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

<pre>             Health Council of the Netherlands
          Ifosfamide
             Evaluation of the effects on reproduction,
             recommendation for classification
2014/08
</pre>

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<pre>Ifosfamide
     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 Ifosfamide
Uw kenmerk            : DGV/MBO/U-932542
Ons kenmerk           : U-8079/HS/cn/543-K14
Bijlagen              :1
Datum                 : 3 april 2014
Geachte minister,
Graag bied ik u hierbij het advies aan over de effecten van ifosfamide 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>Ifosfamide
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. 2014/08, The Hague, April 3, 2014
</pre>

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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. Ifosfamide - Evaluation of the effects on
reproduction, recommendation for classification. The Hague: Health Council of
the Netherlands, 2014; publication no. 2014/08.
all rights reserved
ISBN: 978-90-5549-998-4
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<pre>   Contents
   Samenvatting 9
   Executive summary 11
   Scope 13
.1 Background 13
.2 Committee and procedure 13
.3 Labelling for lactation 14
.4 Data 15
.5 Presentation of conclusions 15
.6 Final remark 16
   Ifosfamide 17
.1 Introduction 17
.2 Human studies 18
.3 Animal studies 22
.4 Conclusions 30
   References 35
   Contents                       7
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<pre>  Annexes 39
A The Committee 41
B The submission letter (in English) 43
C Comments on the public draft 45
D Regulation (EC) 1272/2008 of the European Community 47
E Additional considerations to Regulation (EC) 1272/2008 59
F Fertility and developmental studies in animals 61
  Ifosfamide
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<pre>Samenvatting
In het voorliggende advies heeft de Gezondheidsraad ifosfamide onder de loep
genomen. Ifosfamide wordt veel gebruikt bij chemotherapie voor veel verschil-
lende type tumoren, zoals weke-delensarcomen, ovariumcarcinomen, osteosar-
comen en haematopoietische maligniteiten. 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 voortplanting beoordeelt. Het
gaat vooral om stoffen waaraan mensen tijdens de beroepsuitoefening 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 commissie, kijkt zowel naar effec-
ten op de vruchtbaarheid van mannen en vrouwen als naar effecten op de ontwik-
keling van het nageslacht. Bovendien worden effecten van blootstelling van de
zuigeling via de moedermelk beoordeeld.
Op basis van Verordening (EG) 1272/2008 van de Europese Unie doet de com-
missie een voorstel voor classificatie. Voor ifosfamide komt de commissie tot de
volgende aanbevelingen:
• voor effecten op de fertiliteit adviseert de commissie ifosfamide in categorie
    1B (stoffen waarvan verondersteld wordt dat zij toxisch zijn voor de mense-
    lijke voortplanting) te classificeren en met H360F (kan de vruchtbaarheid
    schaden) te kenmerken
Samenvatting                                                                     9
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<pre>  •   voor effecten op de ontwikkeling adviseert de commissie ifosfamide in cate-
      gorie 1B (stoffen waarvan verondersteld wordt dat zij toxisch zijn voor de
      menselijke voortplanting) te classificeren en met H360D (kan het ongeboren
      kind schaden) te kenmerken
  •   voor effecten op en tijdens de lactatie adviseert de commissie om ifosfamide
      niet te kenmerken wegens onvoldoende geschikte gegevens.
0 Ifosfamide
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<pre>Executive summary
In the present report, the Health Council of the Netherlands reviewed ifosfamide.
Ifosfamide is widely used in chemotherapy for many different tumour types,
such as soft tissue sarcomas, osteosarcomas, ovarian carcinomas and
haematopoietic malignancies. 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 ifosfamide, these recommendations are:
• for effects on fertility, the Committee recommends classifying ifosfamide in
    category 1B (presumed human reproductive toxicant), and labelling with
    H360F (may damage fertility)
• for effects on development, the Committee recommends classifying
    ifosfamide 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
      ifosfamide due to a lack of appropriate data.
2 Ifosfamide
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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 ifosfamide by the Health Council’s
        Subcommittee on the Classification of Reproduction Toxic Substances, hereafter
        Scope                                                                             13
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<pre>    called the Committee. The members of the Committee are 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 organisations 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 received comments,
    and the replies by the Committee, can be found on the website of the Health
    Council.
    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 Labelling for 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
 4  Ifosfamide
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<pre>    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
    independent of dosage), the labelling for effects on or via lactation is based on a
    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 leads to
    exceeding the exposure limit for the general population, e.g. the acceptable daily
    intake (ADI).
1.4 Data
    Literature searches were conducted in the on-line databases XTOXLINE,
    MEDLINE and CAPLUS, up to and including January 2012 without a starting
    date; an update was performed in TOXNET in June 2013. Publications cited in
    the selected articles, but not selected during the primary search, were reviewed if
    considered appropriate. In addition, handbooks and most recent reviews were
    consulted. 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 are considered.
        In the assessment of the potential reproduction toxic effects of ifosfamide,
    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 precludes assessment of the compound for
        reproductive toxicity
    • sufficient data show that no classification for reproductive toxicity 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)16, 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  Ifosfamide
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<pre> hapter 2
        Ifosfamide
2.1     Introduction
        chemical name             : ifosfamide
        IUPAC name                : 3-(2-chloroethyl)-2-[(2-chloroethyl)amino]tetrahydro-
                                    2H-1,3,2-oxazaphosphorine 2-oxide
        CAS name                  : 2H-1,3,2-oxazaphosphorin-2-amine, N,3-bis(2-
                                    chloroethyl)tetrahydro-, 2-oxide
        CAS registry number       : 3778-73-2
        EC/EINECS number          : 223-237-3
        synonyms                  :  3-(2-chloroethyl)-2-[(2-chloroethyl)amino]perhydro-2H-
                                    1,3,2-oxazaphosphorine 2-oxide; N,3-bis(2-chloroethyl)-
                                    tetrahydro-2H-1,3,2-oxazaphosphorin-2-amine 2-oxide;
                                    iphosphamide; isophosphamide; isofosfamide; isoendoxan;
                                    isofosfamidum
        colour and physical state : white crystals
        molecular weight          : 261.1
        molecular formula         : C7H15Cl2N2O2P
        structure                 :            O
                                           O       N
                                               P              Cl
                                               N
                                                          Cl
                                    phosphor is a stereocentre; 2 enantiomers (R/S)
        melting point             : 48-50 °C; 39-41 °C from anhydrous ether; 72 °C
        vapour pressure           : 3.96x10-3 Pa at 25°C (estimated)
        Log P (octanol-water)     : 0.86 (experimental)
        Ifosfamide                                                                          17
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<pre>    solubility                       :   soluble in water (1 in 10) and carbon disulphide (1.5 in 100);
                                         very soluble in dichloromethane
    use                              :   in cancer chemotherapy and always administered as a racemic
                                         mixture. In the Netherlands, it can be prescribed for the
                                         treatment of small-cell and non-small-cell lung carcinomas,
                                         testicular tumours (in case of recurrence after conventional
                                         therapy), ovarian carcinomas, soft tissue sarcomas (especially
                                         leyomyo-, rhabdomyo-, chondrosarcomas) and osteosarcomas,
                                         and as second-line therapy for Hodgkin’s disease and non-
                                         Hodgkin’s lymphomas with an intermediate or high degree of
                                         malignancy.3
    kinetics                         :   Oral bioavailability is close to 100%. No information on
                                         bioavailability after dermal or inhalation exposure is available.
                                         Following intravenous administration, the volume of
                                         distribution of ifosfamide approximates the total body water
                                         volume, suggesting that distribution takes place with minimal
                                         tissue binding. Ifosfamide can cross the blood-brain barrier.
                                         Ifosfamide is a prodrug that needs activation via CYP3A4, a
                                         cytochrome P450 isoenzyme, to 4-hydroxy-ifosfamide. Further
                                         oxidation results in formation of ifosforamide mustard with
                                         concurrent formation of acrolein. The metabolite ifosforamide
                                         mustard can alkylate DNA and is believed to be the major cause
                                         of the cytotoxicity observed. Dechloroethylation of ifosfamide
                                         with concurrent formation of chloroacetaldehyde is also a major
                                         metabolic route accounting for 25-60% of the metabolism of
                                         ifosfamide in humans.8
    regulations and advisories       :   In 2008, the Health Council of the Netherlands was of the
                                         opinion that ifosfamide should be considered as carcinogenic to
                                         humans, a recommendation comparable to the EU classification
                                         as a category 2 carcinogen (according to Guideline 93/21/EC of
                                         the European Union ).6 A harmonised EU GSH classification
                                         for carcinogenicity is not available.
                                         IARC concluded that there was limited evidence for the
                                         carcinogenicity of ifosfamide in experimental animals. No
                                         conclusion was drawn for the carcinogenicity in humans in the
                                         absence of data. Therefore, according to the IARC guidelines,
                                         ifosfamide was considered to be not classifiable as to its
                                         carcinogenicity to humans (Group 3).7
    Data from1,7,20 unless otherwise noted.
2.2 Human studies
    Numerous studies are available in which ifosfamide was administered as part of
    multidrug chemotherapy. However, in most of the studies, the effect of
    ifosfamide on fertility or on developmental toxicity can or has not been
    evaluated. The few studies that give an evaluation of reproductive effects of
    ifosfamide mainly focus on male fertility.
 8  Ifosfamide
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<pre>Fertility studies
Longhi et al. investigated fertility in 96 male patients by extracting information
on individual drugs by comparing different combinations of drugs. In a
retrospective study in male patients treated for osteosarcoma at the Rizzoli
Orthopaedic Institute in Bologna (Italy) from 1976 to 1996, the persons alive in
2001 could be divided into six groups based on different chemotherapy
protocols. The median age at time of chemotherapy was 17 years (range: 10-42
years) and the median follow-up was nine years (range: 4-17 years) after the end
of chemotherapy. Four drugs were administered (methotrexate, cisplatin,
doxorubicin, ifosfamide) at different doses. Forty patients received combinations
of methotrexate, cisplatin and doxorubicin, but no ifosfamide, and 56 patients
combinations of methotrexate, cisplatin, doxorubicin and ifosfamide (44 patients
ifosfamide doses of 24 to 42 g/m2 (≈690-1,200 mg/kg bw*; 12 doses of 60 to 72
g/m2 (≈1,710-2,060 mg/kg bw)). Twenty-six of the 96 patients (age: 21-46 years)
had semen analysis after the interview (no semen analysis performed before
chemotherapy). Following combination therapy including ifosfamide (median:
42 g/m2, range: 24-72 g/m2; ≈1200 mg/kg bw, range: ≈690-2,060 mg/kg bw), an
increase in the incidence of azoospermia was found compared to treatment with
combinations without ifosfamide (14/16 vs. 5/10; p=0.005). It should be noted
that no tests were performed to exclude other causes of sterility. Of the other
drugs used, none demonstrated a statistically significant association between
dosage and the occurrence of azoospermia. Of the six patients who were
normospermic, five had received no ifosfamide and one ifosfamide doses of 24
g/m2 (≈690 mg/kg bw). Impaired libido or sexual dysfunction was not reported
by any of the patients. For testosterone, LH and FSH values measured in five
patients and fathering of children by seven patients, results could not be linked to
ifosfamide specifically.9
Garolla et al. compared the effects of ifosfamide and cyclophosphamide
treatment on semen characteristics in 33 males who had received chemotherapy
for childhood cancer at the Oncoematology Paediatric Clinic in Padova (Italy)
between 1980 and 2000. All patients had received chemotherapy at pre- or
postpubertal age for soft tissues or bone sarcomas by several multidrug
protocols, including cyclophosphamide (eight subjects) or ifosfamide (25
All dose conversions in this study are rough estimations as an unknown number of children were
present in each group; all conversions are based on a bw of 70 kg and a body surface of 2 m2 for an
adult.17
Ifosfamide                                                                                          19
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<pre>  subjects). Ten patients of the ifosfamide group had received a higher dose than
  60 g/m2 (≈2,110 mg/kg bw*) and 15 a lower dose. Slow (24 hours) and rapid
  infusion (1-3 hours) protocols were divided equally between the low- and high-
  dose group. The time between end of treatment and evaluation is not exactly
  clear: at least two years is stated in the title of a table and at least seven years is
  stated in the text. Results for the ifosfamide group consisted of normal values for
  seminal volume, total sperm count and sperm concentration, a normal percentage
  of sperm aneuploidies, and normal plasma concentrations of FSH, LH,
  testosterone, inhibin B (marker of spermatogenesis), prolactin and oestradiol,
  when evaluated according to 1999 World Health Organization guidelines. When
  the ifosfamide group was divided based on pubertal stage at time of treatment
  (13 prepubertal vs. 12 postpubertal) or based on protocol used (13 slow vs. 12
  rapid infusion), no differences in terms of semen concentrations, testicular size,
  inhibin B, LH and testosterone plasma levels were seen either. The FSH plasma
  concentration was increased in the rapid infusion group compared to the slow
  infusion group (p<0.05). Evaluation of sperm chromosomes by FISH
  (fluorescence in situ hybridization) analysis showed a slightly higher mean
  percentage of sperm aneuploidies compared to normozoospermic controls
  (1.8±0.7% vs. 1.6%; not statistically significant).5
  Ridola et al. investigated gonadal function in adult male survivors who had
  received ifosfamide treatment in a multidrug protocol during childhood. Male
  patients were eligible for the study if they had received ifosfamide as the only
  alkylating agent. The median age at treatment was 12 years (range: 0.5-20.7
  years) and the median age at investigation was 22.5 years with a median time of
  8.5 years after treatment. The median cumulative dose of ifosfamide was 54 g/m2
  (range: 18-114 g/m2; ≈1,720 mg/kg bw**, range: 570-3,640 mg/kg bwa).
  Elevated FSH values were seen in 5/60 patients having received ifosfamide at a
  cumulative dose of ≥48 g/m2 (≈1,530 mg/kg bwa); no association with
  ifosfamide dose was obvious. All but two males had normal testosterone levels.
  LH was elevated in 14/100 patients. At the time of hormone measurement, 6/100
  patients had fathered at least one child.19
  Williams et al. evaluated gonadal function of patients enrolled in multidrug
  protocols for treatment of Ewing’s sarcoma and soft tissue sarcoma containing
  Dose conversion in this study based on a bw of 42 kg and body surface of 1.34 m2 for a 12-year-old
  boy (median age).15
* All dose conversions in this study based on a bw of 42 kg and body surface of 1.34 m2 for
  a 12-year-old boy (median age).15
0 Ifosfamide
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<pre>ifosfamide as the only potential gonadotoxic agent in twenty-two Children’s
Cancer Study Group Centres in the UK in the 1980-1990s. All patients were
event-free survivors for more than two years, were post-pubertal and more than
15 years of age at the time of study. Boys were treated at a median age of 11.8
years (range: 5.4-21.3 years) and girls at a median age of 12.1 years (range: 3.6-
15.6 years). Pubertal development, menstrual history in the girls and semen
analysis in the boys were investigated after a median follow-up of ten years. All
32 boys progressed normally through puberty. No gonadal dysfunction was seen
at a total ifosfamide dose of <60 g/m2 (≈1,910 mg/kg bw*). In those with a dose
>60 g/m2 (≈1,910 mg/kg bw), 8/11 who underwent semen analysis were
subfertile, 8/26 had elevated FSH levels and 13/26 showed decreased inhibin B,
supporting evidence of germ cell failure. All 13 girls progressed through puberty
normally and had regular menses. Biochemical results were in line with
published data except for anti-Müllerian hormone (AMH) levels, which were
lower compared with an age-matched reference group. AMH is an age-
dependent hormone in pre-menopausal women and declines with age as follicle
numbers fall. Nine patients not recruited into the study were known to have had
11 live births.22
Developmental toxicity studies
No premature deliveries or malformations were reported in 11 children fathered
by seven patients having received ifosfamide in multidrug therapy.9
Mir et al. (2012) reported on 11 cases with high-grade sarcomas diagnosed
during the third trimester of pregnancy and receiving chemotherapy until
delivery. Five of these cases were retrieved from medical records from their own
institute. These patients were treated with doxorubicin and ifosfamide. Of the
remaining six cases, which were obtained from literature, two received a similar
treatment while the other four had received ifosfamide in combination with
several other chemotherapy agents. Abnormalities during pregnancy included,
amongst others, oligo/anhydramnios (5/11), neutropenia (5/11) and intrauterine
growth retardation (4/11). All deliveries were premature; six were by Caesarean
section. One (female) newborn, delivered by emergency Caesarean section at 29
weeks had a birth weight of 720 g, Apgar scores of 3 and 7 at one and five
minutes, anuria and intraventricular haemorrhage, and died at day 7. Another one
All dose conversions in this study based on a bw of 31 kg and a body surface of 1.09 m2 for a nine-
year-old boy.15
Ifosfamide                                                                                          21
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<pre>    (male), submitted to neonatal intensive care, had anaemia and thrombopenia at
    day 8, and was ‘normal’ at 12 weeks. Seven other babies had Apgar scores ≥7
    while no data were available for the two remaining cases. All these nine babies
    appeared ‘normal’ at follow-up examination.10
        The Committee notes that this may be a selected case series, as population-
    based denominators for exposure and effects were not available.
    Lactation
    No studies are available regarding ifosfamide levels in breast milk or the effects
    of ifosfamide on or via human lactation.
2.3 Animal studies
    Fertility and developmental toxicity studies in laboratory animals are
    summarized in Annex F.
    Fertility studies
    In a preliminary experiment, Nagaoka et al. administered daily ifosfamide doses
    of 0, 5, 10 and 15 mg/kg bw to male Sprague-Dawley rats (n=10/group)
    intravenously for three weeks before mating and during mating and to female
    Sprague-Dawley rats (n=10/group) for three weeks before mating, during mating
    and until gestational day seven. Females were sacrificed at gestational day 14.
    No clear signs of toxicity were observed in the animals treated but body weight
    gain was decreased in the two higher dose groups. There were no statistically
    significant differences in mating and pregnancy rates between the groups, but
    treatment caused dose-related, statistically significant increases in embryol-
    ethality (see also below Developmental toxicity studies).14
        Based on these results (and those of a subchronic toxicity study), Nagaoka
    et al. intravenously administered daily ifosfamide doses of 0, 1.25, 2.5 and
    5 mg/kg bw to male Sprague-Dawley rats (n=20/group) from nine weeks before
    cohabitation and during cohabitation and to female rats (n=20/group) for two
    weeks before cohabitation, during habitation and until gestational day 7. During
    treatment, body weight and food and water consumption recordings and general
    condition observations were regularly made. Female animals were sacrificed at
    gestational day 20, and uterine contents were examined and maternal primary
    organs were weighed.
 2  Ifosfamide
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<pre>    Treatment did not affect the percentage of males and females mating, the
percentage of pregnant females, the number of corpora lutea, the number of
implantation sites and the implantation ratio, and absolute or relative testis or
ovary weights. There was no effect on male body weights while body weights of
females given 2.5 or 5 mg/kg bw/day were decreased during gestation (no
statistical analysis presented). At autopsy, male relative spleen weights were
decreased at 1.25 mg/kg bw (p<0.05) and relative and absolute spleen weights at
2.5 and 5 mg/kg bw (all p<0.01). Male relative and absolute lung weights were
increased at 5 mg/kg bw (both p<0.05). In females, body weights were decreased
at 5 mg/kg bw (p<0.05), relative spleen weights at 2.5 mg/kg bw (p<0.05) and
absolute spleen weights at 2.5 and 5 mg/kg bw (both p<0.01), while relative lung
weights were increased at 5 mg/kg bw (p<0.01).14
Quinto et al. investigated the effect of ifosfamide on sperm cells. Male mice of
genotype (C3H x C57BL/6)F1 (n=6), 11-15 weeks old, were administered
intraperitoneal ifosfamide doses of 0, 12.5, 25, 50 or 100 mg/kg bw/day in
distilled water for five consecutive days. Mice were killed 35 days after the first
injection. Testis weights, sperm count and sperm morphology were evaluated.
    No effect on sperm count was observed. The only effects observed were
decreased testis weights (p<0.05) and increased sperm abnormalities (p<0.05;
type not specified) at 100 mg/kg bw/day.18
Ypsilantis et al. studied the effect of ifosfamide on testes and semen
characteristics in rabbits. Six-month-old male New Zealand white rabbits
(n=10/group) were given single intravenous ifosfamide doses of 0, 60, 90, 120
or 240 mg/kg bw in distilled water. Semen was collected weekly, three weeks
prior to treatment until the day of sacrifice, and examined for volume, sperm
concentration and total sperm count. Sperm motility and morphology was only
examined in the first ejaculate. Five rabbits per group were sacrificed one week
and the remaining five 18 weeks after administration. Testes, epididymides and
accessory sex glands were weighed. Testicular preparations were examined
histologically.
    Paired testis weights were – not dose relatedly – decreased at 90, 120 and
240 mg/kg bw, while accessory sex gland weights were decreased at 240 mg/kg
bw one week after administration. Eighteen weeks after administration, no
effects were observed on reproductive organ weights. No effects were observed
on seminiferous tubule diameter, percentage of the most advanced germ cell
type in seminiferous tubule cross section or number of germ cells per stage 1
seminiferous tubule cross section at post-treatment weeks 1 or 18. Total sperm
Ifosfamide                                                                          23
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<pre>  counts were decreased at post-treatment week 5 at 60, 90 and 120 mg/kg bw and
  from post-treatment weeks 5 to 7 at 240 mg/kg bw when compared to pre-
  treatment values (p<0.05). Primary sperm defects (i.e. small, double or deformed
  heads; swollen, abaxial or double midpieces; proximal droplets; double or coiled
  tails) were increased at post-treatment week 2 and 3 at 240 mg/kg bw (p<0.05),
  while secondary sperm defects (i.e. flagging or detached heads; bent, flagging or
  detached midpieces; distal droplets; bent tails) were increased at the 1st, 2nd and
  6th week post-treatment at 120 mg/kg bw and from post-treatment weeks 3 to 7 at
  240 mg/kg bw (p<0.05). Sperm motility was decreased from post-treatment
  weeks 2 to 5 at 240 mg/kg bw (p<0.05). The changes noted in sperm defects for
  each treatment group were higher than the corresponding changes in the control
  group over time, while changes in sperm count and motility were similar.23
  Ehling et al. (1998) performed dominant lethal mutation tests in 13-14-week-old
  (102/E1xC3H/E1)F1 male mice following a standard protocol. Groups of mice
  were given single intraperitoneal injections of ifosfamide doses of 0, 300 and 600
  mg/kg bw (n=40/group) or of 0, 250 and 600 mg/kg bw (n=35/group). After
  successful matings, females were sacrificed at gestational day 14-17, and uterine
  contents were inspected for the number of corpora lutea, preimplantation loss,
  and the number of total, live and dead implantations.
       Apart from increased percentages of dead implants, no statistically
  significant changes were found in the aforementioned end points. Ifosfamide
  induced dominant lethal mutations in spermatozoa at doses of 300 and 600
  mg/kg bw and in spermatids and spermatocytes at 600 mg/kg bw.4
  Ehling et al. also conducted specific-locus mutation assays by treating 9-16-
  week-old mice with single intraperitoneal doses of 600 mg/kg bw (number of
  animals treated not presented) followed by sequential mating with 10-13-week-
  old untreated test-stock virgin females.
       Ifosfamide induced specific-locus mutations in post-spermatogonial germ-
  cell stages but not in spermatogonial stem cells.4
  Developmental toxicity studies
  In the afore-mentioned preliminary study of Nagaoka et al., intravenous injection
  of doses of ifosfamide of 0, 5, 10 and 15 mg/kg bw into male Sprague-Dawley
  rats (n=10/group) for three weeks before mating and during mating and into
  female Sprague-Dawley rats (n=10/group) for three weeks before mating, during
  mating and until gestational day 7, caused increased embryolethality of 20%
4 Ifosfamide
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<pre>(p<0.05), 86% (p<0.01) and 100% (p<0.01) at 5, 10 and 15 mg/kg bw,
respectively, when compared to controls (4%). Maternal body weights were
decreased at the two higher doses.14
    In the main study in which intravenous doses of ifosfamide of 0, 1.25, 2.5
and 5 mg/kg bw were given to groups of 20 male rats from nine weeks before
cohabitation and during cohabitation and to 20 female rats two weeks before
cohabitation, during habitation and until gestational day 7, decreases were
observed concerning foetal viability (at 2.5 and 5 mg/kg bw; 91 and 78% vs.
96% in controls; p<0.05 and <0.01, respectively), the number of living foetuses
per pregnant rat (at 5 mg/kg bw; 11.3 vs. 13.3 in controls; p<0.05) and mean
foetal body weight (at 5 mg/kg bw; 3.13±0.25 g vs. 3.50±0.19 in controls;
p<0.05). Treatment did not cause increases in the number of foetuses with
visceral or skeletal abnormalities. Maternal toxicity included decreased body
weights at 5 mg/kg bw (p<0.05), decreased relative spleen weights at 2.5 mg/kg
bw (p<0.05) and absolute spleen weights at 2.5 and 5 mg/kg bw (both p<0.01)
and increased relative lung weights were increased at 5 mg/kg bw (p<0.01).14
In preliminary experiments, Nagaoka and Narama exposed groups of 10-13
female Sprague-Dawley rats to intravenous ifosfamide doses of 1.25, 2.5, 5 and
10 mg/kg bw from gestational day 7-17. Animals were sacrificed at gestational
day 20. Increased foetal mortality (39 and 100% at 5 and 10 mg/kg bw,
respectively) and decreased foetal body weights (at 2.5 and 5 mg/kg bw) were
observed, but no external abnormalities. Treatment did not induce clear
symptoms of toxicity but maternal body weights were decreased in the late
stages of gestation.12
    In the main study, groups of 30 pregnant rats were treated with intravenous
doses of ifosfamide of 0, 1.25, 2.5 and 5 mg/kg bw/day from gestational day
7-17. Body weight and food and water consumption recordings and general
condition observations were regularly made. Twenty animals/group were
autopsied on gestational day 20 and uterine contents were examined and primary
organs weighed. The remaining ten animals/group were allowed to litter and
sacrificed at postnatal day 21. Pups were observed for up to 77 days (function,
motor coordination, behaviour, fecundity by mating to produce an F2
generation).
    No clear symptoms of toxicity were observed in the pregnant rats. Maternal
body weights during gestation were decreased in the animals injected with 2.5
and 5 mg/kg bw compared to those in controls; in the postnatal period, the body
weights of the high-dose animals were increased. Food and water consumption
did not differ between groups during gestation but were decreased in the high-
Ifosfamide                                                                      25
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<pre>  dose animals from postnatal days 10-14 onwards. At autopsy on gestational
  day 20, dose-response related changes in absolute weights of the heart (increase;
  at 5 mg/kg bw: p<0.05), spleen (decrease; at 5 mg/kg bw: p<0.05) and the liver
  (decrease: at 2.5 mg/kg bw: p<0.05; at 5 mg/kg bw: p<0.01) and in relative
  weights of the heart (increase; at 2.5 and 5 mg/kg bw: p<0.01), the lung
  (increase; at 5 mg/kg bw: p<0.01) and the kidneys (increase; at 5 mg/kg bw:
  p<0.01) were seen. At autopsy on postnatal day 21, organ weight changes
  observed were increased absolute heart (p<0.05), increased absolute ovary
  (p<0.05) and decreased relative liver weights (p<0.01) all in animals treated with
  5 mg/kg bw.
       Treatment did not affect the number of corpora lutea, the number of corpora
  lutea/litter, the number of implantations, the number of implantations/litter and
  the sex ratio. At 5 mg/kg bw, the numbers of placental remnants, early, late and
  total deaths and foetal deaths/implantations were increased (all p<0.01) and the
  number of live foetuses/litter decreased (p<0.01). Mean body weights, body
  lengths, tail lengths and placental weights were decreased at all doses (p<0.01).
  Treatment with 5 mg/kg bw caused an increase in the number of foetuses with
  external abnormalities (4/169 (2.4% vs. 0/265 in controls; p<0.05) and in the
  number of litters with foetuses with external abnormalities (3 vs. 0; n.s. (not
  statistically significant)). Skeletal effects observed included increased numbers
  of foetuses with cervical rib (1.25 mg/kg bw: 10/22 (p<0.01); 2.5 mg/kg bw:
  7/32 (n.s.); 5 mg/kg bw: 6/19 (p<0.05; controls: 1/21). At all doses, there were
  signs of delayed ossification.
       As to the groups that were allowed to deliver, there was no effect on
  gestational period. The total number of implantations was dose-relatedly
  decreased as was the number of live pups (both n.s.). At 5 mg/kg bw, the number
  of live pups/litter, the rate of birth and the stillborn rate, body weights of male
  and female pups (at birth, four-day old and one-, two- and three-week old) and
  viability, when four-day and three-week old, were decreased and the number of
  pups with skeletal abnormalities increased (all p<0.01). At post-mortem
  examinations of four-day-old pups, no external abnormalities were seen.
  Recording of primary organs showed decreases in absolute weights of almost all
  these organs (p<0.05) and in absolute and relative weights of testis p<0.01) in
  high-dose males and of absolute weights of lungs (p<0.05) and testes (p<0.01) in
  mid-dose males and increases in relative heart weights (p<0.05) in mid-dose
  females. In three-week-old pups, there were increases in the number of pups with
  skeletal abnormalities at 5 mg/kg bw (1/7 (15%) vs. 0/58 in controls; p<0.01), in
  the number of pups with cervical ribs at 2.5 and 5 mg/kg bw (6/59 (10%) and 1/7
  (15%), respectively, vs. 0/58; p<0.05 and 0.01, respectively). In seven-week-old
6 Ifosfamide
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<pre>pups, absolute weights of all organs recorded were decreased in high-dose male
pups (n=3; p<0.01) and of brain (p<0.05), spleen (p<0.01) and kidneys (p<0.05)
in high-dose females (n=2). In these females, relative ovary weights were
increased (p<0.01). Postnatal functional, behavioural and sexual development
was generally not affected, apart from a dose-related impaired performance in
the water multiple T-maze test in six-weeks-old pups (at 5 mg/kg bw: p<0.05)
and decreased gestational periods in pregnant F1 offspring at 1.25 and 5 mg/kg
bw (p<0.05).12
    Nagaoka and Narama also tested the effects of single intravenous doses of
ifosfamide of 0, 5 and 10 mg/kg bw administered on gestational day 7, 10 or 13
(n=10/group). Injection of 10 mg/kg bw on gestational day 7 caused increases in
the number of foetal deaths (p<0.05) and decreases in mean live foetal mean
body weights, body lengths and tail lengths (all p<0.01) and of placental weights
(p<0.05); injection of 5 mg/kg bw also induced decreased body weights
(p<0.05), body lengths (p<0.01) and tail lengths (p<0.05).
    Injection of 10 mg/kg bw on gestational day 10 resulted in similar effects as
seen when given at gestational 7 and further increases in the number of foetuses
and litters with external, visceral and skeletal abnormalities (p<0.01); after
injection of 5 mg/kg bw, decreases in mean body weights, tail lengths and
placental weights and increases in the number of foetuses with external and
skeletal abnormalities were reported (all p<0.01).
    Injection of 10 mg/kg bw on gestational day 13 caused decreased placental
weights (p<0.01), increased numbers of foetuses with external, visceral and
skeletal abnormalities (p<0.01) and increased numbers of litters with visceral and
skeletal abnormalities (p<0.05) while 5 mg/kg bw increased the number of
foetuses and litters with skeletal abnormalities (p<0.01).12
Nagaoka et al. administered intravenous doses of 0, 2.5, 5 and 10 mg/kg bw to
groups of 20 rats from gestational day 17 to postnatal day 21. Thereafter, dams
were sacrificed and organ weights recorded. The offspring as examined for
external and internal abnormalities, for effects on organ weights, development,
and behaviour and reproductive performance.
    Treatment did not affect maternal body weights but absolute and relative
spleen weights were decreased at all doses (p<0.01) and absolute and relative
liver weights at 5 mg/kg bw (p<0.05 and <0.01, respectively).
    No statistically significant differences between groups were observed with
respect to length of the gestational period, the total number of implantations, the
parturition index, the number of live pups, live litter size, percentage of viability
Ifosfamide                                                                            27
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<pre>  for four days and the weaning rate but the number of stillborns was decreased at
  5 (p<0.05) and 10 mg/kg bw (p<0.01). No external abnormalities were seen.
      Male pup birth weights and male and female pup birth weights were
  decreased at 5 and 10 mg/kg bw, respectively (p<0.05, <0.01 and <0.01,
  respectively), and body weights of male and female pups of the high-dose group
  continued to be lower than those of controls throughout the lactational period
  (p mostly <0.01). Examination of effects on development, puberty, reflexes and
  behavioural tests showed retarded auricle separation at 5 and 10 mg/kg bw (both
  p<0.01), increased ambulation and rearing in the open-field test (both p<0.01)
  and an increased number of errors on the third day in the water T-maze test
  (p<0.05). Upon sacrifice just after weaning, no consistent effects on organ
  weights were seen except for increased relative male and female lung weights
  (p<0.01). Examination of seven-week-old pups showed increases in the number
  of animals with external and visceral abnormalities (mainly hydrocephalus) and
  in the number of litters having rats with external and visceral abnormalities (all
  p<0.01). Upon sacrifice, no consistent changes were observed in relative organ
  weights while absolute weights of the heart, lungs, spleen, kidneys and liver in
  males and females and of the testes were statistically significantly decreased at
  10 mg/kg bw. Mating of groups of 17-20 animals/sex within the same dose
  groups did not reveal consistent effects of ifosfamide treatment of maternal
  animals on the reproductive performance of the F1 generation and the
  development of the F2 generation.13
  Nagaoka and Narama initially treated groups of five pregnant rabbits with
  intravenous doses of ifosfamide of 0, 5, 10, 30 and 50 mg/kg bw from gestational
  days 6-18. Animals were sacrificed at gestational day 29. At 50 mg/kg bw, all
  animals died. At 30 mg/kg bw, body weights were decreased and foetal mortality
  was 100%. In the two lower dose groups, foetal mortality was ‘mildly’ increased.
      Based on these results, Nagaoka and Narama treated groups of ten pregnant
  rabbits with intravenous doses of ifosfamide of 0, 5, 10 and 20 mg/kg bw/day
  from gestational days 6-18. Body weight and food and water consumption
  recordings and general condition observations were performed regularly. Dams
  were sacrificed on gestational day 29 and uterine contents were examined.
      Treatment did not affect maternal body weight gain or water or food
  consumption when compared to controls. At 20 mg/kg bw, 2/10 rabbits had
  anaemia. At autopsy, the only consistent effect on absolute and/or relative organ
  weights were dose-related increases in spleen weights being statistically
  significantly different (p<0.05) at 5 and 10 mg/kg bw.
8 Ifosfamide
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<pre>     Apart from decreased numbers of corpora lutea/litter (10.1±0.9 vs. 12.9±1.7
in controls; p<0.01) and decreased mean foetal body weights (30.9±0.5 g vs.
37.1±5.4 g in controls; p<0.01) at 20 mg/kg bw, no statistically significant
changes were seen in the number of implantations, the number of implantations/
litter, the number of foetal deaths (early and late death, placental remnants, total),
the number of foetal deaths/litter, number of live foetuses, the number of live
foetuses/liter, sex ratio, mean body length, mean placental weight and the
viability of live foetuses at six and 24 hours. Treatment with 20 mg/kg bw caused
increased numbers of foetuses with external malformations, viz. ectrodactyla
(7/82 (9%) vs. 0/98 in controls; p<0.01) of litters with external malformations
(4 vs. 0; n.s.) of foetuses with skeletal abnormalities (22/82 (27%) vs. 1/98 (1%);
p<0.01) of litters with foetuses with skeletal abnormalities (7 vs. 1; p<0.01) and
of foetuses with skeletal variations (62/82 (76%) vs. 39/98 (40%); p<0.01).
Apart from one case of ectrodactyla at 10 mg/kg bw, no effects were observed at
doses of 5 or 10 mg/kg bw.11
Bus and Gibson investigated the teratogenicity of ifosfamide with the knowledge
that the analogue cyclophosphamide is teratogenic. Female Swiss Webster mice
(n=6-7/group) were administered intraperitoneal doses of ifosfamide of 0, 5, 10
or 20 mg/kg bw in saline on gestational day 11. Gravid females were sacrificed
on gestational day 19. The number of live, dead and resorbed foetuses, foetal
weights and gross abnormalities were recorded. Half of the litters was examined
for soft tissue abnormalities and the remaining half for skeletal abnormalities.
     In the control group, no gross, soft tissue or skeletal abnormalities were
observed. At 5 mg/kg bw, the percentages of supernumerary ribs (55±14%;
p<0.05) and of absent or not ossified phalanges (15±10.5% vs. 0% in controls;
n.s. ) were increased. At 10 mg/kg bw, foetal weights and crown-rump lengths
were decreased (p<0.05). Gross abnormalities observed included adactyly
(16±14%; n.s.). With respect to soft tissue and skeletal abnormalities, there were
increased rates of amongst others cleft palate (9±4%; n.s.), internal
hydrocephalus (10±5%; n.s.), cryptorchidism (21±16%; n.s.), fused sternebrae
(22±11%; n.s.), supernumerary ribs (10±8%; n.s.), fused vertebrae (50±16%;
p<0.05), absent or not ossified metatarsals (25±13%; n.s.), metacarpals
(25±13%; n.s.) and phalanges (32±15%; n.s.). At 20 mg/kg bw, the number of
foetuses, foetal weights and crown-rump lengths were decreased (all: p<0.05)
and the number of resorptions increased (p<0.05). The foetuses showed
increased rates of open eyes (90±10%), external hydrocephalus (90±10%),
micromelia (63±19%), adactyly (83±17%), syndactyly (65±16%), microcaudate
(78±15%), and kinky tails (27±19%) (all: p<0.05). Soft tissue examination
Ifosfamide                                                                             29
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<pre>    revealed statistically significant (p<0.05) increases in the rates of internal
    hydrocephalus (90±10%), microphakia (47±23%), kidney ectopia (67±21%) and
    hydronephrosis (77±14.5%) and non-significant increases of cleft palate
    (20±20%), exencephaly (10±10%), and cryptorchidism (40±24.5%). As to
    skeletal abnormalities, incidences of defects such as absent or not ossified skull
    bones, sternebrae, fibula, ulna, radius, metatarsals, metacarpals, and phalanges,
    and fused sternebrae, ribs and vertebrae were increased (p<0.05), ranging from
    44 (fused sternebrae) to 100% (fused ribs/vertebrae; absent or not ossified
    fibula). No information of toxic effects on dams was given.2
    Weigt et al. investigated exposure of zebra fish (Danio rerio) embryos to
    ifosfamide concentrations of 0.25 to 4 mM. Embryos were scored for
    developmental effects and lethality on post-fertilization days 1, 2 and 3. The
    numbers of embryos with developmental effects and of dead embryos were
    statistically significantly increased at concentrations of 1 mM ifosfamide and
    higher. Malformations of the chorda, head, sacculi/otoliths and tail (tip) and
    growth retardation were observed. Several of these developmental effects could
    be detected from two days post fertilization. The authors compared the EC20 of
    916 µM to the human plasma concentration of ~7.6-490 µM after ifosfamide
    treatment in the absence of umbilical cord blood data showing that teratogenic
    effects in zebra fish embryos occur in the same range as human plasma
    concentrations.21
    Lactation
    No studies were available regarding ifosfamide levels in animal milk.
         Nagaoka et al. administered intravenous doses of 0, 2.5, 5 and 10 mg/kg bw
    to groups of 20 rats from gestational day 17 to postnatal day 21. Body weights of
    male and female pups of the high-dose group were decreased throughout the
    lactational period (p mostly <0.01). Examination of effects on development,
    puberty, reflexes and behavioural tests showed some effects on developmental
    and behavioural end points. Upon sacrifice just after weaning, no consistent
    effects on organ weights were seen except for increased relative male and female
    lung weights (p<0.01).13
2.4 Conclusions
    The Committee notes that the doses that caused adverse effects in animals were
    far lower than the human therapeutic doses. However, this does not affect the
 0  Ifosfamide
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<pre>following conclusions and classification proposals since these are based on
hazard evaluation only.
Fertility
No effects were observed in semen and biochemical analyses in male patients
treated for cancer according to multidrug protocols including ifosfamide.5 In a
study in which groups of male patients treated with combinations of drugs with
and without ifosfamide were compared, the incidence of aazospermia was
increased in the groups receiving ifosfamide.9 In two studies among groups of
male patients receiving combinations of drugs with ifosfamide as the only
alkylating/gonadotoxic drug, elevated LH levels (semen characteristics not
investigated)19, decreased sperm counts, increased FSH levels and decreased
inhibin B levels22 were seen. In view of the small group sizes and the
inconsistent results, the Committee is of the opinion that the human studies are
not sufficient for classification.
In laboratory animals, ifosfamide did not affect fertility end points in a study in
which male and female rats were intravenously injected from two weeks before
starting cohabituation, during habituation and until gestational day 7.14 In mice,
ifosfamide caused decreased testes weights and increased sperm abnormalities
following intraperitoneal administration for five days18 and dominant lethal and
specific-locus mutations following single intraperitoneal injections4. In rabbits,
transient decreases in testis and accessory gland weights, sperm counts and
sperm motility, and sperm defects were seen following a single intravenous
injection.23
    Based on animal data, the Committee proposes to classify ifosfamide for
effects on fertility in category 1B.
Developmental toxicity
Only two reports on developmental effects in humans were available: one small
study on developmental toxicity in children fathered by chemotherapy patients
having received ifosfamide9 and one case series on the outcome of pregnancies
of women with high-grade sarcomas diagnosed during the third trimester of
pregnancy and receiving chemotherapy with ifosfamide in combination with
several other agents until delivery10.
    The Committee is of the opinion that this information is not sufficient for
classification.
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<pre>  Ifosfamide intravenously administered to rats before and during cohabitation and
  the subsequent first seven gestational days, from gestational day 7-17 and from
  gestational day 17 to postnatal day 21 induced prenatal and postnatal effects
  including increased foetal mortality, decreased foetal growth, increased
  frequency of external and visceral abnormalities (among which hydrocephalus)
  and skeletal defects (abnormalities, variations, reduced ossification) and
  impaired performance in behavioural tests12-14, and, injected into rabbits from
  gestational day 6-18, increased incidences of external and skeletal abnormalities
  (among which ectrodactyla)11. In these studies, maternal toxicity, viz. decreased
  body weights, decreased spleen and increased lung weights in rats12-14 and
  decreased spleen weights in rabbits11, were reported. However, in one of these
  studies in rats12, retarded ossification was seen in the absence of maternal
  toxicity. In addition, intravenous injection on gestational day 7, 10 or 13 into
  rats12 and intraperitoneal injection on gestational day 11 into mice2 resulted in
  similar developmental effects.
      The Committee is of the opinion that the developmental effects occurred
  independently from maternal toxicity. Therefore, based on the animal data, the
  Committee recommends to classify ifosfamide in category 1B.
  Lactation
  No human or animal data were available on the excretion of ifosfamide in human
  or animal milk nor human data on the effects of ifosfamide on or via lactation.
      In rats, intravenous injection of ifosfamide from gestational day 17 to
  postnatal day 21 caused decreased pup body weights as well as some effects on
  developmental and behavioural end points.13 The effects seen could be caused by
  exposure during lactation, by prenatal exposure through the mother or both, but
  the available data do not allow the Committee to distinguish between these
  possibilities.
      Therefore, the Committee concluded that a lack of appropriate data precludes
  assessment of ifosfamide for effects on or via lactation.
  Proposed classification for fertility
  Category 1B, H360F.
  Proposed classification for developmental toxicity
  Category 1B, H360D.
2 Ifosfamide
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<pre>Proposed labelling for effect on or via lactation
Lack of appropriate data precludes the assessment of ifosfamide for effects on or
via lactation.
Ifosfamide                                                                        33
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<pre>4 Ifosfamide</pre>

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<pre>References
Budavari S, O'Neil M, Smith A, Heckelman P, Obenchain J, editors. Ifosfamide. In: The Merck
Index; an encyclopedia of chemicals, drugs, and biologicals. Whitehouse Station NJ, USA: Merck &
Co, Inc.; 1996.
Bus JS, Gibson JE. Teratogenicity and neonatal toxicity of ifosfamide [3-(2-chloroethyl)-2-[(2-
chloroethyl)amino]tetrahydro-2H-1,2,3-oxazaphosphorine-2-oxide] in mice. Proc Soc Exp Biol Med.
1973;143:965-70.
College voor Zorgverzekeraars (CVZ). Ifosfamide [L01AA06]. In: Farmocotherapeutisch kompas.
[Internet]. [cited 2012 November]. Available from: http://www.fk.cvz.nl/Preparaatteksten/I/
ifosfamide.asp?blPrint=True.
Ehling UH, Favor J, Neuhäuser-Klaus A, Adler ID. Induction of specific-locus and dominant lethal
mutations in male mice by ifosfamide (Holoxan). Genet Res. 1998;72:177-83.
Garolla A, Pizzato C, Ferlin A, Carli MO, Selice R, Foresta C. Progress in the development of
childhood cancer therapy. Reprod Toxicol. 2006;22:126-32.
Health Council of the Netherlands. Ifosfamide; Evaluation of the carcinogenicity and genotoxicity.
[Internet]. [cited 2012 November].The Hague, the Netherlands: Health Council of the Netherlands;
2008: publication no. 2008/06OSH. Available from: http://www.gr.nl/en/publications/ifosfamide-
evaluation-carcinogenicity-and-genotoxicity.
International Agency for Research on Cancer (IARC). Isophosphamide. In: Some antineoplastic and
immunosuppressive drugs. [Internet]. [cited 2012 November]. Lyon, France: IARC; 1981. p. 237-47.
( IARC monographs on the evaluation of the carcinogenic risk of chemicals to humans; Vol. 26).
Available from: http://monographs.iarc.fr/ENG/Monographs/vol1-42/mono26.pdf.
References                                                                                         35
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<pre>  Kerbusch T, de Kraker J, Keizer HJ, van Putten JWG, Groen HJM, Jansen RLH, et al. Clinical
  pharmacokinetics and pharmacodynamics of ifosfamide and its metabolites. Clin Pharmacokinet.
  2001;40:41-62.
  Longhi A, Macchiagodena M, Vitali G, Bacci G. Fertility in male patients treated with neoadjuvant
  chemotherapy for osteosarcoma. J Pediatr Hematol Oncol. 2003;25:292-6.
0 Mir O, Berrada N, Domont J, Cioffi A, Boulet B, Terrier P, et al. Doxorubicin and ifosfamide for
  high-grade sarcoma during pregnancy. Cancer Chemother Pharmacol. 2012;69:357-67.
1 Nagaoka T, Narama I. [Reproductive studies of ifosfamide (Z 4942). Organogenesis administration in
  rabbits]. Kiso to Rinsho 1982; 16: 542-552.
2 Nagaoka T, Narama I. [Reproductive studies of ifosfamide (Z 4942). Organogenesis administration in
  rats.]. Kiso to Rinsho. 1982;16:517-41. Japanese.
3 Nagaoka T, Oishi M, Narama I. [Reproductive studies of ifosfamide (Z 4942). Prenatal and postnatal
  administration in rats.]. Kiso to Rinsho. 1982;16:553-68. Japanese.
4 Nagaoka T, Takatsuka K, Narama I. [Reproductive studies of ifosfamide (Z 4942). Preconceptional
  and early pregnancy administration in rats.]. Kiso to Rinsho. 1982;16:508-16. Japanese.
5 Nederlands Kenniscentrum Farmacotherapie bij Kinderen (NKFK). Kinderformularium; Denekamp
  schaal. [Internet]. [cited 2012 May]. Available from: http://www.kinderformularium.nl/search/
  index.php?content=denekamp.
6 Niessink R, de Vries J, Hoolinger M. Toxicology principles and applications. Boca Raton, FL, USA:
  CRC Press; 1995.
7 Paulussen J, Mahieu C, Bos P. Default values in occupational risk assessment. Zeist, the Netherlands:
  TNO Nutrition and Food Research Institute; 1998. Report No.: V98.390.
8 Quinto I, De Marinis E, De Dominicis G, Della Morte R, Staiano N. Induction of sperm abnormalities
  in mice by ifosfamide and trofosfamide. Mutat Res. 1988;201:113-6.
9 Ridola V, Fawaz O, Aubier F, Bergeron C, de Vathaire F, Pichon F, et al. Testicular function of
  survivors of childhood cancer: A comparative study between ifosfamide- and cyclophosphamide-
  based regimens. Eur J Cancer. 2009;45:814-8.
0 US National Library of Medicine (NLM), ed. Ifosfamide. In: ChemIDplus. [Internet]. [cited May
  2013]. Available from: http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?CHEM.
1 Weigt S, Huebler N, Strecker R, Braunbeck T, Broschard TH. Zebrafish (Danio rerio) embryos as a
  model for testing proteratogens. Toxicology. 2011;281:25-36.
2 Williams D, Crofton PM, Levitt G. Does ifosfamide affect gonadal function? Pediatr Blood Cancer.
  2008;50:347-51.
3 Ypsilantis P, Papaioannou N, Psalla D, Politou M, Simopoulos C. Effects of single dose
  administration of ifosfamide on testes and semen characteristics in the rabbit. Reprod Toxicol.
  2003;17:237-45.
6 Ifosfamide
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<pre>Literature consulted but not cited
Brennemann W, Stoffel-Wagner B, Helmers A, Mezger J, Jäger N, Klingmüller D. Gonadal function
of patients treated with cisplatin based chemotherapy for germ cell cancer. J Urol. 1997;158:844-50.
Corapcioglu F, Dillioglugil O, Sarper N, Akansel G, Caliskan M, Arisoy AE. Spinal cord
compression and lung metastasis of Wilms’ tumor in a pregnant adolescent. Urology. 2004;64:
807-10.
Decanter C, Morschhauser F, Pigny P, Lefebvre C, Gallo C, Dewailly D. Anti-Mullerian hormone
follow-up in young women treated by chemotherapy for lymphoma: preliminary results. Reprod
BioMed Online. 2010;20:280-5.
Fernandez H, Diallo A, Baume D, Papiernik E. Anhydramnios and cessation of fetal growth in a
pregnant mother with polychemotherapy during the second trimester. Prenat Diagn. 1989;9:681-2.
Ishikawa T, Kamidono S, Fujisawa M. Fertility after high-dose chemotherapy for testicular cancer.
Urology. 2004;63:137-40.
Janeway KA, Grier HE. Sequelae of osteosarcoma medical therapy: a review of rare acute toxicities
and late effects. Lancet Oncol. 2010;11:670-8.
Lewis LD. Ifosfamide pharmacokinetics. Invest New Drugs. 1991;9:305-11.
Merimsky O, Le Chevalier T, Missenard G, Lepechoux C, Cojean-Zelek I, Mesurolle B, et al.
Management of cancer in pregnancy: a case of Ewing’s sarcoma of the pelvis in the third trimester.
Ann Oncol. 1999;10:345-50.
Pectasides D, Pectasides E, Papaxoinis G, Skondra M, Gerostathou M, Karageorgopoulou S, et al.
Testicular function in poor-risk nonseminomatous germ cell tumors treated with methotrexate,
paclitaxel, ifosfamide, and cisplatin combination chemotherapy. J Androl. 2009;30:280-6.
Sharon N, Neumann Y, Kenet G, Schachter J, Rechavi G, Toren A. Successful pregnancy after high-
dose cyclophosphamide and ifosfamide treatment in two postpubertal women. Pediatr Hematol
Oncol. 2001;18:247-52.
Wang JJ, Lu H, Chan KK. Stereoselective pharmacokinetics of ifosfamide in male and female rats.
AAPS Pharmsci. 2000;2:98-108.
Ypsilantis P, Papaioannou N, Psalla D, Politou M, Pitiakoudis M, Simopoulos C. Effects of
subchronic ifosfamide-mesna treatment on testes and semen characteristics in the rabbit. Reprod
Toxicol. 2003;17:699-708.
References                                                                                           37
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<pre>8 Ifosfamide</pre>

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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
                                                         39
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<pre>0 Tellurium</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 Center, Utrecht
     •  N. Roeleveld
        Reproductive Epidemiologist, Radboud university medical centre, 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 first draft of this report was prepared by Dr. H.M. Barentsen, from the
     Regulatory Affairs Department of WIL Research Europe BV (Den Bosch,
     The Committee                                                               41
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<pre>  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.
2 Ifosfamide
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<pre>nnex B
     The submission letter (in English)
     Subject         : Submission of the advisory report Ifosfamide
     Your reference  : DGV/MBO/U-932542
     Our reference   : U-8079/HS/cn/543-K14
     Enclosed        :1
     Date            : April 3, 2014
     Dear Minister,
     I hereby submit the advisory report on the effects of ifosfamide 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)                                                43
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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
4 Ifosfamide
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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, D. Murray, S. Rengasamy, K. Krajnak, C. B’Hymer;
         National Institute for Occupational Safety and Health, 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                                              45
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<pre>6 Ifosfamide</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                                                      47
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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.
8 Ifosfamide
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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                                                        49
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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.
0 Ifosfamide
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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                                                         51
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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.
2 Ifosfamide
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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                                                       53
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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
4 Ifosfamide
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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
Regulation (EC) 1272/2008 of the European Community                                                       55
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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.
  6           Ifosfamide
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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                                                        57
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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
 8            Ifosfamide
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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                              59
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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.
0 Ifosfamide
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<pre> nnex         F
              Fertility and developmental studies
              in animals
 able 1 Fertility studies with ifosfamide in animals.
 uthors           species             experimental period/         dose/route    general     effects on reproductive organs/
                                      design                                     toxicity    effects on reproduction
Nagaoka et al. Sprague-Dawley males: 3 wk before                   0, 5, 10, 15 see below    preliminary study
1982)             rats (n=10/sex/     mating, during mating        mg/kg         Table 2     no effect on mating and pregnancy
                  group               females: 3 wk before         bw/d; iv                  rates
                                      mating, during mating,
                                      until gd 7; sacrifice: gd 14
Nagaoka et al. Sprague-Dawley males: 9 wk before                   0, 1.25, 2.5, see below   main study
1982)             rats (n=20/sex/     cohabitation, during         5 mg/kg       Table 2     no effect on percentage of males
                  group               cohabitation                 bw/d; iv                  and females mating; percentage of
                                      females: 2 wk before                                   pregnant females; number of
                                      cohabitation, during                                   corpora lutea; number of
                                      cohabitation,, until gd 7;                             implantation sites; implantation
                                      sacrifice: gd 20;                                      ratio; relative/absolute testis wt;
                                      examination of uterine                                 relative/absolute ovary wt
                                      contents, weighing
                                      primary organs
Quinto et al.     male                5 d; sacrifice: 35 d after   0, 12.5, 25, not reported sperm count (n x 106/epididymis):
1988)             (C3H xC57BL/        1st injection; testis wt,    50, 100                   4.7±0.5, 2.5±0.2, 4.4±1.2, 2.0±0.5,
                  6)F1 mice (n=6/     sperm count and              mg/kg bw/                 2.5±0.9, at 0, 12.5, 25, 50, 100 mg/
                  group) (11-15-wk morphology (500                 d; ip                     kg bw/d, respectively
                  old)                spermatozoa/animal)                                    testis weight (mg/10 g bw):
                                      evaluated                                              76.9±1.6, 72.8±2.9, 74.7±2.5,
                                                                                             69.4±2.5, 62.4±1.8*, respectively
                                                                                             sperm abnormalities (%):
                                                                                             1.6±0.14, 2.1±0.35, 1.6±0.17,
                                                                                             2.6±0.53, 3.9±0.60*, respectively
              Fertility and developmental studies in animals                                                                     61
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<pre> psilantis et al. male New Zealand single dose;                0, 60, 90,             libido: no effect
2003)             white rabbits    sacrifice: n=5/group 1 wk   120, 240               semen quality: total sperm count:
                  (n=10/group) (6- and n=5/group 18 wk after   mg/kg bw;              decreased (vs. pre-treatment values;
                  mo old)          injection;                  iv                     p<0.05;) at wk 5 post-treatment at
                                   parameters evaluated:                              60, 90, 120 mg/kg bw and from wk
                                   reproductive organ wt                              5-7 post-treatment at 240 mg/kg
                                   (testes, epididymides,                             bw; sperm defects: primary defects
                                   accessory glands);                                 (i.e. small, double, or deformed
                                   testicular histology                               heads, swollen, abaxial, or double
                                   (seminiferous tubule                               mid pieces, proximal droplets, and
                                   diameter, percentage of the                        double or coiled tails): increased
                                   most advanced germ cell                            (vs. controls; p<0.05) at wk 2 and 3
                                   type in seminiferous                               post-treatment at 240 mg/kg bw;
                                   tubule cross section,                              secondary defects (i.e. flagging or
                                   number of germ cells per                           detached heads, bent, flagging or
                                   stage 1 seminiferous                               detached midpieces, distal droplets,
                                   tubule cross section);                             and bent tails) increased (vs.
                                   semen quality (weekly                              controls; p<0.05) at wk 1, 2, 6 post-
                                   collection - 4 times on 1 d                        treatment at 120 mg/kg bw, at wk 3-
                                   – for 3 wk prior to                                7 at 240 mg/kg bw; sperm motility:
                                   treatment until day of                             decreased (vs. controls; p<0.05) at
                                   sacrifice: examined for                            wk 2-5 post-treatment at 240 mg/kg
                                   volume, sperm                                      bw.
                                   concentration, total sperm                         post-mortem:
                                   count, sperm motility,                             1 wk post-treatment:
                                   sperm morphology); libido                          bw: no effect;
                                   (willingness to mount a                            testis weight: decreased (vs.
                                   teaser doe in oestrus;                             controls; p<0.05) paired testis
                                   ability to ejaculate)                              weight at 90, 120 and 240 mg/kg
                                                                                      bw (not dose related); no effect on
                                                                                      left or right testis weight
                                                                                      paired epididymis weight: no effect
                                                                                      on head, tail or total wt
                                                                                      accessory sex gland wt: decreased
                                                                                      (vs. controls; p<0.05) at 240 mg/kg
                                                                                      bw
                                                                                      testicular histology: no effect
                                                                                      18 wk post-treatment:
                                                                                      bw: no effect
                                                                                      reproductive organ wt: no effect
                                                                                      testicular histology: no effect
bbreviations: bw=body weight; d=day(s); ip=intraperitoneal; iv=intravenous; mo=month(s); wk=week(s); wt=weight(s).
: p<0.05; **: p<0.01.
 2            Ifosfamide
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<pre> able 2 Developmental toxicity studies with ifosfamide in animals.
 uthors     species     experimental dose/           general toxicity                      developmental toxicity
                        period/ design route
Nagaoka et Sprague- see above           0, 5, 10, 15 no clear signs of toxicity;           preliminary study
 l. (1982)  Dawley      Table 1         mg/kg bw/ decreased bw gain at 10, 15 mg/kg        embryolethality: 4%, 20%*,
            rats (n=10/                 d; iv        bw                                    86%**, 100%**
            sex/
            group
Nagaoka et Sprague- see above           0, 1.25,     male bw (g): 500±41, 494±28,          main study
 l. (1982)  Dawley      Table 1         2.5, 5 mg/ 498±35, 495±39, female bw (g):          foetal viability (%): 96, 93, 91*,
            rats (n=20/                 kg bw/d; iv 395±24, 397±34, 385±26, 377±24*        78**
            sex/ group                               male organ wt, absolute wt of lung    number of living foetuses/pregnant
                                                     (g): 1.71±0.16, 1.74±0.22,            rat: 13.3, 13.1, 11.8, 11.3*
                                                     1.73±0.17, 1.83±0.18*, of spleen      mean foetal bw (g): 3.50±0.19,
                                                     (mg): 991±148, 901±145,               3.43±0.19, 3.42±0.23, 3.13±0.25*
                                                     824±130**, 763±83**, relative wt      no increases in numbers of foetuses
                                                     of lung (mg/100 g bw): 337±27,        with visceral or skeletal
                                                     340±36, 336±30, 364±41*, of spleen    abnormalities
                                                     (mg/100 g bw): 196±32, 176±26*,
                                                     159±18**, 151v14**; female organ
                                                     wt, absolute wt of spleen (mg):
                                                     705±86, 714±116, 633±61**,
                                                     634±72**, relative wt of lung (mg/
                                                     100 g bw): 344±36, 333±20,
                                                     355±42, 384±44**, of spleen
                                                     178±17, 180±25, 165±11*, 169±20,
                                                     of liver (g/100 g bw): 3.88±0.23,
                                                     4.05±0.17*, 3.88±0.22, 3.99±0.19,
                                                     of kidney (mg/100 g bw): 499±39,
                                                     516±41, 520±34, 544±67*
Nagaoka/    female      gd 7-17;        0, 1.25,     no clear symptoms of toxicity;        preliminary study
Narama      Sprague- sacrifice: gd 2.5, 5, 10 decreased bw in late stages of               increased foetal mortality at 5 and
1982)       Dawley      20              mg/kg bw/ gestation                                10 mg/kg bw; 39 and 100%, resp.
            rats (n=10-                 d; iv                                              decreased foetal bw at 2.5 and 5
            13/group)                                                                      mg/kg bw
Nagaoka/    female      gd 7-17;        0, 1.25,     no clear signs of toxicity; decreased main study
Narama      Sprague- sacrifice: gd 2.5, 5 mg/ bw at 2.5 and 5 mg/kg bw during              groups sacrificed on gd 20
1982)       Dawley      20 (n=20/       kg bw/d; iv gestation; in postnatal period         no effect on number of corpora
            rats (n=30/ group)                       increased bw at 5 mg/kg bw; no        lutea; number of corpora lutea/
            group)      examination                  effect on food, water consumption     litter; number of implantations;
                        of uterine                   during gestation, decreased at 5 mg/  number of implantations/litter; sex
                        contents,                    kg bw from pnd 10-14 onwards;         ratio
                        weighing                     at autopsy on gd 20:                  parameters affected:
                        primary                      absolute wt of heart (mg): 924±99,    number of early deaths: 13, 10, 11,
                        organs                       927±84, 957±82, 993±91*               55**
                        pnd 21 (n=10/                of spleen (mg): 631±73, 594±67,       number of placental remnants: 2, 4,
                        group) pup                   601±81, 586±91*                       1, 32**
                        observation                  of liver (g): 14.53±1.32, 14.15±1.61, number of late death: 0, 1, 4, 26**
                        for up to 77 d               13.66±1.27*, 12.65±1.43**             number of total death: 15, 15, 16,
                        (function,                   relative wt of heart (mg/100 g bw):   113**
                        motor                        238±20, 244±18, 258±17**,             foetal death/implantations (%): 5.4,
                        coordination,                291±17**                              5.2, 5.5, 40.1**
                        behaviour,                   of lung (mg/100 g bw): 344±40,        mean number of live foetuses/litter:
           Fertility and developmental studies in animals                                                                    63
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<pre>           fecundity by  340±20, 365±30, 396±59**            13.3±2.0, 13.6±2.7, 13.8±2.8,
           mating to    kidneys (mg/100 g bw): 485±28,       8.5±5.8**
           produce F2   492±39, 500±57, 570±59**             mean bw (g): 3.53 no clear signs of
           generation)  at autopsy on pnd 21:                toxicity; decreased bw gain at 10,
                        absolute wt of heart (g): 1.01±0.09, 15 mg/kg bw 0.22, 3.21±0.22**,
                        1.01±0.08, 1.00±0.10, 1.17±0.11*     3.05±0.21, 1.84±0.34**
                        of ovaries (mg): 90.1±7.5, 96.2±9.8, mean body length (cm): 3.6±0.1,
                        103.6±29, 106.5±14.4*                3.5±0.1**, 3.5±0.1**.1, 2.9±0.2**
                        relative wt of liver (g/100 g bw):   mean tail length (cm): 1.4±0.1,
                        4.25±0.20, 4.21±0.33, 3.89±0.50,     1.3±0.0**, 1.3±0.0**, 1.1±0.1**
                        3.61±0.34**                          mean placental wt (g): 0.45±0.06,
                                                             0.38±0.05**,
                                                             0.32±0.03**, 0.16±0.04**
                                                             number of foetuses with external
                                                             abnormalities (%): 0/265, 0/272, 0/
                                                             275, 4/169 (2.4*
                                                             number of litters with foetuses with
                                                             external abnormalities: 0, 0, 0, 3
                                                             number of foetuses with cervical
                                                             rib: 1/21, 10/22**, 7/32, 6/19*
                                                             groups allowed to deliver
                                                             no effect on gestational period
                                                             total number of implantations: 154,
                                                             146, 142, 133
                                                             number of live pups: 142, 133, 129,
                                                             60
                                                             mean number of live pups/litter:
                                                             14.2±1.1, 13.3±1.8, 12.9±3.0,
                                                             6.7±5.3**
                                                             mean rate of birth (%): 92.5±6.7,
                                                             94.7±6.0, 91.8±7.1, 50.5±36.9
                                                             stillborn rate (%): 0, 3.6*, 1.5,
                                                             13.0**
                                                             mean male pup bw (g): at birth:
                                                             6.2±0.5, 6.0±0.6, 5.7±0.4*,
                                                             3.7±0.4**; pnd 4: 9.0±0.8, 8.8±1.3,
                                                             8.8±1.1, 5.1±1.2**; pnd 7:
                                                             14.7±1.1, 14.0±2.3, 14.3±2.1,
                                                             8.2±0.7**; pnd 14: 29.9±1.3,
                                                             27.5±2.6*, 28.0±2.4*, 19.9±2.7**;
                                                             pnd 21: 45.7±2.5, 43.4±6.9,
                                                             42.3±4.1*, 34.9±5.0**
                                                             mean female pup bw (g): at birth:
                                                             5.8±0.4, 5.6±0.6, 5.4±0.3*,
                                                             3.2±0.1**; pnd 4: 8.5±1.0, 8.2±1.3,
                                                             8.2±1.1, 4.6±0.9**; pnd 7:
                                                             14.0±1.5, 13.4±2.4, 13.3±1.4,
                                                             7.0±1.5; pnd 14: 28.7±2.5,
                                                             25.9±3.5, 27.5±2.9, 17.1±4.4**;
                                                             pnd 21: 43.9±3.3, 41.2±5.9,
                                                             39.8±3.8*, 30.3±6.0**
                                                             pup viability (%): pnd 4: 93.7,
                                                             95.5, 97.7, 45.0**; pnd 21 male:
                                                             100, 92.0* 100, 82.4**/female:
                                                             98.0, 96.0, 100, 82.4**
4 Ifosfamide
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<pre>                                               post-mortem examinations:
                                               pnd 4: no external abnormalities;
                                               organ wt in males: absolute wt of
                                               lung (mg): 465±45, 442±48,
                                               421±45*, 393±60*, of spleen (mg):
                                               208±41, 177±45, 184±31,
                                               141±50*, of liver (g): 1.95±0.15,
                                               1.85±0.35, 1.82±0.26, 1.49±0.46*,
                                               of kidneys (g): 553±36, 526±91,
                                               509±69, 423±130*, of testis (mg):
                                               259±28, 227±52, 221±21**,
                                               148±38**
                                               relative wt of testis (mg/100 g bw):
                                               561±49, 523±78, 519±42,
                                               392±40**
                                               organ wt in females: relative wt of
                                               heart (mg/100 g bw): 511±45,
                                               541±56, 554±32* (no data for high
                                               dose)
                                               pnd 21: no external/internal
                                               abnormalities; number of pups
                                               with skeletal abnormalities (%): 0/
                                               58, 0/55, 1/59 (2), 1/7 (15)**;
                                               number of pups with cervical ribs
                                               (%): 0/58 (0), 2/54 (4), 6/59* (10),
                                               1/7 (15)**
                                               pnd 49: organ wt in males: absolute
                                               wt of brain (g): 1.76±0.06,
                                               1.73±0.06, 1.68±0.10*,
                                               1.51±0.13**, of heart (mg):
                                               958±96, 898±127, 917±128,
                                               709±71**, of lung (g): 1.24±0.18,
                                               1.16±0.11, 1.18±0.33,
                                               0.84±0.03**, of spleen (mg):
                                               635±90, 594±113, 580±108,
                                               491±23**, of liver (g): 12.66±1.04,
                                               11.63±1.88, 12.32±1.92,
                                               9.23±0.36**, of kidneys (g):
                                               2.18±0.14, 2.21±0.22, 2.12±0.36,
                                               1.61±0.26**, of testis (g):
                                               2.72±0.28, 2.72±0.42, 2.57±0.59,
                                               1.85±0.26**
                                               relative wt of brain (mg/100 g bw):
                                               694±31, 733±63, 700±76,
                                               858±11**, of heart (mg/100 g bw):
                                               377±31, 378±23, 380±41, 403±3*
                                               organ wt in females: absolute wt of
                                               brain (g): 1.66±0.07, 1.64±0.08,
                                               1.58±0.10, 1.51±0.12*, of spleen
                                               (mg): 474±83, 478±85, 448±90,
                                               385±1**, of kidneys (g):
                                               1.54±0.12, 1.59±0.13, 1.52±0.16,
                                               1.26±0.11*, of ovaries (mg):
                                               63.2±9.5, 74.4±13.3*, 61.6±13.3,
                                               60.0±9.9
Fertility and developmental studies in animals                                  65
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<pre>                                                                           relative wt of ovaries (mg/100 mg
                                                                           bw): 35.6±4.5, 41.3±6.8*,
                                                                           36.2±6.5, 41.3±0.8**
                                                                           postnatal development
                                                                           no effect on mean day of separation
                                                                           of auricle, emergence of abdominal
                                                                           hear, eruption of lower incisors,
                                                                           separation of eye lid, descent of
                                                                           testes, opening of vagina;
                                                                           at pnd 20: no effect on vision,
                                                                           hearing, pain sense, motility
                                                                           at pnd 28: open-field behaviour:
                                                                           mean ambulation/pup: 50.0±23.7,
                                                                           62.8±24.9, 69.5±23.0, 72.8±32.0;
                                                                           mean rearing/pup: 7.7±4.5,
                                                                           9.6±5.0, 12.1±6.4*, 11.0±3.5;
                                                                           mean grooming/pup: 1.0±1.6,
                                                                           0.2±0.4*, 0.7±1.4, 0.6±0.9; no
                                                                           effect on preening, defecation,
                                                                           urination
                                                                           at pnd 35: no effect on rotarod
                                                                           performance
                                                                           at pnd 42: straight channel
                                                                           swimming test, mean time to reach
                                                                           goal (s): 77.8±21.6, 76.4±36.3,
                                                                           60.2±20.4*, 55.5±11.9*; no effect
                                                                           on water T-maze test (performed 1
                                                                           and 2 d later)
                                                                           at pnd 77: reproductive
                                                                           performance of F1: mean gestation
                                                                           period (d): 21.9±0.3, 21.2±0.8*,
                                                                           20.9±1.1, 21.3±0.5*
                                                                           mean pup bw at birth: 6.5±0.3,
                                                                           6.2±0.5, 6.1±0.3*, 6.1±0.4
                                                                           reproductive performance (of F1 to
                                                                           produce F2): no effect on
                                                                           number of males mated,
                                                                           copulating, impregnating, %
                                                                           copulating/mating, %
                                                                           impregnation/mating,
                                                                           number of females mated,
                                                                           copulating, pregnant, %
                                                                           copulating/mating, % pregnant/
                                                                           mating
                                                                           number of implantations, mean
                                                                           implantations/litter, number live
                                                                           pups, mean live pups/litter, number
                                                                           of stillborns, stillborn rate, mean
                                                                           birth rate, sex ratio, number of pups
                                                                           with external abnormalities
Nagaoka/ female      single         0, 5, 10  no data on maternal toxicity gd 7:
Narama   Sprague-    administration mg/kg bw;                              total number of foetal deaths: 5, 11,
1982)    Dawley      on gd 7, 10 or iv                                     17*
         rats (n=10/ 13                                                    mean bw live foetuses (g):
         group)                                                            3.55±0.17, 3.24±0.41*,
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<pre>                                               3.05±0.25**
                                               mean body length live foetuses
                                               (cm): 3.7±0.07, 3.5±0.17*,
                                               3.4±0.13**
                                               mean tail length of liver foetuses
                                               (cm): 1.4±0.03, 1.3±0.10*,
                                               1.3±0.07**
                                               mean placental wt (g): 0.47±0.05,
                                               0.43±0.05, 0.43±0.03*
                                               gd 10:
                                               total number of foetal deaths:4, 10,
                                               55**
                                               mean bw live foetuses (g):
                                               3.56±0.22, 3.15±0.26**,
                                               2.91±0.42**
                                               mean body length live foetuses
                                               (cm): 3.6±0.08, 3.5±0.13,
                                               3.3±0.24**
                                               mean tail length of liver foetuses
                                               (cm): 1.4±0.05, 1.3±0.08**,
                                               1.2±0.15**
                                               mean placental wt (g): 0.43±0.05,
                                               0.35±0.06**, 0.28±0.08*
                                               number of foetuses with external
                                               abnormalities (%): 0, 13 (10)**, 62
                                               (63)**; number of litters with
                                               abnormal foetuses: 0, 2, 6**;
                                               meningocele, encephalocele (%):
                                               0, 12 (9)**, 54 (55)**; short/curly
                                               tail (%): 0, 1 (0.8), 11 (11)**,
                                               systematic oedema: 0, 0, 35
                                               (367)**, cleft palate (%); 0, 0, 1 (1
                                               number of foetuses with visceral
                                               abnormalities (%): 0, 2 (4), 20
                                               (61)**; number of litters with
                                               abnormal foetuses: 0, 2, 6**;
                                               enlargement of subdural space with
                                               encephalon deformation (%): 0, 1
                                               (2), 10 (30)**; deformation nasal
                                               cavities, olfactory bulb (%): 0, 0, 3
                                               (9)*; deformation eye ball, retina,
                                               cornea (%): 0, 0, 15 (46)**;
                                               hydronephrosis defect, incomplete
                                               kidney development (%): 0, 0, 11
                                               (33)**, subcutaneous oedema (%):
                                               0, 0, 12 (36)**
                                               number of foetuses with skeletal
                                               abnormalities (%): 0, 11 (13)**, 45
                                               (69)**; number of litters with
                                               abnormal foetuses: 0, 3, 7**; rib
                                               abnormalities (%): 0, 10 (11)**, 39
                                               (60)**, fusion, deformation of
                                               vertebrae (%): 0, 3 (3), 29 (45)**,
                                               partial cranium defect (%): 0, 9
                                               (10)**, 38 (59)**
Fertility and developmental studies in animals                                   67
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<pre>                                                                                      gd 13:
                                                                                      mean placental wt (g): 0.43±0.03,
                                                                                      0.42±0.04, 0.38±0.04**
                                                                                      number of foetuses with external
                                                                                      abnormalities (%): 0, 0, 35 (25)**;
                                                                                      number of litters with abnormal
                                                                                      foetuses: 0, 0, 3; meningocele,
                                                                                      encephalocele (%): 0, 0, 35 (25)**
                                                                                      number of foetuses with visceral
                                                                                      abnormalities (%): 1 (1), 3 (7), 9
                                                                                      (20)**; number of litters with
                                                                                      abnormal foetuses: 1, 2, 5**;
                                                                                      enlargement of subdural space with
                                                                                      encephalon deformation (%): 0, 0,
                                                                                      7 (15)**
                                                                                      number of foetuses with skeletal
                                                                                      abnormalities (%): 0, 11 (13)**, 26
                                                                                      (28)**; number of litters with
                                                                                      abnormal foetuses: 0, 5**, 4*; rib
                                                                                      abnormalities (%): 0, 10 (12)**, 7
                                                                                      (7)**, partial cranium defect: 0, 0,
                                                                                      21 (22)**
Nagaoka et female      gd 17-pnd21; 0, 2.5, 5,  F0: no effect on bw; absolute wt of no effect on length of the
 l. (1982) Sprague-    sacrifice       10 mg/kg spleen (mg): 641±113, 511±76**,       gestational period, total number of
           Dawley      dams: pnd 21; bw; iv     460±69**, 438±83, of liver (g):       implantations, parturition index,
           rats (n=20/ primary organ            14.0±1.5, 13.1±1.9, 13.1±1.8,         number of live pups, live litter size,
           group)      weighing;                12.7±1.7*; relative wt of spleen (mg/ percentage of viability for 4 d, the
                       observation F1           100 g bw): 212±30, 171±22**,          weaning rate; no external
                       for 77 d                 154±20**, 147±23**, of liver (g/100 abnormalities
                       (function,               g bw): 4.63±0.38, 4.39±0.43,          number of stillborns: 0, 2, 6**,
                       motor                    4.41±0.54, 4.24±0.34**                11**
                       coordination,                                                  mean male pup bw: at birth:
                       behaviour,                                                     6.4±0.5, 6.3±0.4, 6.1±0.3*,
                       fecundity by                                                   5.8±0.3**, at pnd 4: 9.3±1.2,
                       mating to                                                      9.1±1.0, 8.7±0.8, 8.6±0.7*, at pnd
                       produce F2                                                     7: 14.5±1.6, 14.3±1.5, 13.8±0.9,
                       generation,                                                    13.3±1.0**, at pnd 14: 28.2±2.9,
                       organ wt) and                                                  28.2±1.9, 26.7±1.5*, 25.9±2.1**,
                       F2 (part of the                                                at pnd 21: 44.0±4.3, 44.2±3.2,
                       pregnant F1                                                    42.1±2.4, 39.1±3.3**
                       females                                                        mean female pup bw: at birth:
                       sacrificed at                                                  5.9±0.5, 6.0±0.3, 5.8±0.5,
                       gd 20; the                                                     5.5±0.4**, at pnd 4: 8.8±1.3,
                       remaining                                                      8.9±1.0, 8.5±0.9, 8.1±0.8*, at pnd
                       females                                                        7: 13.6±1.5, 14.1±1.0, 13.4±1.0,
                       allowed to                                                     12.6±1.1*, at pnd 14: 26.8±2.2,
                       litter; F2 pups                                                27.8±1.7, 26.0±1.8, 24.8±1.4**, at
                       sacrificed at                                                  pnd 21: 42.0±3.9, 43.2±2.5,
                       weaning)                                                       41.0±2.6, 37.6±2.2
                                                                                      pnd 21: male pup organ wt:
                                                                                      absolute wt of liver (g): 1.77±0.29,
                                                                                      1.88±0.20, 1.78±0.16, 1.61±0.17*;
                                                                                      relative wt of lung (mg/100 g bw):
                                                                                      1012±64, 992±56, 1011±82,
                                                                                      1109±127**, of liver (g/100 g bw):
  8        Ifosfamide
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<pre>                                               4.14±0.26, 4.32±0.24*, 4.34±23*,
                                               4.14±0.24, of kidney (mg/100 g
                                               bw): 1145±76, 1190±70, 1192±75,
                                               1210±91*
                                               female pup organ wt: absolute wt
                                               of heart (mg): 219±28, 223±22,
                                               218±26, 201±19*, of liver (g):
                                               1.77±0.26, 1.90±0.23, 1.82±0.22,
                                               1.60±0.16*, of kidney (mg):
                                               501±69, 533±60, 516±56,
                                               463±48*, of ovary (mg): 114±27,
                                               132±22*, 119±24, 111±23; relative
                                               wt of lung (mg/100 g bw):
                                               1002±40, 1014±77, 1015±85,
                                               1109±92**, of ovary (mg/100 g
                                               bw): 27.7±5.1, 31.0±4.6*,
                                               29.1±5.2, 30.3±6.5
                                               postnatal development
                                               no effect on mean day of eruption
                                               of lower incisors, separation of eye
                                               lid, descent of testes, opening of
                                               vagina;
                                               mean day of auricular separation:
                                               2.1±0.2, 2.0±0.3, 2.4±0.4**,
                                               2.5±0.4**, of emergence of
                                               abdominal hear: 0.9±0.5, 8.8±0.5,
                                               8.6±0.6, 8.9±0.5
                                               at pnd 20: no effect on vision,
                                               hearing, pain sense, motility
                                               open-field behaviour: mean
                                               ambulation/pup: 47±18, 41±21,
                                               52±18, 60±22**; mean rearing/
                                               pup: 8.3±4.0, 7.4±5.3, 8.5±5.2,
                                               11.5±6.4**; no effect on preening,
                                               grooming defecation, urination, on
                                               rotarod performance, on straight
                                               channel swimming test (1st day),
                                               water T-maze test (2nd day); water
                                               T-maze test 3rd day: time (s):
                                               130±73, 139±61, 140±71,
                                               257±401, error: 3.9±4.8, 5.5±6.2,
                                               4.5±4.1, 24.5±55.1
                                               at pnd 49: external examination:
                                               number of abnormal rats (%): 0, 0,
                                               0, 39** (49), number of litters with
                                               abnormal rats: 0, 0, 0, 15**,
                                               hydrocephalic change: 0, 0, 0, 39;
                                               visceral examination: number of
                                               abnormal rats (%): 1 (3), 2 (5), 23
                                               (62)**, number of litters with
                                               abnormal rats: 1, 2, 0, 16**,
                                               internal,external hydrocephalus
                                               (%): 0, 0, 0, 22 (60)**, unilateral
                                               hydronephrosis (%): 1 (3), 2 (5), 0,
                                               1 (3); male organ wt: absolute wt of
Fertility and developmental studies in animals                                   69
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<pre>             heart (mg): 922±103, 922±85,
             888±81, 825±82*, of lung (g):
             1.20±0.15, 1.23±0.14, 1.15±0.13,
             1.06±0.11**, of spleen (mg):
             650±99, 685±97, 591±65*,
             580±92*, of liver (g): 12.3±1.6,
             12.3±1.4, 11.3±2.0, 10.3±2.0**, of
             kidney (g): 2.09±0.26, 2.08±0.22,
             2.03±0.22, 1.79±0.19**, of testis
             (g): 2.60±0.23, 2.65±0.31,
             2.61±0.25, 2.47±0.27**, relative
             wt of brain (mg/100 g bw):
             713±66, 711±61, 757±62*,
             798±86**, of kidney (mg/100 g
             bw: 837±79, 851±68, 889±63*,
             823±47, of testis (mg/100 g bw):
             1083±75, 1081±54, 1144±82*,
             1137±102; female organ wt:
             absolute wt of heart (mg): 695±60,
             711±79, 715±88, 638±75*, of lung
             ( g): 0.96±0.11, 0.97±0.11,
             0.97±0.08, 0.84±0.12**, of spleen
             (mg): 468±87, 479±85, 472±83,
             393±96**, of liver (g): 8.5±1.0,
             8.7±1.1, 8.6±1.1, 7.2±1.6**, of
             kidney (g): 1.48±0.13, 1.55±0.22,
             1.48±0.14, 1.30±0.17**, relative
             wt of brain (mg/100 g bw):
             956±71, 924±61, 953±61,
             1175±321, of heart (mg/100 g bw):
             395±35, 393±25, 411±36,
             430±43**
             reproductive performance (of F1 to
             produce F2):
             no effect on % males copulating,
             impregnating, % females
             copulating, pregnant;
             F1 pregnant females sacrificed at
             gd 20: no effect on mean number of
             corpora lutea/litter, mean number
             of implantations/ litter,
             implantation ratio, mean number of
             live foetuses/litter, foetal viability,
             mean foetal bw, number of external
             abnormalities; number of foetal
             deaths: 8, 6, 2, 11
             F1 pregnant females allowed to
             litter: mean gestation period (d):
             21.1±0.3, 21.3±0.4, 21.1±0.4,
             20.8±0.5*; number of stillborns: 0,
             1, 1, 3; no effect on mean litter size,
             viability on pnd 4, weaning rate,
             number of external abnormalities.
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<pre>Nagaoka/   female     gd 6-18;      0, 5, 10,    50 mg kg bw: 100% mortality          preliminary study
Narama     Japanese   sacrifice: gd 30, 50 mg/ 30 mg kg bw: decreased bw              30 mg/kg bw: 100% foetal
1982)      white      29            kg bw/d; iv                                       mortality
           rabbits                                                                    5, 10 mg/kg bw: ‘mildly’ increased
           (n=5/                                                                      foetal mortality (not quantified)
           group)
Nagaoka/   female     gd 6-18;      0, 5, 10, 20 no effect on maternal bw, food and   main studies
Narama     Japanese   sacrifice: gd mg/kg bw/ water consumption                       no effects on mean number of
1982)      white      29            d; iv        at 20 mg/kg bw: anaemia in 2/10      implantations/ litter, foetal deaths
           rabbits                               animals;                             (early and late death, placental
           (n=10/                                at autopsy: organ wt: absolute wt of remnants, total), foetal deaths/litter,
           group)                                lung (g): 11.9±1.7, 13.1±1.6,        live foetuses, and live foetuses/
                                                 13.8±1.4*,12.2±1.7, of spleen (9):   liter, on sex ratio, mean body
                                                 1.38±0.51, 1.81±0.67, 1.90±0.44*,    length, mean placental weight,
                                                 2.09±0.74*, relative wt of lung (mg/ viability of live foetuses at 6 and 24
                                                 100 g): 313±36, 352±47, 362±27**,    hr;
                                                 332±51, of spleen (mg/100 g bw):     mean number of corpora lutea/litter
                                                 35.6±10.4, 52.5±22.4, 50.0±12.4*,    : 12.9±1.7, 11.2±2.4, 11.6±1.7,
                                                 57.0±21.5*, of kidney (mg/100g       10.1±0.9**; mean foetal bw (g):
                                                 bw): 485±29, 548±77, 505±54,         37.1±5.4, 36.1±5.0, 33.1±3.1,
                                                 514±44                               30.9±5.0*
                                                                                      number of foetuses with external
                                                                                      abnormalities (%): 0, 0, 1 (1), 7 (9);
                                                                                      number of litters with foetuses with
                                                                                      external abnormalities: 0, 0, 1, 4;
                                                                                      ectrodactyla (%): 0, 0, 1, 7 (9)**;
                                                                                      number of litters with skeletal
                                                                                      abnormalities (%): 1 (1), 0, 1 (1),
                                                                                      22 (27)**, number of litters with
                                                                                      foetuses with skeletal
                                                                                      abnormalities: 1, 0, 1, 7**;
                                                                                      vertebrae fusion: 0, 0, 0, 4*, rib
                                                                                      deformation, shortening, defect: 0,
                                                                                      0, 0, 14**, sternebrae deformation,
                                                                                      fusion, separation: 1, 0, 0, 5*,
                                                                                      digitus defect: 0, 0, 1, 7**; number
                                                                                      of foetuses with skeletal variations
                                                                                      (%): 39 (40), 20 (25), 48 (49), 62
                                                                                      (76)**, limbar rib: 35, 18, 48,
                                                                                      58**, 7th sternebrae: 0, 3*, 0, 7**;
                                                                                      no visceral abnormalities
Bus/Gibson female     gd 11;        0, 5, 10, 20 not reported                         gross abnormalities (%):
 1973)     Swiss      sacrifice: gd mg/kg bw;                                         open eyes: 0, 0, 7.8±5.0,
           Webster    19            ip                                                90.0±10.0*; external
           mice (n=6-                                                                 hydrocephalus, 0, 1.3±1.3, 1.3±1.3,
           7/group)                                                                   90.0±90.0*;
                                                                                      micromelia: 0, 3.9±3.9, 0,
                                                                                      63.3±18.6*; adactyly: 0, 0,
                                                                                      15.8±13.9, 83.3±16.7*; syndactyly:
                                                                                      0, 1.0±1.0, 2.4±2.4, 65.0±15.9*;
                                                                                      microcaudate: 0, 0, 0, 78.3±14.8*;
                                                                                      kinked tail: 0, 1.0±1.0, 1.3±1.3,
                                                                                      26.7±19.4
                                                                                      soft tissue abnormalities (%):
                                                                                      cleft palate: 0, 0, 9.3±4.4,
           Fertility and developmental studies in animals                                                                71
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<pre>                                                                                         20.0±20.0; internal hydrocephalus:
                                                                                         0, 0, 9.9±4.9, 90.0±10.0*;
                                                                                         microphakia: 0, 0, 7.1±7.1,
                                                                                         46.7±22.0*; kidney ectopia: 0, 0,
                                                                                         2.4±2.4, 66.7±21.1*;
                                                                                         hydronephrosis: 0, 0, 0,
                                                                                         76.7±14.5*; exencephaly: 0, 0, 0,
                                                                                         10.0v10.0; cryptorchidism: 0, 0,
                                                                                         20.8±16.3, 40.0±24.5
                                                                                         skeletal abnormalities (%):
                                                                                         absent, not ossified bones: 0, 0, 0,
                                                                                         66.7±33.3*;
                                                                                         absent, not ossified vertebrae: 0,
                                                                                         4.8±4.8, 6.5±4.4, 55.6±29.4*;
                                                                                         fused sternebrae: 0, 3.3±3.3,
                                                                                         22.0±10.8, 44.4±29.4*;
                                                                                         supernumerary ribs: 0, 55.2±14.1*,
                                                                                         10.4±8.2, 0; fused ribs: 0, 0, 0,
                                                                                         100*; absent, not ossified ribs: 0, 0,
                                                                                         2.1±2.1, 16.7±16.7; fused
                                                                                         vertebrae: 0, 0, 49.8±16.1*, 100*;
                                                                                         absent, not ossified fibula: 0, 0, 0,
                                                                                         100*; absent, not ossified ulna: 0,
                                                                                         0, 0, 61.1±20.0*; absent, not
                                                                                         ossified radius: 0, 0, 0, 61.1±20.0*;
                                                                                         absent, not ossified metatarsals: 0,
                                                                                         2.4±2.4, 25.3±13.0, 61.1±20.0*;
                                                                                         absent, not ossified metacarpals: 0,
                                                                                         0, 25.3±13.0, 77.8±22.2*; absent,
                                                                                         not ossified phalanges: 0,
                                                                                         15.2±10.5, 32.1±15.1, 61.1±20.0*;
                                                                                         exostosis: 0, 0, 4.2±4.2, 16.7±16.7
bbreviations: bw=body weight; d=day(s); gd=gestational day(s); hr=hour(s); ip=intraperitoneal; iv=intravenous; n=number(s);
nd=postnatal day(s); s=second(s); wt=weight(s).
: p<0.05; **: p<0.01
2           Ifosfamide
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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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