<b>Bijsluiter</b>. De hyperlink naar het originele document werkt niet meer. Daarom laat Woogle de tekst zien die in dat document stond. Deze tekst kan vreemde foutieve woorden of zinnen bevatten en de opmaak kan verdwenen of veranderd zijn. Dit komt door het zwartlakken van vertrouwelijke informatie of doordat de tekst niet digitaal beschikbaar was en dus ingescand en vervolgens via OCR weer ingelezen is. Voor het originele document, neem contact op met de Woo-contactpersoon van het bestuursorgaan.<br><br>====================================================================== Pagina 1 ======================================================================

<pre>             Health Council of the Netherlands
          β-Estradiol
             Evaluation of the carcinogenicity and genotoxicity
2013/23
</pre>

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<pre>β-Estradiol
    Evaluation of the carcinogenicity and genotoxicity
</pre>

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<pre></pre>

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<pre>Aan de minister van Sociale Zaken en Werkgelegenheid
Onderwerp                 : aanbieding advies β-Estradiol
Uw kenmerk                : DGV/BMO-U-932542
Ons kenmerk               : U-7912/SV/fs/246-A19
Bijlagen                  :1
Datum                     : 18 oktober 2013
Geachte minister,
Graag bied ik u hierbij het advies aan over de gevolgen van beroepsmatige blootstelling
aan β-oestradiol.
Dit advies maakt deel uit van een uitgebreide reeks waarin kankerverwekkende stoffen
worden geclassificeerd volgens richtlijnen van de Europese Unie. Het gaat om stoffen
waaraan mensen tijdens de beroepsmatige uitoefening kunnen worden blootgesteld.
      Dit advies is opgesteld door een vaste subcommissie van de Commissie Gezondheid
en beroepsmatige blootstelling aan stoffen (GBBS), de Subcommissie Classificatie van
carcinogene stoffen. Het advies is getoetst door de Beraadsgroep Gezondheid en omgeving
van de Gezondheidsraad.
Ik heb dit advies vandaag ter kennisname toegezonden aan de staatssecretaris van
Infrastructuur en Milieu en aan de minister van Volksgezondheid, Welzijn en Sport.
Met vriendelijke groet,
prof. dr. W.A. van Gool,
voorzitter
Bezoekadres                                                      Postadres
Rijnstraat 50                                                    Postbus 16052
2515 XP Den               Haag                                   2500 BB Den     Haag
E - m a i l : s r. v i n k @ g r. n l                            w w w. g r. n l
Te l e f o o n ( 0 7 0 ) 3 4 0 5 5 0 8
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<pre></pre>

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<pre>β-Estradiol
Evaluation of the carcinogenicity and genotoxicity
Subcommittee on the Classification of Carcinogenic Substances of the
Dutch Expert Committee on Occupational Safety,
a Committee of the Health Council of the Netherlands
to:
the Minister of Social Affairs and Employment
No. 2013/23, The Hague, October 18, 2013
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<pre>The Health Council of the Netherlands, established in 1902, is an independent
scientific advisory body. Its remit is “to advise the government and Parliament on
the current level of knowledge with respect to public health issues and health
(services) research...” (Section 22, Health Act).
     The Health Council receives most requests for advice from the Ministers of
Health, Welfare & Sport, Infrastructure & the Environment, Social Affairs &
Employment, Economic Affairs, and Education, Culture & Science. The Council
can publish advisory reports on its own initiative. It usually does this in order to
ask attention for developments or trends that are thought to be relevant to
government policy.
     Most Health Council reports are prepared by multidisciplinary committees of
Dutch or, sometimes, foreign experts, appointed in a personal capacity. The
reports are available to the public.
                 The Health Council of the Netherlands is a member of the European
                 Science Advisory Network for Health (EuSANH), a network of science
                 advisory bodies in Europe.
                 The Health Council of the Netherlands is a member of the International Network
                 of Agencies for Health Technology Assessment (INAHTA), an international
                 collaboration of organisations engaged with health technology assessment.
 I NA HTA
This report can be downloaded from www.healthcouncil.nl.
Preferred citation:
Health Council of the Netherlands. β-Estradiol. Evaluation of the carcinogenicity
and genotoxicity. The Hague: Health Council of the Netherlands, 2013; publication
no. 2013/23.
all rights reserved
ISBN: 978-90-5549-975-5
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<pre>   Contents
   Samenvatting 9
   Executive summary 11
   Scope 13
.1 Background 13
.2 Committee and procedures 13
.3 Data 14
   General information 15
.1 Identity and physico-chemical properties 15
.2 IARC classification 16
   Carcinogenicity studies 17
.1 Observations in humans 17
.2 Carcinogenicity studies in animals 18
.3 Cell transformation assays 25
.4 Conclusion 25
   Genotoxicity 27
.1 Gene mutation assays 27
.2 Cytogenicity assays 28
   Contents                                    7
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<pre> .3 Miscellaneous 29
 .4 Conclusion 31
    Classification 33
 .1 Evaluation of data on carcinogenesis and genotoxicity 33
 .2 Recommendation for classification 34
 .3 Additional consideration 34
    References 35
    Annexes 41
A   Request for advice 43
B   The Committee 45
C   The submission letter 47
D   Comments on the public review draft 49
E   IARC evaluation and conclusion 51
F   Animal studies 57
G   Carcinogenic classification of substances by the Committee 63
    β-Estradiol
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<pre>Samenvatting
Op verzoek van de minister van Sociale Zaken en Werkgelegenheid evalueert en
beoordeelt de Gezondheidsraad de kankerverwekkende eigenschappen van
stoffen waaraan mensen tijdens de beroepsmatige uitoefening kunnen worden
blootgesteld. In het voorliggende advies neemt de Subcommissie Classificatie
van carcinogene stoffen van de Commissie Gezondheid en beroepsmatige bloot-
stelling aan stoffen van de Raad, die deze evaluatie en beoordeling verricht,
β-oestradiol onder de loep. β-Oestradiol is een natuurlijk vrouwelijk geslachthor-
moon en wordt onder andere gebruikt in anticonceptiemiddelen en hormonale
therapie. Deze evaluatie betreft alleen externe blootstelling aan β-oestradiol.
Op basis van de beschikbare gegevens concludeert de commissie dat β-oestradiol
kankerverwekkend is voor de mens en beveelt zij aan de stof te classificeren in
categorie 1A*. De commissie concludeert dat β-oestradiol werkt via een niet-sto-
chastisch genotoxisch mechanisme, er zijn echter ook aanwijzingen voor een sto-
chastisch genotoxisch mechanisme.
Volgens het classificatiesysteem van de Gezondheidsraad (zie bijlage G).
Samenvatting                                                                       9
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<pre>0 β-Estradiol</pre>

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<pre>Executive summary
At request of the Minister of Social Affairs and Employment, the Health Council
of the Netherlands evaluates and judges the carcinogenic properties of
substances to which workers are occupationally exposed. The evaluation is
performed by the Subcommittee on Classification of Carcinogenic Substances of
the Dutch Expert Committee on Occupational Safety of the Health Council. In
this report, the Committee evaluated β-estradiol. β-Estradiol is a natural female
sex hormone that is used in among others contraceptives and hormonal therapy.
This evaluation only concerns the external exposure to β-estradiol.
Based on the available information, the Committee concludes that β-estradiol
is known to be carcinogenic to man, and recommends to classify the substance
in category 1A*. The Committee concludes furthermore that β-estradiol acts by a
non-stochastic genotoxic mechanism, however, there are also indications for a
stochastic genotoxic mechanism.
According to the classification system of the Health Council (see Annex G).
Executive summary                                                                 11
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<pre>2 β-Estradiol</pre>

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<pre> hapter 1
        Scope
1.1     Background
        In the Netherlands a special policy is in force with respect to occupational use
        and exposure to carcinogenic substances. Regarding this policy, the Minister of
        Social Affairs and Employment has asked the Health Council of the Netherlands
        to evaluate the carcinogenic properties of substances and to propose a
        classification (see Annex A). In addition to classifying substances, the Health
        Council also assesses the genotoxic properties of the substance in question. The
        assessment and proposal for a classification are expressed in the form of standard
        sentences (see Annex G).
        This report contains the evaluation of the carcinogenicity of β-estradiol.
1.2     Committee and procedures
        The evaluation is performed by the Subcommittee on Classification of
        Carcinogenic Substances of the Dutch Expert Committee on Occupational Safety
        of the Health Council, hereafter called the Committee. The members of the
        Committee are listed in Annex B. The submission letter can be found in Annex C.
            In 2013, the President of the Health Council released a draft of the report for
        public review. The individuals and organisations that commented on the draft are
        Scope                                                                               13
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<pre>    listed in Annex D. The Committee has taken these comments into account in
    deciding on the final version of the report.
1.3 Data
    The evaluation and recommendation of the Committee is standardly based
    on scientific data, which are publicly available. The starting points of the
    Committees’ reports are, if possible, the monographs of the International
    Agency for Research on Cancer (IARC). This means that the original sources of
    the studies, which are mentioned in the IARC-monograph, are reviewed only by
    the Committee when these are considered most relevant in assessing the
    carcinogenicity and genotoxicity of the substance in question. In the case of
    β-estradiol, such monograph and several updates exist.1-3 In the 1987 supplement,
    β-estradiol was reviewed as part of a group of steroidal estrogens.3 In two
    subsequent monographs, the carcinogenicity of β-estradiol-containing
    contraceptives and hormonal therapies were evaluated.4,5 The summaries and
    conclusions of the IARC monographs specifically addressing β-estradiol are
    inserted in Annex E.
         As evaluations of 19994 and 20075 did not specifically address β-estradiol
    as such, the literature search for carcinogenic and genotoxic properties of
    β-estradiol was performed starting from 1987.
    More recently published data were retrieved from the online databases Medline,
    Toxline, Chemical Abstracts, and RTECS. The last updated online search was in
    April 2013. The new relevant data were included in this report.
 4  β-Estradiol
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<pre> hapter 2
        General information
2.1     Identity and physico-chemical properties
        β-Estradiol (CAS number 50-28-2) relates to the beta-isomeric form of estradiol,
        17β-estradiol. The α-isomeric form is not addressed in this report. The data have
        been retrieved from the European Substance Information System (ESIS*), and
        the Hazardous Substances Data Bank (HSDB**).
        The relevant physico-chemical properties of β-estradiol are presented below.
        Chemical name              :  Estradiol
        CAS registry number        :  50-28-2
        EINECS number              :  200-023-8
        Synonyms                   :  Dihydrofollicular hormone; dihydrofolliculin; dihydromenformon;
                                      dihydrotheelin; dihydroxyestrin; 3,17β-dihydroxyestra-1,3,5(10)-
                                      triene; 3,17-epidihydroxyestratriene; β-estradiol; 17β-estradiol;
                                      3,17β-estradiol; (D)-3,17β-estradiol; oestradiol-
                                      17β; 17β-oestradiol
        Appearance                 : White or slightly yellow, small crystals or crystalline powder
        Use                        : Medicine (estrogenic hormone)
        ESIS can be accessed via the ECB-site: http://esis.jrc.ec.europa.eu/.
 *      HSDB can be accessed via http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?HSDB.
        General information                                                                             15
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<pre>    Chemical formula         : C18H24O2
    Structural formula
    Molecular weight         : 272.39 g/mol
    Boiling point            : -
    Melting point            : 173 - 179 ˚C
    Vapour pressure          : -
    Vapour density (air = 1) : -
    Solubility               : In water: 60 mg/L at 27 ˚C. Freely soluble in alcohol; soluble in
                               acetone, dioxane, other organic solvents; solutions of fixed alkali
                               hydroxides; sparingly soluble in vegetable oils.
    Conversion factor        : 1 mg/m3 = 0.09 × ppm
    EU Classification        : Not classified
    (100% solution)
2.2 IARC classification
    In 1974, IARC concluded that estrogens for treatment of the menopausal
    syndrome and related conditions had not been shown to be associated with a risk
    of cancer.1 IARC concluded in 1979 and 1987 that there was sufficient evidence
    for carcinogenicity of β-estradiol in experimental animals.2,3 The summaries of
    these IARC evaluations are included in Annex E of this report.
         β-Estradiol is primarily used as a component of oral contraceptives and
    post-menopausal therapies. In 1999, post-menopausal estrogen therapy was
    considered carcinogenic to humans (Group 1) and post-menopausal estrogen-
    progestogen therapy possibly carcinogenic to humans (Group 2B).4 Combined
    estrogen-progestogen menopausal therapy was considered carcinogenic to
    humans (Group 1) in 2007.5 However, for the 1999 and 2007 monographs, no
    specific data on β-estradiol were considered by IARC.
 6  β-Estradiol
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<pre> hapter 3
        Carcinogenicity studies
3.1     Observations in humans
        No human studies on β-estradiol were evaluated by IARC.2,3
        Cohort studies
        A large number of cohort studies demonstrating a relationship between increased
        cancer risk and β-estradiol-containing pharmaceuticals (contraceptives, meno-
        pausal therapy etc.) was included in the IARC evaluations of 1999 and 2007.4,5
        However, in nearly all cases co-exposure to other estrogens and/or progestogens
        occurred, precluding the establishment of the relationship between exposure to
        β-estradiol only and cancer incidence in humans.
        Thomas et al. performed a meta-analysis of 29 epidemiologic papers (covering
        the period of January 1966 to July 1996).6 The criterion for considera-tion was
        that the study reported a point estimate for the measurement of β-estradiol in
        blood (or estrogen in urine) in postmenopausal breast cancer cases (retrospective
        or prospective) and controls. The ratio of the average estrogen concentration in
        the women with breast cancer to that in the women without breast cancer (and its
        95% confidence interval (CI) was calculated for each study, and the results were
        summarised by calculating weighted averages of the log ratios. In six prospective
        studies on serum β-estradiol concentration, 329 women who subsequently
        Carcinogenicity studies                                                           17
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<pre>    developed breast cancer had, overall, a 15% (CI = 6-24%, p = 0.0003) higher
    mean concentration of β-estradiol in their blood than the 1,105 women who
    remained free of cancer. The results of these prospective studies did not differ
    significantly from each other (χ2 for heterogeneity = 8.7; degrees of freedom = 5;
    p > 0.1). Out of 16 case-control studies that compared the serum concentration of
    β-estradiol in breast cancer cases and controls, 7 reported a significantly higher
    mean concentration of β-estradiol than the controls. Over-all, the breast cancer
    cases had a 24% higher mean concentration of β-estradiol than the controls. There
    was, however, significant heterogeneity among the results of these studies
    (χ2 = 57.2, degrees of freedom = 15; p < 0.00001).
    Beral et al.7 reported on the results of the Million Women Study, a cohort study
    conducted between 1996 and 2001 that included 1,084,110 UK women aged
    50-64 years. Half of the women in the study had used hormone-replacement
    therapy, who were compared to never users, after stratification by age, time
    since menopause, parity and age at first birth, family history of breast cancer,
    body-mass index, region, and deprivation index. All current users of hormone-
    replacement therapy at recruitment, but not past users, were more likely to
    develop, or die from invasive breast cancer. Results are summarised for users
    of preparations containing only oestrogen (including β-estradiol). The relative
    risk (RR) of breast cancer incidence was statistically significantly raised for all
    current oestrogen-only users versus all never users (RR = 1.30; 95% CI 1.22-
    1.38, p < 0.0001). This increased risk was dependent on treatment duration with
    the highest risk observed for women with a use of ≥ 10 years (RR = 1.37; 95% CI
    1.22-1.54). Risks varied little between specific estrogens used (equine estrogen
    and ethinylestradiol), and doses applied. The relative risks were statistically
    significantly increased separately for oral, transdermal, and implanted oestrogen-
    only formulations (RR = 1.32; 95% CI 1.21-1.45, RR = 1.24; 95% CI 1.11-1.39,
    and RR = 1.65; 95% CI 1.26-2.16, respectively).
3.2 Carcinogenicity studies in animals
    IARC reported on several animal carcinogenicity studies, e.g. with oral and
    subcutaneous administration, in various species.
        Mammary, pituitary, uterine, cervical, vaginal, testicular, lymphoid and bone
    tumours were observed in mice. Also in rats, β-estradiol induced mammary and/
    or pituitary tumours. In hamsters malignant kidney tumours were noted and in
    guinea pigs, premalignant lesions were reported.3 These studies are summarised
    in Annex F.
 8  β-Estradiol
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<pre>    No inhalation studies are available.
Oral administration
Studies with mice
Several studies on the tumourigenic activity of β-estradiol have been conducted
as part of the International Life Sciences Institute’s (ILSI)/Health and Environ-
mental Science Institute Alternatives to Carcinogenicity Testing (ACT) project).
McClain et al. reported the results of a neonatal mouse model in two oral gavage
studies using CD-1 mice (following ILSI protocol).8 The neonatal mouse model
is known to be very sensitive for the detection of genotoxic carcinogens, and
irresponsive to chemicals that act via epigenetic mechanisms. The two main
target tissues for this model are considered to be the liver and the lungs. Animals
are treated on days 8 and 15 of age, weaned around 22 days of age and then
maintained until 1 year of age at which time they were sacrificed. Six litters
containing 4 neonates/sex/litter were dosed for each group.
    In one of the oral gavage studies, mice were treated with doses of 1, 2 and 4
mg/kg bw. No dose-dependent increase in adenoma or carcinoma of the liver or
lung was reported. In the other study, after treatment with 2, 3 and 4 mg/kg bw,
positive results were reported. A clear increase was observed in liver tumours in
male mice, particularly carcinomas (5 in male mice, versus 0 in controls) and in
lung tumours (6 lung adenomas in males and 4 in females, versus 0 in controls).
Van Kreijl et al. reported the results of β-estradiol tested in DNA repair deficient
Xpa-/- and Xpa-/- / p53+/- knock-out mice in a C57BL/6 genetic background
(further referred to as XPA and XPA/p53 model).9 Groups of male and female
mice (15 mice/sex/group) received β-estradiol by gavage at dose levels of
0 (control), 1, 2.5 and 5 mg/kg bw/day (XPA group) or 0 (control) and 5 mg/kg
bw/day (XPA/p53 group), 7 days/week for 9 months (39 weeks), after which the
animals were sacrificed and subjected to gross patho-logical and histological
examinations. The observed spontaneous tumour formation in the XPA mice
after 9 months was comparable to that of wild-type mice (total 6%); in the
XPA/p53 mice it was somewhat higher (9% males; 13% females).
    A positive tumour response was observed for β-estradiol in XPA/p53 group,
but not in the XPA group. This conclusion was based on the increased incidence
of osteosarcomas (3 in males receiving 5 mg/kg/bw, compared to none in the
controls). The authors noted that although this increase was not statistically
Carcinogenicity studies                                                              19
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<pre>  significant, the response was considered positive in view of the rare nature of the
  tumour, and the presence of supporting fibro-osseous lesions.
  Also within the ILSI framework, tumourigenicity of β-estradiol was evaluated in
  two 26-week short-term carcinogenicity studies in the p53+/- mice.10 The
  protocol included administration by gavage of three dose levels of β-estradiol
  (0.1, 0.5 and 2.0 mg/kg bw/day and 0.5, 2.0 and 5.0 mg/kg bw/day, in two studies
  respectively), to groups of 15 mice/sex/dose for 26 weeks.
      In the study using dose levels up to 2 mg/kg bw/day no treatment-related
  neoplastic effects were observed in any tissue. In the second study, testing up to a
  2.5-fold higher dose (5.0 mg/kg bw/day) resulted in increased incidences of
  pituitary hyperplasias (in 12 out of 15 mice; 1 case of focal hyperplasia) and a
  single adenoma in the female high-dose mice. Although an incidence of 1/15 for
  adenoma was not statistically significant, if evaluated together with the evidence
  of a hyperplastic response, it was considered an equivocal response. High-dose
  wild-type mice in the same study showed a similar pattern, but at a lower
  incidence, of hyperplastic pituitary lesions (in 5 out of 15 mice).
  β-Estradiol (0.1-5 mg/kg/day) has also been tested in the CB6F1-rasH2 mouse
  model, used in the ILSI project as a model that is responsive to genotoxic
  carcinogens.11 Animals were dosed daily at levels of 0.1, 0.5, 2.0, and 5.0 mg/kg
  β-estradiol in an ethanol/methocel formulation for 6 months, followed by a
  2-week recovery/respite from treatment. Control and high-dose groups with non-
  transgenic mice were included. The response to β-estradiol was reported to be
  negative, both in CB6F1-rasH2 animals and the non-transgenic control group.
  Subcutaneous injection
  Studies with mice
  Martinez et al. studied the response of the inbred BALB/cCrgl newborn female
  mice to neonatal treatment with β-estradiol.12 In mice, neonatal exposure to
  potent natural and synthetic estrogens results in the development of cervico-
  vaginal tumours, some of which resemble tumours in human females exposed to
  DES in utero. Beginning within 16 hours after birth, groups of 35 (control) and
  43 (test group) mice were given 5 daily subcutaneous injections of either 5 µg
  β-estradiol or 20 µL sesame oil. Animals were weaned 21 days of age. All mice
  that survived 20 months of age were sacrificed and subjected to gross
  pathological and histopathological examinations. The only tumour seen in
0 β-Estradiol
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<pre>control mice was 1 lymphoma. The incidence of malignant tumours was
significantly greater in animals exposed to β-estradiol. The incidence of
cervicovaginal tract carcinomas was 43%, and, for any tumour, 49% (some mice
had more than one type of tumour detected). Also two cases of cholangiocar-
cinoma of the gallbladder, 1 case of ovarian granulosa cell tumour, 1 case of
mammary gland carcinoma and 1 case of bronchoalveolar adenoma of the lung
were detected.
The tumourigenicity in mice of β-estradiol was also studied by Newbold and
Liehr.13 The study was conducted to determine the potency of estrogens and
estrogen metabolites to induce uterine adenocarcinoma in CD-1 mice, treated for
the first 5 days of life. Outbred female CD-1 mice were treated with β-estradiol
on days 1-5 of neonatal life (2 µg/pup/day) as subcutaneous injections in corn
oil. Corn oil alone was used as a control. Animals were weaned at 21 days of age
and sacrificed at 12 or 18 months of age.
     One out of 15 mice treated with β-estradiol developed an uterine tumour. No
tumours were observed in controls treated with corn oil.
Fujii reported the results of a newborn mouse tumourigenesis assay (NMTA)
on 45 chemicals, including β-estradiol.14 Forty male and female newborn ICR
mice were injected subcutaneously within 24 hours of birth with 0.67 mg/kg bw
β-estradiol. Control animals received 1% gelatin. The treated animals were
weaned at 1 month, separated by sex and observed for 1 year. Animals were
necropsied completely when moribund or dead.
     All animals died within 30 months. In total 9 males and 2 females developed
tumours (tumour incidence of 39% and 13%, respectively). A statistically
significant increase in the number of liver tumours (4; 17% tumour incidence)
was observed in male rats.
Subcutaneous implantation
Studies with rats
Mense et al. administered 3 mg of β-estradiol (only the total dose was reported)
subcutaneously to female ACI rats in a form of cholesterol pellets (3 mg
β-estradiol and 17 mg cholesterol).15 All animals were divided into 4 subgroups,
containing at least 10 rats each, which underwent their respective treatments for
7, 15, 120 or 240 days. At the end of each of these time periods, animals were
Carcinogenicity studies                                                           21
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<pre>  euthanised and subjected to gross pathological and histopatho-logical
  examination.
      No mammary tumours were observed in 10 control rats, while the incidence
  of mammary tumours in rats treated with β-estradiol was 82% following 240
  days (9 out of 11 rats). The first palpable breast tumour appeared after 128 days.
  The tumours were classified as adenocarcinoma and showed evidence of
  invasion. No significant morphological changes were noted in kidneys, uteri,
  lungs or brains. Treatment with the antioxidant vitamin C reduced both tumour
  incidence and latency, whereas the estrogen metabolic inhibitor α-
  naphthoflavone completely abrogated breast cancer development.
  Singh et al.16 investigated the effects of a phytoestrogen quercetin and its co-
  exposure with β-estradiol on the breast via subcutaneous implantation in female
  ACI rats. Feeding the rats (n = 6) with the quercetin-enriched diet (2.5 g/kg food)
  for 8 months did not induce breast tumours. However, when rats were implanted
  with β-estradiol pellets (3 mg β-estradiol, 17 mg cholesterol; only the total dose
  was reported) and fed the same diet (n = 10), 100% of the animals developed
  breast tumours within 8 months. A separate group of rats was implanted with
  β-estradiol in cholesterol pellets alone (n = 11) and not co-exposed to quercetin.
  Rats in the quercetin + β-estradiol group displayed an increased mammary
  tumour incidence relative to animals from the β-estradiol treatment group, where
  tumour incidence was equal to 82%. However, the increase in tumour incidence
  in the quercetin + β-estradiol group compared to the β-estradiol group was not
  statistically significant. Average tumour latency was significantly shorter for
  animals in the quercetin + estradiol group versus animals in the β-estradiol
  group, indicating that breast tumours appeared earlier in the quercetin + β-
  estradiol group compared to animals exposed to β-estradiol alone. No tumours
  were found in the control group (n = 10), which received the implantation of the
  vehicle alone.
  Inoh et al. studied the carcinogenic effect of 5 mg β-estradiol (implanted
  subcutaneously as a pellet in female W/Fu rats), alone and in combination with
  nitrosobutylurea (NBU).17 NBU alone (250 ppm in drinking water) for 14 days
  did not induce carcinogenic effects. Twelve out of 13 rats treated with β-estradiol
  alone developed mammary tumours (p < 0.005), while pituitary tumours were
  observed in 6 out of 13 animals (p < 0.005). No untreated control group was
  included.
2 β-Estradiol
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<pre>Mills et al. implanted two groups of female August Copenhagen Irish (ACI)
rats (26 and 20 animals, experiments I and II) subcutaneously under the back
skin with a pellet containing 18 µmol of β-estradiol, corresponding to 4.9 mg of
β-estradiol (only the total dose was reported in the paper).18 Two separate groups
of animals (24 for experiment 1, 10 for experiment 2) were used as controls and
implanted with a vehicle alone (cholesterol).
    All 46 rats developed large pituitary tumours within 7 months after the
implantation and the experiment had to be terminated early. Histopathologically,
major changes included the presence of extensive hyperplasia/adenoma, which
were often coupled with multifocal hemorrhage, cystic change, and apoplectic
necrosis. Histopathology suggested that they were essentially pituitary
adenomas. In addition, 48% of all animals developed mammary tumours. In
experiment I with 26 rats, the first palpable mammary tumour was observed at
126 days after the implantation. At the time of death, 9 animals (34.6%) were
found to have one or multiple palpable mammary tumours and 4 other had early-
stage mammary tumours (ductal carcinoma in situ), which brought the final
mammary tumour incidence to 50%. A similar total mammary tumour incidence
(45%) was observed in experiment II.
Turan et al. studied the potential induction of mammary tumours in female ACI
rats (7-8 weeks of age) subcutaneously implanted with cholesterol pellets
containing 1, 2 or 3 mg β-estradiol (only the total doses were reported).19 Female
rats were divided into 2 groups. The animals were implanted with a single 20 mg
pellet containing cholesterol as an inert vehicle and β-estradiol (8 rats/dose). The
rats were palpated for tumours twice weekly and terminated when a mammary
tumour reached approximately 3 cm2 in size, or at 36 weeks.
    A 50% incidence of palpable mammary tumours was observed after 18 weeks
for 3 mg β-estradiol, 19 weeks for 2 mg β-estradiol and 36 weeks for 1 mg
β-estradiol in the first group. At termination after 36 weeks in the first group, the
incidence of palpable mammary tumours was 100% for 3 mg β-estradiol, 73% for
2 mg β-estradiol and 50% for 1 mg β-estradiol. A 100% incidence of mammary
tumours was observed after 24 weeks of treatment with 3 mg β-estradiol. No
tumours were detected in controls.
Studies with hamsters
Li et al. studied the carcinogenic activity of β-estradiol, included in a panel of
synthetic and natural estrogens, in the hamster kidney tumour model.20 This
model is one of the primary experimental systems to evaluate the carcinogenic
Carcinogenicity studies                                                               23
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<pre>  properties of estrogens.21 Adult castrated-male Syrian (LAK:LVG outbred)
  hamsters exposed for approximately 9 months by subcutaneous implantation of
  112 (± 15) mg β-estradiol pellets. Hormone pellets were renewed each 3 months.
  Renal tumour foci were distinguished microscopically. β-Estradiol induced
  bilateral and multiple renal tumours in 6/6 animals, with a combined number of
  tumour nodules in both kidneys of 18.0 ± 3.
  In a study with a similar design, Li et al. studied the carcinogenic activity of
  β-estradiol as a 20 mg pellet.22 The release rates of the pellets were adjusted so
  that mean daily absorptions were ca. 111 ± 11 µg (only the total dose was
  reported) After 9 months treatment, again a 100% kidney tumour incidence was
  observed in animals implanted with β-estradiol.
  Liehr et al. confirmed the positive findings of β-estradiol in the kidney tumour
  model in Syrian hamsters, reporting incidences of 80-90%.23,24
      Zhu and Liehr subsequently studied the influence of co-exposure to
  quercetin, an inhibitor of estradiol-2-hydroxylase, on the tumourigenicity of
  (25 mg) β-estradiol in male Syrian hamsters.25 Whereas quercetin itself did not
  induce tumours, it increased the mean tumour size, the mean number of large
  tumour nodules and the incidence of abdominal metastases of kidney tumours as
  after treatment with β-estradiol.
  Bhat et al. have investigated the role of oxidative stress in estrogen carcino-
  genesis using the hamster renal tumour model.26 The authors implanted groups
  of 10 male Syrian hamsters subcutaneously with 25 mg pellets (of various
  estrogens, including β-estradiol). Hamsters were killed after 7 months and
  inspected macroscopically for tumour nodules. A 90% tumour incidence was
  found in the group treated with β-estradiol.
  Other routes
  Studies with mice
  Mc Clain et al. reported the results of a neonatal mouse model with C56BL/6N
  mice receiving β-estradiol i.p. (following a protocol by National Center for
  Toxicological Research (NCTR)).8 In this study, mice were administrated a total
  dose of 50 or 100 nmol (13.6 or 27.3 µg) β-estradiol. No increased incidence of
  neoplasms in liver or lung was reported.
4 β-Estradiol
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<pre>3.3 Cell transformation assays
    A number of cell transformation studies with β-estradiol have been published
    mainly using female cells, all reporting positive results. The cell transformation
    assays include the assessment of anchorage-independent growth of the
    immortalised but non-transformed human mammary epithelial cell line MCF-
    10A27, phenotypical changes in immortalised human breast epithelial cells MCF-
    10F28-31, microsatellite instability and neoplastic transformation of immortalised
    human endometrial (EM) glandular cells32, anchorage independent growth in
    EM cells32, and cellular transformation and genetic effects in Syrian hamster
    embryo (SHE) cells33.
        Transformation capability has also been observed in mammalian cells from
    male origin. β-Estradiol induced neoplastic transformation of rat prostatic
    epithelial NRP-152 cells, as was demonstrated by the expression of tumour
    markers and their capacity of forming colonies in soft agar and tumors in
    immunodeficient nude mice.34 Transformation induction by β-estradiol, in the
    presence of testosterone, has also been described in a model for prostate
    carcinogenesis with human prostate stem-progenitor cells.35
3.4 Conclusion
    The value of the available human data considers the Committee limited, as in
    virtually all cases, the use of oral contraceptives or hormone-replacement therapy
    involved other estrogens and/or progestogens besides β-estradiol present in the
    preparations. A positive correlation has been found between the mean serum
    concentrations of endogenous β-estradiol in women and the development of
    breast cancer. Furthermore, use of preparations containing estrogen only
    (including β-estradiol) is associated with an increased risk of breast cancer.
        No animal inhalation carcinogenicity studies are available. From the animal
    data on other routes, the Committee concludes that β-estradiol is able to induce
    tumours in rats, mice and hamsters.
    Carcinogenicity studies                                                            25
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<pre>6 β-Estradiol</pre>

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<pre> hapter 4
        Genotoxicity
4.1     Gene mutation assays
        In vitro
        Bacterial gene mutation assays have been consistently negative.36,37
             In mammalian gene mutation assays, positive and negative results have been
        obtained. In several of these studies, V79 cells were applied. Drevon et al. co-
        cultured V79 cells with freshly isolated liver cells and β-estradiol (0, 25, 75 and
        100 µM) for 48 h, before plating and assessment of azaguanine and ouabain
        resistance.38 No increase in mutant colonies was observed.
             Rajah and Pento also used the Chinese hamster lung fibroblast V79 cell line
        to study the mutagenicity of β-estradiol.39 Cells were exposed for 4 days to 0.1, 1
        and 10 nM concentrations of β-estradiol, in the absence of metabolic activation.
        At the higher doses (1 and 10 nM) β-estradiol was not mutagenic at the
        hypoxanthine-guanine phosphoribosyltransferase (hprt) locus of V79 cells, but
        at the lower dose (0.1 nM) it caused a 2-fold enhancement over the control. The
        survival of the cells did not vary between the dose levels.
             Kong et al. found that β-estradiol significantly increased the mutant
        frequency of the hprt gene of Chinese hamster V79 cells at both physiological
        and pharmacological concentrations (2.57-, 3.45-, 2.63- and 8.78-fold, at
        concentrations of 0.01, 0.1 and 100, 1,000 nM, respectively), in the absence of
        metabolic activation.40
        Genotoxicity                                                                        27
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<pre>        Cuendet et al. treated Chinese hamster V79 cells with concentrations of
    10, 100 or 1,000 nM in the absence of a metabolic activation system.27 No
    significant cytotoxicity was observed. At 10 and 100 nM, a concentration-
    dependent increase in the colony formation and a statistically significant
    difference in the colony formation in comparison to the solvent control was
    observed (p < 0.0005).
        Tsutsui et al. allowed Syrian Hamster Embryo (SHE) cells to grow in the
    presence of β-estradiol for an expression time of 4 days, without metabolic
    activation, and plated and incubated cells for 7 days for colony formation.41
    Concentrations of 1, 3 and 10 µg/mL (3.7, 11.0 and 37 µM) β-estradiol did not
    induce the number of mutant colonies.
        In a mouse lymphoma assay with L 5178Y tk +/- cells, equivocal responses
    were reported by Richold.42 β-estradiol was tested at a concentration range of
    25-255 µg/mL, in the presence or absence of S9-mix.
    In vivo
    In their genotoxicity evaluation of estradiol, Dhillon and Dhillon included an
    host-mediated assay (HMA) in mice.43 Animals were treated with 0.1, 1 or
    10 mg/kg bw estradiol (two i.p. injections with a 12-hour interval). DMSO
    (5 ml/kg bw) and 2-aminofluorene (20 mg/kg bw) served as negative and
    positive control, respectively. After 4 hours, 1.5 mL culture of S. typhimurium
    was injected into the tail vein. After an additional 2-hour incubation, mice were
    killed and livers removed, minced, washed and homogenised. The homogenates
    were transferred to agar plates and incubated for 48-hour to allow the growth of
    His+ revertant colonies. No statistically significant increase in mutant colonies
    compared to concurrent controls was observed.
4.2 Cytogenicity assays
    In vitro
    Joosten et al. and Liehr reviewed the genotoxicity data of β-estradiol.36,37 Several
    in vitro (structural) chromosomal aberration tests, both with positive43-47 and
    negative results33,41,48-51 have been reported in these reviews.
        Positive results were reported in the in vitro micronucleus assay.52-54 One in
    vitro sister chromatid exchange (SCE) assay was reported negative50 and one
    positive46. In the review by Joosten et al., also six numerical chromosomal
    aberration tests were reported (both for polyploidy47,48,55 and for
 8  β-Estradiol
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<pre>    aneuploidy33,41,50,51), all showing positive results. In two Comet assays, positive
    results were reported.54,56
    In addition, Eckert and Stopper57 examined the ability of β-estradiol to induce
    micronucleus formation in the Chinese hamster V79 cell line, by exposing cells
    to concentrations up to and including 100 µM for 4 hours in the absence of a
    metabolic activation system. β-Estradiol induced an increase in cells with
    micronuclei in V79 cells in a concentration-dependent manner.
    In vivo
    In the majority of the in vivo micronucleus assays reviewed by Joosten et al.36
    (i.e. two performed in mouse bone marrow cells58,59, one in mouse pheripheral
    lymphocytes60, one in mouse spermatids52, and two in rat bone marrow
    cells58,59), no induction of micronucleated cells was observed. In one bone
    marrow micronucleus assay in mice, positive results were reported.43
         One in vivo chromosomal aberration test in Syrian hamster renal cells was
    reported to be positive.45
4.3 Miscellaneous
    In vitro
    Several unscheduled DNA synthesis (UDS) tests in rat hepatocytes showed
    negative results50,61,62, whereas in an UDS test by Althaus et al., positive results
    were reported.63
    Han and Liehr (cited in Joosten et al.), reported no increase in adducts in Syrian
    hamster DNA incubated with liver microsomes.64
         Yagi et al.65 studied the ability of β-estradiol and metabolites to induce DNA
    adducts in Syrian hamster embryo (SHE) cells using a 32P-post-labeling assay.
    DNA adducts were detected in cells treated with 2- and 4-hydroxyestradiol. In
    contrast, DNA adducts were not detected in SHE cells treated with β-estradiol.
         Van Aswegen et al. examined in vitro genetic toxicity of β-estradiol and its
    catechol metabolites, 2-hydroxy- and 4-hydroxyestradiol, using the Chromotest,
    in the absence of the liver homogenate S9.66 The toxicity of estradiol and the
    catechol-estradiols was determined using the Toxi-Chromotest by measuring the
    activity of β-galactosidase in terms of absorbance. An increase in absorbance at
    low concentrations of estrogens was detected, reaching approximately a 2-fold
    Genotoxicity                                                                         29
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<pre>  increase in absorption at 5 nM β-estradiol and 15 nM 4-hydroxyestradiol, while
  above these concentrations a decrease in enzyme production was observed,
  indicating cell damage due to toxicity.
      Fernandez et al.28,29 tested microsatellite instability and loss of
  heterozygosity of chromosomes 13 and 17 in MCF-10F cells after treatment with
  β-estradiol. MCF-10F cells treated with β-estradiol or 4-hydroxyestradiol alone
  or in combination with antiestrogen ICI182780 exhibited a loss of heterozygosity
  in the region 13q12.3 with the marker D13S893 in comparison to the parental
  cell line. Cells treated with β-estradiol or 4-hydroxyestradiol at doses of 0.007
  and 70 nM and 2-hydroxyestradiol only at a higher dose (3.6 µM) showed a
  complete loss of 1 allele with D13S893. No further microsatellite changes
  were detected with any of the other 24 markers used for chromosome 13. For
  chromosome 17, differences were found using the marker TP53-Dint located in
  exon 4 of the tumour suppressor gene p53. Cells treated with β-estradiol or
  4-hydroxyestradiol at doses of 0.007 nM and 70 nM and 2-hydroxyestradiol at a
  higher dose of 3.6 µM exhibited a 5 bp deletion in p53 exon 4.
      Continuing the work of Fernandez et al.28,29, Russo and co-workers67 demon-
  strated that the treatment of MCF-10F cells with 70 nM β-estradiol resulted in
  loss of genetic material. The first loss detected in β-estradiol treated cells was in
  chromosome 9p11-13. The same loss was maintained in the β-estradiol-treated
  transformed (invasive) cells, in the tumours formed by these cells in SCID mice,
  and in all cell lines derived from these tumours. β-Estradiol treated invasive cells
  also exhibited loss of chromosome 4p, which expanded to the loss of the
  complete chromosome in the tumours derived from these cells as well as in the
  cell lines derived from the tumours. Four additional losses appeared in all the
  tumours and in their derived cells, that included chromosomes 3p12.3-13,
  8p11.1-21, 18q, and 9p21-pter, whereas the loss of chromosome 9p11-13
  observed in previous cell lines was no longer evident. Gains in 1p and 5p15-qter
  were observed in the four tumours formed by β-estradiol treated invasive cells in
  SCID mice and the cell lines derived from them.
  In vivo
  In Joosten et al. several studies have been noted in which adduct formation was
  reported in Syrian hamster liver and kidney in vivo.68-70
  Cavalieri et al.71 treated female Sprague-Dawley rats at each of four mammary
  glands with the metabolites of β-estradiol, catechol estrogene-3,4-quinone and
  4-hydroxyestradiol by intra-mammillary injection (200 nmol). The 4-hydroxy-
0 β-Estradiol
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<pre>    estradiol-1(α,β)-N7Glua depurinating adduct was detected at a level of 2.3 or
    1.4 µmol/mol DNA-phosphate for treatment with the quinone and hydroxy
    derivative, respectively.
4.4 Conclusion
    In several in vitro studies, an ability of β-estradiol to induce gene mutations
    and/or chromosome aberrations or aneuploidy in mammalian cell systems has
    been reported. From the available in vivo genotoxicity studies, there is evidence
    that β-estradiol can induce structural and numerical chromosome aberrations.
    The Committee concludes that β-estradiol is genotoxic.
    Genotoxicity                                                                      31
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<pre>2 β-Estradiol</pre>

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<pre> hapter 5
        Classification
5.1     Evaluation of data on carcinogenesis and genotoxicity
        No human data are available on the carcinogenicity of exclusive exposure to
        β-estradiol. Although a positive correlation has been observed between the mean
        serum concentrations of endogenous β-estradiol in women and the development
        of breast cancer, the Committee cannot establish a direct relationship between
        the exposure to β-estradiol and increased human cancer risk.
            Only animal data on oral and subcutaneous routes of administration are
        available. These studies demonstrate that β-estradiol can induce the development
        of tumours, including mammary, pituitary, kidney and liver tumours in rats, mice
        and hamsters. Although these studies do not comply with the current guidelines,
        the Committee considers that there is sufficient evidence of carcinogenicity of
        β-estradiol in animals.
            The Committee concludes that β-estradiol is genotoxic. In vitro, several
        studies are available that report the ability of β-estradiol to induce gene
        mutations, chromosome aberrations or aneuploidy. From the available in vivo
        genotoxicity studies, there is evidence that β-estradiol can induce structural and
        numerical chromosome aberrations. These observations are in line with a non-
        stochastic genotoxic mechanism. The weak mutagenic effects observed in vitro
        however, also indicate a stochastic genotoxic mechanism.
            The Working Group of IARC classified steroidal estrogens, oral contracep-
        tives (combined) and post-menopausal estrogen therapy as carcinogenic to
        Classification                                                                     33
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<pre>    humans (Group 1) and post-menopausal oestrogen-progestogen therapy as
    possibly carcinogenic to humans (Group 2B), in one or more of its evaluations
    published in 19873, 19994 and 20075. In all cases, β-estradiol was listed as one of
    the substances used in these preparations.
        Based on the available data on carcinogenicity and mechanism of action of
    β-estradiol, and the available data on hormonal therapies including β-estradiol,
    the Committee considers β-estradiol to be carcinogenic in humans.
5.2 Recommendation for classification
    Based on the available information, the Committee concludes that β-estradiol
    is known to be carcinogenic to man, and recommends to classify the substance
    in category 1A*. The Committee concludes furthermore that besides a non-
    stochastic genotoxic mechanism, there are also indications for a stochastic
    genotoxic mechanism.
5.3 Additional consideration
    The Committee notes that β-estradiol is an essential endogeneous hormone,
    which should be taken into account when assessing the risk of external exposure
    to β-estradiol.
    According to the classification system of the Health Council (see Annex G).
 4  β-Estradiol
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  References                                                                                              37
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<pre>4 Ahmad ME, Shadab GG, Hoda A, Afzal M. Genotoxic effects of estradiol-17beta on human
  lymphocyte chromosomes. Mutat Res 2000; 466(1): 109-115.
5 Banerjee SK, Banerjee S, Li SA, Li JJ. Induction of chromosome aberrations in Syrian hamster renal
  cortical cells by various estrogens. Mutat Res 1994; 311(2): 191-197.
6 Kochhar TS. Inducibility of chromosome aberrations by steroid hormones in cultured Chinese
  hamster ovary cells. Toxicol Lett 1985; 29(2-3): 201-206.
7 Sato Y, Sakakibara Y, Oda T, izu-Yokota E, Ichinoseki K. Effect of estradiol and ethynylestradiol on
  microtubule distribution in Chinese hamster V79 cells. Chem Pharm Bull (Tokyo ) 1992; 40(1): 182-
  184.
8 Banduhn N, Obe G. Mutagenicity of methyl 2-benzimidazolecarbamate, diethylstilbestrol and
  estradiol: structural chromosomal aberrations, sister-chromatid exchanges, C-mitoses, polyploidies
  and micronuclei. Mutat Res 1985; 156(3): 199-218.
9 Stenchever MA, Jarvis JA, Kreger NK. Effect of selected estrogens and progestins on human
  chromosomes in vitro. Obstet Gynecol 1969; 34(2): 249-251.
0 Tsutsui T, Suzuki N, Fukuda S, Sato M, Maizumi H, McLachlan JA et al. 17beta-Estradiol-induced
  cell transformation and aneuploidy of Syrian hamster embryo cells in culture. Carcinogenesis 1987;
  8(11): 1715-1719.
1 Tsutsui T, Tamura Y, Hagiwara M, Miyachi T, Hikiba H, Kubo C et al. Induction of mammalian cell
  transformation and genotoxicity by 2-methoxyestradiol, an endogenous metabolite of estrogen.
  Carcinogenesis 2000; 21(4): 735-740.
2 Pylkkänen L, Jahnukainen K, Parvinen M, Santti R. Testicular toxicity and mutagenicity of steroidal
  and non-steroidal estrogens in the male mouse. Mutat Res 1991; 261(3): 181-191.
3 Schnitzler R, Foth J, Degen GH, Metzler M. Induction of micronuclei by stilbene-type and steroidal
  estrogens in Syrian hamster embryo and ovine seminal vesicle cells in vitro. Mutat Res 1994; 311(1):
  84-93.
4 Yared E, McMillan TJ, Martin FL. Genotoxic effects of oestrogens in breast cells detected by the
  micronucleus assay and the Comet assay. Mutagenesis 2002; 17(4): 345-352.
5 Schuler M, Hasegawa L, Parks R, Metzler M, Eastmond DA. Dose-response studies of the induction
  of hyperdiploidy and polyploidy by diethylstilbestrol and 17beta-estradiol in cultured human
  lymphocytes using multicolor fluorescence in situ hybridization. Environ Mol Mutagen 1998; 31(3):
  263-273.
6 Anderson D, Dobrzynska MM, Basaran N. Effect of various genotoxins and reproductive toxins in
  human lymphocytes and sperm in the Comet assay. Teratog Carcinog Mutagen 1997; 17(1): 29-43.
7 Eckert I, Stopper H. Genotoxic effects induced by beta-oestradiol in vitro. Toxicol In Vitro 1996;
  10(5): 637-642.
8 Ashby J, Fletcher K, Williams C, Odum J, Tinwell H. Lack of activity of estradiol in rodent bone
  marrow micronucleus assays. Mutat Res 1997; 395(1): 83-88.
9 Shelby MD, Tice RR, Witt KL. 17-beta-estradiol fails to induce micronuclei in the bone marrow cells
  of rodents. Mutat Res 1997; 395(1): 89-90.
8 β-Estradiol
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<pre>0 Morita T, Asano N, Awogi T, Sasaki YF, Sato S, Shimada H et al. Evaluation of the rodent
  micronucleus assay in the screening of IARC carcinogens (groups 1, 2A and 2B) the summary report
  of the 6th collaborative study by CSGMT/JEMS MMS. Collaborative Study of the Micronucleus
  Group Test. Mammalian Mutagenicity Study Group. Mutat Res 1997; 389(1): 3-122.
1 Blakey DC, White IN. Unscheduled DNA synthesis caused by norethindrone and related
  contraceptive steroids in short-term male rat hepatocyte cultures. Carcinogenesis 1985; 6(8): 1201-
  1205.
2 Oshiro Y, Balwierz PS, Piper CE. Absence of a genotoxic response from steroids in the rat primary
  hepatocyte unscheduled DNA synthesis assay. Environ Mutagen 1986; 8(3): 461-465.
3 Althaus FR, Lawrence SD, Sattler GL, Longfellow DG, Pitot HC. Chemical quantification of
  unscheduled DNA synthesis in cultured hepatocytes as an assay for the rapid screening of potential
  chemical carcinogens. Cancer Res 1982; 42(8): 3010-3015.
4 Han X, Liehr JG. Microsome-mediated 8-hydroxylation of guanine bases of DNA by steroid
  estrogens: correlation of DNA damage by free radicals with metabolic activation to quinones.
  Carcinogenesis 1995; 16(10): 2571-2574.
5 Yagi E, Barrett JC, Tsutsui T. The ability of four catechol estrogens of 17beta-estradiol and estrone to
  induce DNA adducts in Syrian hamster embryo fibroblasts. Carcinogenesis 2001; 22(9): 1505-1510.
6 Aswegen CH van, Nieuwoudt LB, van Rensburg HG, Steyn PL, du Plessis DJ. Estradiol and
  catecholestradiols as possible genotoxic carcinogens. Clin Physiol Biochem 1989; 7(1): 34-39.
7 Russo J, Fernandez SV, Russo PA, Fernbaugh R, Sheriff FS, Lareef HM et al. 17-Beta-estradiol
  induces transformation and tumorigenesis in human breast epithelial cells. FASEB J 2006; 20(10):
  1622-1634.
8 Han X, Liehr JG. 8-Hydroxylation of guanine bases in kidney and liver DNA of hamsters treated with
  estradiol: role of free radicals in estrogen-induced carcinogenesis. Cancer Res 1994; 54(21): 5515-
  5517.
9 Han X, Liehr JG. DNA single-strand breaks in kidneys of Syrian hamsters treated with steroidal
  estrogens: hormone-induced free radical damage preceding renal malignancy. Carcinogenesis 1994;
  15(5): 997-1000.
0 Liehr JG, Hall ER, Avitts TA, Randerath E, Randerath K. Localization of estrogen-induced DNA
  adducts and cytochrome P-450 activity at the site of renal carcinogenesis in the hamster kidney.
  Cancer Res 1987; 47(8): 2156-2159.
1 Cavalieri EL, Stack DE, Devanesan PD, Todorovic R, Dwivedy I, Higginbotham S et al. Molecular
  origin of cancer: catechol estrogen-3,4-quinones as endogenous tumor initiators. Proc Natl Acad Sci
  U S A 1997; 94(20): 10937-10942.
2 Health Council of the Netherlands. Guideline to the classification of carcinogenic compounds. The
  Hague: Health Council of the Netherlands, 2010; publication no. A10/07E.
  References                                                                                               39
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<pre>A Request for advice
B The Committee
C The submission letter
D Comments on the public review draft
E IARC Monograph
F Animal studies
G Classification of substances with respect to carcinogenicity
  Annexes
                                                               41
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<pre>2 β-Estradiol</pre>

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<pre>nnex A
     Request for advice
     In a letter dated October 11, 1993, ref DGA/G/TOS/93/07732A, to, the State
     Secretary of Welfare, Health and Cultural Affairs, the Minister of Social Affairs
     and Employment wrote:
     Some time ago a policy proposal has been formulated, as part of the simplification of the
     governmental advisory structure, to improve the integration of the development of recommendations
     for health based occupation standards and the development of comparable standards for the general
     population. A consequence of this policy proposal is the initiative to transfer the activities of the
     Dutch Expert Committee on Occupational Standards (DECOS) to the Health Council. DECOS has
     been established by ministerial decree of 2 June 1976. Its primary task is to recommend health based
     occupational exposure limits as the first step in the process of establishing Maximal Accepted
     Concentrations (MAC-values) for substances at the work place.
     In an addendum, the Minister detailed his request to the Health Council as
     follows:
     The Health Council should advice the Minister of Social Affairs and Employment on the hygienic
     aspects of his policy to protect workers against exposure to chemicals. Primarily, the Council should
     report on health based recommended exposure limits as a basis for (regulatory) exposure limits for air
     quality at the work place. This implies:
     •    A scientific evaluation of all relevant data on the health effects of exposure to substances using a
          criteria-document that will be made available to the Health Council as part of a specific request
     Request for advice                                                                                        43
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<pre>      for advice. If possible this evaluation should lead to a health based recommended exposure limit,
      or, in the case of genotoxic carcinogens, a ‘exposure versus tumour incidence range’ and a
      calculated concentration in air corresponding with reference tumour incidences of 10-4 and 10-6
      per year.
  •   The evaluation of documents review the basis of occupational exposure limits that have been
      recently established in other countries.
  •   Recommending classifications for substances as part of the occupational hygiene policy of the
      government. In any case this regards the list of carcinogenic substances, for which the
      classification criteria of the Directive of the European Communities of 27 June 1967 (67/548/
      EEG) are used.
  •   Reporting on other subjects that will be specified at a later date.
  In his letter of 14 December 1993, ref U 6102/WP/MK/459, to the Minister of
  Social Affairs and Employment the President of the Health Council agreed to
  establish DECOS as a Committee of the Health Council. The membership of the
  Committee is given in Annex B.
4 β-Estradiol
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<pre>nnex B
     The Committee
     •  R.A. Woutersen, chairman
        Toxicologic Pathologist, TNO Quality of Life, Zeist, and Professor of
        Translational Toxicology, Wageningen University and Research Centre,
        Wageningen
     •  J. van Benthem
        Genetic Toxicologist, National Institute for Public Health and the
        Environment, Bilthoven
     •  P.J. Boogaard
        Toxicologist, SHELL International BV, The Hague
     •  G.J. Mulder
        Emeritus Professor of Toxicology, Leiden University, Leiden
     •  Ms. M.J.M. Nivard
        Molecular Biologist and Genetic Toxicologist, Leiden University Medical
        Center, Leiden
     •  G.M.H. Swaen
        Epidemiologist, Dow Chemical NV, Terneuzen (until April 1, 2013);
        Exponent, Menlo Park, United States (from August 15, 2013)
     •  E.J.J. van Zoelen
        Professor of Cell Biology, Radboud University Nijmegen, Nijmegen
     •  S.R. Vink, scientific secretary
        Health Council of the Netherlands, The Hague
     The Committee                                                              45
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<pre>  The Health Council and interests
  Members of Health Council Committees are appointed in a personal capacity
  because of their special expertise in the matters to be addressed. Nonetheless, it
  is precisely because of this expertise that they may also have interests. This in
  itself does not necessarily present an obstacle for membership of a Health
  Council Committee. Transparency regarding possible conflicts of interest is
  nonetheless important, both for the chairperson and members of a Committee
  and for the President of the Health Council. On being invited to join a
  Committee, members are asked to submit a form detailing the functions they
  hold and any other material and immaterial interests which could be relevant for
  the Committee’s work. It is the responsibility of the President of the Health
  Council to assess whether the interests indicated constitute grounds for non-
  appointment. An advisorship will then sometimes make it possible to exploit the
  expertise of the specialist involved. During the inaugural meeting the
  declarations issued are discussed, so that all members of the Committee are
  aware of each other’s possible interests.
6 β-Estradiol
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<pre>nnex C
     The submission letter
     Subject            : Submission of the advisory report β-Estradiol
     Uw kenmerk         : DGV/BMO-U-932542
     Ons kenmerk        : U-7912/SV/fs/246-A19
     Enclosed           :1
     Date               : October 18, 2013
     Dear Minister,
     I hereby submit the advisory report on the effects of occupational exposure to
     β-estradiol.
     This advisory report is part of an extensive series in which carcinogenic
     substances are classified in accordance with European Union guidelines. This
     involves substances to which people can be exposed while pursuing their
     occupation.
     The advisory report was prepared by the Subcommittee on the Classification of
     Carcinogenic Substances, a permanent subcommittee of the Health Council’s
     Dutch Expert Committee on Occupational Safety. The advisory report has been
     The submission letter                                                          47
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<pre>  assessed by the Health Council’s Standing Committee on Health and the
  Environment.
  I have today sent copies of this advisory report to the State Secretary of
  Infrastructure and the Environment and to the Minister of Health, Welfare and
  Sport, for their consideration.
  Yours sincerely,
  (signed)
  Professor W.A. van Gool,
  President
8 β-Estradiol
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<pre>nnex D
     Comments on the public review draft
     A draft of the present report was released in 2013 for public review. The
     following organisations and persons have commented on the draft document:
     •   Mr. T.J. Lentz, National Institute for Occupational Safety and Health, USA.
     Comments on the public review draft                                             49
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<pre>nnex E
     IARC evaluation and conclusion
     OESTRADIOL-17b
     VOL.: 6 (1974) (p. 99)
     Summary of Data Reported and Evaluation
     (N.B.: This section should be read in conjunction with the section ‘General
     Conclusions on Hormones’.)
     5.1 Animal carcinogenicity data
     Oestradiol-17b was tested in mice, rats, hamsters and guinea-pigs by
     subcutaneous injection or implantation. Its administration resulted in an
     increased incidence of mammary, pituitary, uterine, cervical, vaginal and
     lymphoid tumours and interstitial-cell tumours of the testis in mice. In rats, there
     was an increased incidence of mammary and pituitary tumours. In hamsters,
     malignant kidney tumours occurred with a high incidence in intact or castrated
     males and in ovariectomized but not in intact females. In guinea-pigs, diffuse
     fibromyomatous abdominal lesions of uncertain histological interpretation were
     observed. Subcutaneous injections in neonatal mice resulted in precancerous and
     cancerous vaginal lesions in later life.
     IARC evaluation and conclusion                                                       51
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<pre>  The studies in monkeys could not be assessed since they were limited in group
  size and duration.
      Oestradiol-17b treatment increased the incidence of mammary and pituitary
  tumours in strains of mice having a spontaneous incidence of these tumours. The
  spontaneous occurrence can be related either to the presence of a virus or to a
  particular genetic susceptibility. No evidence of the possible role of a virus has
  been ascertained for rats.
      The role of the hormonal balance in the development and persistence of these
  tumours and possible synergisms with other carcinogenic factors in increasing
  the incidence of lymphoid tumours has been discussed (see section, "General
  Remarks on the Sex Hormones", in this volume).
  5.2 Human carcinogenicity data
  No case reports or epidemiological studies were available to the Working Group.
  Epidemiological studies on steroid hormones used in oestrogen treatment have
  been summarized in the section, "Oestrogens and Progestins in Relation to
  Human Cancer" in this volume.
  Subsequent evaluations: Vol. 21 (1979); Suppl. 7 (1987)
  OESTRADIOL-17b, OESTRADIOL 3-BENZOATE, OESTRADIOL
  DIPROPIONATE, OESTRADIOL-17b-VALERATE AND
  POLYOESTRADIOL PHOSPHATE
  VOL.: 21 (1979) (p. 279)
  5. Summary of Data Reported and Evaluation
  (N.B. - This section should be read in conjunction with the General Remarks on
  Sex Hormones and with the General Conclusions on Sex Hormones.)
  5.1 Experimental data
  Oestradiol-17b and its esters were tested in mice, rats, hamsters, guinea-pigs and
  monkeys by subcutaneous injection or implantation and in mice by oral
  administration. Its subcutaneous administration resulted in increased incidences
  of mammary, pituitary, uterine, cervical, vaginal and lymphoid tumours and
  interstitial-cell tumours of the testis in mice. In rats, there was an increased
2 β-Estradiol
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<pre>incidence of mammary and/or pituitary tumours. In hamsters, a high incidence
of malignant kidney tumours occurred in intact and castrated males and in
ovariectomized females, but not in intact females. In guinea-pigs, diffuse
fibromyomatous uterine and abdominal lesions were observed. Oral adminis-
tration of oestradiol-17b in mice led to an increased mammary tumour incidence.
Subcutaneous injections in neonatal mice resulted in precancerous and cancerous
cervical and vaginal lesions in later life and an increased incidence of mammary
tumours.
     Oestradiol-17b has teratogenic actions on the genital tract and possibly on
other organs and impairs fertility.
5.2 Human data
No case reports or epidemiological studies on oestradiol-17b alone were
available to the Working Group Epidemiological studies on steroid hormones
used in oestrogen-progestin oral contraceptive preparations have been
summarized in the section, 'Oestrogens and Progestins in Relation to Human
Cancer'.
5.3 Evaluation
There is sufficient evidence for the carcinogenicity of oestradiol-17b in
experimental animals. In the absence of adequate data in humans, it is
reasonable, for practical purposes, to regard oestradiol-17b as if it presented a
carcinogenic risk to humans. Studies in humans strongly suggest that the
administration of oestrogens is causally related to an increased incidence of
endometrial carcinoma; there is no evidence that oestradiol-17b is different from
other oestrogens in this respect.
     For definition of the italicized terms, see Preamble Evaluation.
Previous evaluation: Vol. 6 (1974)
Subsequent evaluation: Suppl. 7 (1987) (Steroidal oestrogens)
Excerpt from Volume: Supplement 7 (1987) (p. 280)
IARC evaluation and conclusion                                                    53
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<pre>  STEROIDAL OESTROGENS (Group 1*)
  Oestradiol-17b and esters
  A. Evidence for carcinogenicity to animals (sufficient)
  Oestradiol-17b and its esters were tested in mice, rats, hamsters and guinea-pigs
  by oral and subcutaneous administration. Administration to mice increased the
  incidences of mammary, pituitary, uterine, cervical, vaginal, testicular, lymphoid
  and bone tumours [ref: 1-5]. In rats, there was an increased incidence of
  mammary and/or pituitary tumours [ref: 1,6]. Oestradiol-17b produced a
  nonstatistically significant increase in the incidence of foci of altered hepatocytes
  and hepatic nodules induced by partial hepatectomy and administration of N-
  nitrosodiethylamine in rats [ref: 7]. In hamsters, a high incidence of malignant
  kidney tumours occurred in intact and castrated males [ref: 1,8-10] and in
  ovariectomized females, but not in intact females [ref: 1]. In guinea-pigs, diffuse
  fibromyomatous uterine and abdominal lesions were observed [ref: 1].
  B. Other relevant data
  No data were available on the genetic and related effects of oestradiol-17b in
  humans.
      Oestradiol-17b did not induce chromosomal aberrations in bone-marrow
  cells of mice treated in vivo. Unusual nucleotides were found in kidney DNA of
  treated hamsters. It induced micronuclei but not aneuploidy, chromosomal
  aberrations or sister chromatid exchanges in human cells in vitro. In rodent cells
  in vitro, it induced aneuploidy and unscheduled DNA synthesis but was not
  mutagenic and did not induce DNA strand breaks or sister chromatid exchanges.
  Oestradiol-17b was not mutagenic to bacteria [ref: 11].
  This evaluation applies to the group of chemicals as a whole and not necessarily to ail individual
  chemicals within the group.
4 β-Estradiol
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<pre>  References
  IARC Monographs, 21, 279-326, 1979.
  Huseby, R.A. (1980) Demonstration of a direct carcinogenic effect of estradiol on Leydig cells of the
  mouse. Cancer Res., 40, 1006-1013.
  Highman, B., Roth, S.I. & Greenman, D.L. (1981) Osseous changes and osteosarcomas in mice
  continuously fed diets containing diethylstilbestrol or 17b-estradiol. J. natl Cancer Inst., 67, 653-662.
  Highman, B., Greenman, D.L., Norvell, M.J., Farmer, J. & Shellenberger, T.E. (1980) Neoplastic and
  preneoplastic lesions induced in female C3H mice by diets containing diethylstilbestrol or 17b-
  estradiol. J. environ. Pathol. Toxicol., 4, 81-95.
  Nagasawa, H., Mori, T. & Nakajima, Y. (1980) Long-term effects of progesterone or
  diethylstilbestrol with or without estrogen after maturity on mammary tumorigenesis in mice. Eur. J.
  Cancer, 16, 1583-1589.
  Inoh, A., Kamiya, K., Fujii, Y. & Yokoro, K. (1985) Protective effects of progesterone and tamoxifen
  in estrogen-induced mammary carcinogenesis in ovariectomized W/FU rats. Jpn. J. Cancer Res.
  (Gann), 76, 699-704.
  Yager, J.D., Campbell, H.A., Longnecker, D.S., Roebuck, B.D. & Benoit, M.C. (1984) Enhancement
  of hepatocarcinogenesis in female rats by ethinyl estradiol and mestranol but not estradiol. Cancer
  Res., 44, 3862-3869.
  Li, J.J., Li, S.A., Klicka, J.K., Parsons, J.A. & Lam, L.K.T. (1983) Relative carcinogenic activity of
  various synthetic and natural estrogens in the Syrian hamster kidney. Cancer Res., 43, 5200-5204.
  Li, J.J. & Li, S.A. (1984) Estrogen-induced tumorigenesis in hamsters: roles for hormonal and
  carcinogenic activities. Arch. Toxicol., 55, 110-118 .
0 Liehr, J.G., Stancel, G.M., Chorich, L.P., Bousfield, G.R. & Ulubelen, A.A. (1986) Hormonal
  carcinogenesis: separation of estrogenicity from carcinogenicity. Chem.-biol. Interactions, 59,
  173-184 .
1 IARC Monographs, Suppl. 6, 437-439, 1987.
  IARC evaluation and conclusion                                                                            55
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<pre> nnex        F
             Animal studies
             Animal carcinogenicity data reported by IARC.2
Species, strain (sex) Dose              Exposure duration/ Carcinogenic effects              References cited in
                                        frequency          Other                             IARC
Oral administration
Mouse, C3H/HeJ        0.5 mg/L          19 months          Mammary tumours                   Welsch et al. (1977)
(MTV+), (females)     in drinking water                    (27/99 compared with 11/100 in
N=100                                                      controls)
Mouse, C3H/HeJ,       0, 100, 1,000 and 24 months          Mammary adenomas:                 Highman et al.
(MTV+),(females)      5,000 µg/kg diet                     4/47 (controls)                   (1977)
N=48                                                       0/35 (100 µg/kg)
                                                           6/36 (1,000 µg/kg)
                                                           8/48 (5,000 µg/kg)
                                                           Other tumours:
                                                           1 adenocarcinoma of the cervix
                                                           (100 µg/kg)
                                                           1 osteosarcoma of the cranium-
                                                           (100 µg/kg)
                                                           2 adenomacarcinomas of the uterus
                                                           (5,000 µg/kg)
                                                           3 adenocarcinomas of the cervix
                                                           (5,000 µg/kg)
                                                           1 adenoacanthoma of the uterus
                                                           (5,000 µg/kg)
                                                           None other in controls
             Animal studies                                                                                   57
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<pre>Subcutaneous and/or intramuscular administration
Mouse, Marsh-        80 µg s.c. or i.m.    Twice weekly     Mammary tumours:                      Bischoff et al.
Buffalo, (MTV+)      (total dose 3.3-4.2   for 6 months     No increased incidence in intact and  (1942)
(females)            mg)                                    ovariectomized animals compared
N=40                                                        to controls.
                                                            Lymphosarcomas:
                                                            • 28% (intact animals)
                                                            • 47% (ovariectomised animals)
                                                            • 10% (controls)
Mouse, Marsh-                                               Lymphoid tumours:
Buffalo, (males)                                            • 8% (intact animals)
N=36-43                                                     • 34% (castrated animals)
                                                            • 5% (controls)
Mouse, 7 strains,                          Once a week s.c. β-estradiol resulted in a greater     Gardner et al.
N=822 (all strains)                        for 10 weeks     incidence of lymphoid tumours         (1944)
                                                            compared with other oestrogens
                                                            tested (further not specified)
Subcutaneous implantation
Mouse,               0.5-1 mg in paraffin                   Mammary tumours:                      Rudali et al. (1971)
(C3HxRIII)F1                                                • 15/16 (implant at 10 days)
(MTV+) (castrated                                           • 18/18 (implant at 70 days)
males)                                                      • 7/41 (castrated controls)
Mouse, BALB/C        5 mg in cholesterol   Every 3-4 months Precancerous lesions:                 Munos (1973)
(females)            mixture               for 20 months    • 2/17
N=20                                                        Squamous cell carcinoma of the
                                                            cervix/vagina:
                                                            • 5/17
                                                            No cervical or vaginal lesions were
                                                            observed in the 16 surviving controls
Mouse,               0.64-0.85 mg in                        Mammary tumours:                      Rudali et al. (1971)
(C3HxRIII)F1         paraffin                               • 15/16 females
(MTV+)                                                      • 15/16 males (castrated)
(both sexes)                                                • 28/34 female controls
                                                            • 10/61 male controls
Mouse,               0, 1, 2.5, 5, 10, and                  Mammary tumours:
(C3HxRIII)F1         100 µg in paraffin                     • 11/33, 11/31, 23/27, 24/27,
(castrated males)                                               27/2 and 23/24 (increasing doses)
 8           β-Estradiol
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<pre>Rats,               Two pellets of   At 4 weeks and 1-3 Mammary tumours (adenocarcinomas, Gillman and Gilbert
several species     5-6 mg           months of age       papillary carcinomas, aplastic          (1955)
(females):                                               carcinomas):
Wistar albino                                            10/27
Albino of the Royal                                      2/38
Cancer Hospital
strain
Hooded rats                                              6/19
originally derived
from MRC
                                                         No equivalent numbers were observed
                                                         in breeding or control rats
Hamster             20 mg            One or more pellets Renal tumours                           Kirkman (1959)
(males and females)                  every 21 weeks      15/15 (intact males)
                                                         12/12 (castrated males)
                                                         10/16 ovariectomised females)
                                                         0/6 (intact females)
                                                         0/145 (intact controls; either sex)
                                                         0/72 (castrated controls)
Guinea-pig          20 or 50 mg                          Fibromyomas in the uterine corpus,      Lipschütz and
(females)                                                mesentery and other abdominal sites,    Vargas (1939)
                                                         3 months after ovariectomy
Monkey, rhesus      575-825 mg       At intervals of 5-6 No tumours were found                   Engle et al. (1943)
(females)                            weeks, for 24-28
N=5                                  months
Monkey, Capuchin    250-700 µg/day   29-145 weeks        A high degree of cystic and polypous    Iglesias and
(females)           (total amount of                     glandular hyperplasia of the uterine    Lipschütz (1947)
N=5                 oestrogen; not                       mucosa developed in all animals
                    specified for
                    estradiol
                    dipropionate/
                    β-estradiol)
Neonatal exposure
Mouse, MTV+         NS               NS                  Increased tumourigenesis                Bern et al. (1975,
                                                                                                 1976); Mori
                                                                                                 (1968a,b)
Mouse, C3H/MS       NS               NS                  Hyperplastic nodules or metaplastic     Mori (1967)
(males)                                                  lesions in various accessory sex organs
             Animal studies                                                                                       59
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<pre>Mouse, A/Crgl;-    5 µg            5 consecutive days Hyperplastic and epidermoid vaginal   Takasugi and Bern
BALB/cCrgl;                        after birth        lesions, mostly with vaginal          (1964)
C57BL/Crgl; RIII/                                     concretions:
Crgl; C3H/Crgl                                        • 16/23 (A/Crgl);
(females)                                             • 6/14 (BALB/cCrgl);
                                                      • 4/16 (C57BL/cCrgl);
                                                      • 3/15 (RIII/Crgl)
                                                      • 0/6 (C3H/Crgl)
                                                      • 1/5 (C57BL/Crgl controls)
Mouse, BALB/cCrgl 25, 5 and 0.1 µg 5 consecutive days Persistent vaginal cornification:     Kimura and Nandi
(females)                          after birth        100% in 25, 5 µg group;               (1967)
                                                      37/42 in 0.1 µg group;
                                                      Vaginal epithelial downgrowths:
                                                      • 16/16 (25 µg; intact)
                                                      • 11/11 (5 µg; intact)
                                                      • 16/19 (0.1 µg; intact)
                                                      • 15/16 (25 µg; ovariectomised)
                                                      • 5/10 (5 µg; ovariectomised)
                                                      • 0/9 (0.1 µg; ovariectomised)
                                                      • 5/10 (intact controls)
                                                      Hyperplastic vaginal lesions:
                                                      • 19/27 (25 or 5 µg; intact)
                                                      • 8/26 (25 or 5 µg; ovariectomised)
                                                      • 3/19 (0.1 µg; intact)
                                                      • 0/9 (0.1 µg; ovariectomised)
                                                      • 0/10 (intact control)
                                                      Increased ovarian weight was observed
                                                      in intact animals treated with
                                                      β-estradiol
Mouse, BALB/       5 and 20 µg     5 consecutive days Mammary tumours:                      Mori (1976)
cfC3H (MTV+),                                         • 8/35 (5 µg)
BALB/c (MTV-),                                        • 32/64 (20 µg)
C57BL (MTV-)                                          • 0/40 (controls)
(females)
Mouse, BALB/       5 and 20 µg     5 consecutive days Mammary adenocarcinoma:               Jones and Bern
cfC3H                                                 • 17/19 (5 µg)                        (1977)
                                                      • 20/32 (5 µg + progesterone)
                                                      • 4/11 (20 µg)
                                                      • 33/44 (20 µg + progesterone)
                                                      • 5/17 (controls)
Mouse, BALB/c      40 µg           5 consecutive days More and different types of mammary Warner and Warner
(MTV-) (females)                   after birth        dysplasias compared to controls given (1975)
                                                      DMBA later in life.
 0         β-Estradiol
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<pre>Rat,             10-40 µg 30 consecutive   Treatment with β-estradiol completely Nagasawa et al.
Sprague-Dawley            days after birth inhibited DMBA-induced mammary (1974)
(females)                                  tumourigenesis (none vs 15/27 in
                                           DMBA controls), but did lead to
                                           complete mammary gland regression
Rat,             100 µg   Single dose      Treatment with β-estradiol did        Yoshida and
Sprague-Dawley                             not influence the incidence of        Fukunishi (1977)
                                           DMBA-induced ear-duct tumours
Rat,             100 µg   Single dose      DMBA-induced mammary dysplasia Yoshida and
Sprague-Dawley                             was significantly accelerated in rats Fukunishi (1978)
                                           given β-estradiol; mammary
                                           carcinoma incidence was significantly
                                           reduced
          Animal studies                                                                         61
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<pre>2 β-Estradiol</pre>

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<pre> nnex        G
             Carcinogenic classification of
             substances by the Committee
             The Committee expresses its conclusions in the form of standard phrases:
 ategory      Judgement of the committee (GRGHS)                                    Comparable with EU Category
                                                                                    67/548/EEC         EC No 1272/2008
                                                                                    (before            (as from
                                                                                    12/16/2008         12/16/2008
A             The compound is known to be carcinogenic to humans.                   1                  1A
              • It acts by a stochastic genotoxic mechanism.
              • It acts by a non-stochastic genotoxic mechanism.
              • It acts by a non-genotoxic mechanism.
              • Its potential genotoxicity has been insufficiently investigated.
                 Therefore, it is unclear whether the compound is genotoxic.
B             The compound is presumed to be as carcinogenic to humans.             2                  1B
              • It acts by a stochastic genotoxic mechanism.
              • It acts by a non-stochastic genotoxic mechanism.
              • It acts by a non-genotoxic mechanism.
              • Its potential genotoxicity has been insufficiently investigated.
                 Therefore, it is unclear whether the compound is genotoxic.
              The compound is suspected to be carcinogenic to man.                  3                  2
3)            The available data are insufficient to evaluate the carcinogenic      Not applicable     Not applicable
              properties of the compound.
4)            The compound is probably not carcinogenic to man.                     Not applicable     Not applicable
ource: Health Council of the Netherlands. Guideline to the classification of carcinogenic compounds. The Hague: Health
 ouncil of the Netherlands, 2010; publication no. A10/07E.72
             Carcinogenic classification of substances by the Committee                                                63
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<pre>4 β-Estradiol</pre>

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<pre>Health Council of the Netherlands
Advisory Reports
The Health Council’s task is to       In addition, the Health Council
advise ministers and parliament on    issues unsolicited advice that
issues in the field of public health. has an ‘alerting’ function. In some
Most of the advisory opinions that    cases, such an alerting report
the Council produces every year       leads to a minister requesting
are prepared at the request of one    further advice on the subject.
of the ministers.
Areas of activity
Optimum healthcare                    Prevention                          Healthy nutrition
What is the optimum                   Which forms of                      Which foods promote
result of cure and care               prevention can help                 good health and
in view of the risks and              realise significant                 which carry certain
opportunities?                        health benefits?                    health risks?
Environmental health                  Healthy working                     Innovation and
Which environmental                   conditions                          the knowledge
influences could have                 How can employees                   infrastructure
a positive or negative                be protected against                Before we can harvest
effect on health?                     working conditions                  knowledge in the
                                      that could harm their               field of healthcare,
                                      health?                             we first need to
                                                                          ensure that the right
                                                                          seeds are sown.
www.healthcouncil.nl
</pre>

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