<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>Advisory Reports Areas of activity The Health Council’s task is to advise ministers and parliament on issues in the field of public health. Most of the advisory reports that the Council produces every year are prepared at the request of one of the ministers. In addition, the Health Council issues unsolicited advice that has an ‘alerting’ function. In some cases, such an alerting report leads to a minister requesting further advice on the subject. Health Council of the Netherlands www.healthcouncil.nl Optimum healthcare What is the optimum result of cure and care in view of the risks and opportunities? Environmental health Which environmental influences could have a positive or negative effect on health? Prevention Which forms of prevention can help realise significant health benefits? Healthy working conditions How can employees be protected against working conditions that could harm their health? Healthy nutrition Which foods promote good health and which carry certain health risks? Innovation and the knowledge infrastructure Before we can harvest knowledge in the field of healthcare, we first need to ensure that the right seeds are sown. Health Council of the Netherlands Hydroxyurea Evaluation of the effects on reproduction, recommendation for classification 2014/10 Hydroxyurea 2014/10 623447_V23_Om_Hydroxyurea_GR_EN.indd Alle pagina's 17-03-14 14:12</pre>

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<pre>Hydroxyurea
    Evaluation of the effects on reproduction,
    recommendation for classification
</pre>

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<pre></pre>

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<pre>Aan de minister van Sociale Zaken en Werkgelegenheid
Onderwerp             : Aanbieding advies Hydroxyurea
Uw kenmerk            : DGV/MBO/U-932542
Ons kenmerk           : U-8076/HS/cn/543-J14
Bijlagen              :1
Datum                 : 3 april 2014
Geachte minister,
Graag bied ik u hierbij het advies aan over de effecten van hydroxyureum op de
vruchtbaarheid en het nageslacht; het betreft ook effecten op de lactatie en via de
moedermelk op de zuigeling.
      Dit advies maakt deel uit van een uitgebreide reeks waarin voor de voortplanting giftige
stoffen worden geclassificeerd volgens richtlijnen van de Europese Unie. Het gaat om
stoffen waaraan mensen tijdens de beroepsuitoefening kunnen worden blootgesteld.
Dit advies is opgesteld door een vaste commissie van de Gezondheidsraad, de
Subcommissie Classificatie reproductietoxische stoffen. Het is vervolgens 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 il : h . st o u t e n @ g r. n l                          w w w. g r. n l
Te l e f o o n ( 0 7 0 ) 3 4 0 7 0 0 4
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<pre></pre>

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<pre>Hydroxyurea
Evaluation of the effects on reproduction,
recommendation for classification
Subcommittee on the Classification of Reproduction Toxic Substances,
a Committee of the Health Council of the Netherlands
to:
the Minister of Social Affairs and Employment
No. 2014/10, The Hague, April 3, 2014
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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.
This report can be downloaded from www.healthcouncil.nl.
Preferred citation:
Health Council of the Netherlands. Hydroxyurea - Evaluation of the effects on
reproduction, recommendation for classification. The Hague: Health Council of
the Netherlands, 2014; publication no. 2014/10.
all rights reserved
ISBN: 978-90-5549-994-6
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<pre>   Contents
   Samenvatting 9
   Executive summary 11
   Scope 13
.1 Background 13
.2 Committee and procedure 13
.3 Effects on or via lactation 14
.4 Data 15
.5 Presentation of conclusions 15
.6 Final remark 16
   Hydroxyurea 17
.1 Introduction 17
.2 Human studies 18
.3 Animal studies 20
.4 Conclusions 28
   References 31
   Contents                       7
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<pre>  Annexes 35
A The Committee 37
B The submission letter (in English) 39
C Comments on the public draft 41
D Regulation (EC) 1272/2008 of the European Community 43
E Additional considerations to Regulation (EC) 1272/2008 55
F Fertility and developmental toxicity studies 57
  Hydroxyurea
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<pre>Samenvatting
In het voorliggende advies heeft de Gezondheidsraad hydroxyureum onder de
loep genomen. Hydroxyureum is een geneesmiddel dat wordt gebruikt voor de
behandeling van patiënten met chronische myeloïde leukemie, met essentiële
thrombocytemie en polycythaemia vera of met sikkelcelanemie. Dit advies past
in een reeks adviezen waarin de Gezondheidsraad op verzoek van de minister
van Sociale Zaken en Werkgelegenheid de effecten van stoffen op de voortplan-
ting beoordeelt. Het gaat vooral om stoffen waaraan mensen tijdens de beroep-
suitoefening kunnen worden blootgesteld. De Subcommissie Classificatie
reproductietoxische stoffen van de Commissie Gezondheid en beroepsmatige
blootstelling aan stoffen (GBBS) van de raad, hierna aangeduid als de commis-
sie, kijkt zowel naar effecten op de vruchtbaarheid van mannen en vrouwen als
naar effecten op de ontwikkeling van het nageslacht. Daarnaast worden effecten
op de lactatie en via de moedermelk op de zuigeling beoordeeld.
Op basis van Verordening (EG) 1272/2008 van de Europese Unie doet de com-
missie een voorstel voor classificatie. Voor hydroxyureum komt de commissie
tot de volgende aanbevelingen:
• voor effecten op de fertiliteit adviseert de commissie om hydroxyureum te
     classificeren in categorie 1B (stoffen waarvan verondersteld wordt dat zij
     toxisch zijn voor de menselijke voortplanting) en te kenmerken met H360F
     (kan de vruchtbaarheid schaden)
Samenvatting                                                                    9
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<pre>  •  voor effecten op de ontwikkeling adviseert de commissie hydroxyureum te
     classificeren in categorie 1B (stoffen waarvan verondersteld wordt dat zij
     toxisch zijn voor de menselijke voortplanting) en te kenmerken met H360D
     (kan het ongeboren kind schaden)
  •  voor effecten op en via lactatie adviseert de commissie om hydroxyureum
     niet te kenmerken wegens onvoldoende geschikte gegevens.
0 Hydroxyurea
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<pre>Executive summary
In the present report, the Health Council of the Netherlands reviewed
hydroxyurea. Hydroxyurea is a drug used in the treatment of patients with
chronic myeloid leukaemia, with essential thrombocytosis and polycytaemia
vera or with sickle-cell anaemia This report is part of a series, in which the
Health Council evaluates the effects of substances on reproduction, at the request
of the Minister of Social Affairs and Employment. It mainly concerns substances
to which man can be occupationally exposed. The Subcommittee on the
Classification of Reproduction Toxic Substances of the Dutch Expert Committee
on Occupational Safety (DECOS) of the Health Council, hereafter called the
Committee, evaluates the effects on male and female fertility and on the
development of the progeny. Moreover, the Committee considers the effects of a
substance on lactation and on the progeny via lactation.
The Committee recommends classification according to Regulation (EC) 1272/
2008 of the European Union. For hydroxyurea, these recommendations are:
• for effects on fertility, the Committee recommends classifying hydroxyurea
    in category 1B (presumed human reproductive toxicant) and labelling with
    H360F (may damage fertility)
• for effects on development, the Committee recommends classifying
    hydroxyurea in category 1B (presumed human reproductive toxicant) and
    labelling with H360D (may damage the unborn child)
Executive summary                                                                  11
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<pre>  •  for effects on or via lactation, the Committee recommends not labelling
     hydroxyurea due to a lack of appropriate data.
2 Hydroxyurea
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<pre> hapter 1
        Scope
1.1     Background
        As a result of the Dutch regulation on registration of compounds toxic to
        reproduction that came into force on 1 April 1995, the Minister of Social Affairs
        and Employment requested the Health Council of the Netherlands to classify
        compounds toxic to reproduction. This classification is performed by the Health
        Council’s Subcommittee on the Classification of Reproduction Toxic Substances
        of the Dutch Expert Committee on Occupational Safety (DECOS). The
        classification is performed according to European Union Regulation (EC) 1272/
        2008 on classification, labelling and packaging (CLP) of substances and
        mixtures. The CLP guideline is based on the Globally Harmonised System of
        Classification and Labelling of Chemicals (GHS). The Subcommittee’s advice
        on the classification will be applied by the Ministry of Social Affairs and
        Employment to extend the existing list of compounds classified as reproductive
        toxicant (category 1A and 1B and 2) or compound with effects on or via
        lactation.
1.2     Committee and procedure
        This document contains the classification of hydroxyurea by the Health
        Council’s Subcommittee on the Classification of Reproduction Toxic
        Substances, hereafter called the Committee. The members of the Committee are
        Scope                                                                             13
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<pre>    listed in Annex A. The submission letter (in English) to the Minister can be
    found in Annex B.
         In 2013, the President of the Health Council released a draft of the report for
    public review. The individuals and organizations that commented on the draft
    report are listed in Annex C. The Committee has taken these comments into
    account in deciding on the final version of the report.
    The classification is based on the evaluation of published human and animal
    studies concerning adverse effects with respect to fertility and development as
    well as lactation of the above-mentioned compound.
    Classification for reproduction (fertility (F) and development (D)):
    Category 1                      Known or presumed human reproductive toxicant (H360(F/D))
       Category 1A                  Known human reproductive toxicant
       Category 1B                  Presumed human reproductive toxicant
    Category 2                      Suspected human reproductive toxicant (H361(f/d))
    No classification for effects on fertility or development
    Classification for lactation:
                                    Effects on or via lactation (H362)
                                    No labelling for lactation
    The classification and labelling of substances is performed according to the
    guidelines of the European Union (Regulation (EC)1272/2008) presented in
    Annex D. The classification of compounds is ultimately dependent on an
    integrated assessment of the nature of all parental and developmental effects
    observed, their specificity and adversity, and the dosages at which the various
    effects occur. The guideline necessarily leaves room for interpretation, dependent
    on the specific data set under consideration. In the process of using the
    regulation, the Committee has agreed upon a number of additional considerations
    (see Annex E).
1.3 Effects on or via lactation
    The recommendation for classifying substances for effects on or via lactation is
    also based on Regulation (EC) 1272/2008. The guideline defines that substances
    which are absorbed by women and have been shown to interfere with lactation or
    which may be present (including metabolites) in breast milk in amounts
    sufficient to cause concern for the health of a breastfed child, shall be classified
    and labelled. Unlike the classification of substances for fertility and
    developmental effects, which is based on hazard identification only (largely
 4  Hydroxyurea
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<pre>    independent of dosage), the labelling for effects on or via lactation is based on
    risk characterization and therefore, it also includes consideration of the level of
    exposure of the breastfed child.
        Consequently, a substance should be labelled for effects on or via lactation
    when it is likely that the substance would be present in breast milk at potentially
    toxic levels. The Committee considers a concentration of a compound as
    potentially toxic to the breastfed child when this concentration exceeds the
    exposure limit for the general population, e.g. the acceptable daily intake (ADI).
1.4 Data
    Literature searches were conducted in the online databases Current Contents and
    Medline, starting from 1966 up to September 2012 and by searches on the
    Internet; an update was performed in TOXNET in June 2013. Literature was
    selected primarily on the basis of the text of the abstracts. Publications cited in
    the selected articles, but not selected during the primary search, were reviewed if
    considered appropriate. In addition, handbooks and a collection of most recent
    reviews were consulted as well as several websites regarding (publications on)
    toxicology and health. References are divided into literature cited and literature
    consulted, but not cited.
        The Committee describes both human and animal studies in the text. The
    animal data are described in more detail in Annex F as well. Of each study, the
    quality of the study design (performed according to internationally
    acknowledged guidelines) and the quality of documentation is considered.
        In the assessment of the potential reproduction toxic effects of hydroxyurea,
    the Committee also used data on adverse effects related to its application as a
    therapeutic agent.
1.5 Presentation of conclusions
    The classification is given with key effects, species and references specified. In
    case a substance is not classified as toxic to reproduction, one of two reasons is
    given:
    • lack of appropriate data preclude assessment of the compound for
        reproductive toxicity
    • sufficient data show that no classification for toxic to reproduction is
        indicated.
    Scope                                                                               15
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<pre>1.6 Final remark
    The classification of compounds is based on hazard evaluation only (Niesink et
    al., 1995)23, which is one of a series of elements guiding the risk evaluation
    process. The Committee emphasizes that for derivation of health-based
    occupational exposure limits these classifications should be placed in a wider
    context. For a comprehensive risk evaluation, hazard evaluation should be
    combined with dose-response assessment, human risk characterization, human
    exposure assessment and recommendations of other organizations.
 6  Hydroxyurea
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<pre>  hapter       2
               Hydroxyurea
2.1            Introduction
 ame                       : hydroxyurea
  AS registry number       : 127-07-1
  AS name                  : urea, hydroxy-
 ynonyms                   : hydroxycarbamide; N-(aminocarbonyl)hydroxylamine; carbamohydroxamic acid;
                             carbamohydroximic acid; carbamoyl oxime; hydroxycarbamine; hydroxylurea
 olour and physical state  : white, crystalline powder
molecular weight           : 76.06
molecular formula          : CH4N2O2
 tructural formula         :
melting point              : 141 oC
 oiling point              : decomposes
 apour pressure            : 0.3 Pa (at 25 oC; estimated)
  og Poctanol/water        : -1.80
 olubility                 : very soluble in water; soluble in hot alcohol; insoluble in ethanol, benzene
 se                        : In the Netherlands, hydroxyurea is registered for treatment of patients with chronic myeloid
                             leukaemia, with essential thrombocytosis and polycytaemia vera, and with sickle-cell
                             anaemia9; the starting doses: for sickle-cell anaemia 15 mg/kg bw/day usually followed by
                             maintenance doses of 15-30 mg/kg bw/day; for chronic myeloid leukaemia 40 mg/kg; for
                             essential thrombocytosis 15 mg/kg bw/day; for polycytaemia vera 15-20 mg/kg bw/day; in
                             the latter three cases, maintenance doses are adjusted based on haematologic values.10
               Hydroxyurea                                                                                             17
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<pre> eneral toxicity           :   In humans, the major treatment-limiting and dose-related adverse effect of hydroxyurea is
                               suppression of the bone marrow, resulting in neutropenia, myelosuppression,
                               thrombocytopenia and anaemia. Hydroxyurea is cytotoxic.17
mechanism                  :   Hydroxyurea is cytostatic by inhibition of ribonucleotide reductase, an enzyme important in
                               creating deoxynucleosides for DNA replication in proliferating cells, which results in S-phase
                               cytotoxicity. An increased sensitivity to radiation therapy is thought to be due to the arrest of
                               malignant cells in G1 phase. In the treatment of sickle cell disease, hydroxyurea induces the
                               production of foetal haemoglobin, which results in prevention of the formation of sickle-
                               shaped red blood cells. Additionally, it can reduce the frequency of painful crises by
                               improving the movement of the sickle-shaped red blood cells through the blood vessels and
                               the need for blood transfusions.17
 inetics                   :   In humans, hydroxyurea is well absorbed after oral dosing and peak plasma levels are
                               detected after 1-4 hours. Hydroxyurea is distributed in a volume that is similar to total body
                               water, is concentrated in blood cells, such as erythrocytes and leukocytes, and it enters the
                               cerebrospinal fluid and breast milk. Hydroxyurea is thought to be excreted via the hepatic
                               metabolism and via renal excretion (unchanged hydroxyurea).
                               In animals, hydroxyurea is well absorbed throughout the body after oral or intraperitoneal
                               dosing. Hydroxyurea or its metabolites are distributed to the embryo in pregnant animals. The
                               main metabolite of hydroxyurea is urea, which is present in the urine. The main route of
                               elimination is by urinary excretion and occurs rapidly, with a half-life of <0.5 hours in rats
                               and mice.17
Data from HSDB22 unless otherwise noted.
2.2          Human studies
2.2.1        Fertility studies
             Male fertility
             In a retrospective multicentre study, Berthaut et al. (2008) studied the potential
             effects of hydroxyurea treatment on sperm parameters of patients with sickle cell
             disease. Semen samples were collected and analysed according to WHO criteria;
             parameters assessed included ejaculate volume, sperm concentration, total sperm
             count, motility, vitality and morphology. In 76 samples obtained from 34 patients
             before treatment, the percentages of abnormal values were 26%, 37%, 40%,
             84%, 64% and 43%, respectively. In only three patients, all parameters were
             normal. In six samples obtained from five patients during treatment, percentages
             of abnormal values were 50%, 100%, 100%, 80%, 67% and 50%, respectively.
             All patients had abnormal parameters but none had azoospermia. In 26 samples
             obtained from eight patients after treatment, percentages of abnormal values
             were 36%, 76%, 68%, 88%, 75% and 77%, respectively. Seven patients had
             abnormal parameters and one patient was azoospermic, four years after
             treatment.6
 8           Hydroxyurea
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<pre>      In a few case reports, effects of hydroxyurea treatment on sperm parameters of
      patients with sickle cell disease (n=7), polycythaemia rubra vera (n=1) or
      thrombocythaemia (n=1) were described based on analyses of semen samples
      obtained during and after treatment. Data from semen samples taken before
      treatment were not available. Parameters assessed in samples obtained during
      treatment were generally impaired and did not always improve after cessation of
      therapy.13,18,19
      Female fertility
      There are no data on the fertility of women after treatment with hydroxyurea.
2.2.2 Developmental toxicity studies
      In a clinical trial on the effect of hydroxyurea on reduction of painful crises in
      153 female and 146 male patients with sickle cell anaemia, several pregnancies
      occurred. These patients were taken off further treatment, but the pregnancy
      outcomes were followed for up to 17 years in a descriptive manner (no statistics
      were performed). Out of a total of 52 pregnancy outcomes reported for female
      participants, six had known hydroxyurea usage at conception and sometime
      during gestation resulting in three elective abortions for unknown reasons, one
      full-term live birth, one premature live birth and one miscarriage. Three
      pregnancies had probable hydroxyurea usage throughout the entire pregnancy
      and resulted in two elective abortions and one miscarriage. Out of a total of 42
      pregnancy outcomes reported for partners of male participants, ten had known
      hydroxyurea usage during conception resulting in two elective abortions, four
      full-term live births, one live birth at gestational age >37 weeks, one premature
      live birth and two miscarriages.4
      Thauvin-Robinet et al. (2001) evaluated data of pregnancy outcome among 31
      women treated with hydroxyurea (dose ranged from 0.5-6 g/day) for either
      essential thrombocythaemia (n=22), chronic myeloid leukaemia (n=6), chronic
      myeloid splenomegaly (n=2) or sickle cell disease (n=1), of which three received
      hydroxyurea throughout pregnancy, 22 during the first trimester, two during the
      first and second trimester and two during the third trimester (of the remaining
      two, the exposure time was not known). The 31 pregnancies resulted in 24 live-
      born infants (one twin), five induced abortions, one miscarriage and two in utero
      foetal deaths. Intrauterine growth retardation was found in 2/31 cases by
      ultrasound. Among the 24 live-born infants, nine were premature and three had
      Hydroxyurea                                                                        19
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<pre>      abnormalities including hip dysplasia, unilateral renal dilatation and pilonidal
      sinus. Five had neonatal respiratory distress considered to be the result of
      prematurity rather than pulmonary malformation. No malformations were seen in
      the two in utero foetal deaths. Pre- or postnatal chromosomal analysis was
      normal in 6/7 cases studied; the remaining case showed inherited inversion of
      chromosome 9.30
      In a comprehensive report on the reproductive and developmental toxicity of
      hydroxyurea by the Center for the Evaluation of Risks to Human Reproduction
      (CERHR) of the (US) National Toxicological Program (NTP), case reports are
      presented and discussed concerning an additional 26 pregnancies in women to
      whom hydroxyurea was prescribed for the treatment of haematological
      malignancies, essential thrombocythaemia and sickle cell disease. Of the seven
      outcomes that were not normal, two were stillbirths, two were preterm deliveries
      and two were cases of intrauterine growth retardation, while one outcome was
      unknown.17
          For further details, the Committee refers to the NTP-CERHR review17.
      The Committee is of the opinion that no conclusions concerning the potential
      developmental effects of hydroxyurea can be drawn from the studies presented
      above because of methodological deficiencies and the unknown influence of the
      underlying maternal illnesses.
2.2.3 Lactation
      Sylvester et al. (1987) reported one case of excretion of hydroxyurea into breast
      milk. A patient with chronic myeloid leukaemia was treated orally during
      lactation with 500 mg hydroxyurea three times a day. Milk samples were
      collected at the start of the treatment and during seven days of lactation (two
      hours after the last dose of hydroxyurea each day). Due to methodological
      difficulties, hydroxyurea could be detected in only a few samples. The three
      reliable hydroxyurea milk concentrations were: day 1; 6.1 mg/L, day 3; 3.8 mg/L
      and day 4; 8.4 mg/L (mean 6.1±2.3 mg/L).29
2.3   Animal studies
      Fertility and developmental toxicity studies in laboratory animals are
      summarized in Annex F.
 0    Hydroxyurea
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<pre>2.3.1 Fertility studies
      Male reproductive system
      Mecklenburg et al. (1975) administered hydroxyurea at amounts of 3 mg/mL
      drinking water (equivalent to 300 mg hydroxyurea/kg bw/day, assuming a water
      intake of 100 mL/kg bw/day) to sexually mature rats (Holtzmann; n=90;
      controls: n=18) for 70 days, followed by a 30-day recovery period. Body weight
      at the end of the treatment was reduced in the treatment group. Germinal cell
      depletion was noted from 14 days after the start of the treatment and the severity
      increased with the duration of the treatment. This effect was ascribed to the
      inhibition of DNA synthesis by hydroxyurea. After cessation of the treatment,
      the germinal epithelium was re-established in most of the seminiferous tubules.20
      Rich and De Kretser (1977) exposed rats (Sprague-Dawley; 60 days old; n=10/
      group) to amounts of hydroxyurea of 3 mg/mL drinking water (equivalent to 300
      mg/kg bw/day) for three months. In treated rats, absolute testis weights were
      statistically significantly reduced (by 40%), destruction of the seminiferous
      epithelium occurred, and serum LH and FSH levels were significantly elevated.24
      Jones et al. (2009) treated adult transgenic sickle cell mice (n=6/group/stage) by
      gavage with 0 or 25 mg hydroxyurea/kg bw/day for 28 or 56 days. Monthly body
      weights were similar between treated mice and controls. Hydroxyurea treatment
      statistically significantly decreased absolute testis weight on day 28 and 56.
      Concomitant with a 52% shrinkage of testis dimensions on day 56, testes from
      treated mice exhibited atrophic degeneration in the seminiferous tubules.
      Epididymides from treated mice showed a 25% shrinkage, along with 69%
      reduction in stored sperm density and 95% reduction in sperm motility on day
      56.15
      Wiger et al. (1995) injected mice (B6C3/F1/BOM M; six to eight weeks old;
      n=5/group) intraperitoneally with 0 or 200 mg hydroxyurea/kg bw/day for five
      days. Testes were examined at various stages after treatment. Atrophy of
      seminiferous tubules was seen in the treated mice five and ten days after the last
      exposure. The absolute and relative testis weights were reduced (40-45% lower
      than controls on days 27 and 33 after treatment). In addition, reduction in the
      proportion of the various spermatid stages and an alteration in sperm chromatin
      Hydroxyurea                                                                        21
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<pre>  structure were noted. Wiger et al. discussed that the primary cause of these
  findings was inhibition of DNA synthesis in the testes.31
  Evenson and Jost (1993) treated mice (C57B/6JxC3H/HeJ F1; 13 to 15 weeks
  old; n≥6/group) with intraperitoneal doses of hydroxyurea of 0, 25, 50, 100, 200,
  400 or 500 mg/kg bw/day for five days. Whole testis, minced testicular cell
  suspensions and caudal epidydimal sperm cells were obtained eight or 29 days
  after treatment. Treatment did not affect body weights. At day 8, absolute testis
  weights were statistically significantly decreased at 400 and 500 mg/kg bw/day
  and testicular cell population ratios were altered at doses ≥100 mg/kg bw. At day
  29, these testis changes were seen at doses ≥50 mg/kg bw/day. Evenson and Jost
  concluded that hydroxyurea inhibited DNA synthesis, causing maturation
  depletion of pachytene spermatocytes and, subsequently, depletion of meiotic
  daughter cells and differentiated cell types leading to mature sperm.11
  Shin et al. (1999) administered single intraperitoneal doses of hydroxyurea of 0,
  100, 200 or 400 mg/kg bw to mice (ICR; six to seven weeks old; n=3/group).
  Testes were examined at 0, 4, 8, 12, 24 and 48 hours after treatment. Both the
  number of apoptotic cells and the level of DNA fragmentation increased
  depending on the dose. The number of apoptotic cells increased continuously,
  peaked at 12 hours and reached control levels by 48 hours. Shin and Shiota
  discussed that apoptosis of damaged testicular cells is apparently a common
  response to toxicants, therefore protecting the next generation of germ cells from
  the damaged cell population.27
  Ficsor and Ginsberg (1980) treated mice (CF1; 12 to 16 weeks old; n=3-4/group)
  intraperitoneally with doses of 0, 125, 250, 500 or 1,000 mg/kg bw/day for five
  days. Examinations were conducted 35 days after the last treatment. Terminal
  body weights were decreased at 1,000 mg/kg bw and absolute testis weights were
  statistically significantly decreased at 500 and 1,000 mg/kg bw. In all dose
  groups, the number of sperm extracted from the cauda was decreased and sperm
  motility was dose-dependently decreased.12
  Singh and Taylor (1981) treated hamsters (inbred PD4 strain; ten to 12 weeks
  old; n=6-9/ group) with intraperitoneal doses of 0, 10, 50 or 250 mg/kg bw/day
  for five days. One, four and 10 weeks after treatment, two or three hamsters of
  each dose were examined. After an initial increase, body weight gradually
  declined with increasing levels of hydroxyurea. A progressive decline in sperm
  number with exposure to increasing dose levels of hydroxyurea occurred, which
2 Hydroxyurea
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<pre>      was already evident at 10 mg/kg bw. No sperm abnormalities were induced at
      doses as high as 250 mg/kg bw.28
      Female reproductive system
      Sampson et al. (2010) investigated the effects of hydroxyurea on ovulation rate
      and embryo development in groups of 20 C57BL/6J female mice. Animals were
      treated with oral doses (gavage) of hydroxyurea of 30 mg/kg bw/day for up to 28
      days; controls received saline (vehicle). Five days prior to cessation of treatment,
      mice were subjected to folliculogenesis induction with pregnant mare serum
      gonadotropin. Forty-eight hours after this induction, five mice/group were
      anaesthetized to collect blood for oestradiol-17ß (E2) measurement; in the
      remaining mice, ovulation was induced with human chorionic gonadotropin
      (hCG) after which they were immediately caged with males for mating. Five
      plugged females/group were sacrificed for ovulation rate determination (about
      15 hours post hCG); the remaining mice were sacrificed about 27 hours post
      hCG, ovaries excised and weighed and embryos harvested. Compared to
      controls, treated mice had decreased ovary weights, ovulation rates and
      circulating E2 levels (p<0.05) and fewer embryos developing to the blastocyst
      stage (32% vs 60% in controls; p<0.05).26
2.3.2 Developmental toxicity studies
      Since the original reports by Murphy and Chaube (1964)21 and Chaube and
      Murphy (1966)8, who showed that single intraperitoneal doses of hydroxyurea of
      250 mg/kg bw or more given to Wistar rats on one of gestational days 9-12
      produced a high proportion of foetuses with malformations, numerous studies on
      the developmental toxicity of hydroxyurea in a variety of animal species have
      been published. In many of these studies, single, often relatively high doses of
      hydroxyurea were administered at single gestational days and in some studies
      even as a positive control.
          The Committee presents here only multi-dose studies; for a complete
      overview, the Committee refers to the comprehensive report of CERHR17.
      Aliverti et al. (1980) administered oral doses of hydroxyurea of 0, 50, 150, 300
      or 450 mg/kg bw/day to female Sprague-Dawley rats (n=8-10/group; 2% Arabic
      gum in water-treated controls: n=27) during gestational days 6-15. The rats were
      killed on gestational day 21 and foetuses were subjected to external, visceral and
      skeletal examinations. There was no information on maternal toxicity. Foetal
      Hydroxyurea                                                                          23
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<pre>  body weights were reduced at levels ≥150 mg/kg bw. At levels ≥300 mg/kg bw,
  hydroxyurea induced postimplantation loss and developmental effects (most
  commonly: craniofacial abnormalities, abdominal wall defects, limb malrotation,
  hydrocephalus and ocular defects).1
  Roll and Bär (1969) exposed female mice by gavage to doses of hydroxyurea of
  0, 5, 10, 15 or 20 mg/animal (according to Roll and Bär ca. 0, 200, 400, 600 or
  800 mg /kg bw/day) from gestational days 6-17. These doses were stated to be
  ‘relatively non-toxic for the maternal animals’ but no data were provided.
      Twenty-one, 19 and 16 dams treated with 0, 400 and 800 mg/kg bw/day,
  respectively, underwent Caesarean section on gestational day 18, and
  implantation sites were examined and foetuses were assessed for skeletal
  abnormalities. Treatment caused statistically significant increases in the total
  number of resorptions (10, 23, 95%, respectively), particularly early (9, 12, 36%,
  respectively) and mid-term (1, 8, 57%, respectively) resorptions. At 400 mg/kg
  bw, foetal body weight was statistically significantly decreased. Skeletal
  evaluation showed an increased number of malformations including sternum
  defects (17% vs. 1% in controls), encephalocele (13% vs. 0.5%), thoracic
  vertebral defects (8% vs. 0%), cervical vertebrae fusion (5.9% vs 1%) and costal
  fusion (5.3% vs.1%). In the few surviving foetuses of the 800 mg/kg bw group,
  no malformations were observed but development was severely retarded.
      Some dams (n=18, 29, 9 at 0, 200, 400 mg/kg bw, respectively; unspecified
  at higher levels) were allowed to deliver and pups were examined for external
  malformations, viability at birth and body weights until the end of the lactation
  period. At 600 and 800 mg/kg bw, complete resorption or abortion occurred. At
  200 and 400 mg/kg bw, the number of stillbirths and pup mortality during the
  lactation period were increased and pup body weights were slightly decreased.
  External malformations were cleft palate (1.2%) and kinked tails (0.8%) at 200
  mg/kg bw and cleft palate (1.5%) and encephalocele (3%) at 400 mg/kg bw (no
  malformation rates provided for the control group).
      Roll and Bär also described treatment of dams during specific stages of
  pregnancy at similar and higher dose levels. Effects on resorptions and foetal
  weight were consistent with those observed after exposure during gestational
  days 6-17. Malformations commonly observed after hydroxyurea treatment on
  gestational days 6 or 7 included cleft palates, sternum defects, encephaloceles
  and vertebral defects. In addition to these effects, limb and tail defects occurred
  with exposures on gestational days 10 or 11.25
4 Hydroxyurea
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<pre>Khera (1979) gave hydroxyurea by capsules to female cats (n=17/group) at daily
doses of 0, 50 or 100 mg/kg bw/day during gestational days 10-22. The cats were
necropsied on gestational day 43, and foetuses were examined for external,
visceral and skeletal malformations. At 100 mg/kg bw, maternal body weight
gain was statistically significantly decreased. Only one cat of the 100 mg/kg bw
group survived until necropsy. No maternal effects were noted at 50 mg/kg bw.
     At 100 mg/kg bw/day, hydroxyurea induced a high number of non-pregnancy
(ten not pregnant vs. five in controls) and resorptions with, consequently, few
live foetuses. The one cat surviving until necropsy had two stunted live foetuses,
one had no apparent anomaly, the other cyclopia. At 50 mg/kg bw, the number of
litters with malformations and the overall number of malformed foetuses was
higher than in controls (which was, according to Khera, of ‘borderline statistical
significance’). The malformations in this group were of various types but cleft
palate and microphthalmia were most frequent.16
Asano and Okaniwa (1987) administered intraperitoneal doses of hydroxyurea of
0, 100 or 200 mg/kg bw/day to Sprague-Dawley and Wistar rats during
gestational day 9-12. Information on maternal toxicity was not provided.
     Groups of 15 to 16 Sprague-Dawley and five Wistar rats were sacrificed on
gestational day 21 and examined for implantations, resorptions and live foetuses
and the foetuses for sex, body weight and malformations. Compared to controls,
there were no differences in the number of implantation sites, resorptions or life
foetuses. In both strains, the weight of live foetuses was statistically significantly
decreased at 200 mg/kg bw. At 200 mg/kg bw, the percentages of visceral
malformations were statistically significantly increased (Sprague-Dawley:
44-51% vs. 1% controls; Wistar: 87-89% vs. 10% in controls). Malformations
observed most commonly in both strains were dilatation of lateral ventricle,
anophthalmia, microphthalmia and ventricular septal defect. In Wistar rats, also
exencephaly, cleft palate and micrognathia were seen. At 100 mg/kg bw, no
adverse effects were produced. Asano and Okaniwa noted that morphological
effects of hydroxyurea were less severe in Sprague-Dawley rat foetuses than in
Wistar rat foetuses.
     Groups of 12 to 22 Sprague-Dawley rats were allowed to deliver
spontaneously. Pups were reared by their biological mothers and observed up to
postnatal day 21. At 200 mg/kg bw, there were statistically significant decreases
in mean weights of male and female pups at birth and at postnatal day 21 and in
viability index at postnatal day 4 and statistically significant increases in the
number of male and female pups with malformations (53 and 43%, respectively;
none in controls). Malformations most commonly observed were anophthalmia
Hydroxyurea                                                                            25
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<pre>  (31% in males; 29% in females), hydrocephaly (39 and 11%, respectively) and
  microphthalmia (13 and 25%, respectively).3
  Asano et al. (1983) treated Wistar rats with doses of hydroxyurea of 0, 25, 50 and
  100 mg/kg bw/day (n=10-12/group) (‘first study’) or 0, 100 and 200 mg/kg
  bw/day (n=8-10/group) (‘second study’) during gestational days 9-12. Dams
  were allowed to deliver spontaneously. Litters were reared by biological mothers
  and observed for up to about eight weeks. No information on maternal toxicity
  was provided.
       There were no differences in delivery index, number of stillbirth, body
  weight, postnatal growth and viability index at levels up to 100 mg/kg bw. At
  200 mg/kg bw (second study), the frequency of stillbirth was increased (p<0.05)
  and the male body weight at birth was decreased (p<0.05). In addition, the
  frequency of malformations (eye defects, dilation of ventricles, cleft lip/palate) at
  birth and postnatal days 4, 14, 21 and 56 was statistically significantly (p<0.01)
  increased (to 20%, 69%, 88%, 63% and 69% of examined pups, respectively). At
  100 mg/kg bw/day, eye defects, dilated ventricles and cranial enlargement were
  noted; the proportion of abnormalities in this group was: at postnatal day 4: 18%
  (first study) or 5.3% (second study); at postnatal day 14: 5.9% (second study); at
  postnatal day 21: 17% (first study) or 0% (second study); and at postnatal day 56:
  7.5% (second study). At 25 and 50 mg/kg bw/day (first study), only a few cases
  of eye defects and dilated ventricles were observed versus none in controls,
  namely at postnatal day 4: dilated ventricle 4.4% and 7.7%, and at postnatal day
  21: microphthalmia 1.3% and 1.2% of examined pups (n= 35-93 pups per sex) at
  25 and 50 mg/kg bw/day, respectively. Delayed development of the female
  righting reflex was noted at two days of age, but statistical significance was
  obtained only at 25 mg/kg bw/day. The male free fall reflex was delayed between
  postnatal days 15-25; statistical significance was obtained at 100 and 200 mg/kg
  bw/day. The number of rearing in the open field test was increased (p<0.05) in
  females at 100 mg/kg bw/day. Rotorod performance and the acquisition rate of
  conditioned avoidance response were not affected.2
  Chahoud and Paumgartten (2009) injected doses of 0, 250, 300, 350, 400, 450,
  500 or 550 mg/kg bw intraperitoneally into Wistar rats (n=13-34 litters/group;
  controls: n=53 litters) on gestational day 11. Caesarean sections were performed
  on gestational day 21 and the foetuses were subjected to skeletal examinations.
  No information on maternal toxicity was given but Chahoud and Paumgartten
  stated that the single treatment in mid-gestation was an attempt to attenuating
  maternal toxicity and to avoiding marked embryo lethality.
6 Hydroxyurea
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<pre>           Dose-related variations were observed at doses ≥250 mg/kg bw (p<0.05);
      at ≥250 mg/kg bw: increased percentages of dumbbell-shaped and bipartite
      ossification centres in thoracic and lumbar vertebrae (14% up to 87% at high-
      dose); at ≥300 mg/kg bw: increased occurrence of zygomatic bone fused to os
      maxilla (19% up to 84% at high-dose). Dose-related malformations occurred at
      doses ≥300 mg/kg bw (p< 0.05); at ≥300 mg/kg bw/d: absent tympanic bone
      (4.2% up to 91% at high-dose); at ≥400 mg/kg bw/d: cleft palate (4.8% up to
      34% at high-dose), absent tibia (3.5% up to 44% at high-dose); at ≥450 mg/kg
      bw/d: bent ribs (0.8% up to 1.9 % at high-dose), bent clavicle (5.6% up to 20% at
      high-dose).7
      Barr and Beaudoin (1981) administered intraperitoneal doses of hydoxyurea of
      200-375 mg/kg bw to two stocks of Wistar rats (n=8-10 litters/group) at one or
      several six-hour intervals on gestational days 9-10.75. Caesarean sections were
      performed on gestational day 21. No information on maternal toxicity was given.
      Foetal and placental weights were decreased and malformations were increased
      in the treated rats. Statistically significant (p<0.05) increases in malformations
      observed most commonly included anopthalmia/microphtalmia (1.7-94%)*,
      hydrocephaly (1.9-35%), exencephaly (0-32%), maxillary hypoplasia (0-41%),
      cleft lip/palate (0-11.5%), protruding tongue (0-27%), hydronephrosis 7-58%),
      tail displasia (0-30%) and anal atresia (0-20%).5
      Gupta and Jaffe (1982) injected Sprague-Dawley rats (n=5/group)
      subcutaneously with 0 or 160 mg hydroxyurea/kg bw/day on gestational days
      17-20. Randomly selected female offspring were followed to their reproductive
      development. Treatment did not affect appearance or body weight of the dams.
      Offspring of hydroxyurea-treated rats did not show effects with respect to age of
      vaginal opening and first appearance of oestrus (n=20) or significant effects on
      oestrus cycle (n=6). Fertility of female offspring (n=9), determined by mating
      with untreated males, was not significantly affected.14
2.3.3 Lactation
      No relevant animal studies on effects of hydroxyurea during lactation were
      available.
      Figures in brackets are ranges of percentages affected pups, depending on stock of rats and exposure
      stage.
      Hydroxyurea                                                                                          27
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<pre>2.4 Conclusions
    Fertility
    One multicentre study6 and three case reports of men with sickle cell
    disease13,18,19 suggest that hydroxyurea therapy reduces sperm counts and
    impairs sperm motility and morphology.
        There were no data on the functional fertility of laboratory animals following
    treatment with hydroxyurea. Oral or intraperitoneal administration of
    hydroxyurea caused decreased testis weights and histological seminiferous
    tubular abnormalities in rats13,20,24 and mice11,12,15,27,31, decreased sperm counts
    in mice12 and hamsters28 and affected sperm morphology or motility in
    mice12,15,31. Oral administration to mice resulted in decreased ovary weights,
    ovulation rates and circulating E2 levels and fewer embryos developing to
    blastocyst stage.26
        Overall, the Committee concludes that the human data are not sufficient for
    classification. Based on the effects observed in laboratory animals, the
    Committee proposes to classify hydroxyurea for effects on fertility in category
    1B (presumed human reproductive toxicant).
    Developmental toxicity
    No adequately designed human studies on developmental toxicity effects of
    hydroxyurea were available.
        In various animal species, repeated oral or intraperitoneal administration
    induced increased numbers of resorptions, stillbirths and postnatal deaths,
    reduced pup weights and external, visceral or skeletal malformations.1-3,16,25 For
    most studies, no or only limited information on maternal toxicity was available.
    However, the Committee considers that the nature and severity of the effects
    observed indicates that they occurred independently from maternal toxicity.
        Therefore, based on the data from laboratory animal studies, the Committee
    proposes to classify hydroxyurea for effects on fertility in category 1B
    (presumed human reproductive toxicant).
    Lactation
    Hydroxyurea was excreted in human breast milk in an amount of 6.1±2.3 mg/L.29
    This value is based on a few observations in one subject only. Since there is no
 8  Hydroxyurea
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<pre>information about a safe/acceptable daily intake of hydroxyurea either, it was not
possible to calculate a safe level for hydroxyurea in human breast milk.
    The Committee proposes not labelling hydroxyurea for effects on or via
lactation due to a lack of appropriate human and animal data.
Proposed classification for fertility
Category 1B; H360F.
Proposed classification for developmental toxicity
Category 1B; H360D.
Proposed labelling for effects during lactation
Lack of appropriate human and animal data precludes assessment of
hydroxyurea for effects on or via lactation.
Hydroxyurea                                                                        29
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<pre>0 Hydroxyurea</pre>

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  psychotoxicity evaluation of FD and C red dye #3 (erythrosine) in rats. Arch Toxicol. 1983;53:253-
  64.
  Vorhees CV, Butcher RE, Brunner RL, Wootten V, Sobotka TJ. Developmental toxicity and
  psychotoxicity of FD and C red dye No. 40 (allura red AC) in rats. Toxicology. 1983;28:207-17.
  Ware RE. Hydroxycarbamide: clinical aspects. C R Biol. 2013;336:177-82.
  Woo GH, Katayama K, Bak EJ, Ueno M, Yamauchi H, Uetsuka K, et al. Effects of prenatal
  hydroxyurea-treatment on mouse offspring. Exp Toxicol Pathol. 2004;56:1-7.
  Woo GH, Bak EJ, Nakayama H, Doi K. Hydroxyurea (HU)-induced apoptosis in the mouse fetal
  lung. Exp Mol Pathol. 2005;79:59-67.
  Wyrobek AJ, Bruce WR. Chemical induction of sperm abnormalities in mice. Proc Natl Acad Sci
  USA. 1975;72:4425-9.
  Yan J, Hales BF. Depletion of glutathione induces 4-hydroxynonenal protein adducts and
  hydroxyurea teratogenicity in the organogenesis stage mouse embryo. J Pharmacol Exp Ther.
  2006;319:613-21.
4 Hydroxyurea
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<pre>A The Committee
B The submission letter (in English)
C Comments on the public draft
D Regulation (EC) 1272/2008 of the European Community
E Additional considerations to Regulation (EC) 1272/2008
F Fertility and developmental toxicity studies
  Annexes
                                                         35
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<pre>6 Hydroxyurea</pre>

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<pre>nnex A
     The Committee
     •  A.H. Piersma, Chairman
        Professor of Reproductive and Developmental Toxicology, Utrecht
        University, Utrecht and National Institute of Public Health and the
        Environment, Bilthoven
     •  D. Lindhout
        Professor of Medical Genetics, Paediatrician (not practising), Clinical
        Geneticist, University Medical Centre, Utrecht
     •  N. Roeleveld
        Reproductive Epidemiologist, Radboud university medical center, Nijmegen
     •  J.G. Theuns-van Vliet
        Reproductive Toxicologist, TNO Triskelion BV, Zeist
     •  D.H. Waalkens-Berendsen
        Reproductive Toxicologist, Zeist
     •  P.J.J.M. Weterings
        Toxicologist, Weterings Consultancy BV, Rosmalen
     •  A.S.A.M. van der Burght, Scientific Secretary
        Health Council of the Netherlands, Den Haag
     •  J.T.J. Stouten, Scientific Secretary
        Health Council of the Netherlands, Den Haag
     The Committee                                                               37
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<pre>  The first draft of the present document was prepared by Dr. B.A.R. Lina and
  Dr. M.J.W. van den Hoven from TNO Triskelion BV, Zeist, the Netherlands, by
  contract with the Ministry of Social Affairs and Employment.
  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.
8 Hydroxyurea
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<pre>nnex B
     The submission letter (in English)
     Subject         : Submission of the advisory report Hydroxyurea
     Your reference  : DGV/MBO/U-932542
     Our reference   : U-8076/HS/cn/543-J14
     Enclosed        :1
     Date            : April 3, 2014
     Dear Minister,
     I hereby submit the advisory report on the effects of hydroxyurea on fertility and
     on the development of the progeny; it also concerns effects on lactation and on
     the progeny via lactation. This advisory report is part of an extensive series in
     which reproduction toxic substances are classified in accordance with European
     guidelines. This involves substances to which people may be exposed
     occupationally.
     The advisory report was prepared by a permanent committee of the Health
     Council of the Netherlands, the Subcommittee on the Classification of
     Reproduction Toxic Substances. The advisory report was consequently reviewed
     by the Health Council’s Standing Committee on Health and the Environment.
     The submission letter (in English)                                                 39
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<pre>  Today I 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
  information.
  Yours sincerely,
  (signed)
  Prof. dr. W.A. van Gool,
  President
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<pre>nnex C
     Comments on the public draft
     A draft of the present report was released in 2013 for public review. The
     following organisation and persons have commented on the draft document:
     • T.J. Lentz, K. Krajnak, D. Murray, S. Rengasamy. National Institute for
         Occupational Safety and Health (NIOSH), Cincinnati OH, USA.
     The received comments, and the reply by the Committee can be found on the
     website of the Health Council.
     Comments on the public draft                                              41
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<pre>2 Hydroxyurea</pre>

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<pre>nnex D
     Regulation (EC) 1272/2008 of the
     European Community
     3.7            Reproductive toxicity
     3.7.1          Definitions and general considerations
     3.7.1.1        Reproductive toxicity includes adverse effects on sexual function and fertility in adult
     males and females, as well as developmental toxicity in the offspring. The definitions presented
     below are adapted from those agreed as working definitions in IPCS/EHC Document No 225, Princi-
     ples for Evaluating Health Risks to Reproduction Associated with Exposure to Chemicals. For classi-
     fication purposes, the known induction of genetically based heritable effects in the offspring is
     addressed in Germ Cell Mutagenicity (section 3.5), since in the present classification system it is con-
     sidered more appropriate to address such effects under the separate hazard class of germ cell muta-
     genicity.
     In this classification system, reproductive toxicity is subdivided under two main headings:
     (a) adverse effects on sexual function and fertility;
     (b) adverse effects on development of the offspring.
     Some reproductive toxic effects cannot be clearly assigned to either impairment of sexual function
     and fertility or to developmental toxicity. Nonetheless, substances with these effects, or mixtures con-
     taining them, shall be classified as reproductive toxicants.
     Regulation (EC) 1272/2008 of the European Community                                                      43
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<pre>  3.7.1.2         For the purpose of classification the hazard class Reproductive Toxicity is differentiated
                  into:
  •     adverse effects
        •    on sexual function and fertility, or
        •    on development;
  •     effects on or via lactation.
  3.7.1.3         Adverse effects on sexual function and fertility
  Any effect of substances that has the potential to interfere with sexual function and fertility. This
  includes, but is not limited to, alterations to the female and male reproductive system, adverse effects
  on onset of puberty, gamete production and transport, reproductive cycle normality, sexual behaviour,
  fertility, parturition, pregnancy outcomes, premature reproductive senescence, or modifications in
  other functions that are dependent on the integrity of the reproductive systems.
  3.7.1.4         Adverse effects on development of the offspring
  Developmental toxicity includes, in its widest sense, any effect which interferes with normal devel-
  opment of the conceptus, either before or after birth, and resulting from exposure of either parent
  prior to conception, or exposure of the developing offspring during prenatal development, or postna-
  tally, to the time of sexual maturation. However, it is considered that classification under the heading
  of developmental toxicity is primarily intended to provide a hazard warning for pregnant women, and
  for men and women of reproductive capacity. Therefore, for pragmatic purposes of classification,
  developmental toxicity essentially means adverse effects induced during pregnancy, or as a result of
  parental exposure. These effects can be manifested at any point in the life span of the organism. The
  major manifestations of developmental toxicity include (1) death of the developing organism, (2)
  structural abnormality, (3) altered growth, and (4) functional deficiency.
  3.7.1.5         Adverse effects on or via lactation are also included in reproductive toxicity, but for
  classification purposes, such effects are treated separately (see Table 3.7.1 (b)). This is because it is
  desirable to be able to classify substances specifically for an adverse effect on lactation so that a spe-
  cific hazard warning about this effect can be provided for lactating mothers.
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<pre>3.7.2        Classification criteria for substances
3.7.2.1      Hazard categories
3.7.2.1.1    For the purpose of classification for reproductive toxicity, substances are allocated to
one of two categories. Within each category, effects on sexual function and fertility, and on develop-
ment, are considered separately. In addition, effects on lactation are allocated to a separate hazard cat-
egory.
Table 3.7.1(a) Hazard categories for reproductive toxicants.
Categories                      Criteria
CATEGORY 1                      Known or presumed human reproductive toxicant
                                Substances are classified in Category 1 for reproductive toxicity when
                                they are known to have produced an adverse effect on sexual function
                                and fertility, or on development in humans or when there is evidence
                                from animal studies, possibly supplemented with other information, to
                                provide a strong presumption that the substance has the capacity to
                                interfere with reproduction in humans. The classification of a sub-
                                stance is further distinguished on the basis of whether the evidence for
                                classification is primarily from human data (Category 1A) or from
                                animal data (Category 1B).
                Category 1A Known human reproductive toxicant
                                The classification of a substance in Category 1A is largely based on
                                evidence from humans.
                Category 1B Presumed human reproductive toxicant
                                The classification of a substance in Category 1B is largely based on
                                data from animal studies. Such data shall provide clear evidence of an
                                adverse effect on sexual function and fertility or on development in
                                the absence of other toxic effects, or if occurring together with other
                                toxic effects the adverse effect on reproduction is considered not to be
                                a secondary non-specific consequence of other toxic effects. However,
                                when there is mechanistic information that raises doubt about the rele-
                                vance of the effect for humans, classification in Category 2 may be
                                more appropriate.
CATEGORY 2                      Suspected human reproductive toxicant
                                Substances are classified in Category 2 for reproductive toxicity when
                                there is some evidence from humans or experimental animals, possi-
                                bly supplemented with other information, of an adverse effect on sex-
                                ual function and fertility, or on development, and where the evidence
                                is not sufficiently convincing to place the substance in Category 1. If
                                deficiencies in the study make the quality of evidence less convincing,
                                Category 2 could be the more appropriate classification.
                                Such effects shall have been observed in the absence of other toxic
                                effects, or if occurring together with other toxic effects the adverse
                                effect on reproduction is considered not to be a secondary non-specific
                                consequence of the other toxic effects.
Regulation (EC) 1272/2008 of the European Community                                                        45
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<pre>  Table 3.7.1(b) Hazard category for lactation effects.
  EFFECTS ON OR VIA LACTATION
  Effects on or via lactation are allocated to a separate single category. It is recognised that for many
  substances there is no information on the potential to cause adverse effects on the offspring via lacta-
  tion. However, substances which are absorbed by women and have been shown to interfere with lac-
  tation, or which may be present (including metabolites) in breast milk in amounts sufficient to cause
  concern for the health of a breastfed child, shall be classified and labelled to indicate this property
  hazardous to breastfed babies. This classification can be assigned on the:
  (a) human evidence indicating a hazard to babies during the lactation period; and/or
  (b) results of one or two generation studies in animals which provide clear evidence of adverse effect
  in the offspring due to transfer in the milk or adverse effect on the quality of the milk; and/or
  (c) absorption, metabolism, distribution and excretion studies that indicate the likelihood that the sub-
  stance is present in potentially toxic levels in breast milk.
  3.7.2.2        Basis of classification
  3.7.2.2.1      Classification is made on the basis of the appropriate criteria, outlined above, and an
  assessment of the total weight of evidence (see 1.1.1). Classification as a reproductive toxicant is
  intended to be used for substances which have an intrinsic, specific property to produce an adverse
  effect on reproduction and substances shall not be so classified if such an effect is produced solely as
  a non-specific secondary consequence of other toxic effects.
  The classification of a substance is derived from the hazard categories in the following order of pre-
  cedence: Category 1A, Category 1B, Category 2 and the additional Category for effects on or via lac-
  tation. If a substance meets the criteria for classification into both of the main categories (for example
  Category 1B for effects on sexual function and fertility and also Category 2 for development) then
  both hazard differentiations shall be communicated by the respective hazard statements. Classifica-
  tion in the additional category for effects on or via lactation will be considered irrespective of a clas-
  sification into Category 1A, Category 1B or Category 2.
  3.7.2.2.2      In the evaluation of toxic effects on the developing offspring, it is important to consider
  the possible influence of maternal toxicity (see section 3.7.2.4).
  3.7.2.2.3      For human evidence to provide the primary basis for a Category 1A classification there
  must be reliable evidence of an adverse effect on reproduction in humans. Evidence used for classifi-
  cation shall ideally be from well conducted epidemiological studies which include the use of appro-
  priate controls, balanced assessment, and due consideration of bias or confounding factors. Less
  rigorous data from studies in humans shall be supplemented with adequate data from studies in
  experimental animals and classification in Category 1B shall be considered.
6 Hydroxyurea
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<pre>3.7.2.3         Weight of evidence
3.7.2.3.1       Classification as a reproductive toxicant is made on the basis of an assessment of the
total weight of evidence, see section 1.1.1. This means that all available information that bears on the
determination of reproductive toxicity is considered together, such as epidemiological studies and
case reports in humans and specific reproduction studies along with sub-chronic, chronic and special
study results in animals that provide relevant information regarding toxicity to reproductive and
related endocrine organs. Evaluation of substances chemically related to the substance under study
may also be included, particularly when information on the substance is scarce. The weight given to
the available evidence will be influenced by factors such as the quality of the studies, consistency of
results, nature and severity of effects, the presence of maternal toxicity in experimental animal stud-
ies, level of statistical significance for inter-group differences, number of endpoints affected, rele-
vance of route of administration to humans and freedom from bias. Both positive and negative results
are assembled together into a weight of evidence determination. A single, positive study performed
according to good scientific principles and with statistically or biologically significant positive results
may justify classification (see also 3.7.2.2.3).
3.7.2.3.2       Toxicokinetic studies in animals and humans, site of action and mechanism or mode of
action study results may provide relevant information which reduces or increases concerns about the
hazard to human health. If it is conclusively demonstrated that the clearly identified mechanism or
mode of action has no relevance for humans or when the toxicokinetic differences are so marked that
it is certain that the hazardous property will not be expressed in humans then a substance which pro-
duces an adverse effect on reproduction in experimental animals should not be classified.
3.7.2.3.3       If, in some reproductive toxicity studies in experimental animals the only effects
recorded are considered to be of low or minimal toxicological significance, classification may not
necessarily be the outcome. These effects include small changes in semen parameters or in the inci-
dence of spontaneous defects in the foetus, small changes in the proportions of common foetal vari-
ants such as are observed in skeletal examinations, or in foetal weights, or small differences in
postnatal developmental assessments.
3.7.2.3.4       Data from animal studies ideally shall provide clear evidence of specific reproductive
toxicity in the absence of other systemic toxic effects. However, if developmental toxicity occurs
together with other toxic effects in the dam, the potential influence of the generalised adverse effects
shall be assessed to the extent possible. The preferred approach is to consider adverse effects in the
embryo/foetus first, and then evaluate maternal toxicity, along with any other factors which are likely
to have influenced these effects, as part of the weight of evidence. In general, developmental effects
that are observed at maternally toxic doses shall not be automatically discounted. Discounting devel-
Regulation (EC) 1272/2008 of the European Community                                                         47
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<pre>  opmental effects that are observed at maternally toxic doses can only be done on a case-by-case basis
  when a causal relationship is established or refuted.
  3.7.2.3.5      If appropriate information is available it is important to try to determine whether devel-
  opmental toxicity is due to a specific maternally mediated mechanism or to a non-specific secondary
  mechanism, like maternal stress and the disruption of homeostasis. Generally, the presence of mater-
  nal toxicity shall not be used to negate findings of embryo/foetal effects, unless it can be clearly dem-
  onstrated that the effects are secondary non-specific effects. This is especially the case when the
  effects in the offspring are significant, e.g. irreversible effects such as structural malformations. In
  some situations it can be assumed that reproductive toxicity is due to a secondary consequence of
  maternal toxicity and discount the effects, if the substance is so toxic that dams fail to thrive and there
  is severe inanition, they are incapable of nursing pups; or they are prostrate or dying.
  3.7.2.4        Maternal toxicity
  3.7.2.4.1      Development of the offspring throughout gestation and during the early postnatal stages
  can be influenced by toxic effects in the mother either through non-specific mechanisms related to
  stress and the disruption of maternal homeostasis, or by specific maternally-mediated mechanisms. In
  the interpretation of the developmental outcome to decide classification for developmental effects it
  is important to consider the possible influence of maternal toxicity. This is a complex issue because
  of uncertainties surrounding the relationship between maternal toxicity and developmental outcome.
  Expert judgement and a weight of evidence approach, using all available studies, shall be used to
  determine the degree of influence that shall be attributed to maternal toxicity when interpreting the
  criteria for classification for developmental effects. The adverse effects in the embryo/foetus shall be
  first considered, and then maternal toxicity, along with any other factors which are likely to have
  influenced these effects, as weight of evidence, to help reach a conclusion about classification.
  3.7.2.4.2      Based on pragmatic observation, maternal toxicity may, depending on severity, influ-
  ence development via non-specific secondary mechanisms, producing effects such as depressed foe-
  tal weight, retarded ossification, and possibly resorptions and certain malformations in some strains
  of certain species. However, the limited number of studies which have investigated the relationship
  between developmental effects and general maternal toxicity have failed to demonstrate a consistent,
  reproducible relationship across species. Developmental effects which occur even in the presence of
  maternal toxicity are considered to be evidence of developmental toxicity, unless it can be unequivo-
  cally demonstrated on a case-by-case basis that the developmental effects are secondary to maternal
  toxicity. Moreover, classification shall be considered where there is a significant toxic effect in the
  offspring, e.g. irreversible effects such as structural malformations, embryo/foetal lethality, signifi-
  cant post-natal functional deficiencies.
8 Hydroxyurea
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<pre>3.7.2.4.3      Classification shall not automatically be discounted for substances that produce devel-
opmental toxicity only in association with maternal toxicity, even if a specific maternally-mediated
mechanism has been demonstrated. In such a case, classification in Category 2 may be considered
more appropriate than Category 1. However, when a substance is so toxic that maternal death or
severe inanition results, or the dams are prostrate and incapable of nursing the pups, it is reasonable
to assume that developmental toxicity is produced solely as a secondary consequence of maternal
toxicity and discount the developmental effects. Classification is not necessarily the outcome in the
case of minor developmental changes, when there is only a small reduction in foetal/pup body weight
or retardation of ossification when seen in association with maternal toxicity.
3.7.2.4.4      Some of the end points used to assess maternal effects are provided below. Data on
these end points, if available, need to be evaluated in light of their statistical or biological signifi-
cance and dose response relationship.
Maternal mortality:
an increased incidence of mortality among the treated dams over the controls shall be considered evi-
dence of maternal toxicity if the increase occurs in a dose-related manner and can be attributed to the
systemic toxicity of the test material. Maternal mortality greater than 10 % is considered excessive
and the data for that dose level shall not normally be considered for further evaluation.
Mating index
(no. animals with seminal plugs or sperm/no. mated × 100) (*)
Fertility index
(no. animals with implants/no. of matings × 100)
Gestation length
(if allowed to deliver)
Body weight and body weight change:
Consideration of the maternal body weight change and/or adjusted (corrected) maternal body weight
shall be included in the evaluation of maternal toxicity whenever such data are available. The calcula-
() It is recognised that the Mating index and the Fertility index can also be affected by the male.
Regulation (EC) 1272/2008 of the European Community                                                       49
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<pre>  tion of an adjusted (corrected) mean maternal body weight change, which is the difference between
  the initial and terminal body weight minus the gravid uterine weight (or alternatively, the sum of the
  weights of the foetuses), may indicate whether the effect is maternal or intrauterine. In rabbits, the
  body weight gain may not be useful indicators of maternal toxicity because of normal fluctuations in
  body weight during pregnancy.
  Food and water consumption (if relevant):
  The observation of a significant decrease in the average food or water consumption in treated dams
  compared to the control group is useful in evaluating maternal toxicity, particularly when the test
  material is administered in the diet or drinking water. Changes in food or water consumption need to
  be evaluated in conjunction with maternal body weights when determining if the effects noted are
  reflective of maternal toxicity or more simply, unpalatability of the test material in feed or water.
  Clinical evaluations (including clinical signs, markers, haematology and clinical chemistry studies):
  The observation of increased incidence of significant clinical signs of toxicity in treated dams relative
  to the control group is useful in evaluating maternal toxicity. If this is to be used as the basis for the
  assessment of maternal toxicity, the types, incidence, degree and duration of clinical signs shall be
  reported in the study. Clinical signs of maternal intoxication include: coma, prostration, hyperactivity,
  loss of righting reflex, ataxia, or laboured breathing.
  Post-mortem data:
  Increased incidence and/or severity of post-mortem findings may be indicative of maternal toxicity.
  This can include gross or microscopic pathological findings or organ weight data, including absolute
  organ weight, organ-to-body weight ratio, or organ-to-brain weight ratio. When supported by find-
  ings of adverse histopathological effects in the affected organ(s), the observation of a significant
  change in the average weight of suspected target organ(s) of treated dams, compared to those in the
  control group, may be considered evidence of maternal toxicity.
  3.7.2.5        Animal and experimental data
  3.7.2.5.1      A number of internationally accepted test methods are available; these include methods
  for developmental toxicity testing (e.g. OECD Test Guideline 414), and methods for one or two-gen-
  eration toxicity testing (e.g. OECD Test Guidelines 415, 416).
  3.7.2.5.2      Results obtained from Screening Tests (e.g. OECD Guidelines 421 — Reproduction/
  Developmental Toxicity Screening Test, and 422 — Combined Repeated Dose Toxicity Study with
0 Hydroxyurea
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<pre>Reproduction/Development Toxicity Screening Test) can also be used to justify classification,
although it is recognised that the quality of this evidence is less reliable than that obtained through
full studies.
3.7.2.5.3      Adverse effects or changes, seen in short- or long-term repeated dose toxicity studies,
which are judged likely to impair reproductive function and which occur in the absence of significant
generalised toxicity, may be used as a basis for classification, e.g. histopathological changes in the
gonads.
3.7.2.5.4      Evidence from in vitro assays, or non-mammalian tests, and from analogous substances
using structure-activity relationship (SAR), can contribute to the procedure for classification. In all
cases of this nature, expert judgement must be used to assess the adequacy of the data. Inadequate
data shall not be used as a primary support for classification.
3.7.2.5.5      It is preferable that animal studies are conducted using appropriate routes of administra-
tion which relate to the potential route of human exposure. However, in practice, reproductive toxic-
ity studies are commonly conducted using the oral route, and such studies will normally be suitable
for evaluating the hazardous properties of the substance with respect to reproductive toxicity. How-
ever, if it can be conclusively demonstrated that the clearly identified mechanism or mode of action
has no relevance for humans or when the toxicokinetic differences are so marked that it is certain that
the hazardous property will not be expressed in humans then a substance which produces an adverse
effect on reproduction in experimental animals shall not be classified.
3.7.2.5.6      Studies involving routes of administration such as intravenous or intraperitoneal injec-
tion, which result in exposure of the reproductive organs to unrealistically high levels of the test sub-
stance, or elicit local damage to the reproductive organs, including irritation, must be interpreted with
extreme caution and on their own are not normally the basis for classification.
3.7.2.5.7      There is general agreement about the concept of a limit dose, above which the produc-
tion of an adverse effect is considered to be outside the criteria which lead to classification, but not
regarding the inclusion within the criteria of a specific dose as a limit dose. However, some guide-
lines for test methods, specify a limit dose, others qualify the limit dose with a statement that higher
doses may be necessary if anticipated human exposure is sufficiently high that an adequate margin of
exposure is not achieved. Also, due to species differences in toxicokinetics, establishing a specific
limit dose may not be adequate for situations where humans are more sensitive than the animal
model.
3.7.2.5.8      In principle, adverse effects on reproduction seen only at very high dose levels in animal
studies (for example doses that induce prostration, severe inappetence, excessive mortality) would
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<pre>              not normally lead to classification, unless other information is available, e.g. toxicokinetics informa-
              tion indicating that humans may be more susceptible than animals, to suggest that classification is
              appropriate. Please also refer to the section on maternal toxicity (3.7.2.4) for further guidance in this
              area.
              3.7.2.5.9      However, specification of the actual ‘limit dose’ will depend upon the test method that
              has been employed to provide the test results, e.g. in the OECD Test Guideline for repeated dose tox-
              icity studies by the oral route, an upper dose of 1 000 mg/kg has been recommended as a limit dose,
              unless expected human response indicates the need for a higher dose level.
              3.7.3          Classification criteria for mixtures
              3.7.3.1        Classification of mixtures when data are available for all ingredients or only for some
              ingredients of the mixture
              3.7.3.1.1      The mixture shall be classified as a reproductive toxicant when at least one ingredient
              has been classified as a Category 1A, Category 1B or Category 2 reproductive toxicant and is present
              at or above the appropriate generic concentration limit as shown in Table 3.7.2 for Category 1A, Cat-
              egory 1B and Category 2 respectively.
              3.7.3.1.2      The mixture shall be classified for effects on or via lactation when at least one ingredi-
              ent has been classified for effects on or via lactation and is present at or above the appropriate generic
              concentration limit as shown in Table 3.7.2 for the additional category for effects on or via lactation.
 able 3.7.2 Generic concentration limits of ingredients of a mixture classified as reproduction toxicants or foreffects on or via
actation that trigger classification of the mixture.
 ngredient classified as:     Generic concentration limits triggering classification of a mixture as:
                              Category 1A                Category 1B               Category 2                Additional category
                              reproductive toxicant reproductive toxicant reproductive toxicant for effects on or via l
                                                                                                             actation
Category 1A                   ≥ 0,3 %
 eproductive toxicant         [Note 1]
  ategory 1B                                             ≥ 0,3 %
eproductive toxicant                                     [Note 1]
Category 2                                                                         ≥ 3,0 %
 eproductive toxicant                                                              [Note 1]
Additional category                                                                                          ≥ 0,3 %
or effects on or via                                                                                         [Note 1]
actation
  ote The concentration limits in the table above apply to solids and liquids (w/w units) as well as gases (v/v units).
  ote 1 If a Category 1 or Category 2 reproductive toxicant or a substance classified for effects on or via lactation is present in
he mixture as an ingredient at a concentration above 0,1 %, a SDS shall be available for the mixture upon request.
  2           Hydroxyurea
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<pre>3.7.3.2        Classification of mixtures when data are available for the complete mixture
3.7.3.2.1      Classification of mixtures will be based on the available test data for the individual
ingredients of the mixture using concentration limits for the ingredients of the mixture. On a case-by-
case basis, test data on mixtures may be used for classification when demonstrating effects that have
not been established from the evaluation based on the individual components. In such cases, the test
results for the mixture as a whole must be shown to be conclusive taking into account dose and other
factors such as duration, observations, sensitivity and statistical analysis of reproduction test systems.
Adequate documentation supporting the classification shall be retained and made available for review
upon request.
3.7.3.3        Classification of mixtures when data are not available for the complete mixture:
               bridging principles
3.7.3.3.1      Subject to paragraph 3.7.3.2.1, where the mixture itself has not been tested to determine
its reproductive toxicity, but there are sufficient data on the individual ingredients and similar tested
mixtures to adequately characterise the hazards of the mixture, these data shall be used in accordance
with the applicable bridging rules set out in section 1.1.3.
3.7.4          Hazard Communication
3.7.4.1        Label elements shall be used for substances or mixtures meeting the criteria for
               classification in this hazard class in accordance with Table 3.7.3
Regulation (EC) 1272/2008 of the European Community                                                        53
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<pre> able 3.7.3 Label elements for reproductive toxicity.
 lassification              Category 1A or Category 1B              Category 2                              Additional category
                                                                                                            for effects on or via
                                                                                                            lactation
GHS Pictograms                                                                                              No pictogram
 ignal Word                 Danger                                  Warning                                 No signal word
Hazard Statement            H360: May damage fertility or the       H361: Suspected of damaging fertil-     H362: May cause
                            unborn child (state specific effect if  ity or the unborn child (state specific harm to breast-fed
                            known)(state route of exposure if it is effect if known) (state route of expo-  children.
                            conclusively proven that no other       sure if it is conclusively proven that
                            routes of exposure cause the hazard)    no other routes of exposure cause the
                                                                    hazard)
 recautionary Statement     P201                                    P201                                    P201
 revention                  P202                                    P202                                    P260
                            P281                                    P281                                    P263
                                                                                                            P264
                                                                                                            P270
 recautionary Statement     P308 + P313                             P308 + P313                             P308 + P313
 esponse
 recautionary Statement     P405                                    P405
 torage
 recautionary Statement     P501                                    P501
Disposal
 4            Hydroxyurea
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<pre>nnex E
     Additional considerations to
     Regulation (EC) 1272/2008
     The classification and labelling of substances is performed according to the
     guidelines of the European Union (Regulation (EC)1272/2008) presented in
     Annex D. The classification of compounds is ultimately dependent on an
     integrated assessment of the nature of all parental and developmental effects
     observed, their specificity and adversity, and the dosages at which the various
     effects occur. The guideline necessarily leaves room for interpretation, dependent
     on the specific data set under consideration. In the process of using the
     regulation, the Committee has agreed upon a number of additional
     considerations:
     • if there is sufficient evidence to establish a causal relationship between
         human exposure to the substance and impaired fertility or subsequent
         developmental toxic effects in the offspring, the compound will be classified
         in category 1A, irrespective of the general toxic effects (see Annex D,
         3.7.2.2.1.)
     • adverse effects in a reproductive study, occurring without reporting the
         parental or maternal toxicity, may lead to a classification other than category
         1B, when the effects occur at dose levels which cause severe toxicity in
         general toxicity studies
     • clear adverse reproductive effects will not be disregarded on the basis of
         reversibility per se
     Additional considerations to Regulation (EC) 1272/2008                              55
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<pre>  •   the Committee dot not only use guideline studies (studies performed
      according to OECD* standard protocols) for the classification of compounds,
      but non-guideline studies are taken into consideration as well.
   Organisation for Economic Cooperation and Development.
6 Hydroxyurea
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<pre>  nnex         F
               Fertility and developmental toxicity
               studies
 able 1 Fertility studies with hydroxyurea in animals.
 uthors            species          experimental       dose/route        general toxicity effects on reproductive organs/
                                    period/ design                                        effects on reproduction
Male fertility
Mecklenburg        Holtzmann rats 70 d, followed by    0, 3 mg/mL of decreased bw at      germinal cell depletion from 14
 t al. (1975)      (n=90; controls: a 30-d recovery    drinking water the end of the      days after the start of the
                   n=18; mature) period                (ca. 300 mg/kg treatment           treatment, severity increasing with
                                                       bw/d, assuming                     the treatment duration; effect
                                                       a water intake                     ascribed to the arrest of DNA
                                                       of 100 mL/kg                       synthesis by hydroxyurea.
                                                       bw/d)                              after cessation of the treatment,
                                                                                          the germinal epithelium re-
                                                                                          established in most of the
                                                                                          seminiferous tubules
  ich/De Kretser Sprague            3 mo               0, 3 mg/mL of no data presented absolute testis wt (g): 1.65±0.3
1977)              Dawley rats                         drinking water                     (controls), 0.66±0.3**
                   (n=10/ group;                       (ca. 300 mg/kg                     caput epididymal wt (mg):
                   60 d old)                           bw/d, assuming                     178±10, 88±7**
                                                       a water intake of                  serum LH levels (ng/mL):
                                                       100 mL/kg                          1.3±0.1, 2.1±0.1**
                                                       bw/d)                              serum FSH levels (ng/mL):
                                                                                          378±27, 751±28**
                                                                                          destruction of the seminiferous
                                                                                          epithelium
               Fertility and developmental toxicity studies                                                                57
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<pre> icsor/ Ginsberg CF1 mice (n=3- 5 d                    0, 125, 250, 500, decreased terminal mean absolute testis wt (g): 277,
1980)            4/ group; 12-16 sacrifice: 35 d       1000 mg/kg        bw at 1000 mg/kg 223, 242, 163*, 129*, resp.
                 wk old)          after treatment      bw/d; ip          bw/d               number of sperm (x106/mL):
                                                                                            124.8, 77.6*, 65.6*, 49.6*, 26.4*
                                                                                            sperm motility (%): 46.6, 34.8,
                                                                                            38.7, 30.7*, 18.9*
 venson/Jost     (C57B/6J x       5d                   0, 25, 50, 100, no effect on bw      at 8 d: statistically significantly
1993)            C3H/HeJ F1)      sacrifice: 8, 29 d 200, 400, 500                          decreased absolute testis wt at
                 mice (n≥6/       after treatment      mg/kg bw/d; ip                       doses ≥400 mg/kg bw/d; altered
                 group; 13-15                                                               testicular cell population ratios at
                 wk old)                                                                    doses ≥100 mg/kg bw/d
                                                                                            at 29 d: statistically significantly
                                                                                            decreased testis wt, altered
                                                                                            testicular cell population ratios at
                                                                                            doses ≥50 mg/kg bw/d
                                                                                            Evenson/Jost concluded that
                                                                                            hydroxyurea inhibits DNA
                                                                                            synthesis, causing maturation
                                                                                            depletion of pachytene
                                                                                            spermatocytes and, subsequently,
                                                                                            depletion of meiotic daughter cells
                                                                                            and differentiated cell types
                                                                                            leading to mature sperm
Wiger et al.     (B6C3/F1/        5d                   0, 200 mg/kg      bw: no effect      atrophy of seminiferous tubules on
1995)            BOM M) mice      sacrifice: 0, 5, 10, bw/d; ip          during treatment; post-treatment d 5 and 10
                 (n=5/group; 6-8  27, 33, 45 d after                     decreased bw gain decreased absolute and relative
                 wk old)          treatment                              during post-       testis wt (40-45% lower than
                                                                         treatment d 0-5    controls) on post-treatment d 27
                                                                         and at d 45;       and 33
                                                                         during treatment reduced proportion of the various
                                                                         period, animals    spermatid stages and altered
                                                                         showed signs of    sperm chromatin structure
                                                                         weakness           Wiger et al. discussed that
                                                                                            inhibition of DNA synthesis in the
                                                                                            testis was the primary cause of
                                                                                            these findings
 hin et al.      ICR mice (n=3/   1d                   0, 100, 200, 400 no effect on bw     dose-dependent increases in
1999)            group; 6-7 wk    sacrifice: 0, 4, 8, mg/kg bw; ip       and testis wt      numbers of apoptotic cells and in
                 old)             12, 24, 48 h after                                        levels of DNA fragmentation
                                  treatment                                                 continuous increases in numbers
                                                                                            of apoptotic cells, peaking at 12 h
                                                                                            and reaching control levels by 48
                                                                                            h
ones et al.      transgenic       28, 56 d             0, 25 mg/kg       no effect on bw    statistically significantly
2009)            sickle cell mice                      bw/d; gavage                         decreased absolute testis wt on d
                 (n=6/group/                                                                28 and 56
                 stage; adult)                                                              on d 56: 52% shrinkage of testis
                                                                                            dimensions; atrophic degeneration
                                                                                            in the seminiferous tubules; 25%
                                                                                            shrinkage of epididymides; 69%
                                                                                            decrease in stored sperm density;
                                                                                            95% decrease in sperm motility
 8           Hydroxyurea
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<pre> ingh/Taylor     inbred PD4      5d                  0, 10, 50, 250 bw: initial increase progressively decreased sperm
1981)            strain hamsters sacrifice: 1, 4 and mg/kg bw/d; ip to roughly 126,      number with exposure to
                 (n=6-9/group;   10 wk after                        114, 123% of         increasing dose levels of
                 10-12 wk old)   treatment                          controls, resp., at  hydroxyurea occurred, which was
                                                                    post-treatment wk    already evident at 10 mg/kg bw/d.
                                                                    1, followed by       no sperm abnormalities
                                                                    gradual decrease
                                                                    to roughly 90, 86,
                                                                    92% of controls,
                                                                    resp., at wk 12
 emale fertility
 ampson et al.   C57BL/6J mice 28 d                  0, 30 mg/kg    no data presented decreased ovary wt*, ovulation
2010)            (n=20/group)    sacrifice: at       bw/d; gavage                        rates*, circulating E2 levels*
                                 treatment d 25, 26,                                     number of embryos developing to
                                 28                                                      the blastocyst stage: 32%
                                 d 23: ip injection                                      (controls), 60%*
                                 of PSMG to
                                 induce
                                 folliculogenesis
                                 d 25: measurement
                                 of E2 levels (n=5/
                                 group);
                                 ip injection of
                                 hCG and
                                 subsequent mating
                                 (n=15/group)
                                 about 15 h post
                                 hCG:
                                 determination
                                 ovulation rate
                                 (n=5/group)
                                 about 27 h post
                                 hCG: examination
                                 ovaries/embryos
 w=body weight; d=day(s); E2=oestradiol-17ß; h=hour(s); hCG=human chorionic gonadotropin; ip=intraperitoneal;
mo=month(s); PSMG=pregnant mare serum; wk=week(s); wt=weight(s); *: p<0.05; **: p<0.001.
             Fertility and developmental toxicity studies                                                               59
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<pre> able 2 Developmental toxicity studies with hydroxyurea in female animals.
 uthors         species      experimental dose/route      general       developmental toxicity
                             period/design                toxicity
Aliverti et al. Sprague-     gd 6-15         0, 50, 150,  no data on    mean foetal bw (g): 5.34±0.31, 5.31±0.41,
1980)           Dawley rats sacrifice: gd 300, 450 mg/ maternal         5.08±0.63, 3.85±0.50, 3.28±0.76, resp.
                (n=8-10/     21; foetuses kg bw/d; oral toxicity        number of resorptions+dead foetuses: 21, 9, 7, 52, 79
                treatment    examined for                 presented     post-implantation loss (%): 5.7, 6.9, 5.5, 50.1, 69.6
                group; n=27 external,                                   number of viable foetuses: 375, 123, 121, 51, 30
                controls)    visceral,                                  number of foetuses with external abnormalities: 0/
                             skeletal                                   375, 0/123, 0/121, 4/51, 12/30; with visceral
                             abnormalities                              abnormalities: 0/196, 0/63, 0/63, 6/30, 15/15; with
                                                                        skeletal abnormalities: 0/179, 0/60, 0/58, 3/21, 11/14
                                                                        most commonly observed abnormalities at 300 and
                                                                        450 mg/kg bw/d
                                                                        external: cranial 0/51, 3/30, resp.; facial 1/51, 4/30;
                                                                        craniofacial dysgenia 3/51, 2/30; absent pinnae 0/51,
                                                                        3/30; amelia/ phocomelia 0/51, 4/30; limb
                                                                        malrotation 0/51, 5/30
                                                                        visceral: hydrocephalus 5/30, 7/16; eye 4/30, 13/16
                                                                        skeletal: markedly reduced orbital bones 0/21, 5/14;
                                                                        reduced/absent/misshapen mandula 2/21, 6/14;
                                                                        vertebrae/sternebrae/ ribs dysgenesia 1/21, 10/14
 oll/Bär        NMRI mice gd 6-17            0, 200, 400, no data on    600 and 800 mg/kg bw/d: complete resorption or
1969)           (number      dams allowed 600, 800 mg/ maternal         abortion
                exposed: see to litter; pups kg bw/d      toxicity      number of pups: 154, 260, 66 at 0, 200, 400 mg/kg
                ‘dose’)      examined for (n=18, 29, 9, presented;      bw/d, resp.
                             external        unspecified, stated to be number of pups/dam: 8.6, 9.0, 7.3, resp.
                             malformation unspecified, ‘relatively      % of stillbirths: 3.3, 8.1, 12.1
                             s, viability at resp.);      non-toxic for pup mortality until pnd 21 (%): 9.7, 20.1 (p=0.0003),
                             birth, bw       gavage       the maternal 24.3 (p=0.0001)
                             until weaning                animals’      mean pup bw at birth: 1.48±0.01, 1.39±0.01
                                                                        (p=0.0002), 1.30±0.02 (p=0.0002)
                                                                        mean pup wt at weaning: 9.60±0.18, 9.39±0.13,
                                                                        9.37±0.22
                             gd 6-17         0, 400, 800                number of implantations: 217, 200, 150, resp.
                             sacrifice: gd mg/kg bw                     total number of resorptions (%): 10.1, 33.5
                             18; dams        (n=21, 19,                 p=0.0007), 94.7; of early resorptions (%): 8.7, 11.5,
                             examined for 16, resp.);                   36.0; of mid-term resorptions (%): 0.9, 7.5, 57.4; of
                             number of       gavage                     late resorptions: 0.5, 4.5, 1.3
                             implantation                               mean foetal bw (g): 1.17±0.01, 0.85±0.02
                             sites; foetuses                            (p<0.0002), no data
                             for skeletal                               abnormalities observed at 0 and 400 mg/kg bw (%):
                             abnormalities                              sternebrae defects: 1.1, 17.1, resp.; encepalocele: 0.5,
                                                                        12.5; missing/shortened tail: 0, 2.0; costal fusion:
                                                                        1.1, 5.3; cervical vertebrae fusion: 1.1, 5.9; thoracic
                                                                        vertebrae defects: 0, 7.9; lumbar vertebrae defects: 0,
                                                                        1.3
                                                                        800 mg/kg bw: in the few surviving foetuses, no
                                                                        malformations but severe retardation of development
 0             Hydroxyurea
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<pre>               gd 6-7          0, 600, 1200   number of implantations: 217, 188, 139
               sacrifice: gd   mg/kg bw/d     total number of resorptions (%): 10.1, 56.4, 72.7; of
               18              (n=21, 18,     early resorptions (%): 8.7, 48.4, 38.8; of mid-term
               dams            12, resp.)     resorptions (%): 0.9, 7.5, 29.4; of late resorptions:
               examined for                   0.5, 0.5, 4.5
               number of                      mean foetal bw (g): 1.17±0.01, 1.00±0.02, 0.88±0.02
               implantation                   abnormalities observed (%):
               sites; foetuses                cleft palate: 0.5, 3.0, 23.7; sternebrae defects: 1.1,
               for skeletal                   16.7, 47.4; encephalocele: 0.5, 0, 15.8; missing/
               abnormalities                  shortened tail: 0, 0, 2.6; costal fusion: 1.1, 1.5, 2.6;
                                              cervical vertebrae fusion: 1.1, 6.1, 7.9; thoracic
                                              vertebrae defects: 0, 0, 15.8; lumbar vertebrae
                                              defects: 0, 0, 10.5
               gd 10-11        0, 600, 1200   number of implantations: 217, 321, 222
               sacrifice: gd   mg/kg bw/d      total number of resorptions (%): 10.1, 8.1, 45.1; of
               18              (n=21, 31,     early resorptions (%): 8.7, 10.9, 6.8; of mid-term
               dams            23, resp.)     resorptions (%): 0.9, 6.9, 36.5; of late resorptions:
               examined for                   0.5, 0.3, 1.8
               number of                      mean foetal bw (g): 1.17±0.01, 1.08±0.01, 1.00±0.01
               implantation                   abnormalities observed (%):
               sites; foetuses                cleft palate: 0.5, 8.0, 28.7; sternebrae defects: 1.1,
               for skeletal                   4.2, 25.4; encephalocele: 0.5, 0.4, 9.8; missing/
               abnormalities                  shortened tail: 0, 8.4, 23.8; costal fusion: 1.1, 1.2,
                                              4.1; cervical vertebrae fusion: 1.1, 0, 4.9; thoracic
                                              vertebrae defects: 0, 13.3, 55.5; lumbar vertebrae
                                              defects: 0, 5.3, 27.0; sacral vertebrae defects: 0, 0,
                                              13.2; hexadactyly hind limb: 0, 0, 2.4; syndactyly
                                              forelimb: 0, 0, 3.2; syndactyly hind limb: 0, 0, 2.4;
                                              tibia aplasia: 0, 0, 9.8; shortened tibia: 0, 0, 4.9
               gd 10           0, 600, 900,   number of implantations: 217, 333, 117, 182
               sacrifice: gd   1200 mg/kg     total number of resorptions (%): 10.1, 12.9, 13.7,
               18              bw (n=21, 32,  25.3; of early resorptions (%): 8.7, 8.7, 8.6, 3.3; of
               dams            13, 21, resp.) mid-term resorptions (%): 0.9, 3.9, 3.4, 22.0; of late
               examined for                   resorptions: 0.5, 0.3, 1.7, 0
               number of                      mean foetal bw (g): 1.17±0.01, 1.15±0.01,
               implantation                   1.09±0.01, 1.03±0.02
               sites; foetuses                abnormalities observed (%):
               for skeletal                   cleft palate: 0.5, 1.7, 6.0, 19.3; sternebrae defects:
               abnormalities                  1.1, 4.7, 13.9, 25.9; encephalocele: 0.5, 0.4, 0, 0;
                                              missing/shortened tail: 0, 0, 13.9, 23.0; costal fusion:
                                              1.1, 0.4, 2.0, 2.2; cervical vertebrae fusion: 1.1, 0.4,
                                              0, 0; thoracic vertebrae defects: 0, 2.1, 26.7, 45.1;
                                              lumbar vertebrae defects: 0, 0, 6.0, 32.5; sacral
                                              vertebrae defects: 0, 0, 0, 25.1; hexadactyly hind
                                              limb: 0, 0, 2.4; syndactyly hind limb: 0, 0, 0, 6.7;
                                              tibia aplasia: 0, 0, 0, 7.4; shortened tibia: 0, 0, 4.9;
                                              ulna aplasia: 0, 0, 0, 1.5
Fertility and developmental toxicity studies                                                         61
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<pre>                         gd 11           0, 600, 900,                  number of implantations: 217, 170, 264, 208
                         sacrifice:      1200 mg/kg                    total number of resorptions (%): 10.1, 7.1, 12.5, 13.9;
                         gd 18           bw (n=21, 17,                 of early resorptions (%): 8.7, 4.7, 9.5, 7.7; of mid-
                         dams            23, 30, resp.)                term resorptions (%): 0.9, 1.8, 2.3, 4.8; of late
                         examined for                                  resorptions: 0.5, 0.6, 0.7, 1.4
                         number of                                     mean foetal bw (g): 1.17±0.01, 1.11±0.01,
                         implantation                                  1.09±0.01, 0.98±0.01
                         sites; foetuses                               abnormalities observed (%):
                         for skeletal                                  cleft palate: 0.5, 0, 0.9, 16.1; sternebrae defects: 1.1,
                         abnormalities                                 5.0, 3.9, 12.8; encephalocele: 0.5, 1.5, 0, 0.6;
                                                                       missing/shortened tail: 0, 0, 0, 5.6; thoracic vertebrae
                                                                       defects: 0, 0, 0, 17.3; lumbar vertebrae defects: 0,
                                                                       0.7, 0, 5.3; hexadactyly hind limb: 0, 0, 3.9, 5.6;
                                                                       syndactyly forelimb: 0, 0, 9.5, 20.1; syndactyly hind
                                                                       limb: 0, 0, 0.9, 10.6; shortened tibia: 0, 0, 0, 1.2
Khera (1979) cats (n=17/ gd 10-22        0, 50, 100     no maternal number of cats aborted: 2/17, 1/17, 1/17, resp.
             group)      sacrifice: gd mg/kg bw/d; toxicity at 50 number cats killed: 0/17, 0/17, 2/17
                         43; foetuses oral              mg/kg bw/d number of cats not pregnant: 5/17, 4/17, 10/17
                         examined for (capsules)        100 mg/kg      number of cast having resorptions and no live
                         external,                      bw/d:          foetuses: 3/17, 3/117, 3/17
                         visceral,                      decreased bw number of cats with live foetuses: 7/17, 8/17, 1/17
                         skeletal                       gain; only     total number of live foetuses: 40, 38, 2; of dead
                         abnormalities                  one cat        foetuses: 3, 0, 0; of resorptions: 20, 16, 13*
                                                        survived until mean foetal wt (g): 11.8±0.3, 11.3±0.4, 9.7±0.9*
                                                        necropsy       number of litters with abnormalities/number
                                                                       examined: 2/7, 5/8, 1/1; of foetuses with
                                                                       abnormalities/number examined: 4/40, 11/38, 1/2; of
                                                                       foetuses with visceral abnormalities/number
                                                                       examined: 1/19, 6/17, 1/1; of foetuses with skeletal
                                                                       abnormalities/number examined: 3/21, 5/21, 0/1
                                                                       abnormalities observed (number of foetuses affected:
                                                                       controls: forked tongue and buccal cavity occupied
                                                                       by undifferentiated mass (1), fused ribs (1),
                                                                       sternebrae: distorded form (1), sternebrae: delayed
                                                                       ossification (1); 50 mg/kg bw: cleft palate (3), cleft
                                                                       palate, exencephaly, microcephaly, split eye lids,
                                                                       microphthalmia (1), cleft palate and lip, rudimentary
                                                                       kidneys, ectrodactyly, hind limb micromelia,
                                                                       taillessness (1), cleft lip and nose (1), bilateral
                                                                       microphthalmia (2), generalized oedema (1), fused
                                                                       ribs/ vertebrae (1), delayed ossification of calvarium
                                                                       (1), delayed ossification of digits/ sternum 91); 100
                                                                       mg/kg bw: cyclopia (single medially located orbit
                                                                       containing globe, rudimentary nose and mandible (1)
 2          Hydroxyurea
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<pre> arr/     two stocks of   at one of 8 6- 0, 200 (at gd  no data on ‘A’ stock:
 eaudoin  Wistar rats     intervals      9.0), 225 at   maternal   resorptions (%): 10.6, 11.5, 9.4, 4.2, 7.3, 7.7, 11.1,
1981)     (n=8-10         from gd 9.0    gd 9.25), 250  toxicity   8.8, at 200, 225, 250, 275, 300, 325, 350, 375 mg/kg
          litters/group); to 10.75       (at gd 9.5),   presented  bw, resp. (no controls included , because ‘purpose of
                          sacrifice:     275 at gd                 the study was not to establish the teratogenicity of
          ‘A’ stock:      gd 21          9.75), 300 (at            hydroxyurea but only to determine if there was a
          colony                         gd 10.0), 325             relation between the time of dosage and fetal
          maintained                     (at gd 10.25),            outcome.’)
          by one of the                  350 (at gd                mean foetal wt (g): 4.91±0.06, 5.09±0.04, 5.26±0.05,
          authors                        10.5), 375 (at            5.51±0.05, 5.39±0.04, 5.15±0.04, 5.35±0.05,
          derived from                   gd 10.75)                 5.16±0.05
          Wistar stock                   mg/kg bw; ip              mean placental wt (g): 438±6, 497±8, 460±8, 465±7,
          originally                                               458±5, 432±8, 481±11, 431±6
          from Albino                                              malformations (%): 3.2, 71.8, 95.0, 71.3, 64.8, 67.6,
          Farms (Red                                               62.0, 62.5, 41.2
          Bank NJ,                                                 % of most commonly (i.e >10% in either ‘A’ or ’B’
          USA)                                                     stock (see below) observed abnormalities:
          ‘B’ stock:                                               anophthalmia/microphthalmia: 55.5, 87.0, 51.3, 53.8,
          CFN Wistar                                               35.3, 5.6, 1.7, 0; hydrocephaly: 13.6, 30.0, 18.3, 6.6,
          purchased                                                1.0, 1.9, 3.3, 0; encephalocele: 3.6, 7.0, 3.5, 0, 0, 0, 0,
          from                                                     0; exencephaly: 8.2, 4.0, 2.6, 0, 0, 0, 0, 0; ear
          Carworth                                                 dysplasia: 6.4, 3.0, 1.7, 0, 0, 0.9, 0, 0; maxillary
          (New York                                                hypoplasia: 11.8, 12.0, 3.5, 0, 0, 0, 0, 0; facial
          NY, USA)                                                 asymmetry: 5.5, 2.0, 1.7, 0, 0, 0, 0, 0; pointed
                                                                   mandible: 3.6, 4.0, 7.0, 0, 0, 0, 0, 0; protruding
                                                                   tongue: 0.9, 5.0, 2.6, 0, 0, 0, 0, 0; cleft lip: 5.5, 3.0,
                                                                   0.9, 0, 0, 0, 0, 0; hydronephrosis: 17.3, 42.0, 37.4,
                                                                   28.6, 34.3, 49.1, 58.3, 34.2; left umbical artery: 8.2,
                                                                   14.0, 11.3, 4.4, 11.8, 13.0, 9.2, 7.0; tail dysplasia:
                                                                   4.5, 1.0, 0, 0, 0, 2.8, 1.7, 0; anal atresia: 0.9, 0, 0, 0, 0,
                                                                   0, 0.8, 0
                                                                   ‘B’ stock:
                                                                   resorptions (%): 6.1, 12.9, 15.9, 9.4, 11.8, 9.9, 12.9,
                                                                   15.8, 10.2, at 0, 200, 225, 250, 275, 300, 325, 350,
                                                                   375 mg/kg bw, resp.
                                                                   mean foetal wt (g): 4.89±0.02, 4.26±0.05, 4.10±0.06,
                                                                   4.29±0.06, 4.45±0.05, 4.41±0.05, 4.44±0.04,
                                                                   4.35±0.04, 4.29±0.04
                                                                   mean placental wt (g): 405±3, 397±5, 373±5, 399±6,
                                                                   392±6, 370±6, 352±6, 355±5, 339±5 (decrease dose
                                                                   dependent: p<0.01)
                                                                   malformations (%): 3.2, 78.1, 91.8, 97.4, 93.7, 86.0,
                                                                   77.9, 62.5, 57.7
                                                                   % of most commonly (i.e. >10% in either ‘A’ (see
                                                                   above) or ’B’ stock) observed abnormalities:
                                                                   anophthalmia/microphthalmia: 66.4, 91.0, 94.0, 90.6,
                                                                   82.0, 47.5, 18.7, 6.2, at 200, 225, 250, 275, 300, 325,
                                                                   350, 375 mg/kg bw, resp.; hydrocephaly: 25.0, 23.0,
                                                                   34.5, 32.3, 14.0, 6.6, 4.7, 3.1; encephalocele: 10.2,
                                                                   13.1, 9.5, 4.7, 0, 0, 0, 0; exencephaly: 8.6, 32.0, 25.0,
                                                                   4.7, 0, 0, 0, 0; ear dysplasia: 8.6, 16.4, 29.3, 12.6, 4.0,
                                                                   4.1, 1.6, 0; maxillary hypoplasia: 8.6, 41.0, 27.6,
         Fertility and developmental toxicity studies                                                                        63
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<pre>                                                                   11.0, 2.0, 0, 0, 0; facial asymmetry: 3.1, 11.5, 5.2,
                                                                   4.7, 1.0, 0, 0, 0; pointed mandible: 3.9, 10.7, 18.1,
                                                                   6.3, 1.0, 0.8, 0.8, 0; protruding tongue: 7.8, 27.0,
                                                                   20.7, 5.5, 1.0, 0, 0, 0; cleft lip: 3.9, 11.5, 3.4, 1.6, 0,
                                                                   0, 0, 0; hydronephrosis: 5.5, 7.4, 12.9, 11.8, 7.0, 21.3,
                                                                   21.1, 23.7; left umbical artery: 17.2, 20.5, 16.4, 11.0,
                                                                   9.0, 13.9, 13.3, 14.4; tail dysplasia: 2.3, 5.7, 4.3, 1.6,
                                                                   4.0, 27.9, 29.7, 27.8; anal atresia: 0, 0.8, 1.7, 0, 2.0,
                                                                   14.8, 19.5, 2.1
Asano et al.  Wistar rats gd 9-12        0, 25, 50, 100 no data on number of implantations: 186, 151, 159, 173, resp.
1983)         (n=10-12/   dams allowed mg/kg bw/d;      maternal   delivery index (% of implantations): 91.4, 88.7, 91.2,
              group)      to litter;     ip             toxicity   83.8
                          sacrifice:                    provided   stillbirths (%): 0, 0, 2.8, 1.4
                          pnd 21                                   pnd 0: mean pup bw (g): males: 5.78±0.44,
                          litters reared                           5.94±0.66, 5.49±0.57, 5.64±0.57; females:
                          by their                                 5.45±0.48, 5.57±0.57, 5.36±0.43, 5.22±0.70
                          biological                               no external malformations
                          mothers; at                              pnd 4: viability index (% of pups survived at birth):
                          pnd 4, culled                            98.8, 93.3, 95.7, 95.1
                          to 4 male and                            number of abnormal pups: 0, 2/45 (4.4%), 4/52
                          4 female                                 (7.7%), 9/50 (18%)
                          pups;                                    malformations observed: dilated ventricular cavity
                          offspring                                (0, 2, 4, 1); anophthalmia (0, 0, 0, 9)
                          observed for                             pnd 21: mean pup bw (g): males: 54.5±4.1, 53.3±4.9,
                          morpholo-                                50.9±4.7, 51.7±3.6; females: 52.7±3.5, 53.0±3.9,
                          gical and                                49.7±4.8, 49.6±5.9
                          behavioural                              viability index (% of survived pups except for pups
                          development                              culled at pnd 4): 100, 97.5, 98.8, 100
                          up to pnd 21;                            number of pups with external malformations: 0, 1/78
                          final                                    (1.3%), 1/83 (1.2%), 14/86 (16.5%)
                          sacrifice:                               malformations observed: microphthalmia: 0, 1,1,0;
                          pnd 21                                   anophthalmia: 0, 0, 0, 13; enlarged cranical vault: 0,
                                                                   0, 0, 10
                                                                   behavioural effects: statistically significantly delayed
                                                                   development of the female righting reflex at pnd 2 at
                                                                   25 mg/kg bw/d; statistically significantly delayed
                                                                   free fall reflex between pnd 15 and 25 in males at
                                                                   100 mg/kg bw;
                                                                   statistically significantly increased numbers of
                                                                   rearing in open field test in postnatal wk 8 in females
                                                                   at 100 mg/kg bw
                                                                   no effects on ‘squares crossed’ number of ‘faecal
                                                                   boluses in open field test, on rotarod performance (in
                                                                   postnatal wk 8), on acquisisation rate of conditioned
                                                                   avoided response
 4           Hydroxyurea
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<pre>         Wistar rats   gd 9-12        0, 100, 200            number of implantations: 125, 160, 160, resp.
         (n=8-10/      dams allowed mg/kg bw/d;              delivery index (% of implantations): 90.4, 83.8, 70.6
         group)        to litter;     ip                     stillbirths (%): 1.8, 4.5, 25.7*
                       offspring                             pnd 0: mean pup bw (g): males: 6.22±0.45,
                       observed for                          5.77±0.61, 5.75±0.54*; females: 5.72±0.51,
                       morpholo-                             5.48±0.67, 5.28±0.51
                       gical and                             number of pups with external malformations: 0, 0,
                       behavioural                           17/84 (20.2%)*
                       development                           malformations observed at 200 mg/kg bw: head
                       up to pnd 56                          (exencephaly, meningocele, dilated ventricular
                                                             cavity, enlarged cranial vault): 12; anotia: 1; cleft lip:
                                                             5; cleft palate: 2; micrognatia: 1; tail (kinky,
                                                             brachyury): 2
                                                             pnd 4: viability index (% of survived pups at birth):
                                                             92.8, 91.4, 64.1
                                                             number of pups with malformations: 0/39, 2/38
                                                             (5.3%), 9/13 (69.2%)*
                                                             malformations observed: head: 0, 2, 4; eye
                                                             (anophthalmia, microphthalmia, pannus, corneal
                                                             opacity, anterior synechia): 0, 1, 8
                                                             pnd 14: viability index (% of survived pups except
                                                             for culled at pnd 4): 100, 94.9, 97.6
                                                             number of pups with malformations: 0/16, 1/17
                                                             (5.9%), 7/8 (87.5%)*
                                                             malformations observed: eye: 0, 1, 6; head: 0, 0, 6
                                                             pnd 21: mean pup bw (g): males: 55.2±4.9, 53.8±6.4,
                                                             49.5±10.4; females: 53.6±4.8, 52.3±4.7, 46.9±14.4
                                                             viability index (% of survived pups for sacrificed
                                                             pups at pnd 14 or 21): 100, 100, 100
                                                             number of pups with malformations: 0/19, 0/18, 5/8
                                                             (62.5%)*
                                                             malformations observed at 200 mg kg/bw: head: 3;
                                                             eye: 6
                                                             pnd 56: viability index (% of survived pups for
                                                             sacrificed pups at pnd 14 or 21): 100, 100, 66.7*
                                                             number of pups with malformations: 0/29, 3/40
                                                             (7.5%), 11/16 (68.8%)*
                                                             malformations observed: head: 0, 1, 9; eyes; 0, 3, 6
                                                             behavioural effects: statistically significantly delayed
                                                             free fall reflex in males at 100 and 200 mg/kg bw
Asano/   Sprague-      gd 9-12        0, 100, 200 no data on number of implantations: 207, 212, 219, resp.
Okaniwa  Dawley rats   sacrifice:     mg/kg bw/d; maternal   number of resorptions: 15, 15, 19
1987)    (n=15-16/     gd 21          ip          toxicity   mean number of live foetuses: 12.8±2.1, 13.1±1.7,
         group)        dams                       presented  12.5±2.9
                       examined for                          mean foetal wt (g): 4.82±0.43, 4.88±0.33,
                       implanta-                             4.48±0.47*
                       tions,                                % of foetuses with skeletal abnormalities:
                       resorptions,                          males: 0, 0, 51.1**; females: 1.1, 1.1, 43.8**
                       number of                             abnormalities most commonly (i.e, >10%) observed
                       life foetuses;                        at 200 mg/kg bw (in males and females, resp.):
                       foetuses for                          dilatation of lateral ventricle: 23/88 (26.1%), 15/112
                       malforma-                             (13.4%); anophthalmia: 16/88 (18.2%), 16/112
                       tions.                                (14.3%); microphthalmia: 20/88 (22.7%), 19/112
        Fertility and developmental toxicity studies                                                                65
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<pre>                                                     (17.0%); ventricular septal defect: 23/88 (26.1%),
                                                     17/112 (15.2%)
                                                     [one control female: ventricular septal defect; one
                                                     low-dose female: dilation of lateral ventricle]
   Wistar rats                            no data on number of implantations: 63, 76, 78, resp.
   (n=5/group)                            maternal   number of resorptions: 9, 4, 5
                                          toxicity   mean number of live foetuses: 10.8±3.3, 14.4±2.3,
                                          presented  14.6±2.1
                                                     mean foetal wt (g): 5.58±0.52, 5.14±0.10,
                                                     4.49±0.33**
                                                     % of foetuses with skeletal abnormalities:
                                                     males: 0, 0, 86.8**; females: 10.0, 6.7, 88.6**
                                                     abnormalities most commonly (i.e >10%) observed
                                                     at 200 mg/kg bw in males and females, resp.:
                                                     exencephaly: 6/38 (15.8%), 0/35 ; dilatation of
                                                     lateral ventricle: 21/38 (55.3%), 18/35 (51.4%);
                                                     anophthalmia: 24/38 (63.2%), 17/35 (48.6%);
                                                     microphthalmia: 8/38 (21.1%), 15/35 (42.9%)
                                                     [one control female: double aortic arch; one low-
                                                     dose female: ventricular septal defect]
   Sprague-    gd 9-12        0, 100, 200 no data on number of implantations: 169, 168, 310, resp.
   Dawley rats dams allowed mg/kg bw/d;   maternal   delivery index (% of implantations): 89.9, 95.2, 79.7
   (n=12-22/   to litter and ip           toxicity   stillbirths (%): 0.7, 0, 5.3
   group)      sacrificed at              presented  pnd 0: mean pup bw (g): males: 5.82±0.49,
               pnd 21;                               5.55±0.45, 5.36±0.66*; females: 5.54±0.48,
               litters reared                        5.31±0.49, 4.99±0.63**
               by their                              pnd 4: viability index (% of pups survived at birth):
               biological                            100, 98.8, 87.6**
               mothers; at                           number of pups malformed: males: 0/25, 0/27, 1/19
               pnd 4, culled                         (5.3%); females: 0/30, 0/35, 5/34 (14.7%)
               to 4 male and                         abnormalities observed: at 200 mg/kg bw in males
               4 female                              and females, resp.: dilation of lateral ventricle: 0/19,
               pups; pups                            1/31 (2.9%); anophthalmia: 0/19, 3/31 (8.8%);
               examined for                          microphthalmia: 0/19, 2/13 (5.9%); ventricular septal
               wt, viability                         defect: 1/19 (5.3%), 1/31 (2.9%)
               and abnorma-                          pnd 21: mean pup bw (g): males: 42.4±3.5, 41.7±2.9,
               lities; final                         39.2±5.4*; females: 41.4±3.4, 40.3±3.2, 37.5±5.0*
               sacrifice:                            weaning index (% of pups survived after culling at
               pnd 21                                pnd 4): 100, 100, 98.7
                                                     number of pups malformed: males: 0/50, 1/46
                                                     (2.2%), 37/70 (52.9%)**; females: 0/50, 1/50 (2%),
                                                     34/80 (42.5)**
                                                     abnormalities observed at 200 mg/kg bw in males
                                                     and females, resp.: hydrocephaly: 27/70 (38.6%), 9/
                                                     80 (11.3%); anophthalmia: 22/70 (31.4%), 23/80
                                                     (28.8%); microphthalmia: 9/70 (12.9%), 10/80
                                                     (25%)
                                                     [one low-dose male: microphthamia; one low-dose
                                                     female: anophthalmia]
6 Hydroxyurea
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<pre>  hahoud/     Wistar rats    gd 11         0, 250, 300,  no data on     number of litters: 53, 18, 17, 21, 34, 17, 15, 13, resp.
 aumgartten   (n=13-34       sacrifice: gd 350, 400,     maternal       number of foetuses: 559, 154, 188, 213, 315, 125,
2009)         litters/group; 21 foetuses   450, 500, 550 toxicity       101, 70
              controls n=53  examined for  mg/kg bw/d;   presented      resorptions (%, implantation): 4.1, 12.7, 6.0, 3.6,
              litters)       skeletal      ip                           13.0, 26.4, 35.2, 51.0
                             abnormalities                              mean foetal bw (g): 4.40±0.37, 4.02±0.37*,
                                                                        4.15±0.30*, 4.00±0.40*, 3.76±0.53*, 3.71±0.54*,
                                                                        3.39±0.61*, 3.07±0.39*
                                                                        variations (%, foetuses): fused zygomatic bone 8.8,
                                                                        10.3, 18.6*, 20.1*, 38.4*, 43.2*, 45.5*, 84.2*;
                                                                        misaligned sternebra sternum 4.3, 2.6, 4.2, 8*, 9.5*,
                                                                        24*, 17.8*, 27.1*; wavy ribs 8.8, 1.3*, 0.5*, 0.5*,
                                                                        2.5*, 2.4*, 3*, 0*; dumbbell-shaped ossification
                                                                        centre in lumbar vertebrae 0, 1.3*, 0.5, 6.1*, 15.2*,
                                                                        25.6*, 10.8*, 12.8*; bipartite ossification centre in
                                                                        lumbar vertebrae 0, 0, 2.6*, 2.3*, 8.8*, 15.2*, 10.8*,
                                                                        15.7*; dumbbell-shaped ossification centre in
                                                                        thoracic vertebrae 0.9, 13.6*, 38.8*, 46.4*, 64.7*,
                                                                        69.6*, 63.3*, 61.4*; bipartite ossification centre in
                                                                        thoracic vertebrae 0.5, 3.9*, 4.8*, 12.2*, 25.3*,
                                                                        26.4*, 36.6*, 61.4*
                                                                        malformations (%, foetuses): cleft palate 0, 0, 0, 0,
                                                                        3.8*, 4.8*, 12.8*, 34.2*; absent tympanic bone 0, 0,
                                                                        4.2*, 5.2*, 35.8*, 52.8*, 64.3*, 91.4*; absent tibia 0,
                                                                        0, 0, 0, 3.5*, 17.6*, 27.7*, 44.2*; bent ribs 0, 0, 0, 0,
                                                                        0, 0.8*, 1.9*, 1.4*; bent clavicle 0, 0, 0, 0, 0, 5.6*,
                                                                        4.0*, 20.0*
Gupta/Jaffe   Sprague-       gd 17-20      0, 160 mg/kg no gross        no effect on age of vaginal opening and first
1982)         Dawley rats examination bw/d; sc           abnormalities appearance of oestrus (n=20) or on oestrus cycle
              (n=5/ group) of                            in offspring (n=6) of female offspring
                             reproductive                               no effect on fertility of female offspring (n=9), after
                             development                                mating with untreated males
                             of randomly
                             selected
                             female
                             offspring
 w=body weight; d=day(s); gd=gestational day(s); hr=hour(s); ip=intraperitoneal; n=number(s); pnd=postnatal day(s);
 c=subcutaneous; wk=week(s); wt=weight(s);*: p<0.05; **: p<0.01.
            Fertility and developmental toxicity studies                                                                      67
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<pre>8 Hydroxyurea</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 reports 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
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<br><br>