPA also indicated that prenatal BPA exposure results in an
increased cell proliferation/apoptosis ratio in normal tissue as well as preneoplastic lesions of
rat mammary gland (Durando et al., 2007; Murray et al., 2007; Vandenberg et al., 2007;
2008). Altogether the data by Jenkins et al. (2009) and Betancourt et al. (2010b) suggest that
either lactational or in utero exposure to BPA may increase the susceptibility of the rat
mammary gland to cancer induction by DMBA. This may be linked to an enhanced cell
proliferation/apoptosis ratio, known to increase the probability of the formation of

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precancerous cells upon a genotoxic challenge (tumour initiation) and in subsequent stages of
carcinogenesis, to accelerate the manifestation of frank neoplasia (tumour promotion and
progression).

In an additional oral study by Moral et al. (2008) the effects of prenatal BPA exposure (25
and 250 µg/kg b.w./day applied by gavage on days 10-21 post-conception) on mammary
gland morphology, proliferation and modification of gene expression were investigated in
Sprague-Dawley CD rats. The architectural modifications induced by the higher dose of BPA
in mammary glands of female offspring were transient increases in the total number of
epithelial structures (day 21 only), terminal ducts (days 21 and 100, but not at days 35 and 50)
and lobule type 1 (day 35 only). The proliferative index in the epithelial structures was not
affected by BPA treatments. Time- and dose-dependent modifications in gene expression
profiles were observed after treatment with both doses of BPA: modulated (mainly up-
regulated) genes related to cell proliferation, apoptosis and differentiation, cell
communication, signal transduction, immunity, protein metabolism and modification.

5.3.5. Conclusions on the endocrine-mediated action of BPA


In vitro- and in vivo-studies (not compliant with the selection criteria in section 3) on
receptors, hormones, immune system, cell proliferation, apoptosis, proteomic, genomic and
epigenetic changes have been presented to compile recent data on potentially relevant
endocrine mechanisms of action of BPA. High doses of BPA (>5 mg/kg b.w./day) may have
biochemical and molecular effects consistent with those observed with other oestrogenic
substances. Effects have also been claimed to occur at low levels of BPA exposure, which
could be independent of the classical hormone receptors. BPA has only weak binding
affinities to these receptors, but its effects may alternatively be induced by cell membrane-
triggered signalling pathways via protein kinases. However, in the absence of clear dose
response curves and due to the shortcomings in experimental design, a conclusion cannot be
reached on the implications of the observed biochemical and molecular changes and to
establish whether they have any impact on human health. Because of the lack of a common
clearly defined mode of action of BPA at low doses, the toxicological relevance of the BPA
effects described cannot be evaluated and the results cannot be taken into consideration for
derivation of a TDI. While low dose effects of BPA are reported for some biochemical
changes the Panel is not aware of any clearly reproducible adverse effect expressed
specifically at low BPA doses only.

EFSA has established an internal task force to initiate the development of a common strategy
towards endocrine active substances. The Panel is aware of EFSA’s ongoing work to monitor
trends and developments in the assessment of health risks of endocrine active substances.

PART II - CONCLUSIONS
The CEF Panel has reviewed toxicological data published between 2007 and July 2010 in the
light of the previous risk assessments of BPA by EFSA in 2006 and 2008. In particular, this
review focuses on toxicokinetic, epidemiological and animal toxicity studies. In the literature
search about 800 articles were retrieved. A full list of the retrieved articles will be made
available by EFSA on request. The study design of all these publications was examined in
first instance. Only studies complying with the inclusion criteria indicated below were
considered for risk assessment purposes.

• Full research papers published after the 2006 opinion in peer-reviewed journals in the
public domain thus covering the period 2007 – July 2010
• Original data (no reviews, discussions or others)

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• Human studies13

• For the animal toxicity studies the focus was on studies having the following
experimental design:

a. Developmental exposure, i.e. pre-, peri-, and/or early post-natal exposure


b. oral route of exposure
c. several tested doses (plus a control) including at least one dose level lower than
the NOAEL of 5 mg/kg b.w./day (low-doses) from which the current TDI has
been derived.

5.4. Conclusions on toxicokinetics


Studies on toxicokinetics of BPA have demonstrated a significantly lower internal exposure
after oral intake as compared to parenteral exposure. This confirms that toxicity studies with
oral administration have higher relevance for human risk assessment of BPA in food than
studies with parenteral administration. In addition, new findings in non human primates (both
adults and newborns) further strengthen the view that BPA is eliminated faster in humans than
in rodents. This fast BPA elimination in primates results in substantially lower internal
exposure to free BPA in humans as compared to rodents. Even human premature infants can
metabolise and excrete BPA efficiently (via glucuronidation and sulfation), and this is also
supported by recent human data and data in young monkeys. The use of the standard
uncertainty factor (UF) of 10 to take into account interspecies differences is therefore
considered quite conservative.

In relation to in utero exposure, studies on transplacental transport of BPA and BPA-


glucuronide in humans and rodents indicated that although transfer may occur, foetal free
BPA levels are highly limited by the efflux pump P-glycoprotein, and placental BPA
glucuronidation may also take place. The Panel noted that exposure to total BPA (the major
constituent being the glucuronide) through lactation is limited to a very low fraction.
Therefore, in utero exposure and exposure through lactation appear to be limited.
The Panel recognises that inter-individual differences occur in expression of the isoenzymes
responsible for the detoxification of BPA. However, even in persons with low expression of
these enzymes, the metabolising capacity is still sufficient to eliminate free BPA from blood
at the low levels of BPA resulting from consumer exposure. Dietary exposures of adults and
infants (aged 3-6 months) were estimated to be up to 1.5 and 13 µg/kg body weight per day,
respectively (EFSA, 2006), based on conservative estimates of food consumption and
migration from food contact materials. These exposures are not anticipated to surpass the
metabolic capacity for BPA in adults or infants.

5.5. Conclusions on human studies


Recent epidemiological studies have suggested some statistically significant associations of
BPA exposure (urine concentrations) and health effects (coronary heart disease, reproductive
disorders) in adults and behavioural changes in young girls. The Panel noted that cross
sectional epidemiological studies such as these can demonstrate statistical associations

13
The Panel excluded from the selection purely biomonitoring studies, which mainly deal with exposure, and
therefore are not useful for TDI setting. The Panel noted that most biomonitoring data obtained in different
biological samples obtained so far have been tabled in a recent FDA report (U.S. FDA, 2010b).

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between BPA exposure and the presence (e.g. coronary heart disease) or absence (e.g. cancer,
asthma) of health outcomes, but the inherent design of cross sectional studies does not allow
establishment of a causal relationship between BPA exposure and health effects (e.g. chronic
diseases). In addition, the Panel has identified some limitations in these studies, which raise
further questions as to the significance of the reported findings. Therefore, the Panel could not
draw any relevant conclusion for risk assessment from these studies.

5.6. Conclusions on studies in animals

5.6.1. Studies on developmental and reproductive toxicity


The studies reporting effects at doses lower than 5 mg/kg b.w./day have severe shortcomings
and were considered to be invalid. The Panel considers that the valid studies do not raise
concern regarding reproductive and developmental toxicity of BPA at doses lower than 5
mg/kg b.w./day.

5.6.2. Developmental neurotoxicity and neurodevelopmental studies


Potentially significant biochemical changes, e.g. altered receptor expression in different brain
regions (see section 5.3), have been reported. However, in the absence of a correlation with a
functional adverse effect, the relevance of these observations for human health cannot be
assessed. The impact of BPA on development of sexually dimorphic behaviour was addressed
in the study by Ryan et al. (2010a), who observed a male-like reduced saccharin preference
and inhibition of lordosis behaviour in female rat offspring from oestrogen-treated but not
from BPA-treated dams. In the study reported by Stump et al. (2009) (See Part I) the effects
of BPA on learning and memory behaviour were inconclusive due to large variability in the
data. Other recent studies have methodological shortcomings. The Panel does not consider the
currently available data as convincing evidence of neurobehavioural toxicity of BPA.

5.6.3. Cell proliferation and apoptosis related to enhancement of tumourigenesis


The study by Jenkins et al. (2009) is the first oral study on a possible BPA-induced
enhancement of sensitivity of the mammary gland to carcinogen-induced breast tumour
formation in rat offspring following lactational BPA exposure of pups. Using the same model
of DMBA-induced mammary carcinogenesis but using in utero BPA exposure, Betancourt et
al. (2010b) also reported an enhancement of susceptibility for mammary gland
carcinogenesis. In consideration of the shortcomings in the design of both studies, in
particular the uncertainty regarding the lactational as well as in utero exposure of the
offspring to BPA, and of the limitations in reporting the Panel concluded that these results
cannot be taken into consideration for derivation of a TDI for BPA. However, the Panel noted
that at the highest dose level studied there is a shift of the ratio between cell proliferation and
apoptosis in favour of cell multiplication in the mammary gland. In view of the mechanistical
data obtained upon in utero exposure in other studies (see section 5.3) and the implications of
an increased cell proliferation/apoptosis ratio in carcinogenesis, the effects reported by
Jenkins and Betancourt deserve further consideration.

5.6.4. Studies on immunotoxicity


Modulation of immune system-related parameters is also an emerging field also in BPA
research. Several studies have reported changes in cytokines, changes in T-cell populations
and other aspects of immune modulation. However, the studies all suffered from
shortcomings in experimental design and reporting. Therefore, at the moment, these studies
cannot be taken into consideration for derivation of a TDI.

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5.7. Conclusions on endocrine-mediated action of BPA


In vitro- and in vivo-studies (not compliant with the selection criteria in section 3) on
receptors, hormones, immune system, cell proliferation, apoptosis, proteomic, genomic and
epigenetic changes have been presented to compile recent data on potentially relevant
endocrine mechanisms of action of BPA. High doses of BPA (>5 mg/kg b.w./day) may have
biochemical and molecular effects consistent with those observed with other oestrogenic
substances. Effects have been claimed to occur at low levels of BPA exposure, which could
be independent of the classical hormone receptors. BPA has only weak binding affinities to
these receptors, but these effects may alternatively be induced by cell membrane-triggered
signalling pathways via protein kinases. However, in the absence of clear dose response
curves and due to the shortcomings in experimental design, a conclusion cannot be reached on
the implications of the observed biochemical and molecular changes and to establish whether
they have any impact on human health. Because of the lack of a common clearly defined
mode of action of BPA at low doses, the toxicological relevance of the BPA effects described
cannot be evaluated and the results cannot be taken into consideration for derivation of a TDI.
While low dose effects of BPA are reported for some biochemical changes the Panel is not
aware of any clearly reproducible adverse effect expressed specifically at low BPA doses
only.

EFSA has established an internal task force to initiate the development of a common strategy
towards endocrine active substances. The Panel is aware of EFSA’s ongoing work to monitor
trends and developments in the assessment of health risks of endocrine active substances.

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PART II - REFERENCES
Adewale HB, Jefferson WN, Newbold RR and Patisaul HB, 2009. Neonatal bisphenol-A
exposure alters rat reproductive development and ovarian morphology without impairing
activation of gonadotropin-releasing hormone neurons. Biology of Reproduction 81, 690-
699.
AFSSA (Agence française de sécurité sanitaire des aliments), 2010. Opinion of the French
Food Safety Agency on the critical analysis of the results of a study of the toxicity of
bisphenol A on the development of the nervous system together with other recently-
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Tyl RW, Myers CB, Marr MC, Sloan CS, Castillo NP, Veselica MM, Seely JC, Dimond SS,
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Wolff MS, Engel S, Berkowitz G, Ye XSM, Zhu C, Wetmur J and Calafat AM, 2008.
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Zhou W, Liu J, Liao L and Han S, 2008. Effect of bisphenol A on steroid hormone production
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PART II - ABBREVIATIONS
AFC Scientific Panel on additives, flavourings, processing aids and
materials in contact with food
AFSSA French Food Safety Agency
Apo-A1 Apolipoprotein A-I precursor
AUC Area under the curve
BASC-2 Behavioural Assessment System for Children
BMI Body mass index
BPA Bisphenol A
BPB 2,2-bis(4-hydroxyphenyl)butane
BSI Behaviour System Index
b.w. Body weight
CNS Central Nervous System
Cmax Maximum serum concentration
CEF Scientific Panel on food contact materials, enzymes, flavourings and
processing aids
CVD Cardiovascular disease
DCs Dendritic cells
DEHP Di(2-ethylhexyl) phthalate
DES Diethylstilbestrol
DMBA Dimethylbenzanthracene
DNA Deoxyribonucleic acid
DPPIII Dipeptidylpeptidase III
DTU Technical University of Denmark
EC European Commission
EE ethinyloestradiol
EFSA European Food Safety Authority
ELISA Enzyme-Linked ImmunoSorbent Assay
ER Oestrogen receptor
ERK Extracellular signal-regulated kinase
EU European Union
EZH2 Enhancer of zeste homolog 2
E2:T oestradiol:testosterone ratio
FCM Food Contact Material(s)
FSH Follicle-stimulating hormone
GABA Gamma-Aminobutyric acid
GD Gestational day
GSH Glutathione
HPLC High performance liquid chromatography
i.c. Intracranial
i.p. Intraperitoneal
i.v. Intravenous
IVF In vitro fertilization
LC/MS Liquid Chromatography/Mass Spectrometry
LOAEL Lowest-Observed-Adverse-Effect-Level
LOD Limit of Detection
LOQ Limit of Quantification
MAPK Mitogen-activated protein kinases
mRNA Messenger Ribonucleic acid
MW Molecular Weight

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N or n Number of subjects/animals
NHANES National Health and Nutrition Examination Survey
NMDA N-methyl D-aspartate
NMDAR N-methyl-D-aspartate receptor
NOAEL No-Observed-Adverse-Effect-Level
NTP National Toxicology Program
OECD Organisation for Economic Co-operation and Development
OVA Ovalbumin
PBPK Physiologically-based pharmacokinetic
PCBs Polychlorinated biphenyls
P-gp P-glycoprotein
PND Postnatal day
s.c. Subcutaneous
SCF Scientific Committee on Food
SD Standard deviation
SG Specific-gravity
SPARC Secreted protein acidic and rich in cysteine
SRC Steroid receptor activator
SULT Sulfotransferases
TDI Tolerable Daily Intake
Th T helper
UDP Uridine 5'-diphosphate
UDPGT Uridine Diphosphate Glucuronyltransferase
UF Uncertainty Factor
UGT UDP-glucuronyl-transferases
U.S. FDA U.S. Food and Drug Administration
U.S. NTP-CERHR National Toxicology Program - Center for the Evaluation of Risks to
Human Reproduction
VAT1 Vesicle amine transporter 1

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PART III - ADVICE ON THE DANISH RISK ASSESSMENT OF BISPHENOL A

PART III - ASSESSMENT

6. Introduction
As of March 27, 2010 Denmark temporarily restricted the use of art. 3 in Commission
Directive 2002/72/EC14 by imposing a ban on bisphenol A (BPA) in materials in contact with
food for children aged 0-3 years until new studies document that low doses of BPA do not
have an impact on development of the nervous system or on the behaviour. The scientific
basis for this ban is a report by the National Food Institute at the Technical University of
Denmark (DTU Food Institute, 2010) mainly focusing on the developmental neurotoxicity
study in rats by Stump (2009). According to the DTU Food Institute, “The reduction in the
learning capacity detected in young male rats in the Stump study can be a sign of low doses
effect of bisphenol A but can also be a matter of coincidence. The results of the study
therefore cannot form a solid NOAEL about the effects on the development of the brain and
on behaviour”.

On 30 March 2010, EFSA received a request from the European Commission to advise by 15
April 2010 on the Danish risk assessment underlying the Danish ban of Bisphenol A in food
contact materials for infants aged 0-3 years and particularly to evaluate whether such risk
assessment provides grounds for the conclusion that the use of a food contact material or
article containing Bisphenol A endangers the health of children up to 3 years old.

At the same time the Scientific Panel on food contact materials, enzymes, flavourings and
processing aids (CEF) was dealing with two other Terms of Reference from the Commission,
one addressing the evaluation of a dietary developmental neurotoxicity study in rats (Stump,
2009), and another one addressing the relevance of a number of recent scientific publications
(2007-2010) to the risk assessment of BPA and their impact on the current tolerable daily
intake (TDI) of 0.05 mg BPA/kg b.w./day.
In order to provide risk managers with a comprehensive and updated opinion on BPA, the
CEF Panel decided to address all three requests on BPA from the Commission in one single
document. The adoption of this scientific document was postponed to 23rd September 2010.

7. Conclusions of the Danish risk assessment


The text shown under this heading is an English translation of the original conclusions of the
DTU Food Institute report. It does not necessarily reflect the opinion of the Panel.

The Danish risk assessment concludes:

“The study (i.e. the Stump study) complies with the recommendations in OECD test guideline
426. The strengths of the study lie in the route of administration via feed, the use of five doses
from a very low to fairly high doses and an adequate number of litters per group (n=23-24).
The weaknesses lie in the absence of measurements of internal BPA levels, the fact that the
behavioural tests carried out are not suited to an assessment of whether BPA can cause
effects that change the normally observed behavioural differences between males and females

14
Commission Directive 2002/72/EC of 6 August 2002 relating to plastic materials and articles intended to come
into contact with foodstuffs

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and the fact that in general there appears to be an extremely high degree of variation in the
behavioural data.
The study does not provide any clear evidence that BPA has harmful effects on the types of
behaviour investigated in the study, but does give rise to a degree of uncertainty with regard
to the effects on learning ability, since impaired learning ability was found in male offspring
with a low dosage of BPA. If this effect had been seen in the highest BPA group, it would have
been concluded that BPA is capable of affecting learning ability. However, the effect was seen
at the lowest BPA dose and not in animals exposed to higher doses, i.e. there is no “normal”
dose-response relationship. Doubts have currently been raised as to whether the effects of all
chemical substances necessarily show a “normal” dose-response curve. For certain effects
which disrupt the endocrine system it has for example been shown that low doses can result in
one type of effect while higher doses have other effects. The underlying mechanism for any
possible effects of BPA on the development of the brain is not known, and it is therefore
difficult to have clear expectations with regard to the dose-response curve. For this reason,
the finding of impaired learning ability in male offspring is considered to be a possible
indication of a low-dose effect of BPA, but it may also be a chance finding. The results of the
study cannot therefore be used to establish a robust NOAEL for effects on the development of
the brain and behaviour.
One fundamental weakness of the study is that its design was not based on the observations of
harmful effects of BPA at low doses on the development of the nervous system or on
behaviour that were found in earlier, limited studies, such as effects on certain aspects of
learning and memory (avoidance learning, schedule-controlled behaviour and
impulsiveness), anxiety-related behaviour and gender-specific behaviour. Consequently, the
study is unable to dispel uncertainty with regard to such effects on the development of the
nervous system.
In summary, in the opinion of the DTU Food Institute, the study does not resolve or change
the uncertainty as to the effects of BPA on the development of the nervous system or on
behaviour in rodents at a low dosage of BPA.”

8. Discussion of the Panel on the Danish risk assessment


The conclusion of the DTU Food Institute is based upon three major lines of arguments:

i. A degree of uncertainty with regard to the effects on learning ability, since in the
study by Stump et al. (2009) impaired learning ability was found in male offspring
with low dosage of BPA.

With respect to the learning and memory endpoint of the Stump study, as examined in the
Biel Maze test, the Panel concluded that the influence of BPA on learning and memory
behaviour cannot be evaluated. With respect to this endpoint, the study is inconclusive and
cannot be used for the risk assessment of BPA (See Part I).

The Panel noted the absence of any effect of BPA on the development of the nervous system
as shown by histopathology and brain morphometry and that where effects were observed
(maternal and offspring body weights, seizures and convulsions in the offspring at PND 11),
these occurred at dose levels above the dose level on which the TDI has been based in
previous evaluations (see Part I).

ii. Doubts on the monotonic (“normal”) dose-response for BPA

It has been argued that BPA may show a non-monotonic dose-response curve. Low dose
effects of BPA have been reported, which might be independent of the effect on the classical
hormone receptors (See Part II). However, most of these studies have several shortcomings

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such as lack of dose responses and limitations in experimental design. The Panel is not aware
of any clearly reproducible adverse effect expressed specifically at low BPA doses only.

iii. Some endpoints which have not been considered, namely certain aspects of learning
and memory (avoidance learning, schedule-controlled behaviour, impulsiveness),
anxiety-related behaviour and gender-specific (i.e., sexually dimorphic) behaviour.

The CEF Panel is aware that the Stump study did not address the endpoints mentioned above
(iii). Nevertheless, the Panel considered that the neurobehavioural studies where these BPA
effects were reported, have major shortcomings in their study design, and this makes the
findings in these studies questionable (see also Part I, section 1.2). In addition, previous
evaluations of these studies revealed no consistent treatment-related effects in the behavioural
endpoints and did not impact on the overall risk assessment (EFSA, 2006; EC, 2008). Also
the review of more recently published studies, which do cover part of the shortcoming as
indicated in this paragraph (in particular sexually dimorphic behaviour in females; see Ryan
et al, 2010; discussed in Part II of this opinion) does not give rise to change the current
position of the Panel. Other more recent studies on developmental neurotoxicity studies as
reviewed in Part II were considered invalid or inadequate for risk assessment purposes.

PART III - CONCLUSIONS


Overall, the Panel concluded that the study by Stump et al. (2009) cannot be used for the
assessment of the effects of BPA on learning and memory due to methodological limitations
(see part I). A number of studies addressing other neurobehavioural endpoints (e.g. learning
and memory behaviour, anxiety-related behaviour and gender-specific behaviour) were
considered invalid or inadequate for risk assessment purposes by the Panel. The Panel does
not consider the currently available data as convincing evidence of neurobehavioural toxicity
of BPA.

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PART III - REFERENCES


DTU Food Institute (DTU Fødevareinstituttets), 2010. Evaluation by the DTU Food Institute
of the industry’s new developmental neurotoxicity study (DNT, OECD TG 426) of
bisphenol A and the significance of the study for the Food Institute’s assessment of the
potential harmful effects of bisphenol A on the development of the nervous system and
behaviour. Available from:
http://www.food.dtu.dk/Admin/Public/Download.aspx?file=Files%2fFiler%2fNyheder%2f
Vurdering_BPA-studie.pdf
EC (European Commission), 2008. Updated Risk Assessment Report of 4'-
Isopropylidenediphenol (Bisphenol-A) (human health). Final approved version awaiting
publication, April 2008. Available from http://ecb.jrc.it/documents/Existing-
Chemicals/RISK_ASSESSMENT/ADDENDUM/bisphenola_add_325.pdf
EFSA (European Food Safety Authority), 2006. Opinion of the Scientific Panel on Food
Additives, Flavourings, Processing Aids and Materials in Contact with Food on a request
from the Commission related to 2,2-bis(4-hydroxyphenyl)propane (Bisphenol A). The
EFSA Journal 428, 1-75. Available from www.efsa.europa.eu.
Ryan BC, Hotchkiss AK, Crofton KM, and Gray LE Jr., 2010. In utero and lactational
exposure to bisphenol A, in contrast to ethinyloestradiol, does not alter sexually dimorphic
behavior, puberty, fertility, and anatomy of female LE rats. Toxicological Sciences 114,
133-148.
Stump (2009) study report. A Dietary Developmental Neurotoxicity Study of Bisphenol A in
rats. October 2009. Submitted by Polycarbonate/BPA Global Group American Chemistry
Council, Arlington, VA, USA.

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PART III - ABBREVIATIONS


BPA Bisphenol A
b.w. Body weight
CEF Scientific Panel on food contact materials, enzymes, flavourings and
processing aids
DTU Technical University of Denmark
EC European Commission
EFSA European Food Safety Authority
NOAEL No-Observed-Adverse-Effect-Level
TDI Tolerable Daily Intake

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PART IV - OVERALL PANEL CONCLUSIONS

9. Background
Bisphenol A [2,2-bis(4-hydroxphenyl)propane, CAS Number 80-05-7] (BPA) is used as a
monomer in the manufacture of polycarbonates and epoxy resins, as an antioxidant in PVC
plastics and as an inhibitor of end polymerisation in PVC. Polycarbonates are used in food
contact plastics such as reusable beverage bottles, infant feeding bottles, tableware (plates and
mugs) and storage containers, whereas epoxy resins are used in protective linings for food and
beverage cans and vats.

Small amounts of BPA can potentially leach out from food containers into foodstuffs and
beverages and therefore be ingested. BPA is permitted for use in food contact plastics in the
European Union with a specific migration limit of 0.6 mg/kg food15.

The No-Observed-Adverse-Effect-Level (NOAEL) of 5 mg/kg body weight (b.w.) per day


identified by the SCF in 2002 has provided the basis for setting the health based guidance
values for BPA both in Europe and the United States (EFSA, 2006; U.S. FDA, 2008). This
NOAEL has been identified in a multi-generation reproductive toxicity study, where the
critical effects were changes in body and organ weights in adult and offspring rats (Tyl et al.,
2002). In a subsequent multi-generation study in mice, the same NOAEL was identified based
on liver effects in adult mice (Tyl et al., 2006). EFSA also concluded that despite some
studies reporting adverse effects at doses lower than 5 mg/kg b.w./day, none of these studies
was sufficiently robust to be used to derive the Tolerable Daily Intake (TDI; EFSA, 2006). On
this basis EFSA derived a TDI of 0.05 mg/kg b.w/day, using an uncertainty factor of 100.
In 2008, EFSA evaluated differences in age-dependent toxicokinetics of BPA in animals and
humans and concluded that these would have no implication for the TDI previously set. EFSA
also concluded that the use of a default uncertainty factor of 100 can be regarded as
conservative for humans given toxicokinetic differences between rats and humans that would
indicate that rats, including neonatal rats, are exposed to higher levels of free BPA (EFSA,
2008).

In October 2009 the Commission asked the European Food Safety Authority (EFSA) to:

- Assess the relevance of a newly available developmental neurotoxicity study of


Bisphenol A in rats (Stump et al, 2009, 2010) and its implication for hazard and risk
assessment of Bisphenol A. and to update, if necessary, the currently applicable
tolerable daily intake for Bisphenol A.
- Furthermore, EFSA should address any new scientific evidence that may affect the
conclusions of the previously adopted opinions on BPA.
- On 30 March 2010, EFSA received an additional request from the European
Commission to advise by 15 April 2010 on the Danish risk assessment underlying the
Danish ban, on 26 March 2010, of Bisphenol A in food contact materials for infants
aged 0-3 years.
In order to provide a global view on the risk assessment of Bisphenol A, the Scientific Panel
on food contact materials, enzymes, flavourings and processing aids (CEF) decided to address

15
Commission Directive 2002/72/EC of 6 August 2002 relating to plastic materials and articles intending to come
into contact with foodstuffs

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the three mandates as given above in a single opinion and postponed the adoption of this
comprehensive document to 23rd September 2010.

The three different questions raised by the Commission are dealt with in three different parts
(PARTS I to III) of the opinion. This PART IV of the opinion presents an overview of the
conclusions from PART I to III, together with an overall conclusion.

10. Conclusions from PART I: Evaluation of the dietary developmental


neurotoxicity study of Bisphenol A in rats
Following earlier evaluations of the impact of BPA on neurologic and behavioural
developmental in rodents indicating overall possible concern for these endpoints (for
references see EC, 2003; 2008; NTP-CERHR, 2008; VKM, 2008), an industrial consortium
has submitted results from a developmental neurotoxicity study with BPA, in accordance with
OECD guideline 426 and in compliance with GLP. On request of the European Commission
to EFSA, the Panel has evaluated the outcome of this study. In the new study (Stump, 2009),
BPA was administered to pregnant rats via the diet at concentration ranging from 0.15 to 2250
mg/kg feed equivalent to 0.01-164 mg/kg b.w./day during gestation and to 0.03-410 mg/kg
b.w./day during lactation. The treatment schedule was relevant to human exposure in utero
and to breastfeeding. Since it can be estimated that the pups in this study were exposed up to
ca. 30 times higher levels of BPA than bottle-fed infants, the BPA exposure and the results
from the study are also regarded as relevant to bottle-fed infants. Dams were evaluated for
general signs of toxicity, and offspring were evaluated for general toxicity including
developmental landmarks and for neurological effects, including behaviour and brain
histopathology.

• General toxicity expressed as reductions in mean body weight and body weight gain in
dams and offspring was detected at levels of 750 and 2250 mg/kg feed, for which a
NOAEL of 75 mg/kg feed (equal to 5.85 mg/kg b.w./day during gestation) could be
derived. This NOAEL is close to that of 5 mg/kg b.w./day identified by the SCF (2002)
for similar effects, and which is the basis for the current TDI of BPA. No effects were
observed on brain development as examined by histopathology.

• In 2 and 4 pups respectively of the 750 and 2250 mg/kg feed (equal to 56.4-164 mg/kg
b.w./day during gestation) dose groups, popcorn seizures and convulsions were
observed on postnatal day (PND) 11. The absence of seizures or convulsions in a
follow-up study and in any study reported in the EU-RAR (EC, 2003; 2008) or in other
recent reviews (e.g. NTP-CERHR, 2008) casts doubt on the relevance of this
observation. The Panel noted that the current Tolerable Daily Intake (TDI) for BPA
(0.05 mg/kg b.w/day) would be sufficiently low to exclude a possible concern for this
effect, seen only at the two highest dose levels.

• The neurodevelopmental toxicity study by Stump (2009) covers motor activity,


learning and memory (spatial behaviour), auditory startle response, brain
histopathology and morphology. The study does not cover some specific aspects of
learning and memory (i.e. avoidance learning, schedule-controlled behaviour, and
impulsiveness), anxiety-related behaviour or sexual dimorphic behaviour, but this does
not invalidate the study. No statistically significant effects were observed in tests on
motor activity or auditory startle or in brain histopathology and morphology.

• According to the statistical analysis by the study authors, in the Biel maze swimming
test for learning and memory on PND 62 the male offspring from the 0.15 mg/kg feed
exposure group showed an increase in the number of errors in Path A trials 1-4, which

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could be interpreted as a delay in learning, because the difference was mainly caused
by an increased number of errors during trials 3 and 4. The effect reached statistical
significance when compared to concurrent controls. No such an effect was observed in
pups studied on PND 22 or in any other exposure group on PND 62. When the
swimming trials were conducted in the reverse path (i.e. the path B) no delay in
learning was observed and also in the repeat Path A trials 11 and 12, no effect was
observed in any exposure group, including the 0.15 mg/kg feed group. Therefore, the
authors concluded that there were no changes in learning and memory.

• In the Biel Maze test only the error counts were reported as a measure of learning and
memory. The animals should have also been evaluated for effects on “time-to-escape”
and checked for long term memory effects. However, this comparison was not made by
the study authors. EFSA’s Assessment and Methodology Unit (AMU) applied a more
appropriate statistical evaluation to the study data. It was realised that the data suffer
from censoring16. Therefore, based on the re analysis of the Biel Maze data the Panel
considered that no conclusion can be drawn from this study on the effect of BPA on
learning and memory behaviour due to large variability in the data.

• Based on the body weight effects on dams and offspring and also taking into account
the occurrence of seizures and convulsions in the two highest dose groups, which were
not observed at the lower dose levels, the study supports the NOAEL which was
derived from multigeneration studies in the past (5 mg/kg b.w./day), leading to a TDI
of 0.05 mg/kg b.w./day. However, the Panel considered that this test on learning and
memory was inconclusive and is of limited value in the risk assessment of BPA

The Panel is aware of ongoing studies by the U.S. Food and Drug Administration which will
address some neurodevelopmental endpoints.

11. Conclusions from PART II: Review of recent scientific literature on the toxicity
of Bisphenol A
The CEF Panel has reviewed toxicological data published between 2007 and July 2010 in the
light of the previous risk assessments of BPA by EFSA in 2006 and 2008. In particular, this
review focuses on toxicokinetic, epidemiological and animal toxicity studies. In the literature
search about 800 articles were retrieved. A full list of the retrieved articles will be made
available by EFSA on request. The study design of all these publications was examined in
first instance. Only studies complying with the inclusion criteria indicated below were
considered for risk assessment purposes.

• Full research papers published after the 2006 opinion in peer-reviewed journals in the
public domain thus covering the period 2007 – July 2010
• Original data (no reviews, discussions or others)
• All human studies17

16
In statistical terminology censoring occurs when the value of an observation is only partially known. In the Biel
water maze test the number of errors made by the rats that did not complete the maze within the 3 minute-limit that
was used, was recorded and the time to escape was recorded as 180 seconds. These experiments are “right
censored”, meaning that the rats could have escaped and made more errors, had they been given sufficient time.
Therefore the time to escape and the number of errors recorded will have been underestimated.
17
The Panel excluded from the selection purely biomonitoring studies, which mainly deal with exposure, and
therefore are not useful for TDI setting. The Panel noted that most biomonitoring data obtained in different
biological samples obtained so far have been tabled in a recent FDA report (U.S. FDA, 2010).

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• For the animal toxicity studies the focus was on studies having the following
experimental design:

a. Developmental exposure, i.e. pre-, peri-, and/or early post-natal exposure


b. Oral route of exposure
c. Several tested doses (plus a control) including at least one dose level lower than
the NOAEL of 5 mg/kg b.w./day (low-doses) from which the current TDI has
been derived.

11.1. Conclusions on toxicokinetics


Studies on toxicokinetics of BPA have demonstrated a significantly lower internal exposure
after oral intake as compared to parenteral exposure. This confirms that toxicity studies with
oral administration have higher relevance for human risk assessment of BPA in food than
studies with parenteral administration. In addition, new findings in non human primates (both
adults and newborns) further strengthen the view that BPA is eliminated faster in humans than
in rodents. This fast BPA elimination in primates results in substantially lower internal
exposure to free BPA in humans as compared to rodents. Even human premature infants can
metabolise and excrete BPA efficiently (via glucuronidation and sulfation), as supported by
recent human data and data in young monkeys. The use of the standard uncertainty factor
(UF) of 10 to take into account interspecies differences is therefore considered quite
conservative.

In relation to in utero exposure, studies on transplacental transport of BPA and BPA-


glucuronide in humans and rodents indicated that although transfer may occur, foetal free
BPA levels are highly limited by the efflux pump P-glycoprotein and placental BPA
glucuronidation may also take place. The Panel noted that exposure to total BPA (the major
constituent being the glucuronide) through lactation is limited to a very low fraction.
Therefore, in utero exposure and exposure through lactation appear to be limited.

The Panel recognises that inter-individual differences occur in expression of the isoenzymes
responsible for the detoxification of BPA. However, even in persons with low expression of
these enzymes, the metabolising capacity is still sufficient to eliminate free BPA from blood
at the low levels of BPA resulting from consumer exposure. Dietary exposures of adults and
infants aged 3-6 months were estimated to be up to 1.5 and 13 µg/kg body weight per day,
respectively (EFSA, 2006), based on conservative estimates of food consumption and
migration from food contact materials. These exposures are not anticipated to surpass the
metabolic capacity for BPA in adults or infants.

11.2. Conclusions on human studies


Recent epidemiological studies have suggested some statistically significant associations of
BPA exposure (urine concentrations) and health effects (coronary heart disease, reproductive
disorders) in adults and behavioural changes in young girls. The Panel noted that cross
sectional epidemiological studies such as these can demonstrate statistical associations
between BPA exposure and the presence (e.g. coronary heart disease) or absence (e.g. cancer,
asthma) of health outcomes, but the inherent design of cross sectional studies does not allow
establishment of a causal relationship between BPA exposure and health effects (e.g. chronic
diseases). In addition, the Panel has identified some limitations in these studies, which raise
further questions as to the significance of the reported findings. Therefore, the Panel could not
draw any relevant conclusion for risk assessment from these studies.

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11.3. Conclusions on studies in animals

11.3.1. Studies on developmental and reproductive toxicity


The studies reporting effects at doses lower than 5 mg/kg b.w./day have severe shortcomings
and were considered to be invalid. The Panel considers that the valid studies do not raise
concern regarding reproductive and developmental toxicity of BPA at doses lower than 5
mg/kg b.w./day.

11.3.2. Developmental neurotoxicity and neurodevelopmental studies


Potentially significant biochemical changes, e.g. altered receptor expression in different brain
regions (see section 5.3), have been reported. However, in the absence of a correlation with a
functional adverse effect, the relevance of these observations for human health cannot be
assessed. The impact of BPA on development of sexually dimorphic behaviour was addressed
in the study by Ryan et al. (2010), who observed a male-like reduced saccharin preference
and inhibition of lordosis behaviour in female rat offspring from oestrogen-treated but not
from BPA-treated dams. In the study reported by Stump et al. (2009) (See Part I) the effects
of BPA on learning and memory behaviour were inconclusive due to large variability in the
data. Other recent studies have methodological shortcomings. The Panel does not consider the
currently available data as convincing evidence of neurobehavioural toxicity of BPA.

11.3.3. Cell proliferation and apoptosis related to enhancement of tumourigenesis


The study by Jenkins et al. (2009) is the first oral study on a possible BPA-induced
enhancement of sensitivity of the mammary gland to carcinogen-induced breast tumour
formation in rat offspring following lactational BPA exposure of pups. Using the same model
of dimethylbenzanthracene (DMBA)-induced mammary carcinogenesis but in utero BPA
exposure, Betancourt et al. (2010) also reported an enhancement of susceptibility for
mammary gland carcinogenesis. In consideration of the shortcomings in the design of both
studies, in particular the uncertainty regarding the lactational as well as in utero exposure of
the offspring to BPA, and of the limitations in reporting the Panel concluded that these results
can not be used to set a new TDI for BPA. However, the Panel noted that at the highest dose
level studied there is a shift of the ratio between cell proliferation and apoptosis in favour of
cell multiplication in the mammary gland. In view of the mechanistical data obtained upon in
utero exposure in other studies (see section 5.3) and the implications of an increased cell
proliferation/apoptosis ratio in carcinogenesis, the effects reported by Jenkins and Betancourt
deserve further consideration.

11.3.4. Studies on immunotoxicity


Modulation of immune system-related parameters is an emerging field also in BPA research.
Several studies have reported changes in cytokines, changes in T-cell populations and other
aspects of immune modulation. However, the studies all suffered from shortcomings in
experimental design and reporting. Therefore, at the moment, these studies cannot be taken
into consideration for derivation of a TDI.

11.4. Conclusions on endocrine-mediated action of BPA


In vitro- and in vivo-studies (not compliant with the selection criteria in section 3) on
receptors, hormones, immune system, cell proliferation, apoptosis, proteomic, genomic and
epigenetic changes have been presented to compile recent data on potentially relevant
endocrine mechanisms of action of BPA. High doses of BPA (>5 mg/kg b.w./day) may have
biochemical and molecular effects consistent with those observed with other oestrogenic
substances. Effects have been claimed to occur at low levels of BPA exposure, which could

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be independent of the classical hormone receptors. BPA has only weak binding affinities to
these receptors, but these effects may alternatively be induced by cell membrane-triggered
signalling pathways via protein kinases. However, in the absence of clear dose response
curves and due to the shortcomings in experimental design, a conclusion cannot be reached on
the implications of the observed biochemical and molecular changes and to establish whether
they have any impact on human health. Because of the lack of a common clearly defined
mode of action of BPA at low doses, the toxicological relevance of the BPA effects described
cannot be evaluated and the results cannot be to set a new TDI. While low dose effects of
BPA are reported for some biochemical changes the Panel is not aware of any clearly
reproducible adverse effect expressed specifically at low BPA doses only.

EFSA has established an internal task force to initiate the development of a common strategy
towards endocrine active substances. The Panel is aware of EFSA’s ongoing work to monitor
trends and developments in the assessment of health risks of endocrine active substances.

12. Conclusions from PART III: Advice on the Danish risk assessment of
Bisphenol A
In March 2010, Denmark temporarily banned BPA in materials in contact with food for
children aged 0-3 years until new studies document that low doses of BPA do not have an
impact on development of the nervous system or on behaviour. The scientific basis for this
ban is a risk assessment report by the Danish National Food Institute (DTU Food Institute,
2010) mainly focusing on the developmental neurotoxicity study in rats by Stump (2009).
EFSA was asked to advise on the Danish risk assessment.

The conclusion of the DTU Food Institute is based upon three major lines of arguments:

iv. A degree of uncertainty with regard to the effects on learning ability, since in the
study by Stump et al. (2009) impaired learning ability was found in male offspring
with low dosage of BPA.

With respect to the learning and memory endpoint of the Stump study as examined in the Biel
Maze study, the Panel concluded that the influence of BPA on learning and memory
behaviour cannot be evaluated. With respect to this endpoint, the study is inconclusive and
cannot be used for the risk assessment of BPA (See Part I).

The Panel noted the absence of any effect of BPA on the development of the nervous system
as shown by histopathology and brain morphometry and that where effects were observed
(maternal and offspring body weights, seizures and convulsions in the offspring at PND 11),
these occurred at dose levels above the dose level on which the TDI has been based in
previous evaluations (see Part I).

v. Doubts on the monotonic (“normal”) dose-response for BPA

It has been argued that BPA may show a non-monotonic dose-response curve. Low dose
effects of BPA have been reported, which might be independent of the effect on the classical
hormone receptors (See Part II). However, most of these studies have several shortcomings
such as lack of dose responses and limitations in experimental design. The Panel is not aware
of any clearly reproducible adverse effect expressed specifically at low BPA doses only.

vi. Some endpoints which have not been considered, namely certain aspects of learning
and memory (avoidance learning, schedule-controlled behaviour, impulsiveness),
anxiety-related behaviour and gender-specific (i.e., sexually dimorphic) behaviour.

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The CEF Panel is aware that the Stump study did not address the endpoints mentioned above
(iii). Nevertheless, the Panel considered the neurobehavioural studies where these BPA
effects were reported as having major shortcomings in their study design, and this makes the
findings in these studies questionable (see also Part I, section 1.2). In addition, previous
evaluations of these studies revealed no consistent treatment-related effects in the behavioural
endpoints and did not impact on the overall risk assessment (EFSA, 2006; EC, 2008). Also
the review of more recently published studies, which do cover part of the shortcoming as
indicated in this paragraph (in particular sexually dimorphic behaviour in females; see Ryan
et al., 2010; discussed in Part II of this opinion) does not give rise to change the current
position of the Panel. Other more recent studies on developmental neurotoxicity studies as
reviewed in Part II were considered invalid or inadequate for risk assessment purposes.

Overall, the Panel concluded that the study by Stump et al. (2009) cannot be used for the
assessment of the effects of BPA on learning and memory due to methodological limitations
(see part I). A number of studies addressing other neurobehavioural endpoints (e.g. learning
and memory behaviour, anxiety-related behaviour and gender-specific behaviour) was
considered invalid or inadequate for risk assessment purposes by the Panel. The Panel does
not consider the currently available data as convincing evidence of neurobehavioural toxicity
of BPA.

13. Overall conclusion


Based on the comprehensive evaluation of recent human and animal toxicity data, the Panel
concluded that no new study could be identified, which would call for a revision of the
current TDI of 0.05 mg/kg b.w./day. This TDI is based on the NOAEL of 5 mg/kg b.w./day
from a multi-generation reproductive toxicity study in rats, and the application of an
uncertainty factor of 100, which is regarded as conservative based on all information on BPA
toxicokinetics.

The Panel noted that some studies conducted on developing animals have suggested other
BPA-related effects of possible toxicological relevance, in particular biochemical changes in
brain, immune-modulatory effects and enhanced susceptibility to breast tumours. These
studies had many shortcomings. At present the relevance of these findings for human health
cannot be assessed, though should any new relevant data become available in the future, the
Panel will reconsider the current opinion.

A minority opinion expressed by a Panel member is presented in an Annex to this opinion.

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PART IV - REFERENCES
Betancourt AM, Eltoum IA, Desmond RA, Russo J and Lamartiniere CA, 2010. In utero
Exposure to Bisphenol A Shifts the Window of Susceptibility for Mammary
Carcinogenesis in the Rat. Environmental Health Perspectives Jul 30. [Epub ahead of
print].
DTU Food Institute (DTU Fødevareinstituttets), 2010. Evaluation by the DTU Food Institute
of the industry’s new developmental neurotoxicity study (DNT, OECD TG 426) of
bisphenol A and the significance of the study for the Food Institute’s assessment of the
potential harmful effects of bisphenol A on the development of the nervous system and
behaviour. Available from:
http://www.food.dtu.dk/Admin/Public/Download.aspx?file=Files%2fFiler%2fNyheder%2f
Vurdering_BPA-studie.pdf
EC (European Commission), 2003. European Union Risk Assessment Report. Bisphenol A,
CAS No: 80-05-7. Institute for Health and Consumer Protection, European Chemicals
Bureau, European Commission Joint Research Centre, 3rd Priority List, Luxembourg:
Office for Official Publications of the European Communities. Available from
http://ecb.jrc.it/Documents/Existing-
Chemicals/RISK_ASSESSMENT/REPORT/bisphenolareport325.pdf
EC (European Commission), 2008. Updated Risk Assessment Report of 4'-
Isopropylidenediphenol (Bisphenol-A) (human health). Final approved version awaiting
publication, April 2008. Available from http://ecb.jrc.it/documents/Existing-
Chemicals/RISK_ASSESSMENT/ADDENDUM/bisphenola_add_325.pdf
EFSA (European Food Safety Authority), 2006. Opinion of the Scientific Panel on Food
Additives, Flavourings, Processing Aids and Materials in Contact with Food on a request
from the Commission related to 2,2-bis(4-hydroxyphenyl)propane (Bisphenol A). The
EFSA Journal 428, 1-75. Available from www.efsa.europa.eu.
EFSA (European Food Safety Authority), 2008. Scientific Opinion of the Panel on Food
additives, Flavourings, Processing Aids and Materials in Contact with Food (AFC) on a
request from the Commission on the toxicokinetics of Bisphenol A . The EFSA Journal
759, 1-10.
Jenkins S, Raghuraman N, Eltoum I, Carpenter M, Russo J and Lamartiniere CA, 2009. Oral
exposure to bisphenol A increases dimethylbenzanthracene-induced mammary cancer in
rats. Environmental Health Perspectives 117, 910-915.
NTP-CERHR (National Toxicological Program-Center for the Evaluation of Risks to Human
Reproduction), 2008. NTP-CERHR Monograph on the Potential Human Reproductive and
Developmental Effects of Bisphenol A. September 2008. NIH Publication No. 08-5994.
Available from http://cerhr.niehs.nih.gov/chemicals/bisphenol/bisphenol.pdf
Ryan BC, Hotchkiss AK, Crofton KM, and Gray LE Jr., 2010. In utero and lactational
exposure to bisphenol A, in contrast to ethinyloestradiol, does not alter sexually dimorphic
behavior, puberty, fertility, and anatomy of female LE rats. Toxicological Sciences 114,
133-148.
SCF (Scientific Committee on Food), 2002. Opinion of the Scientific Committee on Food on
Bisphenol A. Expressed on 17 April 2002. Available from
http://ec.europa.eu/food/fs/sc/scf/out128_en.pdf.
Stump (2009) study report. A Dietary Developmental Neurotoxicity Study of Bisphenol A in
rats. October 2009. Submitted by Polycarbonate/BPA Global Group American Chemistry
Council, Arlington, VA, USA.

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Stump DG, Beck MJ, Radovsky A, Garman RH, Freshwater L, Sheets LP, Marty MS,
Waechter JM, Dimond SS, Van Miller JP, Shiotsuka RN, Beyer D, Chappelle AH and
Hentges SG, 2010. Developmental neurotoxicity study of dietary bisphenol A in Sprague-
Dawley rats. Toxicological Sciences 115, 167-182.
Tyl RW, Myer CB, Marr MC, Thomas BF, Keimowitz AR, Brine DR, Veselica MM, Fail PA,
Chang TY, Seely, JC, Joiner RL, Butala JH, Dimond SS, Cagen SZ, Shiotuka RN, Stropp
GD and Waechter JM, 2002. Three-Generation Reproductive Toxicity Study of Dietary
Bisphenol A in CD Sprague-Dawley Rats. Toxicological Sciences 68, 121-146.
Tyl RW, Myers CB, and Marr MC, 2006. Draft Final Report. Two-generation reproductive
toxicity evaluation of Bisphenol A (BPA; CAS No. 80-05-7) administered in the feed to
CD-1® Swiss mice (modified OECD 416). RTI International Center for Life Sciences and
Toxicology, Research Triangle Park, NC, USA.
U.S. FDA (Food and Drug Administration), 2008. Draft assessment of bisphenol A for use in
food contact applications: draft version 08/14/2008. Available from
http://www.fda.gov/ohrms/dockets/AC/08/briefing/2008-
0038b1_01_02_FDA%20BPA%20Draft%20Assessment.pdf
U.S. FDA (Food and Drug Administration), 2010. Memorandum of 11/16/2009, Summary of
Bisphenol A Biommonitoring Studies [FDA-2010-N-0100-0001] Available from
http://www.regulations.gov/search/Regs/home.html#docketDetail?R=FDA-2010-N-0100
VKM (Norwegian Scientific Committee for Food Safety), 2008. Opinion of the Scientific
Panel on Food Additives, Flavourings, Processing Aids, Materials in Contact with Food
and Cosmetics of the Norwegian Scientific Committee for Food Safety. Assessment of
four studies on developmental neurotoxicity of bisphenol A.

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PART IV - ABBREVIATIONS
AMU Assessment and Methodology Unit
BPA Bisphenol A
b.w. Body weight
CAS Chemical abstracts service
CEF Scientific Panel on food contact materials, enzymes, flavourings and
processing aids
DMBA Dimethylbenzanthracene
DTU Technical University of Denmark
EC European Commission
EU-RAR European Union Risk Assessment Report
EFSA European Food Safety Authority
GLP Good Laboratory Practice
NOAEL No-Observed-Adverse-Effect-Level
NTP-CERHR National Toxicology Program - Center for the Evaluation of Risks to
Human Reproduction
OECD Organisation for Economic Co-operation and Development
PND Postnatal day
PVC Polyvinyl chloride
SCF Scientific Committee on Food
TDI Tolerable Daily Intake
UF Uncertainty Factor
U.S. FDA U.S. Food and Drug Administration
VKM Norwegian Scientific Committee for Food Safety

EFSA Journal 2010; 8(9):1829 110


EFSA Journal 2010; 8(9):1829

Scientific Opinion on Bisphenol A: evaluation of a study investigating its


neurodevelopmental toxicity, review of recent scientific literature on its toxicity
and advice on the Danish risk assessment of Bisphenol A

MINORITY OPINION

This minority opinion from Catherine Leclercq is based on the view that there are significant
uncertainties about the current validity of the NOAEL (5 mg/kg b.w.) used to establish the TDI for
bisphenol A in the EFSA opinion of 2006.
These uncertainties emerge from the results of several new studies performed on animals exposed
during prenatal or postnatal development and published since 2006. These studies indicate that
adverse effects might occur in the developing offspring of laboratory rodents at oral dose levels below
the current NOAEL. The effects of concern include, in particular, brain receptor programming,
immune modulation and enhancement of susceptibility to breast tumours. More limited data hint also
to other potential effects, such as on metabolic programming. Studies on BPA modes of action provide
biological plausibility to such effects. Due to methodological shortcomings, none of the new studies can
be used to derive a more stringent NOAEL that could lead to a newly established numerical TDI
value.
However, due to the overall weight of evidence, the current TDI of 50 µg/kg body weight may not be
confirmed as a full TDI and should be considered as temporary.
The derivation of a temporary TDI generally implies the use of an additional safety factor ranging
from 2 to 10. In this specific case the choice of an additional safety factor would be highly arbitrary
and even an additional factor of 10 might not be sufficient since some effects were observed at dose
levels which are orders of magnitude below the NOAEL of 5 mg/kg bw. Robust data are required to
reduce the specific uncertainties identified and establish a full TDI.
When giving due consideration to the identified uncertainties, it appears that the effects of potential
concern may have specific relevance for prenatal and postnatal development in humans. Exposure to
BPA in pregnant or lactating women and in infants therefore requires attention until more robust data
on the areas of uncertainty will be available. According to the EFSA assessment in 2006, infants are
the population subgroup with potential highest dietary exposure. They also are supposed to have a less
efficient BPA metabolizing capacity as compared to adults. The two main potential sources of dietary
exposure to BPA in infants are can coatings and polycarbonate bottles. In particular, avoiding bottles
made out of polycarbonate for formula-fed infants could significantly reduce their potential exposure.

Suggested citation: EFSA Panel on food contact materials, enzymes, flavourings and processing aids (CEF). Scientific Opinion on
Bisphenol A: evaluation of a study investigating its neurodevelopmental toxicity, review of recent scientific literature on its toxicity
and advice on the Danish risk assessment of Bisphenol A. EFSA Journal 2010;8(9):1829, Annex [6 pp.]
doi:10.2903/j.efsa.2010.1829. Available online: www.efsa.europa.eu/efsajournal.htm

© European Food Safety Authority, 2010 1


Minority Opinion - Bisphenol A

The minority opinion is related to Part II and part IV (overall conclusions) of the opinion. Extracts of the text
of the opinion of the Panel are reported underneath in italics with suggestions for addition or substitution in
bold.

PART II – REVIEW OF RECENT SCIENTIFIC LITERATURE ON THE TOXICITY OF BISPHENOL A

5.2. Animal toxicity studies

5.2.1.2 Other developmental toxicity studies


…..
Developmental neurotoxicity and neurobehavioural studies
…..
Tian et al. (2010) treated 2 pregnant ICR mice per group by oral administration of BPA (0, 100, 500 µg/kg
b.w./day) from GD 7 to PND 21. The 5 week old male and female offspring (9 to 17 pups/litter; not further
specified) were tested PND 22 to PND 36 for spontaneous exploratory activity in the open field, anxiety-
related behaviour in the elevated plus-maze test, spontaneous alteration behaviour in the Y-maze test,
novelty recognition in the novel object test, ligand-binding studies for D1, D2 and dopamine transporters
(involved in anxiety-related behaviour) and NMDA receptors (contribute to memory and learning). There
was no effect on total locomotion in the open field, but central locomotion increased at 100 µg/kg b.w., but
not at 500 µg/kg b.w.. BPA increased time spent in the open arm (significant at 500 µg/kg b.w.) of the
elevated plus-maze but did not change total number of entries of the open arms. Both BPA groups
significantly decreased percentage of alteration behaviour in the Y-maze test. BPA decreased novel object
recognition at 100 µg/kg b.w. but not at 500 µg/kg b.w.. Receptor binding assays did not show an effect on
D1 receptor, a significant increase in D2 receptor binding at 100 µg/kg b.w., an almost identical reduction
in dopamine transporter binding at 100 and 500 µg/kg b.w. and a dose-dependent reduction in NMDA
receptor binding in frontal cortex and hippocampus with 100 and 500 µg/kg b.w.. The Panel considered the
study design by Tian et al. (2010) as not valid (insufficient number of dams per group and inappropriate
statistics not performed on a per dam basis).

Suggestion for modification: substitute “as not valid” with “as having important shortcomings which
renders it unsuitable for consideration in order to set a new TDI.”

Suggestion for addition: However, the effect reported on brain receptor programming deserves
further consideration as its plausibility is supported by a number of mechanistic studies performed in
vitro (Alyea et al., 2009, Choi et al., 2007, Yokosuka et al., 2008) as well as in vivo at low oral doses
(Honma et al., 2006) or subcutaneous doses (Zhou et al., 2009) (see section 5.3). A human study on the
relation between prenatal BPA exposure and behaviour in 2-year old females (Braun et al., 2009)
provides some indication of possible relevance of behavioural changes to human. This area of
uncertainty should be explored through more robust studies.

Studies on metabolic effects

The effects of peri- and postnatal exposure to BPA on adipose tissue mass were investigated by Miyawaki et
al. (2007). Groups of 3 pregnant ICR mice were exposed to BPA in drinking water (0, 1 or 10 µg/ml,
resulting in 0, 0.26, 2.72 mg/kg b.w./day) from GD 10 to end of lactation. Offspring were exposed up to PND
31 and groups of 16 to 25 offspring per sex and dose group were evaluated. Body weights of female offspring
were increased at the low and high dose group, body weights of the males at the high dose group. Adipose
tissue weight was increased significantly in females at the low dose and in males at the high dose group.
Serum leptin was increased only in females of the low dose group. Total cholesterol was increased only in
females with the highest increase in the low dose group. Serum triacylglycerol and non-esterified fatty acid

EFSA Journal 2010; 8(9):1829 2


Minority Opinion - Bisphenol A

levels were increased and serum glucose levels decreased only in males of the low dose group. The low
number of dams per group invalidates this study.

Suggestion for modification: substitute “invalidates this study” with “and the unclear dose-response
relationship of observed effects renders this study unsuitable for consideration in order to set a new
TDI.”

Suggestion for addition: Nevertheless, some mechanistic studies performed in vivo hint that BPA may
alter metabolic programming, including a study using low oral dose levels (Somm et al., 2009).
Considering that metabolic programming is a relevant effect that is insufficiently covered by current
testing guidelines, the potential effect of BPA on metabolic programming is an area of uncertainty that
may deserve further consideration.

Studies on immunologic effects

Yan et al. (2008) examined the response of mice to a bacterial pathogen after exposure to BPA during
development. Mice were housed in polymethylpentene cages, fed commercial diet and drank water from
glass bottles. Bedding was not described. Pregnant BALB/c mice were exposed to BPA in drinking water at
concentrations of 0, 1, 10, or 100 nM (equivalent to 0.03, 0.3 and 3 µg/kg b.w./day) for 2 weeks before
mating and 1 week after mating. At 10 weeks of age, male offspring were given a subcutaneous injection of
Leishmania (L.) major in the hind footpad. The offspring of exposed mothers showed dose-dependent
enhanced footpad swelling at 6 and 8 weeks after infection with L. major antigen. Production of interleukin-
4 (IL-4) and interferon-γ (IFN-γ) by splenocytes was significantly increased in offspring from dams exposed
to 10 and 100 nM BPA but not in those born to 1 nM BPA-treated females compared with the nonexposed
control mice. In addition, diminished occurrence of CD4+CD25+ T cells was detected at the same dose
levels, suggesting that the increased cytokine production could be due to the decreased number of regulatory
T cells. However, the lack of inclusion in the experimental design of the appropriate marker (Foxp3) in
addition to that of CD4+ and CD25+ does not allow the conclusion that the measured cells were indeed
regulatory T cells. In addition, the study has some shortcomings (small experimental groups of 3-4 animals
and evaluations in males only, no indication of the number of exposed dams, or whether animals in the tested
groups were littermates).
In conclusion, due to the limited study reporting, no clear conclusions can be drawn from this study and,
therefore, this study cannot be taken into consideration for derivation of a TDI for BPA.

Suggestion for addition: However, the study lends further support to immunological effects elicited by
BPA, as indicated by several mechanistic studies performed in vivo at low oral dose levels (Oshima et
al., 2007; Yang et al., 2008) as well as in vitro evidence on human monocyte-derived dendritic cells
(Guo et al. 2010). Since a dose-response cannot be confidently established, immune modulation is a
current uncertainty area in BPA risk assessment, considering also that this type of effect is
insufficiently covered by current testing guidelines.

5.2.2. Conclusions on animal toxicity studies


5.2.2.2. Developmental Neurotoxicity and Neurodevelopmental Studies.

Potentially significant biochemical changes, e.g. altered receptor expression in different brain regions (see
section 5.3), have been reported. However, in the absence of a correlation with a functional adverse effect,
the relevance of these observations for human health cannot be assessed. The impact of BPA on development
of sexually dimorphic behaviour was addressed in the study by Ryan et al. (2010a), who observed a male-
like reduced saccharin preference and inhibition of lordosis behaviour in female rat offspring from

EFSA Journal 2010; 8(9):1829 3


Minority Opinion - Bisphenol A

oestrogen-treated but not from BPA-treated dams. In the study reported by Stump et al. (2009) (See Part I)
the effects of BPA on learning and memory behaviour were inconclusive due to large variability in the data.
Other recent studies have methodological shortcomings. The Panel does not consider the currently available
data as convincing evidence of neurobehavioural toxicity of BPA.

Suggestion for substitution of the last sentence: In conclusion, the findings from Ryan et al. (2010a)
and Stump et al. (2009) do not support the hypothesis of behavioural changes due to low BPA
exposure. However, the effect reported by Tian et al (2010) on brain receptor programming deserves
further consideration, as similar effects are indicated in in vitro and in vivo mechanistic studies.

5.2.2.4 Studies on Immunotoxicity

Modulation of immune system-related parameters is an emerging field also in BPA research. Several studies
have reported changes in cytokines, changes in T-cell populations and other aspects of immune modulation
(see section 5.3). However, all the studies including that of Yan et al. (2008) suffered from shortcomings in
experimental design and reporting. Therefore, at the moment, these studies cannot be taken into
consideration for derivation of a TDI.

Suggestion for modification of the last sentence: These studies cannot be taken into consideration for
derivation of a TDI. However, immune effects represent an area of uncertainty deserving further
consideration.

Suggestion for addition of a paragraph:

5.2.2.5. Metabolic effects


The study by Miyawaki et al. (2007) on effects of perinatal and postnatal exposure to BPA on adipose
tissue mass may hint to a possible effect of low doses of BPA on metabolic programming which is
suggested also by other mechanistic studies. Human studies on the relation between BPA and
cardiovascular diseases (Lang et al., 2008 and Melzer et al. 2010) provide some indication of possible
relevance of metabolic effects to humans. Considering also that these effects are insufficiently covered
by current testing guidelines, further consideration may be warranted.

Suggestion for substitution of the conclusions on animal studies (paragraphs 5.6 and 11.3)
Overall, the new animal studies considered valid by the Panel do not raise concern regarding
reproductive and developmental toxicity of BPA and do not support the hypothesis of behavioural
changes due to BPA exposure at doses lower than the current NOAEL. A number of other animal
studies which have shortcomings provide some evidence of possible effects at lower doses on brain
receptor programming, immunity and enhancement of cancer susceptibility in the mammary gland.
More limited data hint also to other potential effects, such as on metabolic programming. Studies on
BPA modes of action provide biological plausibility to such effects. Due to methodological
shortcomings, none of the new studies can be used to derive a TDI. However, the overall weight of the
evidence indicates that these effects represent specific areas of uncertainty and deserve further
consideration.

EFSA Journal 2010; 8(9):1829 4


Minority Opinion - Bisphenol A

5.7. Conclusions on endocrine-mediated action of BPA


In vitro- and in vivo-studies (not compliant with the selection criteria in section 3) on receptors, hormones,
immune system, cell proliferation, apoptosis, proteomic, genomic and epigenetic changes have been
presented to compile recent data on potentially relevant endocrine mechanisms of action of BPA. High doses
of BPA (>5 mg/kg b.w./day) may have biochemical and molecular effects consistent with those observed with
other estrogenic substances. Effects have been claimed to occur at low levels of BPA exposure, which could
be independent of the classical hormone receptors. BPA has only weak binding affinities to these receptors,
but these effects may alternatively be induced by cell membrane-triggered signalling pathways via protein
kinases. However, in the absence of clear dose response curves and due to the shortcomings in experimental
design, a conclusion cannot be reached on the implications of the observed biochemical and molecular
changes and to establish whether they have any impact on human health. Because of the lack of a common
clearly defined mode of action of BPA at low doses, the toxicological relevance of the BPA effects described
cannot be evaluated and the results cannot be taken into consideration for derivation of a TDI. While low
dose effects of BPA are reported for some biochemical changes the Panel is not aware of any clearly
reproducible adverse effect expressed specifically at low BPA doses only.

Suggestion for addition:


Studies on endocrine-mediated action of BPA provide biological plausibility to the existence of areas of
uncertainty identified in animal toxicity studies, as discussed in section 5.3. In particular, in vivo
mechanistic studies support the uncertainties over immune effects (e.g., Oshima et al. 2007) and
mammary tumourigenesis (e.g., Betancourt et al., 2010b) in immature animals.

PART IV – OVERALL PANEL CONCLUSIONS


13. Overall conclusion

Suggestion for substitution of the overall Panel conclusion:

Based on the comprehensive evaluation of recent human and animal toxicity data, there are significant
uncertainties about the current validity of the NOAEL (5 mg/kg bw) used to establish the TDI for
bisphenol A in the EFSA opinion of 2006.
These uncertainties emerge from the results of several new studies performed on animals exposed
during prenatal or postnatal development. These studies indicate that adverse effects might occur in
the developing offspring of laboratory rodents at oral dose levels below the current NOAEL. The
effects of concern include, in particular, brain receptor programming, immune modulation and
enhancement of susceptibility to breast tumours. More limited data hint also to other potential effects,
such as on metabolic programming. Studies on BPA modes of action provide biological plausibility to
such effects. Due to methodological shortcomings, none of the new studies can be used to derive a more
stringent NOAEL that could lead to a newly established numerical TDI value.
However, due to the overall weight of evidence, the current TDI of 50 µg/kg body weight may not be
confirmed as a full TDI and should be considered as temporary.
The derivation of a temporary TDI generally implies the use of an additional safety factor ranging
from 2 to 10. In this specific case the choice of an additional safety factor would be highly arbitrary
and even an additional factor of 10 might not be sufficient since some effects were observed at dose
levels which are orders of magnitude below the NOAEL of 5 mg/kg bw. Robust data are required to
reduce the specific uncertainties identified and establish a full TDI.
When giving due consideration to the identified uncertainties, it appears that the effects of potential
concern may have specific relevance for prenatal and postnatal development in humans. Exposure to
BPA in pregnant or lactating women and in infants therefore requires attention until more robust data
on the areas of uncertainty will be available. According to the EFSA assessment in 2006, infants are

EFSA Journal 2010; 8(9):1829 5


Minority Opinion - Bisphenol A

the population subgroup with potential highest dietary exposure. Moreover simulations taking into
account both age-dependent metabolic differences and specific pattern of exposure, predict for
newborns a 3-fold greater free BPA blood concentration as compared with the adult after oral
exposure to the same quantity of BPA per kg b.w. (see section 4.5). Infants are therefore supposed to
have a less efficient BPA metabolizing capacity as compared to adults. The two main potential sources
of dietary exposure to BPA in infants are can coatings and polycarbonate bottles. In particular,
avoiding bottles made out of polycarbonate for formula-fed infants could significantly reduce their
potential exposure.

EFSA Journal 2010; 8(9):1829 6