<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>www.healthcouncil.nl Innovation and the knowledge infrastructure Before we can harvest knowledge in the field of healthcare, we first need to ensure that the right seeds are sown. Healthy working conditions How can employees be protected against working conditions that could harm their health? Environmental health Which environmental influences could have a positive or negative effect on health? Healthy nutrition Which foods promote good health and which carry certain health risks? Prevention Which forms of prevention can help realise significant health benefits? Optimum healthcare What is the optimum result of cure and care in view of the risks and opportunities? Areas of activity Advisory Reports The Health Council’s task is to advise ministers and parliament on issues in the field of public health. Most of the advisory opinions that the Council produces every year are prepared at the request of one of the ministers. In addition, the Health Council issues unsolicited advice that has an ‘alerting’ function. In some cases, such an alerting report leads to a minister requesting further advice on the subject. Health Council of the Netherlands Health Council of the Netherlands Dexamethasone 2016/15 2016/15 Dexamethasone Evaluation of the effects on reproduction, recommendation for classification</pre>

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<pre>Dexamethasone
    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 Dexamethasone
Uw kenmerk            : DGV/BMO/U-932542
Ons kenmerk           : U-1024028/EvV/cn/543-R16
Bijlagen              :1
Datum                 : 18 oktober 2016
Geachte minister,
Graag bied ik u hierbij het advies aan over de effecten van dexamethason 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 Volksgezondheid van de Gezondheidsraad.
Ik heb dit advies vandaag ter kennisname toegezonden aan de minister van VWS en aan
de staatssecretaris van IenM.
Met vriendelijke groet,
prof. dr. J.L. Severens
vicevoorzitter
Bezoekadres                                                       Postadres
Parnassusplein 5                                                  Postbus 16052
2 5 11 V X      Den Haag                                          2500 BB         Den Haag
E - m a i l : p w. v a n . v l i e t @ 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 3 2 7
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<pre></pre>

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<pre>Dexamethasone
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. 2016/15, The Hague, October 18, 2016
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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 and Sport, Infrastructure and the Environment, Social Affairs
and Employment, and Economic Affairs. 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. Dexamethasone. Evaluation of the effects on
reproduction, recommendation for classification. The Hague: Health Council of
the Netherlands, 2016; publication no. 2016/15.
all rights reserved
ISBN: 978-94-6281-086-0
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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 15
.4 Data 15
.5 Presentation of conclusions 16
.6 Final remark 16
   Dexamethasone 17
.1 Introduction 17
.2 Human studies 19
.3 Animal studies 20
.4 Conclusions 43
   References 47
   Contents                       7
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<pre>  Annexes 55
A The Committee 57
B The submission letter (in English) 59
C Comments on the public draft 61
D Regulation (EC) 1272/2008 of the European Community 63
E Additional considerations to Regulation (EC) 1272/2008 75
F Fertility and developmental toxicity studies 77
  Dexamethasone
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<pre>Samenvatting
In het voorliggende advies heeft de Gezondheidsraad dexamethason onder de
loep genomen. Dexamethason is een synthetisch steroïdhormoon (cortico-
steroïde). Het wordt gebruikt als geneesmiddel voor onderdrukking van ontste-
kingsreacties (aspecifiek anti-inflammatoir effect) en van (auto-)immuunproces-
sen. 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 tij-
dens de beroepsuitoefening kunnen worden blootgesteld. Mensen die werkzaam
zijn in de farmaceutische industrie, in apotheken of in ziekenhuizen kunnen tij-
dens hun werk in aanraking komen met dexamethason.
    De Subcommissie Classificatie reproductietoxische stoffen van de Commis-
sie Gezondheid en beroepsmatige blootstelling aan stoffen (GBBS) van de raad,
hierna aangeduid als de commissie, kijkt zowel naar effecten op de vruchtbaar-
heid van mannen en vrouwen als naar effecten op de ontwikkeling van het nage-
slacht. Daarnaast worden effecten op de lactatie en via de moedermelk op de
zuigeling beoordeeld.
Op basis van Verordening (EG) 1272/2008 van de Europese Unie doet de com-
missie een voorstel voor classificatie. Voor dexamethason komt de commissie tot
de volgende aanbevelingen:
• voor effecten op de fertiliteit adviseert de commissie dexamethason niet te
    classificeren wegens onvoldoende geschikte gegevens
Samenvatting                                                                     9
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<pre>  •  voor effecten op de ontwikkeling adviseert de commissie dexamethason in
     categorie 1B (stoffen waarvan verondersteld wordt dat zij toxisch zijn voor
     de menselijke voortplanting) te classificeren en met H360D (kan het ongebo-
     ren kind schaden) te kenmerken
  •  voor effecten op of via lactatie adviseert de commissie om dexamethason niet
     te kenmerken wegens onvoldoende geschikte gegevens.
0 Dexamethasone
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<pre>Executive summary
In the present report, the Health Council of the Netherlands reviewed
dexamethasone. Dexamethasone is a corticosteroid. It is used as an anti-
inflammatory or immunosuppressive agent. 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. Man can be
occupationally exposed to dexamethasone in the pharmaceutical industry, in
pharmacies or in hospitals.
    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 dexamethasone, these recommendations are:
• for effects on fertility, the Committee recommends not classifying
    dexamethasone due to a lack of appropriate data
• for effects on development, the Committee recommends classifying
    dexamethasone 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
     dexamethasone due to a lack of appropriate data.
2 Dexamethasone
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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 B and 2) or compound with effects on or via lactation.
1.2     Committee and procedure
        This present document contains the classification of dexamethasone by the
        Health Council’s Subcommittee on the Classification of Reproduction Toxic
        Substances. The members of the Committee are listed in Annex A. The
        submission letter (in English) to the Minister can be found in Annex B.
        Scope                                                                             13
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<pre>      In 2015 the President of the Health Council released a draft of the report for
  public review. The individuals and organizations that commented on the draft
  report are listed in Annex C. The Committee has taken these comments into
  account in deciding on the final version of the report.
  The classification is based on the evaluation of published human and animal
  studies concerning adverse effects with respect to fertility and development and
  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
  Hazard statement codes:
  H360F                     May damage fertility.
  H360D                     May damage the unborn child.
  H361f                     Suspected of damaging fertility.
  H361d                     Suspected of damaging the unborn child.
  H360FD                    May damage fertility. May damage the unborn child.
  H361fd                    Suspected of damaging fertility. Suspected of damaging the unborn child.
  H360Fd                    May damage fertility. Suspected of damaging the unborn child.
  H360Df                    May damage the unborn child. Suspected of damaging fertility.
  H362                      May cause harm to breast-fed children.
  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).
4 Dexamethasone
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<pre>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
    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 exceeds 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 of Medline, starting
    from 1966 up to 2008, and by searches on the Internet; updates were performed
    in TOXNET, the latest one in July 2015. Literature was selected primarily on the
    basis of the text of the abstracts. Publications cited in the selected articles, but not
    selected during the primary search, were reviewed if considered appropriate. In
    addition, handbooks and a collection of most recent reviews were consulted as
    well as several websites regarding (publications on) toxicology and health.
    References are divided in literature cited and literature consulted but not cited.
    The Committee describes both the 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
    dexamethasone, the Committee also used data on adverse effects related to its
    application as a therapeutic agent.
    Scope                                                                                    15
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<pre>1.5 Presentation of conclusions
    The classification is given with key effects, species, and references specified. In
    case a substance is not classified as toxic to reproduction, one of two reasons is
    given:
    • Lack of appropriate data preclude assessment of the compound for
        reproductive toxicity.
    • Sufficient data show that no classification for toxic to reproduction is
        indicated.
1.6 Final remark
    The classification of compounds is based on hazard evaluation (Niesink et al.,
    199538) only, 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  Dexamethasone
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<pre> hapter      2
             Dexamethasone
2.1          Introduction
 ame                  : dexamethasone
UPAC name             : (11β,16α)-9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione
  AS name             : pregna-1,4-diene-3,20-dione, 9-fluoro-11,17,21-trihydroxy-16-methyl-, 11β,16α)-
CAS registry number   : 50-02-2
 U/EINECS number      : 200-003-9
 ynonyms              : 16α-methyl-9α-fluoro-1,4-pregnadiene-11β,17α,21-triol-3,20-dione; 16α-methyl-9α-fluoro-
                        11β,17α,21-trihydroxypregna-1,4-diene-3,20-dione; 16α-methyl-9α-fluoroprednisolone; 16α-
                        methyl-9α-fluoro-∆1-hydrocortisone; 1-dehydro-16α-methyl-9α-fluorohydrocortisone; 9-fluoro-
                        11β,17,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione; 9α-fluoro-11β,17α,21-trihydroxy-
                        16α-methyl-1,4-pregnadiene-3,20-dione; 9α-fluoro-16α-methyl-1,4-pregnadiene-11β,17α,21-triol-
                        3,20-dione; 9α-fluoro-16α-methyl-11β,17,21-trihydroxypregna-1,4-diene-3,20-dione; 9α-fluoro-
                        16α-methylprednisolone
 olour/physical state : white to practically white crystalline powder
molecular weight      : 392.5
molecular formula     : C22H29FO5
ormula                :
             Dexamethasone                                                                                         17
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<pre>melting point           :   262-264 oC
 apour pressure         :   1.2 x10-11 Pa (at 25 oC) (estimated)
 og Poctanol/water      :   1.83
 olubility              :   10 mg/100 mL water (at 25 oC); soluble in acetone, ethanol, chloroform
 ses                    :   as a synthetic glucocorticoid with anti-inflammatory and anti-rheumatic activity and
                            immunosuppressant effects in a wide variety of disorders; antenatal administration to pregnant
                            women, for instance to those at risk of delivering a child with congenital adrenal hyperplasia (off-
                            label use in the Netherlands)54
 ccupational exposure       can occur in the pharmaceutical industry, in pharmacies or in hospitals
 inetics                : toxicokinetic studies revealed rapid absorption after intramuscular administration to dogs and rats
                            with peak plasma levels found after 30 minutes and six hours, respectively. Dexamethasone is
                            rapidly excreted in urine and faeces. Dexamethasone esters are rapidly hydrolyzed in serum.
                            Biotransformation in rats and humans is comparable and involves mainly hydroxylation to
                            6-hydroxy- and 20-dihydro-dexamethasone. However, there was additional evidence that at high
                            (therapeutic) doses in people, dexamethasone is metabolized by an additional route involving
                            epoxidation28
 eneral toxicity        : following repeated oral administration of dexamethasone to rats and dogs in short-term toxicity
                            studies, the main target organs were the thymus and the adrenal gland. Corticosteroid
                            concentrations in plasma and hepatic glycogen were reduced, whereas serum lipid levels were
                            increased. In rats given oral doses of 0.0003-0.1 mg dexamethasone/kg bw/day for 90 days, the loss
                            of thymus size, mass and cortical T cells, and morphological changes in the adrenal gland and a
                            decrease in corticosterone and white blood cell counts were observed in male and female rats at
                            doses above 0.01 mg/kg bw/day. Due to the decrease in white blood cell counts in female rats at
                            0.003 mg/kg bw/day, this dose was considered to be a marginal effect level. In a study with rats
                            given oral doses of 0.0005-0.004 mg/kg bw/day dexamethasone for seven days, the corticosterone
                            concentration was reduced in the highest-dose group and the activity of tyrosine aminotransferase
                            in the liver was increased in a dose-related manner at 0.002 and 0.004 mg/kg bw/day. The No
                            Observed Effect Level in this study was 0.0015 mg/kg bw/day28
Data from HSDB57 unless otherwise noted
              The adrenal cortex makes and secretes two different classes of hormones, the
              glucocorticoids and the mineralocorticoids. These hormones are involved in the
              regulation of metabolism and sodium and potassium balance, respectively.
              Dexamethasone is a synthetic derivative of the glucocorticoid hydroxycortisone.
              It is used for its anti-inflammatory and anti-rheumatic activity and its
              immunosuppressant effects. Since dexamethasone can pass the placental barrier
              and perinatal foetal serum concentrations are almost 100% of the maternal
              concentrations, maternal administration of dexamethasone to pregnant women is
              widely used therapeutically to promote lung maturation in human foetuses
              considered at risk of preterm delivery.
 8            Dexamethasone
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<pre>2.2   Human studies
2.2.1 Fertility studies
      No studies were available regarding the effects of exposure to dexamethasone on
      human fertility.
2.2.2 Developmental toxicity studies
      Carmichael and Shaw performed a population-based case-control study
      including 662 cases of orofacial clefts, 207 conotruncal heart defects, 265 neural
      tube defects, 165 limb reduction defects and 734 healthy controls, and collected
      information on medication use from one month before conception through the
      third month of pregnancy in maternal interviews a few years after delivery. The
      mother of one child with an orofacial cleft reported dexamethasone use, versus
      none of the control mothers.9
      Dawes et al. determined the effect of administration of dexamethasone to
      pregnant patients on foetal heart rate and its variation in a retrospective analysis
      of computerized data derived from case studies. Dexamethasone (two doses of
      12 mg intramuscularly, 12 hours apart, one to four occasions per patient) was
      given at weekly intervals to 28 pregnant women at weeks 27 to 32 of pregnancy.
      Foetal heart rate two days before and four days after dexamethasone treatment
      (n=28) and umbilical arterial flow velocity (n=19) were measured and analysed.
      The results showed an increase in foetal heart rate variation for up to one day
      (n=10, p<0.01). In case of foetal distress and reduced umbilical flow (n=18),
      only a small increase in foetal heart rate variation was observed.11
      In a randomised controlled trial of pregnant women at increased risk of preterm
      delivery, Mulder et al. studied the effects of dexamethasone versus
      betamethasone (concentration and route of exposure not described) on foetal
      behaviour and foetal heart rate variation. The women (two groups of 30) were
      between weeks 26 and 33 of pregnancy and were studied daily over five
      successive days. For dexamethasone, an increase in foetal heart rate short term
      variation was found on day 1 (p<0.05). The observed change was transient, as
      the values returned to baseline on day 4.35
      Dexamethasone                                                                        19
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<pre>      Senat et al. also studied the effects of dexamethasone and betamethasone on
      foetal heart rate of foetuses in a clinical trial. Pregnant women with preterm
      labour (n=40) received four intramuscular injections of 4 mg dexamethasone at
      weekly intervals starting between weeks 25 and 33 of pregnancy. No changes in
      foetal heart rate variability were found 1-7 days after dexamethasone treatment.45
      Lee et al. investigated the neurodevelopmental outcome of extremely low birth
      weight infants exposed prenatally to dexamethasone. Dexamethasone was given
      as four 6 mg intramuscular doses administered with 12-h intervals during the
      admission for delivery. In this retrospective cohort study, infants weighing 401-
      1,000 grams at birth were included (n=408). Neonates with similar birth weights
      and receiving no steroids served as controls (n=153). Cerebral palsy, Barley
      scales of infant development, mental development, psychomotor development,
      blindness, hearing, neurodevelopmental impairment and unimpaired
      neurodevelopmental status at corrected gestational age were measured. There
      were no associations between prenatal dexamethasone exposure and any follow-
      up outcome, compared with no prenatal steroid exposure.31
      Amador-Licona et al. investigated the effect of prenatal dexamethasone on the
      renal vascular resistance in preterm infants in a cross-sectional study. Pregnant
      women were treated with 6 mg dexamethasone intramuscularly, repeated every
      12 hours for four doses, 48 hours or more before delivery between weeks 27 and
      34 of pregnancy. The study included 37 infants treated with dexamethasone
      prenatally and 24 infants without treatment with prenatal steroids served as
      controls. Preterm infants who received prenatal dexamethasone and control
      infants showed no differences in birth weight or gestational age. The renal
      resistance measured between 12 and 72 hours after birth was decreased in treated
      infants (right renal artery p=0.001 and left renal artery p=0.01) without affecting
      renal volume and insulin levels.1
2.2.3 Lactation
      No data are available regarding the excretion of dexamethasone in breast milk or
      the effects of exposure to dexamethasone on infants during the lactation period.
2.3   Animal studies
      Fertility and developmental toxicity studies in laboratory animals are
      summarized in Annex F.
 0    Dexamethasone
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<pre>2.3.1 Fertility studies
      Effects on female fertility
      De Greef and Van der Schoot investigated the effects of dexamethasone on the
      ovarian activity in rats. Wistar rats were injected subcutaneously with daily doses
      of 0.1 mL dexamethasone sodium phosphate solution (0.25, 1 or 4 mg/mL
      saline). Several experiments were carried out. A control group was included
      (n=unknown). In the first experiment, rats were treated from the day of ovulation
      with 0.008 (n=6), 0.03 (n=7) or 0.1 (n=14) mg/kg body weight (bw)
      dexamethasone. The animals were killed on the first day following the
      observation of a cornified vaginal smear or eight to 23 days after the start of
      treatment. At autopsy, weights of the ovaries, adrenal glands and uterus were
      recorded. Ovaries were examined microscopically and fallopian tubes were
      examined for eggs. In the second experiment, daily treatment with 0.1 mg/kg bw
      dexamethasone was studied when treatment started two days before expected
      ovulation (n=17). Ten rats were killed two days later (to examine whether
      ovulation had occurred) and the remaining seven rats were killed after eight to 12
      days of treatment. Uterine weight, ovarian weight and adrenal weight were
      recorded and ovaries were examined macroscopically and microscopically. In a
      third experiment (0.1 mg/kg bw dexamethasone from the day of ovulation, killed
      on day 9 of treatment; n=10) pseudopregnancy was investigated. Dexamethasone
      at the highest dose prolonged the ovarian cycle in the first experiment (p≤0.05).
      Histologically, the ovaries showed one generation of corpora lutea with signs of
      luteolysis and numerous ruptured follicles. All three treatment groups showed
      suppression of the weight of the adrenal glands at autopsy after 7-10 days. The
      seven rats killed after eight to 12 days of treatment in the second experiment
      showed a prolonged oestrous cycle. No differences were observed in the third
      experiment, except for an increased number of ovulation follicles after the start
      of treatment close to the expected time of ovulation and in pseudopregnancy
      (p=0.05). General toxicity was not described in the results.12
      Maciel et al. investigated the effect of dexamethasone on ovarian follicular
      development and plasma hormone concentrations. Non-lactating Holstein cows
      were synchronized using prostaglandin F2α and one day after ovulation the
      animals (n=6) received a daily intramuscular injection of 0.044 mg
      dexamethasone/kg bw until the first dominant follicle stopped growing. Control
      animals (n=5) received vehicle injections (1 mL/100 kg bw/day of vehicle
      Dexamethasone                                                                       21
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<pre>  containing ethanol, polyethylene glycol, and sterile-filtered Millipore water).
  Cows were weighed daily and blood samples were collected daily. There was no
  effect on body weight. Dexamethasone increased systemic glucose (p<0.05) and
  insulin concentrations (p<0.05), decreased plasma concentrations of insulin-like
  growth factor-I and II (p<0.05) without affecting insulin-like growth factor
  binding protein levels, did not affect the growth rate of dominant follicles,
  decreased plasma progesterone concentrations (p<0.10) without affecting
  gonadotropin levels and had no effect on plasma leptin concentrations.32
  Effects on male fertility
  Hatamoto et al. studied the effect of dexamethasone treatment on sperm
  parameters, seminal plasma antioxidant enzyme activity. Male adult Rottweiler
  dogs (n=18) were allocated to treatment groups (two groups were included to
  investigate whether oral supplementation with Vitamin E reduced adverse effects
  of dexamethasone). Dexamethasone was given intramuscularly for 7 days at a
  dose of 0.01 mg/kg bw/day. A control group was included. Body weight and food
  consumption were not affected. Semen collections were performed twice weekly
  for 14 weeks and blood samples to measure hormone levels were collected once
  a week. Dexamethasone treatment reduced ejaculate volume (p=0.0002) and
  increased thiobarbituric acid-reactive substances in the seminal plasma
  (p<0.0001). It also increased the number of sperm per ejaculate (p=0.0122), the
  percentage of abnormal sperm (p=0.0002) and the seminal plasma lipid
  peroxidation (p=0.0264). Furthermore, it elevated the activity of one of two
  antioxidant enzymes (p=0.0264).20
  Gür et al. investigated the influence of dexamethasone on sperm characteristics
  and hyaluronidase activity of semen and serum. Akkaraman rams (n=7) were
  injected intramuscularly at a dose of 0.25 mg/kg bw for 2 days. A control group
  (n=7) was included. After the last administration, blood and semen samples were
  taken after 1, 2, 4, 24, 48, 72 and 96 hours. Semen evaluation and enzyme assays
  were performed. The results indicated that dexamethasone increases
  hyaluronidase activity in serum (p<0.001, except for the 1st hour) and semen
  (p<0.001, p<0.01, p<0.05), but decreases sperm concentration (p<0.001 except
  the 96th hour), semen volume (p<0.05) and sperm motility (p<0.05 except for the
  72 and 96th hours) in rams. The rate of abnormal spermatozoa was not affected.
  Information on general toxicity was not provided.18
2 Dexamethasone
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<pre>      Barth et al. investigated the sequential appearance of sperm abnormalities after
      dexamethasone treatment. Bulls (n=8) of mixed Bos Taurus breeds with normal
      semen quality were treated with 20 mg dexamethasone intramuscularly daily for
      seven days. Untreated controls (n=4) were included. Beginning on the first day
      of treatment semen was collected in 4 bulls of each group three times a week for
      the first 25 days and then twice a week until day 42. Semen volume,
      concentration, motility, percent alive and differential counts of sperm
      abnormalities were recorded. Four animals treated for seven days were used to
      determine the effect on blood and testis tissue concentrations of testosterone.
      Control bulls were used for both semen and blood sampling. Dexamethasone
      induced lower basal, peak episodic and mean testosterone concentrations in
      blood (p<0.05). The concentrations in testis tissue were unchanged. There was a
      marked increase in sperm defects such as detached heads, midpiece defects and
      nuclear vacuoles (no statistics mentioned). However, all bulls, recovered to
      approximately pre-treatment levels of sperm defects by six weeks after initiation
      of treatment. General toxicity was not described in the results.5
2.3.2 Developmental toxicity studies
      Teratogenic potential
      No studies were available on the effects of oral administration of dexamethasone.
      The relationship between glucocorticoids and teratogenic potential was studied
      by Jerome and Hendrickx by comparing the effect of triamcinolone acetonide
      and dexamethasone given to pregnant monkeys. Rhesus macaques (n=12) were
      treated intramuscularly with 1.0 or 10.0 mg/kg dexamethasone sodium
      phosphate at different days during pregnancy (between gestation days 23 and
      49). A control group of two concurrent untreated animals and nine untreated
      control animals of a previous study was included. Pregnancies were terminated
      by hysterectomy on gestation day 100 ± 3. All foetuses were weighed,
      photographed, and examined for external and internal abnormalities. No effect
      on body weight was observed. The brain weight and the diameter of the cranial
      fossa were reduced in the highest dose group (p<0.05). The malformations
      observed appeared to be limited to the cranium, i.e. cranium bifidum* and aplasia
       a congenitally incomplete skull, often with an incomplete brain
      Dexamethasone                                                                     23
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<pre>  cutis congenita* (2 out of 6 animals affected at 1.0 mg/kg, 4 out of 6 animals at
  10.0 mg/kg). Presence or absence of maternal toxicity was not reported.27
  Sauerbier studied the effect of prenatal dexamethasone as a function of timing of
  the dose. NMRI mice (n=12/group) were treated intramuscularly at doses of 10
  or 50 mg/kg bw on gestation day 13 at four selected time points: 07:00, 13:00,
  19:00 or 01:00 (according to 4 different breeding periods, i.e. 06:00-08:00;
  12:00-14:00; 18:00-20:00 and 24:00-02:00). All mice were sacrificed at
  gestation day 18. Reproductive parameters were determined and foetuses were
  examined for external and skeletal abnormalities. Control groups (n=10), treated
  similarly with saline, were included. Cleft palate and resorptions were observed
  in all dexamethasone-treated groups. A maximum response, manifested by
  teratogenicity and embryolethality, was observed at 07:00 for both dose levels.
  A minimum response was seen at 19:00. The effects were dose-dependent.
  P-values were not mentioned. In this study it was possible to demonstrate a
  circadian rhythmicity in dexamethasone-induced cleft palate and
  embryolethality. Maternal toxicity was not described in the results.44
  Ballard et al. treated pregnant CD-1 mice (n=unknown) in both eyes with
  1 µl-drops of dexamethasone five times daily during gestation days 10-13. Four
  treatment groups were included: 1) control, saline; 2) low dose, approximately
  the human therapeutic concentration (not mentioned); 3) medium dose, one-half
  log unit above the low dose and 4) high dose, one log unit above the low dose.
  Mice were weighed throughout gestation and sacrificed at gestation day 18.
  Caesarian section was performed and all foetuses were processed for internal
  examination according to the Wilson method58. A dose-related increase in the
  incidence of cleft palate (all dose levels, p<0.05) and a dose-related increase in
  the incidence of sex organ abnormalities (dislocation, in the mid- and high-dose
  groups only, p<0.05) were observed. Maternal toxicity was not described in the
  results.4
  Cardiovascular effects
  No studies were available on cardiovascular effects of oral administration of
  dexamethasone.
  the congenital absence or deficiency of a localized area of skin, usually on the scalp, with the base of
  the defect covered by a thin translucent membrane
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<pre>In a mechanistic study of Torres et al. the maturation of the rat heart was
investigated after prenatal dexamethasone treatment in rats. A subcutaneous,
slow release, dexamethasone pellet (approximately 0.048 mg/day) was inserted
to pregnant rats (n=8) beginning at gestation day 17. Control dams were
unmanipulated (n=8). After 4-5 days of exposure, hearts were collected from
neonatal rats within 24 h after birth. The organs were collected from 7-8 male
and female offspring from treated and control mothers each. The effects on heart
growth, cell number and DNA synthesis, extracellular matrix (ECM) and myosin
heavy chain (MHC) mRNA expression were determined. Prenatal exposure to
dexamethasone produced a higher heart/body weight ratio (p<0.05) and
proliferative index (in females only, p<0.05) in association with a relative
decrease in ECM content (p<0.05) and α-MHC mRNA (in males only, p<0.05),
findings indicative for an immature heart as compared to the control group.
Presence or absence of maternal toxicity was not reported. The average litter size
and mortality was not different between the groups.53
Slotkin et al. investigated the effect of prenatal dexamethasone treatment on the
development of the neonatal heart and kidney. Pregnant Sprague Dawley rats
(n=unknown) were treated subcutaneously with 0.2 or 0.8 mg dexamethasone
phosphate/kg bw/day on gestational days 17, 18 and 19. Controls received
equivalent volumes of saline vehicle (1 ml/kg). Maternal weight gain was
recorded and dams were allowed to litter. Pups were randomized and
redistributed to nursing dams within the treatment group. At autopsy at weaning
the body weight, heart weight and three biochemical markers of cell
development in the heart were assessed: DNA content of the heart as an index of
total cell numbers, DNA content per gram of tissue as an index of cell packing
density and protein/DNA ratio as an index of relative cell size. Body and heart
weights were lower (p<0.01). DNA content of the heart was diminished by
dexamethasone at both doses (p<0.01). Cell packing density was decreased at
first (p<0.01) and cells were enlarged (p<0.01). The conclusion from the results
of this study was that foetal exposure to dexamethasone causes widespread
inhibition of cell proliferation with disruption of parameters of cell
differentiation and growth in the heart. Dexamethasone slowed maternal weight
gain, but did not reduce the proportions of dams delivering pups or litter size at
birth (data not shown).47
Dodic et al. investigated whether the dexamethasone-induced hypertension seen
in a previous study14 was associated with left ventricular hypertrophy and a
reduced cardiac functional reserve (COmax-0). Pregnant ewes (n=unknown) were
Dexamethasone                                                                      25
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<pre>  treated intravenously with 11.5 mg dexamethasone per day for 2 days at 27 days
  of gestation. Six female lambs served as a control group. Lambs were allowed to
  litter and surgery was performed at 50 days of age. Brief prenatal exposure led to
  the development of hypertension (p<0.01), left ventricular hypertrophy (p<0.05),
  and reduced cardiac functional reserve (p<0.05) in adult life. Maternal toxicity
  was not described in the results.15
  Evidence for microvascular dysfunction after prenatal dexamethasone treatment
  in sheep was given by Molnar et al.. Dexamethasone was administered to
  pregnant ewes as three weekly courses of four intramuscular injections of 2 mg
  at 12h-intervals. Dexamethasone (n=7) or saline (n=7) was given at days 103,
  110 and 117 of gestation. All ewes underwent Caesarean section and foetal
  femoral arteries were evaluated using wire myography. Foetal body weight was
  lower (p<0.01). Microvessel dysfunction, as expressed by enhanced endothelin-
  induced vasoconstriction (p=0.003 at the highest concentration), decreased
  endothelium-dependent relaxation (p<0.05) and normal endothelium-
  independent relaxation was observed, a combination which is associated with
  several forms of adult hypertension and thus adult cardiovascular health.
  Maternal toxicity was not described in the results.33
  To determine the effects of a single course of maternally administered
  dexamethasone on foetal sheep in utero, Quaedackers et al. treated pregnant
  Romney/Suffolk ewes at gestation day 103 intramuscularly with two injections
  (24 hours apart) of 12 mg dexamethasone (n=8) or vehicle (n=7). Foetuses were
  continuously monitored for five days. In this study, a transient increase in blood
  pressure in the preterm sheep foetus was observed (p<0.05), with no sustained
  changes in vascular resistance. Maternal toxicity was not described in the
  results.42
  The effect of maternal dexamethasone treatment on the development of foetal
  arteries was investigated by Hai and colleagues. Pregnant ewes were given a 6
  mg intramuscular injection or placebo (0.9% saline) injection every 12h for 48h
  starting at gestation days 104, 105 or 106 (single dose group, n=unknown) and
  on gestation days 76, 84, 91, 98 and 105 of gestation in the repeated-dose group.
  The dose and treatment schedule were based on current treatment practice in
  pregnant women with premature labour. Foetuses were catheterized at 99-101
  days of gestation and foetal arterial samples were collected on days 106-108.
  Thereafter, foetuses were removed and weighed and carotid arteries were
  dissected. Contraction and myosin light chain isoform expression were
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<pre>investigated. Multiple dosing during pregnancy resulted in a reduced pup body
weight (p<0.05). In response to 1 µM phenylephrine, arteries from foetuses of
dexamethasone-treated ewes exhibited biphasic contractions (p<0.0001). The
relaxation rate was higher in arteries from foetuses of dexamethasone-treated
dams than control animals (p<0.05). Repeated maternal administration of
dexamethasone induced an almost twofold increase in myosin LC17a isoform
expression in the foetal arteries (p<0.05). Maternal toxicity was not described in
the results.19
Neurodevelopmental effects
Oral administration
Hauser et al. studied the effect of prenatal dexamethasone treatment on maternal
endocrinology (plasma cortisol and oestrogen titres) and postnatal physical
growth, plasma and urinary adrenocorticotropic hormone and cortisol titres, and
social and maintenance behaviour from birth to weaning in the common
marmoset monkey. Pregnant marmosets received 5 mg dexamethasone/kg bw/day
orally (tablets were crushed and suspended in fruit syrup) during early gestation
(days 42-48) or late gestation (days 90-96). A control group received vehicle. The
route of administration was chosen to reduce stress. Females were allowed to
litter. There was no statistically significant effect of prenatal treatment on
maternal body weight. The infants showed effects from postnatal day 56 on. Early
administration of dexamethasone resulted in increased weight gain in the
presence of normal skeletal growth (p<0.05), increased eating (p<0.05), and
increased sympathetic autonomic nervous system arousal (p<0.05), increased
time spent mobile (p<0.05); increased time spent eating (p<0.05), trends toward
more solitary play (p=0.053) and tail hair piloerection (p=0.085). This phenotype
shows similarity to that of the human metabolic syndrome. Late dexamethasone
exposure (aiming at an equivalent developmental stage to human foetuses at risk
of preterm delivery) was largely without effect on physical, endocrine, and
behavioural measures across infancy.21
Based on the fact that prenatal stress is an important risk factor in schizophrenia,
the effect of prenatal dexamethasone on the development of schizophrenia-like
phenotypes was investigated by Hauser and colleagues. Wistar rats (n=14) were
exposed to 0.1 mg/kg dexamethasone (dissolved in 0.01% ethanol) per day via
drinking water between gestational days 15 and 21. A control group (n=12) was
included receiving 0.01% ethanol. Maternal body weight was recorded during
Dexamethasone                                                                        27
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<pre>  gestation and rats were allowed to litter. During lactation females and pups were
  exposed to tap water only. A second study (n=10 rats/group) was performed
  according to the same procedure. A cross-fostering design (after culling to four
  males and four females) was used to allow dissociation of any direct prenatal
  effects on offspring from effects dependent on dexamethasone exposure from the
  rearing dam. Maternal behaviour was checked on postnatal day 1-14 and
  offspring was weaned on postnatal day 21. The offspring was tested for prepulse
  inhibition (PPI) and latent inhibition (LI), both known to be disrupted in
  schizophrenia patients. Pup birth weight was reduced by prenatal dexamethasone
  treatment (p<0.05) and a reduced body weight in adulthood was observed (in
  male pups only, p<0.05). The effect on maternal body weight was not reported.
  Dexamethasone-treated dams demonstrated an increased pup-directed behaviour
  (p=0.029). The study did not provide support for the hypothesis that prenatal
  dexamethasone exposure leads to schizophrenia-like deficits in PPI or LI.22
  Dupouy et al. studied the effects of chronic dexamethasone exposure of mothers
  on different hormones of the hypothalamo-pituitary-adrenal axis (corticotrophin-
  releasing factor, adrenocorticotropic hormone, and corticosterone) in brain
  pieces (stalk, median eminence, and hypothalamus), pituitary gland, adrenals and
  plasma of 21-day-old rat foetuses. Pregnant rats (n=unknown) were treated with
  either plain tap water or water containing dexamethasone acetate (0.01 mg/ml)
  from gestation day 15 to 21. On day 21 the animals were sacrificed and the
  foetuses analysed. A marked reduction of body weight (-66% vs. controls,
  p<0.001), severe atrophy of the adrenals (-83%, p<0.001) and decreases in
  corticosterone concentrations in the adrenals (-74%, p<0.001) and in plasma
  (reduction to undetectable level, p<0.001) were observed after dexamethasone
  treatment. The adrenal changes correlated well with a drastic decrease in plasma
  adrenocorticotropic hormone (ACTH) concentrations (reduction to undetectable
  level, p<0.001) and pituitary ACTH-content (-93%, p<0.001). This low cortico-
  stimulating activity of the foetal pituitary glands was associated with a decreased
  corticotrophin-releasing factor (CRF) hypothalamic content (-57%, p<0.001) and
  concentration (-67%, p<0.001). These results suggest both pituitary and
  hypothalamic sites for the in vivo inhibiting action of dexamethasone on the rat
  hypothalamic-pituitary-adrenal axis in late gestation. Maternal toxicity was not
  described in the results.17
  Brabham et al. examined the effects of prenatal dexamethasone on spatial
  learning and response to stress. Pregnant Sprague Dawley rats (n=20-24)
  received 0.0025 mg/ml dexamethasone in the evening water daily from gestation
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<pre>day 15 until delivery. A control group receiving vehicle was included. In this
study, a concentration of 0.01 mg/ml resulted in a high incidence of foetal
abortion and a concentration of 0.005 mg/ml resulted in 50% maternal mortality
on spontaneous vaginal delivery. All dexamethasone-treated animals received an
equivalent amount of dexamethasone during 15-21 days of gestation, which
averaged 0.27 ± 0.02 mg/kg/day (mean ± SD). After parturition pups were cross-
fostered and reduced to eight or nine per dam. Spatial visual memory was
evaluated in young adults (days 65-75 of age) with the Morris water maze. The
cortisone response to restraint stress was examined at day 65 of age (naïve
animals to behavioural testing) and expression of glucocorticoid and
mineralocorticoid receptors mRNA was determined by in situ hybridization in
brain tissue derived from 80-days old pups. Exposure to dexamethasone caused
restlessness in mothers (p<0.05), low birth weights (p<0.05) and poor weight
gain in the offspring (p<0.05). Males exposed to dexamethasone during
pregnancy and cross-fostered to treated dams had impaired spatial learning
(p<0.05), inability to rapidly terminate adrenocorticoid response to stress
(p<0.05) and decreased hippocampal glucocorticoid receptor mRNA expression
(p<0.05). Dexamethasone-exposed animals that were raised by vehicle-treated
dams had adequate responses. The dexamethasone-treated dams showed a
smaller increase of body weight at GD 15-18 (p<0.001), a larger one at two
weeks after giving birth (p=0.05) and return to a normal one at three weeks after
giving birth.6
Subcutaneous administration
Carlos et al. studied the mechanism of abnormalities to the nervous system
structure and function after dexamethasone treatment. In this study
dexamethasone was administered subcutaneously to pregnant rats during
gestation days 17, 18 and 19. Dams (n=unknown) were given 0.2 mg/kg bw/day
or 0.8 mg/kg bw/day. Control animals received equivalent volumes of saline
(1 mg/kg). Maternal weight gain was recorded and pups were allowed to litter.
At birth, pups were randomized within their respective treatment groups and
redistributed to the nursing dams with litter size maintained at 9-11 pups.
Animals were weaned at postnatal day 22. At autopsy (day not mentioned) the
brain was investigated for weight, DNA content, protein content and DNA
synthesis. The forebrain showed persistent elevations of DNA (p<0.01) and
reduced protein/DNA (p<0.01), indicative of replacement of neurons with glia.
Because the treatment period coincided with the timing of neuronal cell
replication in the forebrain, but not in the other regions, these results suggest that
Dexamethasone                                                                          29
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<pre>  the critical period for lasting deficits of dexamethasone coincides with the peak
  of neuronal mitosis. Dexamethasone slowed maternal weight gain (data not
  shown).8
  The effect of dexamethasone (0.050 mg/kg bw/day) injected subcutaneously to
  pregnant Sprague Dawley rats during gestation days 16-21 was investigated by
  Nagano and colleagues. A control group receiving vehicle was included; the
  number of dams in each group was not described. At postnatal day 2 the pups
  were randomized and redistributed to the dams within each treatment group and
  the litter size was maintained at six or seven male pups per dam. Behavioural
  tests, quantitative analysis of corticotropin-releasing factor (CRF), corticosterone
  levels and immunohistochemistry and image analysis were included. A reduced
  body weight in the offspring was observed in postnatal weeks 4, 7 and 10
  (p<0.01 or p<0.05). Dexamethasone did not cause any apparent differences in
  pre-weaning maternal care between the dexamethasone-treated and control
  dams. Prenatal dexamethasone exposure decreased corticotropin-releasing factor
  mRNA in the hypothalamus (p<0.01) and disturbed the plasma corticosterone
  response to restraint stress in the offspring at postnatal week 4 (PNW4) (p<0.01).
  In contrast, it was not until PNW10 that increased anxiety-like behaviour
  emerged in the dexamethasone-exposed offspring (p<0.05). In association with
  the acquisition of increased anxiety-like behaviour at PNW10, glucocorticoid
  receptor expression was decreased in the amygdala in dexamethasone-exposed
  offspring at PNW7 (p<0.01) and PNW10 (p<0.001). Thus, prenatal exposure to
  dexamethasone hampered the neuroendocrinological development in the
  offspring during early life and it was suggested that this disturbance could result
  in the induction of increased anxiety-like behaviour in adulthood.
  Dexamethasone treatment had no apparent effect on maternal care during the
  pre-weaning period.36
  The effect of prenatal and postnatal dexamethasone treatment on the serotonergic
  and dopaminergic systems was investigated by Slotkin and colleagues. Pregnant
  Sprague Dawley rats (n=unknown) were treated subcutaneously with doses of
  0.05, 0.2 or 0.8 mg dexamethasone/kg bw/day on gestation days 17-19. Control
  rats received equivalent volumes (1 ml/kg) of saline vehicle. Females were
  allowed to deliver and pups were maintained at a litter size of 10. Randomization
  was repeated every 3-4 days to obviate any differences in maternal caretaking.
  Maternal toxicity was not described. To study the postnatal effects, pups were
  given 0, 0.05, 0.2 or 0.8 mg dexamethasone/kg bw/day on postnatal days 1-3 or
  7-9 and the same randomization procedures were performed. On postnatal day 60
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<pre>animals were decapitated. 5-hydroxytryptamine (5HT) receptor binding, 5HT
concentration, 5-hydroxyindolacetic acid (5HIAA), dopamine (DA)
concentration and turnover, dihydroxyphenylacetic acid (DOPAC) and
homovanillic acid (HVA) were measured in the cerebral cortex and/or
hippocampus. 5HT receptor binding and 5HT transporter binding were increased
in the cerebral cortex during gestation and after birth (p<0.0006 and p<0.0001,
respectively). 5HT concentration in the hippocampus and cerebral cortex were
increased after birth (p<0.002), then decreased till levels below normal
(p<0.0006). 5HT turnover was unchanged. DA concentration in the cerebral
cortex was already increased during gestation (p<0.03), decreased till levels
below normal at postnatal days 1-3 (p<0.0001) and returned to normal again by
postnatal days 7-9. DA turnover in the cerebral cortex was unchanged. Results
indicated that dexamethasone administration during phases of brain
development, analogous to those in preterm infants, produces changes in indices
of 5HT and DA synaptic function.46
Kreider et al. investigated the effect of prenatal dexamethasone at clinical doses
on neurobehavioural development in adolescent and adult rats. Sprague Dawley
rats (n=unknown) were treated subcutaneously with 0.2 mg dexamethasone/kg
bw/day on gestation days 17, 18 and 19. A control group receiving equivalent
volumes of saline (1 ml/kg) was included. Dams were allowed to litter and pups
were randomized to nursing dams every 4 days until weaning. Behavioural tests
were performed at postnatal days 31, 32, and 33 (figure 8 activity) and 45-68
(eight-arm radial maze). Dexamethasone had no statistically significant effect on
maternal weight gain during pregnancy, gestation index, litter size, viability and
sex ratio (data not shown). Dexamethasone treatment ablated the normal
differences between sexes in locomotor activity by lowering the values in
females to the levels of males (p<0.0003). Habituation of activity was similarly
impaired in females to match the profile of males (p<0.01), while males in the
dexamethasone group showed a partially feminized pattern of habituation
(p<0.004). Dexamethasone delayed learning in males (p<0.08), while improving
performance in females in the 8-arm radial maze (p<0.04). Values of
hippocampal [3H]hemicholinium-3 binding, a biomarker for cholinergic
synaptic activity, were increased in males to the levels of females after
dexamethasone treatment (p<0.007). From this study it can be concluded that
dexamethasone given at clinical doses has adverse effects on neurodevelopment,
producing sex selective alterations in activity, learning and memory.29
Dexamethasone                                                                      31
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<pre>  Somm et al. examined the effect of dexamethasone treatment on growth and
  brain metabolism. Pregnant Sprague Dawley OFA rat dams were implanted
  subcutaneously with a minipump delivering either vehicle or dexamethasone at a
  dose of 0.1 mg/kg/day at gestational days 14-21. Litter size was smaller in the
  dexamethasone-treated group (p<0.001). For follow-up, litter size was brought to
  10-12 pups. Perinatal body weight of pups was diminished by dexamethasone at
  postnatal days 1 and 21 (p<0.001). Pup hippocampal metabolism was reduced, as
  evidenced by lower concentrations of several brain metabolites and lower
  hippocampal gene expression at postnatal day 7 (p<0.05). Maternal body weight
  throughout gestation and weight gain during its third week were decreased
  (p<0.001 and p<0.01, respectively).48
  Intramuscular injection
  The effect of repeated low doses of maternally administered dexamethasone on
  growth in sheep during foetal life and the first 2 years of postnatal life was
  investigated by Kutzler and colleagues. Different cohorts were used. They all
  received four intramuscular injections of 29-33 µg/kg bw dexamethasone in
  saline or an equal volume of saline within 48 hours on gestation days 103-104,
  110-111 and 117-118. In the first cohort twenty ewes were administered either
  dexamethasone (n=9) or saline (n=11) and autopsy was performed at gestation
  day 119. Foetal and placental measurements and immunocytochemistry were
  investigated. In cohort two, females were allowed to lamb. Sixteen female ewes
  were administered dexamethasone (n=8) or saline (n=8). Ewes were allowed to
  deliver and neonatal measurements (newborn body weight and organ weight
  within 12 hours after birth) were performed. In the last cohort, twenty-six ewes
  were administered dexamethasone (n=13) or saline (n=13). Ewes were allowed
  to lamb and postnatal measurements were performed biweekly for 8 weeks (body
  weight, biparietal diameter (BPD), crown-to-rump length (CRL), thoracic girth
  circumference (TGC), abdominal girth circumference (AGC), and radial bone
  length (RBL). The study showed that repeated maternal dexamethasone
  treatment at doses threefold lower than what women in preterm labour receive,
  results in decreased foetal body weight (p<0.05), prolonged gestation length
  (p<0.05), normal birth weight, decreased newborn brain weight (p<0.05) and
  biparietal diameter (p<0.05). Other postnatal growth measures were normal.
  Maternal toxicity was not described in the results.30
  Neurotoxic effects induced by prenatal administration of dexamethasone to
  Rhesus monkeys during the early third trimester were investigated by Uno and
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<pre>colleagues. Dexamethasone was given intramuscularly to pregnant monkeys on
gestation day 132 (single injection with doses of 0.5, 5 or 10 mg/kg bw) or on
days 132 and 133 (multiple injections at 12-h intervals with 0.125 x 4, 1.25 x 4 or
2.5 mg/kg x 4). A control group receiving vehicle was included. Maternal
toxicity was not described in the results. Foetuses were delivered by Caesarean
section on day 135 of gestation to study neurotoxic effects and at day 162 of
gestation to investigate whether the effects seen at day 135 were persistent. Brain
sections were investigated. A critical dose of dexamethasone given prenatally
appeared to induce an irreversible reduction of the number of neurons in the
hippocampus (p<0.05) and poorly differentiated hippocampal neurons.56
Miscellaneous routes of administration
The effect of prolonged low-dose dexamethasone treatment in early gestation
was investigated in sheep by Moritz and colleagues. Pregnant merino ewes
(n=18) were infused intravenously with approximately 0.020 mg
dexamethasone/kg bw/day for 20 days. A control group (n=15) received saline.
Ewes were killed on gestation day 45. Maternal and foetal organs were weighed
and preserved. A second cohort of ewes (n=3 controls and n=4 dexamethasone-
treated) were maintained until foetuses were at 130 day of gestation. At autopsy
on gestational day 130 foetal organs were weighed and collected. Brain was
further dissected. A last cohort was allowed to lamb and offspring was studied at
2 months of age. Immediate and permanent effects on the growth of the foetus
and developing organs, and programming effects (gene expression levels of the
angiotensin receptors, angiotensinogen, mineralocorticoid and glucocorticoid
receptor in kidney and brain of foetuses at gestation day 130) were studied using
the three cohorts. There were no persistent, long-term effects of prolonged low-
dose dexamethasone treatment in normal ovine foetuses.34
Uno et al. also performed longitudinal studies of the juvenile monkeys with
induced prenatal hippocampal deficiency. Rhesus monkeys (n=8) were treated
with 5 mg dexamethasone/kg bw/day (route of administration not reported) at
gestation days 132 and 133. A control group (n=3) receiving vehicle was
included. After birth all infants lived with their mother for 1 year. Plasma cortisol
levels were measured at 9 months of age and magnetic resonance images (MRI)
of the brain were made at 20 months of age. Prenatal administration of
dexamethasone induced an irreversible reduction in the size of the hippocampus.
It also induced high plasma cortisol at the circadian baseline and post-stress
Dexamethasone                                                                         33
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<pre>  levels in the juvenile rhesus monkeys. Statistics were not reported. Maternal
  toxicity was not described in the results.55
  Effects on the immune and endocrine systems
  No studies were available on effects of oral administration of dexamethasone on
  the immune and endocrine systems.
  Subcutaneous injection
  Negić et al. investigated whether dexamethasone during pregnancy influenced
  the morphology of the pituitary, luteinising hormone (LH) and follicle
  stimulating hormone (FSH). Pregnant Wistar females were either treated with
  dexamethasone subcutaneously on gestation days 16-19 at doses of 1.0 or 0.5
  mg/kg bw/day. The day when sperm was found was designed as gestation day 1.
  Control groups were injected with equal volumes of 0.9% saline. On gestation
  day 19 (n=8) and on gestation day 21 (n=8) rats were sacrificed. To compare the
  effects of pregnancy, a control group of virgin rats was included too (three daily
  injections and necropsy 24 h after the last treatment (n=8) or necropsy 72 h after
  the last treatment (n=8)). Histological analysis and morphometric analysis on
  pituitary glands was performed. The results demonstrated that pregnancy in rats
  led to a marked reduction of morphometric parameters (cell volume, volume
  density and number of cells) of LH and FSH compared to virgin control values
  (p<0.05). Moreover, daily dexamethasone treatment of pregnant females affected
  the size of LH and FSH cells on day 19 of pregnancy (p<0.05). The decrease in
  cell volume was reversible (normalization of LH and FSH cell function on day
  21 of pregnancy). The results indicate that there are no prolonged effects of
  dexamethasone treatment during pregnancy on LH and FSH cells. Information
  on maternal toxicity was not provided.37
  In a study performed by Stojanoski et al. the effect of prenatal dexamethasone
  treatment on the development of pituitary adrenocorticotrophic (ACTH) cells and
  adrenal glands in the offspring was investigated. Wistar rats (n=unknown) were
  injected subcutaneously with dexamethasone on gestation day 16 (1.0 mg/kg bw),
  17 (0.5 mg/kg bw) and 18 (0.5 mg/kg bw). Control females received the same
  volume of saline vehicle. On day 19 of gestation autopsy was performed and
  foetuses were prepared for histological and morphometric measurements. Results
  showed maternal dexamethasone treatment in the period when foetal
  hypothalamo-pituitary-adrenal (HPA) axis begins to function, inhibited the axis.
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<pre>Pituitary ACTH cells were fewer and had a smaller volume (p<0.05). In the
adrenal organs cortical cells of the zona glomerulosa had reduced proliferative
activity (p<0.05). Thus, reduced pituitary ACTH cell function and mitotic activity
led to the suppression of adrenocortical cell multiplication in the zona
glomerulosa. Information on maternal toxicity was not provided.49
Page et al. investigated whether prenatal exposure of rats alters Leydig cell
steroidogenic capacity in immature and adult rats. Sprague Dawley rats were
treated subcutaneously with 0.1 mg/kg bw per day dexamethasone (n=9) or
saline and 0.4% ethanol (n=9) on gestation days 14-19 (day 0 is defined as the
morning of appearance of the vaginal plug). Immature male offspring were
weaned at postnatal day 21 and studied at day 35 days of age and adult males
were killed and investigated at 90 days of age (n=26 dexamethasone-treated and
n=27 control males for both immature and adult males). Maternal toxicity was
not described. Immature male offspring showed decreased levels of testosterone
in serum (p<0.05) and a reduced testosterone production in Leydig cells
(p<0.001). Their serum ACTH and corticosterone concentrations were reduced
(p<0.001). Mature male offspring demonstrated higher serum levels of these
hormones (p<0.001). They also exhibited higher serum testosterone
concentrations (p<0.05) and testosterone production in Leydig cells (p<0.001).
The results demonstrate that a high level of maternal dexamethasone affects the
steroid output from both the hypothalamic-pituitary-gonadal (HPG) and
hypothalamic-pituitary-adrenal (HPA) axes; Leydig cell testosterone production
was affected in both pubertal and adult rats.41
The effects of prolonged dexamethasone administration to pregnant rats on the
structure and function of the adrenal glands was investigated by Hristić and
colleagues. Pregnant Wistar rats (n=10) were injected subcutaneously with 0.3
mg dexamethasone/kg bw/day during 5 days, starting from day 16 of pregnancy.
A control group (n=10), that received the same volume of saline (0.3 ml/kg
bw/day) was included. The first cohort of dams (number not mentioned) and
foetuses were killed 24 h after the last injection. The second and third cohort
were killed at postnatal days 3 and 14. The adrenal gland was investigated for
morphometric parameters, metaphase index of cortical cells and histological
analysis. The proliferative activity of adrenal cortical cells was inhibited and
atrophic changes in the adrenal glands were seen. The main findings were a
decreased volume of the foetal adrenal gland (p<0.005), its zona glomerulosa
plus capsula (p<0.005) and its inner zone (p<0.005) as the consequence of
atrophic changes in the gland and reduction of the volume and total number of
Dexamethasone                                                                      35
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<pre>  adrenocortical cells (p<0.05 and p<0.005, respectively). These morphometric
  changes were found in 3- and 14-day-old pups. Information on maternal toxicity
  was not provided.26
  Holson et al. studied the effect of prenatal dexamethasone treatment (among
  other stressors) on the sexual behaviour of the male offspring. Pregnant Sprague
  Dawley derived CD rats (n=4/group) were treated subcutaneously with
  dexamethasone at 0 or 0.1 mg/kg bw/day on gestation days 14 to 21. At birth all
  litters were culled to 8 pups (4 ± 1 of each sex) and raised up to weaning. Litter
  size, number of stillbirths and sex ratio were not altered by dexamethasone, but a
  reduced pup birth weight (an effect still seen at postnatal day 85) was observed
  (p<0.01). Male adrenal weight at birth was reduced (p<0.01). Male sexual
  differentiation at birth, as measured by anogenital distance, was also decreased
  (p<0.05). Male sexual behaviour was assessed for three weeks, starting at
  postnatal day 90 by investigating the number of ejaculations per trial and the
  latency to first ejaculation after cohabitation with oestrous females, and the
  lordosis quotient after postgonadectomy exposure to stud males. Dexamethasone
  affected both sexual performance of adult males, as measured by the ejaculation
  parameters mentioned (both p<0.05), and the Lordosis quotient of castrated
  hormone-primed males after exposure to stud males (p<0.05). From the results of
  this study it can be concluded that prenatal dexamethasone exposure
  demasculinizes and feminizes male offspring. Maternal toxicity, demonstrated by
  diminished weight gain, was considerable (p<0.0001).23
  Intraperitoneal injection
  Bakker et al. examined the effects of prenatal dexamethasone treatment on the
  developing thymus, spleen and hypothalamo-pituitary adrenal axis. Female
  Wistar rats were given 400 µg of dexamethasone-21-phosphate (n=7) or saline
  (n=6) intraperitoneally on days 17 and 19 of pregnancy. A third group of
  pregnant rats (n=6) received no injection. After maternal dexamethasone
  treatment, the offspring showed reduced body weights compared to either control
  group, on the first day after birth. Thymus, spleen, hypothalamus and blood
  plasma of offspring were examined at several time intervals (1-20 days) after
  birth. Prenatal exposure to dexamethasone resulted in decreased T cell numbers
  in thymus and spleen on postnatal day 1 (p<0.05 or <0.005). T cell numbers in
  the spleen were reduced at all neonatal ages studied (postnatal days 1, 7 and 20;
  p<0.05 or <0.005). Regarding the hypothalamus, prenatal exposure to
  dexamethasone altered the pattern of neonatal changes in peptide expression in
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<pre>corticotrophin-releasing hormone neurons. No effects were seen on
adrenocorticotrophic hormone and corticosterone levels in plasma. Maternal
toxicity was not described in the results.2
Bakker et al. also investigated whether maternal treatment with dexamethasone
or saline alters the corticosterone response to a mild stressor in the offspring and
whether maternal treatment results in long-term altered in vivo humoral and
cellular immune responsiveness in the offspring. Pregnant Wistar rats (3 animals
per group) were given saline or dexamethasone-21-phosphate at a dose of 1.2
mg/kg body weight intraperitoneally at days 17 and 19 of gestation. A third
group of pregnant rats was left undisturbed. After maternal dexamethasone
treatment, the offspring showed no altered corticosteroid response to a novel
environment at 20 days of age, as compared to either control group. Furthermore,
no effects of maternal dexamethasone exposure were seen on immuno-
globulin-2a production after immunization with a conjugated pneumococcal
polysaccharide at 6 weeks of age. Cellular immune responses, measured by an
oxazolone-induced contact hypersensitivity response at 8 weeks of age, were
lower in offspring of dexamethasone-treated dams as compared to saline-treated
dams (p<0.005). The response was not different from that in offspring of
untreated dams. Information on maternal toxicity was not provided.3
Yu et al. evaluated the immune programming influenced by prenatal
dexamethasone. Pregnant Sprague-Dawley rats (6-8 animals per group) received
saline or dexamethasone at 0.1 mg/kg/day at gestational days 14-20. Male
offspring were killed at day 7 or day 120 after birth. Spleen were collected for
immune analysis. Three out of five inflammation mediators were decreased at
day 7, one of them, matrix metalloproteinase-9, still at day 120 (p<0.05). Upon
concanavalin-A stimulation prenatal dexamethasone treatment reduced tumour
necrosis factor-α production (p<0.05), but not interferon-γ production in spleen
cells at day 120.59
Miscellaneous routes of administration
The effect of dexamethasone on the adrenal gland in foetal and neonatal rats was
investigated by Hristić and colleagues. Wistar rats (n=5) were treated with a
single dose (route unknown) of 1.5 mg dexamethasone /kg bw on day 16 of
gestation. Group 2 included five females which received an equivalent volume of
saline (1.5 ml) on the same day of gestation. Dams and foetuses were sacrificed
at gestation day 21. Some neonatal animals were sacrificed on days 4 or 15 of
Dexamethasone                                                                        37
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<pre>  life. Adrenal glands of 20-day old foetuses, 3-day old neonatal rats and 14-day
  old neonatal rats were investigated with light and electron microscopy and
  stereological measurements were performed. Special attention was paid to
  necrotic changes and the presence of macrophages, multinuclear giant cells and
  lymphocytes. Findings were a decrease of both adrenal weight and volume,
  volume of the zona glomerulosa plus capsule, and volume of the inner zone, both
  in foetuses and 3-day-old rats (p-values are <0.05, <0.025 or <0.005). The
  number, but not the total volume of cells was decreased (p<0.05 or p<0.005).
  Morphological signs of necrosis were observed. In 14-day-old animals, the
  degree of atrophic changes in the adrenal cortex was reduced. The data
  demonstrated that even a single dose applied to pregnant rats during the period
  critical for the development of the hypothalamo-pituitary-adrenal system in
  foetuses leads to prominent changes in the structure of the adrenal cortex of the
  offspring that are partially maintained up to postnatal day 14. The changes
  suggest that cortical function was inhibited. Maternal toxicity was not described
  in the results.25
  Effects on renal function
  No studies were available on effects of oral administration of dexamethasone on
  renal function.
  The effects of prolonged low-dose dexamethasone treatment in early gestation on
  blood pressure and renal function in adult sheep were investigated by Dodic and
  colleagues. Pregnant Mereno ewes were treated intravenously with
  dexamethasone (0.020 mg/kg bw/day) from gestation days 25 to 45 (n=11) or
  with saline (n=9). Ewes were allowed to lamb and blood pressure and renal
  function was studied in offspring at 2 years of age. From the results of this study
  there was no evidence of altered renal function or predisposition to adult
  hypertension.16
  Ortiz et al. investigated whether prenatal exposure to dexamethasone adversely
  affects renal development and predisposes rats to develop renal disease and
  hypertension in later life. Pregnant Sprague Dawley rats (n=unknown) were
  given two daily intraperitoneal injections of 0.2 mg/kg bw dexamethasone on
  gestational days 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20 or 20 and
  21. A control group injected intraperitoneally with vehicle was included. Blood
  pressure, glomerular number and insulin clearance were measured in offspring at
  postnatal day 60 or 90. Length of gestation, litter size, body weight and kidney
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<pre>weight at postnatal day 1 were not affected. In the offspring of dams treated at
gestation days 15 and 16 the glomerular number was 30% reduced (p<0.01) and
the systolic blood pressure was higher (p<0.05). After treatment at gestation days
17 and 18 a 20% reduction in the number of glomeruli was measured (p<0.01).
No effects were seen in other treatment regimens. The results from this study
showed that two daily doses of dexamethasone did not produce intrauterine
growth retardation and that adult offspring of rats receiving dexamethasone at
different times during pregnancy have a reduced number of nephrons and
hypertension. Information on maternal toxicity was not provided.39
In a second study performed by Ortiz et al. the same experimental method was
used. Pregnant rats (number and strain not reported) were treated
intraperitoneally twice daily with 0 or 0.2 mg/kg bw dexamethasone on
gestational days 11 and 12, 13 and 14, 15 and 16, 17 and 18, or 19 and 20. In this
study the effect on neonates and the long-term effect at 6 to 9 months of age was
measured. In the offspring of dams treated at gestation days 15 and 16 the
glomerular number was 20% reduced at 6 to 9 months of age (p<0.05). This was
similar to the reduction observed at 3 weeks of age. After treatment at gestation
days 17 and 18 a 17% reduction in the number of glomeruli was measured at
6 months of age (p<0.05). After treatment at days 13 and 14, 15 and 16, or 17 and
18 of gestation blood pressure was elevated at 6 months of age (p<0.05). The day
13-14 group did not show a reduced glomerular number. Thus, prenatal treatment
in rats at specific points during gestation resulted in a reduction in glomerular
number, glomerulosclerosis, and hypertension. Hypertension was observed in
animals that had a reduction in glomeruli as well as animals that did not have a
reduction in glomerular number. Information on maternal toxicity was not
provided.40
Slotkin et al. investigated the effect of prenatal dexamethasone treatment on the
development of the neonatal heart (see earlier section) and kidney. Pregnant
Sprague Dawley rats (n=unknown) were treated subcutaneously with 0.2 or
0.8 mg dexamethasone phosphate/kg bw/day on gestational days 17, 18 and 19.
Controls received equivalent volumes of saline vehicle (1 ml/kg). Maternal
weight gain was recorded and dams were allowed to litter. Pups were randomized
and redistributed to nursing dams within the treatment group. At autopsy at
weaning the body weight, kidney weight and three biochemical markers of cell
development in the kidney were assessed: DNA content as an index of total cell
numbers, DNA content per gram of tissue as an index of cell packing density and
protein/DNA ratio as an index of relative cell size. Body and kidney weights
Dexamethasone                                                                      39
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<pre>  were lower (p<0.01). DNA content of the kidney was diminished (p<0.01). Cell
  packing density was increased (p<0.01 or p<0.05, depending on the dose of
  dexamethasone) and cells were enlarged, after an initial decrease in size
  (p<0.01). It was concluded from the results of this study that foetal exposure to
  dexamethasone causes widespread inhibition of cell proliferation with disruption
  of parameters of cell differentiation and growth in the kidney. Dexamethasone
  slowed maternal weight gain (data not shown).47
  Tain et al. investigated the effect of dexamethasone on blood pressure and
  kidney. The purpose of the study was to examine whether maternal melatonin
  administration could attenuate the effects of dexamethasone. Sprague Dawley
  rats were injected subcutaneously with 0 or 0.1 mg/kg/day of dexamethasone on
  gestational days 16-22. Male pups (n=12/group) were examined and sacrificed at
  16 weeks of age. Blood pressure, kidney histology and expression of a range of
  genes relevant to kidney development were investigated. Hypertension and
  reduced nephron numbers were observed (p<0.05). No changes were found in
  kidney expression of genes involved in apoptosis or nephrogenesis, except for a
  gene coding for one of the class I histone deacetylases. Some genes of the renin-
  angiotensin system playing a part in blood pressure control were upregulated
  (p<0.05). Maternal toxicity was not described in the results.51
  Tain et al. investigated the effect of dexamethasone on blood pressure and kidney
  once more, to examine whether maternal citrulline administration could prevent
  the effects of dexamethasone. Sprague Dawley rats were injected subcutaneously
  with 0 or 0.2 mg/kg/day of dexamethasone on gestational days 15 and 16. Male
  pups (n=8-10/group) were examined and sacrificed at 16 weeks of age. Blood
  pressure and expression of a range of genes relevant to kidney development were
  investigated. Hypertension was observed (p<0.05). Various genes involved in
  apoptosis or nephrogenesis were upregulated (p<0.05). Maternal toxicity was not
  described in the results.52
  Rogers et al. examined the effect of dexamethasone on blood pressure and
  kidney. Pregnant Sprague Dawley rats were injected subcutaneously with 0.6
  mg/kg/day of dexamethasone on gestational days 16-20. Controls received
  vehicle. Litters were standardized to 10-12 pups per litter at birth. Both male and
  female offspring had reduced birth weights (p<0.05). They also had increased
  blood pressure at 7 weeks (males only), 37 weeks (females only, males not
  examined) and 65 weeks of age (p<0.05). Males had reduced nephron counts and
  increased glucocorticoid receptor gene expression in their kidneys (p<0.05);
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<pre>female kidneys were not examined. Maternal weight gain during pregnancy
(gestational days 19-21) was reduced (p<0.05).43
Effects on skeletal development
No studies were available on effects of oral administration of dexamethasone on
skeletal development.
Pregnant Wistar rats (n=5) were treated intramuscularly with 0.1 mg/kg during
gestation at days 9, 11 and 13 (i.e. the sensitive period of early foetal brain
development) by Swolin-Eide and colleagues. Five control animals were injected
intramuscularly with saline. Blood samples were collected 4 h after injection, to
determine corticosterone concentrations. The dose level of 0.1 mg/kg was
supposed to suppress corticosterone. Litters were weighed at birth and litters
were adjusted to the same ratio of male/female pups. Six male and six female
pups were killed at 6 weeks, the remaining males at 10 weeks of age and the
remaining females at 12 weeks of age. Body weight, food intake, tissue weights,
bone measurements and hormone analysis were performed. Dexamethasone did
not induce maternal toxicity. Male pups showed transient increases in crown-
rump length and tibia and femur lengths at 3-6 weeks of age (p<0.05 or <0.001).
Cortical bone dimensions were altered in 12-week old females (p<0.05 or
<0.001). Areal bone mineral densities of the long bones and the spine were
unchanged in both male and female offspring.50
Effects on hearing
No studies were available on effects of oral administration of dexamethasone on
hearing.
The effect of dexamethasone injected subcutaneously to pregnant Wistar rats
(n=20-22) at a dose of 0.1 mg/kg/day during gestation days 14-21 was
investigated by Hougaard and colleagues. Control animals received vehicle. The
dose level and exposure period were based on reports of increased noise-induced
hearing loss in offspring of rat dams treated during the last week of gestation
with a different route of exposure (Canlon et al.7). Females were allowed to litter
and in a subset of the male offspring, hearing was measured before, one day after,
and one month after exposure to noise, to assess noise-induced hearing loss. The
body weight gain during pregnancy of dams treated with dexamethasone was
lower from gestation day 19 onwards (p<0.001). The number of live pups per
Dexamethasone                                                                       41
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<pre>  litter and the pup weight at postnatal day 3 (day 0 not reported) were lower than
  in the control group (p<0.05 and p<0.001, respectively). At weaning the body
  weight had returned to normal (data not shown). With respect to hearing loss,
  these data do not support the previous report of Canlon et al.7, since the prenatal
  exposure to dexamethasone was not associated with enhanced noise-induced
  hearing loss compared to controls.24
  Canlon et al. studied the effect of intraperitoneally injected dexamethasone
  (0.1 mg/kg bw) or vehicle once daily in rats from gestation day 14 until
  parturition. Auditory brain stem responses, the effects of moderate and intense
  noise exposure, the percentage of apoptotic nuclei after acoustic trauma and the
  effects of free radicals on acoustic exposure were measured. Prenatal treatment
  with dexamethasone increased the susceptibility of the inner ear to acoustic noise
  trauma in adult life. This acoustic trauma was reduced after treatment with a free
  radical scavenger. Information on maternal toxicity was not provided.7
  Various effects measured in nonhuman primates
  Oral administration
  De Vries et al. studied the effect of prenatal dexamethasone exposure on the
  cardiometabolic and hypothalamic-pituitary-adrenal axis function. African
  vervet monkeys (Chlorocebus aethiops) were exposed to dexamethasone by diet
  at dosages of 0.050, 0.120 or 0.200 mg/kg bw/d from mid-gestation (gestational
  day not mentioned) up to birth (n=10/group). A control group was included and
  maternal parameters (weight, urine volume, blood pressure, plasma electrolytes)
  were measured. Monkeys were allowed to litter. Weight, head circumference,
  head length, biparietal diameter, crown-heel length, crown-rump length, tibia
  length, forearm length and hip width were measured at birth and at 2, 4, 6, 8, and
  12 months of age. In addition motor function, ponderal index (weight/height3),
  glucose tolerance test, dexamethasone suppression test, blood pressure
  measurements and 24-hour urine collections were performed and after necropsy
  organ weights, liver activity and pancreas histology were determined and
  investigated. Prenatal exposure to the tested, low doses of dexamethasone had no
  effect on maternal parameters and foetal birth weight. A disordered glucose-
  insulin homeostasis (p<0.02 or p=0.051, depending on dexamethasone dose),
  reduced pancreatic β cell mass (p<0.0001), and elevated blood pressure (p<0.04)
  and cortisol levels (p<0.05) in juvenile offspring were observed.13
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<pre>      Coe et al. summarized the developmental consequences of prenatal
      dexamethasone treatment in nonhuman primates. A review of literature in
      primates dating from 1977 to 2005 was undertaken. Most studies were performed
      with Rhesus monkeys (Macaca mulatta); in some studies Macaca nemestrina or
      Papio monkeys were used. Initiation of prenatal exposure varied from gestation
      days 120 to 143 with a duration of 2 to 42 days. Exposure typically involved
      intramuscular injections at different dose levels. Duration of treatment varied
      from 1 day to 6 weeks, with a typical exposure lasting 2 days. Across studies
      dosages varied from 0.1 to 15 mg/kg maternal bw/day. Endocrine physiology,
      foetal, placental and uterine physiology, physical growth, hepatic and pancreatic
      effects, lung function and brain alterations and immune consequences were
      investigated. The most prominent effects observed were:
      • reduced cortisol levels in maternal blood (with a rapid recovery, returning to
          normal levels within 1-2 days after treatment)
      • dexamethasone rapidly crosses the placenta (not bound to transporter
          proteins, not easily converted to cortisol by placental enzymes)
      • transient increase in maternal and foetal blood pressure
      • post-treatment effects on foetal adrenal glands (smaller) and cortisol levels in
          umbilical cord blood (reduced)
      • reduced foetal body size and smaller head circumferences (with no effect on
          length of long limb bones)
      • although airway functioning is usually improved by prenatal dexamethasone
          treatment, it appeared that very specific regimens are required (in this species
          0.5 mg/kg bw for 4 times at 12 h intervals prior to delivery).
      • reduced total absolute brain and cerebellum weights at preterm delivery and
          delivery at term
      • morphological changes in the hippocampus.
          Some physiological effects were found at the lowest concentration
      (suppression of adrenal activity), but more serious disturbances of growth and
      neural development occurred at dosages of > 2 mg/kg bw/day.10
2.4   Conclusions
2.4.1  Fertility
      No human studies on fertility effects of dexamethasone were available.
      Animal studies with respect to fertility were performed with small numbers of
      male or female animals from a variety of species.5,18,20 The findings in males
      Dexamethasone                                                                        43
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<pre>      were limited to sperm changes, such as a reduced sperm volume18,20 and effects
      on sperm morphology5,18. Some of these effects diminished after a recovery
      period of 6 weeks. In female fertility studies12,32 effects on hormone levels and
      ovulation were observed. Neither mating behaviour, nor pregnancy outcomes
      were investigated in animals exposed during adulthood. The Committee
      interprets the effects on sexual behaviour of male animals that had been exposed
      in utero to be related to developmental alterations rather than a direct effect on
      fertility.23 Thus, functional fertility data are considered not to be available.
      Therefore, the Committee proposes not to classify dexamethasone for fertility
      due to a lack of appropriate data.
2.4.2 Developmental toxicity
      Studies on the teratogenic potential of dexamethasone in humans included both
      prospective and retrospective ones.1,9,11,31,35,45 Confounding by indication did not
      seem to play a role. Effects of limited significance, on foetal heart rate and renal
      resistance, were found in some of the studies.1,11,35 It is uncertain whether these
      treatment-related effects are adverse. Together, the human studies do not allow
      classification of dexamethasone for developmental toxicity due to a lack of
      appropriate data.
      A large number of animal studies with dexamethasone has been carried out.
      These experiments involve various animal species (nonhuman primates, rats,
      mice and sheep) and routes of administration (oral, intraperitoneal, sub-
      cutaneous, intravenous, intramuscular). They show a range of pre- and postnatal
      developmental effects, including cardiovascular and neurodevelopmental effects,
      as well as effects on the immune and endocrine systems, renal function, skeletal
      development and hearing.
          The Committee considers the studies with oral administration as the most
      relevant ones: two studies in nonhuman primates and three in rats.6,13,17,21,22 The
      primate studies demonstrate neurodevelopmental effects and effects on glucose-
      insulin and cardiovascular systems, without any evidence of maternal
      toxicity.13,21 The rat studies also show neurodevelopmental effects.6,17,22 In one
      of those studies maternal toxicity was detected6, in the other ones no information
      on maternal toxicity was provided17,22.
          Some non-oral studies provide supporting evidence for developmental
      toxicity of dexamethasone. Among these are three studies in nonhuman primates
      with intramuscular administration.27,55,56 The nature of the morphological effects
      in one of the studies27, together with the neurodevelopmental effects seen in the
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<pre>      other ones55,56, is suggestive for effects on the offspring, although a role of
      maternal toxicity cannot be ruled out. The other studies providing supporting
      evidence are three rat studies with subcutaneous or intramuscular administration
      in which maternal toxicity was absent.29,36,50 They show neurodevelopmental
      and skeletal effects.
          In some of the non-oral studies in which developmental toxicity was
      observed, that were carried out in rats, mice or sheep, maternal toxicity was
      reported to be present.8,23,24,43,47,48 In the remaining studies no information on
      maternal toxicity was provided.2-4,7,15,19,26,30,33,37,39-42,44,46,49,51-53,59
      Rat and mouse are common species for toxicity testing of substances. However,
      their relevance as predictive models for humans can be questioned in the case of
      dexamethasone, because rodents are typically viewed as “corticoid-sensitive”
      species, and their newborn pups are extremely immature at birth. The
      development of the rat kidney for example, is significantly different from that of
      the human kidney. Nephrogenesis ends by about 34 weeks of gestation in the
      human, but rats continue to form new nephrons until approximately 1 week after
      birth.40 Although rodent studies contribute to the evidence on the developmental
      toxicity of dexamethasone, the crucial studies include two well-performed oral
      studies in primates. Moreover, similar types of adverse effect have been reported
      in primates and rodents. Therefore, the Committee takes both primate and rodent
      studies into account.
      In conclusion, pre- and postnatal developmental effects have been observed in
      several studies in nonhuman primates and rodents in the absence of maternal
      toxicity. Consequently, the Committee proposes to classify dexamethasone in
      category 1B (presumed human reproductive toxicant) and to label it H360D
      (may damage the unborn child).
2.4.3 Lactation
      No human or animal data were available regarding the excretion of
      dexamethasone in breast milk or the effects of exposure to dexamethasone on
      infants during the lactation period.
      Proposed classification for fertility
      A lack of appropriate data precludes assessment of dexamethasone for fertility.
      Dexamethasone                                                                      45
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<pre>  Proposed classification for developmental toxicity
  Category 1B, H360D.
  Proposed labelling for effects on or via lactation
  A lack of appropriate data precludes assessment of dexamethasone for lactation.
6 Dexamethasone
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8 Dexamethasone
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0 Dexamethasone
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2 Dexamethasone
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4 Dexamethasone
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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
                                                         55
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<pre>6 Dexamethasone</pre>

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<pre>nnex A
     The Committee
     •  D. Lindhout, chairman
        Emeritus Professor of Medical Genetics, Paediatrician (not practising),
        Clinical Geneticist, University Medical Center, Utrecht
     •  N. Roeleveld
        Reproductive Epidemiologist, Radboud university medical center, Nijmegen
     •  J.G. Theuns-van Vliet
        Reproductive Toxicologist, Triskelion BV, Zeist
     •  T.G.M. Vrijkotte
        Epidemiologist, AMC, Amsterdam
     •  D.H. Waalkens-Berendsen
        Reproductive Toxicologist, Zeist
     •  P.J.J.M. Weterings
        Toxicologist, Weterings Consultancy BV, Rosmalen
     •  A.H. Piersma, structurally consulted expert
        Professor of Reproductive and Developmental Toxicology, Utrecht
        University, and National Institute of Public Health and the Environment,
        Bilthoven
     •  P.W. van Vliet, scientific secretary
        Health Council of the Netherlands, Den Haag
     The Committee                                                               57
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<pre>  The first draft of this report was prepared by M.M. Tegelenbosch-Schouten MSc
  (TNO Quality of Life, Zeist, The Netherlands) by contract with the Ministry of
  Social Affairs and Employment.
  The Health Council and interests
  Members of Health Council Committees are appointed in a personal capacity
  because of their special expertise in the matters to be addressed. Nonetheless, it
  is precisely because of this expertise that they may also have interests. This in
  itself does not necessarily present an obstacle for membership of a Health
  Council Committee. Transparency regarding possible conflicts of interest is
  nonetheless important, both for the chairperson and members of a Committee
  and for the President of the Health Council. On being invited to join a
  Committee, persons 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 Health Council to assess
  whether or not someone can become a member. An expert who has no financial
  but another clearly definable interest, can become a member under the restriction
  that he will not be involved in the debate on the subject to which his interest
  relates. If a person’s interest is not clearly definable, he can sometimes be
  consulted as an expert. Experts working for a ministry or governmental
  organisation can be structurally consulted. During the inaugural meeting the
  declarations issued are discussed, so that all members of the Committee are
  aware of each other’s possible interests.
8 Dexamethasone
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<pre>nnex B
     The submission letter (in English)
     Subject          : Submission of the advisory report Dexamethasone
     Your reference   : DGV/BMO/U-932542
     Our reference    : U-1024028/EvV/cn/543-R16
     Enclosure(s)     :1
     Date             : October 18, 2016
     Dear Minister,
     I hereby submit the advisory report on the effects of dexamethasone 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. It concerns
     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 Public Health.
     The submission letter (in English)                                                  59
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<pre>  Today I sent copies of this advisory report to the Minister of Health, Welfare and
  Sport and tot the State Secretary of Infrastructure and the Environment, for their
  information.
  Yours sincerely,
  (signed)
  Professor J.L. Severens
  Vice President
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<pre>nnex C
     Comments on the public draft
     A draft of the present report was released in 2015 for public review. The
     following persons and organisation have commented on the draft document:
     • T.J. Lentz, A.F. Hubbs. National Institute for Occupational Safety and Health
         (NIOSH), Cincinnati, OH, USA.
     The comments received, and the reply by the Committee can be found on the
     website of the Health Council.
     Comments on the public draft                                                    61
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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                                                      63
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<pre>  3.7.1.2         For the purpose of classification the hazard class Reproductive Toxicity is differentiated
                  into:
  •     adverse effects
        •    on sexual function and fertility, or
        •    on development;
  •     effects on or via lactation.
  3.7.1.3         Adverse effects on sexual function and fertility
  Any effect of substances that has the potential to interfere with sexual function and fertility. This
  includes, but is not limited to, alterations to the female and male reproductive system, adverse effects
  on onset of puberty, gamete production and transport, reproductive cycle normality, sexual behaviour,
  fertility, parturition, pregnancy outcomes, premature reproductive senescence, or modifications in
  other functions that are dependent on the integrity of the reproductive systems.
  3.7.1.4         Adverse effects on development of the offspring
  Developmental toxicity includes, in its widest sense, any effect which interferes with normal devel-
  opment of the conceptus, either before or after birth, and resulting from exposure of either parent
  prior to conception, or exposure of the developing offspring during prenatal development, or postna-
  tally, to the time of sexual maturation. However, it is considered that classification under the heading
  of developmental toxicity is primarily intended to provide a hazard warning for pregnant women, and
  for men and women of reproductive capacity. Therefore, for pragmatic purposes of classification,
  developmental toxicity essentially means adverse effects induced during pregnancy, or as a result of
  parental exposure. These effects can be manifested at any point in the life span of the organism. The
  major manifestations of developmental toxicity include (1) death of the developing organism, (2)
  structural abnormality, (3) altered growth, and (4) functional deficiency.
  3.7.1.5         Adverse effects on or via lactation are also included in reproductive toxicity, but for
  classification purposes, such effects are treated separately (see Table 3.7.1 (b)). This is because it is
  desirable to be able to classify substances specifically for an adverse effect on lactation so that a spe-
  cific hazard warning about this effect can be provided for lactating mothers.
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<pre>3.7.2        Classification criteria for substances
3.7.2.1      Hazard categories
3.7.2.1.1    For the purpose of classification for reproductive toxicity, substances are allocated to
one of two categories. Within each category, effects on sexual function and fertility, and on develop-
ment, are considered separately. In addition, effects on lactation are allocated to a separate hazard cat-
egory.
Table 3.7.1(a) Hazard categories for reproductive toxicants.
Categories                      Criteria
CATEGORY 1                      Known or presumed human reproductive toxicant
                                Substances are classified in Category 1 for reproductive toxicity when
                                they are known to have produced an adverse effect on sexual function
                                and fertility, or on development in humans or when there is evidence
                                from animal studies, possibly supplemented with other information, to
                                provide a strong presumption that the substance has the capacity to
                                interfere with reproduction in humans. The classification of a sub-
                                stance is further distinguished on the basis of whether the evidence for
                                classification is primarily from human data (Category 1A) or from
                                animal data (Category 1B).
                Category 1A Known human reproductive toxicant
                                The classification of a substance in Category 1A is largely based on
                                evidence from humans.
                Category 1B Presumed human reproductive toxicant
                                The classification of a substance in Category 1B is largely based on
                                data from animal studies. Such data shall provide clear evidence of an
                                adverse effect on sexual function and fertility or on development in
                                the absence of other toxic effects, or if occurring together with other
                                toxic effects the adverse effect on reproduction is considered not to be
                                a secondary non-specific consequence of other toxic effects. However,
                                when there is mechanistic information that raises doubt about the rele-
                                vance of the effect for humans, classification in Category 2 may be
                                more appropriate.
CATEGORY 2                      Suspected human reproductive toxicant
                                Substances are classified in Category 2 for reproductive toxicity when
                                there is some evidence from humans or experimental animals, possi-
                                bly supplemented with other information, of an adverse effect on sex-
                                ual function and fertility, or on development, and where the evidence
                                is not sufficiently convincing to place the substance in Category 1. If
                                deficiencies in the study make the quality of evidence less convincing,
                                Category 2 could be the more appropriate classification.
                                Such effects shall have been observed in the absence of other toxic
                                effects, or if occurring together with other toxic effects the adverse
                                effect on reproduction is considered not to be a secondary non-specific
                                consequence of the other toxic effects.
Regulation (EC) 1272/2008 of the European Community                                                        65
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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.
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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                                                         67
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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 postnatal functional deficiencies.
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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                                                       69
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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
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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.
Regulation (EC) 1272/2008 of the European Community                                                       71
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<pre>  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
  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.
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<pre> 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.
              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                                                                 73
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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
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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                              75
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<pre>  •   The Committee does not only use guideline studies (studies performed
      according to OECD* standard protocols) for the classification of compounds,
      but non-guideline studies are taken into consideration as well.
   Organisation for Economic Cooperation and Development.
6 Dexamethasone
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<pre> nnex         F
              Fertility and developmental toxicity
              studies
 able 1 Fertility studies in animals.
Authors       Species        Experimental      Dose and route  General       Developmental toxicity           Remarks
                             period/design                     toxicity
De Greef      Wistar rats Ovarian activity     0 mg/rat        Not described Induction of prolactin-dependent
1987)                        was measured      (n=unknown),                  luteal activity and large number
                             with different    0.008 mg/kg bw                of eggs after high doses
                             methods           (n=6), 0.03
                                               mg/kg bw (n=7),
                                               or 0.1 mg/kg bw
                                               (n=14)
                                               subcutaneously
Maciel et al. Non-           Cows were         0 or 0.044      No effect on Decrease plasma progesterone
2001)         lactating      treated from one mg/kg bw         bw. Increase (p<0.10). No effect on the growth
              Holstein       day after         intramuscularly systemic      rate of dominant follicles
              cows (n=6) ovulation until                       glucose and
              and controls the first                           insulin
              (n=5)          dominant follicle                 (p<0.05).
                             stopped growing.                  Decrease IGF-
                                                               I and II
                                                               (p<0.05)
              Fertility and developmental toxicity studies                                                            77
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<pre>Hatamoto Male adult          Dogs were          0 or 0.01         No effect on   Reduced ejaculate volumes
 t al. (2006) Rottweiler     treated for 7      mg/kg bw/day      body weight    (p=0.0002).
              dogs           days.              intramuscularly   or food intake Increased thiobarbituric acid-
              (n=18)         Semen collection                     (data not      reactive substances (p<0.0001).
                             twice weekly for                     reported)      Increased number of sperm per
                             14 weeks. Blood                                     ejaculate (p=0.0122), increased
                             collection once a                                   percentage of abnormal sperm
                             week                                                (p=0.0002), increased seminal
                                                                                 plasma lipid peroxidation
                                                                                 (p=0.0264). Elevated activity of
                                                                                 one of two antioxidant enzymes
                                                                                 (p=0.0264)
Gür et al.    Akkaraman Rams were               0 or 0.25         Not described Increased hyaluronidase activity
2005)         rams (n=7/ treated for 2          mg/kg bw                         in serum (p<0.01, except after 1
              group)         days.              intramuscularly                  hr) and sperm (p<0.001-0.05)
                             After last dose                                     Decreased sperm concentration
                             blood and semen                                     (p<0.001, except at 96 hrs),
                             samples taken                                       semen volume (p<0.05) and
                             after 1, 2, 4, 24,                                  sperm motility (p<0.05, except at
                             48, 72 and 96 hrs                                   72 and 96 hrs).
                                                                                 No effect on rate of abnormal
                                                                                 spermatozoa
  arth et al. Mixed Bos Bulls were              0 or 20 mg/bull Not described Lower basal, peak episodic and         Recovery to
1994)         Taurus Bulls treated for 7        intramuscularly                  testosterone concentrations in      pre-treatment
              (n=8) and      days. Sperm                                         blood (p<0.05).                     levels of
              controls       abnormalities                                       Concentration in testis tissue      sperm defects
              (n=4)          were analysed                                       unchanged.                          after 6 wks
                                                                                 Increase in sperm defects such as
                                                                                 detached heads, midpiece defects
                                                                                 and nuclear vacuoles (no statistics
                                                                                 mentioned)
Abbreviations: bw=body weight; hr(s)=hour(s); n=number; wk(s)=week(s).
 able 2 Developmental toxicity studies in animals.
Authors         Species           Experimental period/      Dose and route    General         Developmental toxicity
                                  design                                      toxicity
 eratogenic potential
 erome and Pregnant               Monkeys were treated at   0, 1.0 or 10.0    Not described No effect on body weight; brain
Hendrikx        Rhesus            different days during GD  mg/kg bw                          weight reduced at 10.0 mg/kg
1988)           monkeys           23-49                     intramuscularly                   (p<0.05); cranial fossa diameter
                (n=12).                                                                       reduced at 10.0 mg/kg (p<0.05);
                Sacrifice on                                                                  cranium malformations (2/6
                GD100                                                                         animals affected at 1.0 mg/kg, 4/6
                                                                                              at 10.0 mg/kg)
 auerbier       NMRI Mice         Mice were treated on      10 or 50          Not described Cleft palate and resorptions at all
 t al. (1986)   (n=12/group).     GD13 at 07:00, 13:00,     mg/kg bw                          dose levels. Effect dose-dependent
                and control       19:00 or 01:00            intramuscularly                   (p-values not mentioned)
                (n=10)
                Sacrifice on
                GD 18
  8           Dexamethasone
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<pre>  allard et al. Pregnant CD-1 Mice were treated five     Therapeutic       Not described Increased incidence of cleft palate.
1977)           mice           times daily during        concentration                   Increased incidence of sex organ
                (n=unknown). GD10-13                     (not specified)                 abnormalities
                Sacrifice on                             or vehicle,
                GD 18                                    ocular
                                                         application
  ardiovascular effects
 orres et al. Pregnant rats    Rats were treated from    0.0048 mg/day     Not described Higher heart/body weight ratio
1997)           (n=8/group)    GD17 for 4-5 days.        subcutaneously,                 (p<0.05) and proliferative index
                               Foetal heart was          (slow release                   (p<0.05).
                               investigated              pellet); controls               Decrease in ECM-content (p<0.05)
                                                         unmanipulated                   and α-MHC mRNA (p<0.05)
 lotkin et al. Pregnant        Rats were treated on GD 0, 0.2 or 0.8       Maternal body Lower body weight (p<0.01); lower
1991)           Sprague        17, 18 and 19. Foetal     mg/kg bw/day      weight gain   heart weight, DNA-content, cell
                Dawley rats    heart and kidney were     subcutaneously    slower, but   packing density (all p<0.01) and
                (n=unknown)    investigated                                specifics not enlarged cells (p<0.01)
                                                                           reported
Dodic et al.    Pregnant       Ewes were treated for 2   11.5 mg/day       Not described Hypertension (p<0.01), left
2001)           treated ewes   days at 27 days of        intraveneously                  ventricular hypertrophy (p<0.05),
                (n= unknown)   gestation. Allowed to                                     and reduced cardiac functional
                and control    litter and surgery at PND                                 reserve (p<0.05) in adult life
                (n=6)          50
Molnar et. al. Pregnant ewes   Ewes were administered    0 or 2 mg/ewe Not described     Body weight lower (p<0.01).
2002)           (n=7) and      as three weekly courses   intramuscularly                 Microvessel dysfunction: enhanced
                controls (n=7) of four intramuscularly                                   endothelin-induced
                               injections at 12 h-                                       vasoconstriction (p=0.003 at
                               intervals                                                 highest concentration), decreased
                                                                                         endothelium-dependent relaxation
                                                                                         (p<0.05); normal endothelium-
                                                                                         independent relaxation
Quaedackers Pregnant           Ewes were treated with    2 injections of 0 Not described Transient increase in blood pressure
 t al. (2005) Romney/          two injections (24 h      or 12 mg                        (p<0.05).
                Suffolk ewes   apart) at GD 103.         (24 h apart)                    No effect on vascular resistance
                (n=8) and      Allowed to litter and     intramuscularly
                control (n=7)  litter monitored for 5
                               days
Hai et al.      Pregnant ewes Ewes were treated with     0 or 6 mg/day Not described     Reduced pup body weight
2002)           (n=unknown) single doses (starting GD    intramuscularly                 (p<0.05).
                               104, 105, or 106) or                                      Biphasic contractions in foetal
                               multiple doses (starting                                  arteries (p<0.05);
                               GD 76, 84, 91, 98 and                                     Increased relaxation rate in foetal
                               105). Foetuses                                            arteries (p<0.05);
                               catheterized at GD 99-                                    Increased myosin LC17a isoform
                               101. Foetal arteries                                      expression in fetal arteries (p<0.05)
                               investigated
               Fertility and developmental toxicity studies                                                                79
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<pre>  eurodevelopmental effects
Hauser et al. Pregnant        Monkeys were       0 or 5          No effect on       Early treatment: Increased weight gain
2007)           Marmoset      treated during     mg/kg bw/day    maternal body      in the presence of normal skeletal
                monkey        early              orally          weight             growth (p<0.05), increased eating
                              (GD42-48) or late                                     (p<0.05), increased sympathetic
                              (GD90-96)                                             autonomic nervous system arousal
                              gestation                                             (p<0.05); increased time spent mobile
                                                                                    (p<0.05); increased time spent eating
                                                                                    (p<0.05), trends toward more solitary
                                                                                    play (p=0.053) and tail hair piloerection
                                                                                    (p=0.085).
                                                                                    Late treatment: no effect
Hauser et al. Wister rats     Rats were treated 0 or 0.1         Effect on          Reduced pup birth weight (p<0.05)and
2006)           (n=14) and    between GD15-21. mg/kg/day in      maternal body      adult body weight (p<0.05, male pups
                controls      Offspring tested   drinking water  weight not         only).
                (n=12)        for prepulse                       reported.          No effect on PPI and LI
                              inhibition (PPI)                   Increased pup-
                              and latent                         directed
                              inhibition (LI)                    behaviour
                                                                 (p=0.029)
Dupouy et al. Wistar rats     Rats were treated  0 or 0.01 mg/ml Not described      Reduction of body weight (p<0.001),
1987)           (n=unknown) from GD 15-21.       in drinking                        severe atrophy of the adrenals
                              Effect on          water                              (p<0.001), and decreases in adrenal and
                              hormones of the                                       plasma corticosterone concentrations
                              hypothalamo-                                          (p<0.001).
                              pituitary-adrenal                                     Decrease in plasma adrenocorticotrophic
                              axis measured in                                      hormone (ACTH) concentration and
                              hypothalamus,                                         pituitary ACTH content (p<0.001).
                              pituitary gland,                                      Decreased corticotrophin-releasing
                              adrenals and                                          factor hypothalamic content and
                              plasma in the                                         concentration (p<0.001)
                              offspring
  rabham        Pregnant      Rats were treated  0 or 0.0025     Maternal body      Restlessness in mothers (p<0.05).
 t al. (2000) Sprague         daily from GD15    mg/ml in the    weight showed a    Low birth weights and poor weight gain
                Dawley rats   until delivery     evening water   smaller increase   in the offspring (p<0.05).
                (n=20-24)                                        at GD 15-18        Impaired spatial learning (p<0.05),
                                                                 (p<0.001), a       inability to rapidly terminate
                                                                 larger one at 2    adrenocorticoid response to stress
                                                                 wks after giving   (p<0.05) and decreased hippocampal
                                                                 birth (p=0.05)     glucocorticoid receptor mRNA
                                                                 and returned to    expression (p<0.05).
                                                                 normal levels at 3 Dexamethasone exposed animals reared
                                                                 wks after giving   to vehicle dams had adequate reponses
                                                                 birth
  arlos et al.  Pregnant rats Rats were treated 0, 0.2 or 0.8    Maternal weight    The forebrain showed persistent
1992)           (n=unknown) during GD17, 18 mg/kg bw/day gain slowed (data          elevations of DNA (p<0.01) and reduced
                              and 19. Pups after subcutaneously not reported)       protein/DNA (p<0.01), indicative of
                              weaning                                               replacement of neurons with glia
                              investigated for
                              brain defects
  0            Dexamethasone
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<pre>Nagano et al. Pregnant       Rats were treated 0 or 0.005        No effect on    Reduced body weight in the offspring at
2008)          Sprague       during GD16-21. mg/kg bw/day        maternal care   PNW 4, 7 and 10 (p<0.01 or p<0.05).
               Dawley rats   Neuroendocrinolo subcutaneously     during          Decreased corticotropin-releasing factor
               (n=unknown)   gical development                   preweaning      mRNA in hypothalamus (p<0.01),
                             pups investigated.                                  disturbed plasma corticosterone
                                                                                 response to restraint stress at PNW 4
                                                                                 (p<0.01), increased anxiety-like
                                                                                 behaviour (p<0.05), decreased
                                                                                 glucocorticoid receptor expression
                                                                                 amygdale at PNW7 (p<0.01) and PW10
                                                                                 (p<0.001).
                                                                                 Neuroendocrinological development
                                                                                 altered in pups
 lotkin et al. Pregnant      Dams were treated   Dams; 0, 0.05, Not described    5HT receptor binding and 5HT
2006)          Sprague       during GD17-19.     0.2 or 0.8                      transporter binding increased in the
               Dawley rats   Pups were treated   mg/kg bw/day                    cerebral cortex during gestation and
               (n=unknown)   during PND1-3 or    subcutaneously.                 after birth (p<0.0006 and p<0.0001,
                             7-9.                Pups; 0, 0.05,                  respectively). 5HT concentration in
                             Pups were           0.2 or 0.8                      hippocampus and cerebral cortex
                             sacrificed on       mg/kg bw/day                    increased after birth (p<0.002), then
                             PND60 and brain                                     decreased till levels below normal
                             was investigated                                    (p<0.0006). 5HT turnover unchanged.
                                                                                 DA concentration in cerebral cortex
                                                                                 already increased during gestation
                                                                                 (p<0.03), decreased till levels below
                                                                                 normal at PND 1-3 (p<0.0001) and
                                                                                 returned to normal by PND 7-9. DA
                                                                                 turnover in cerebral cortex unchanged
Kreider et al. Pregnant      Rats were treated 0 or 0.2          No effect on    No effect on gestation index, litter size,
2005)          Sprague       on GD17, 18 and mg/kg bw/day        maternal weight viability and sex ratio.
               Dawley rats   19. Behavioural     subcutaneously  gain during     Normal sex differences in locomotor
               (n=unknown)   tests at PND31, 32,                 pregnancy       activity ablated by lowering values in
                             and 33                                              females to levels of males (p<0.0003).
                                                                                 Habituation of activity similarly
                                                                                 impaired in females to match profile of
                                                                                 males (p<0.01), while males showed a
                                                                                 partially feminized pattern of
                                                                                 habituation (p<0.004). Delayed learning
                                                                                 in males (p<0.08), while improving
                                                                                 performance in females in the 8-arm
                                                                                 radial maze (p<0.04). Values of
                                                                                 hippocampal [3H]hemicholinium-3
                                                                                 binding increased in males to levels of
                                                                                 females (p<0.007)
              Fertility and developmental toxicity studies                                                               81
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<pre> omm et al.    Pregnant          Rats were treated 0 or 0.1             Maternal body    Litter size smaller (p<0.001). Perinatal
2014)          Sprague           on GD14-21.          mg/kg bw/day      weight           body weight diminished at PND1 and 21
               Dawley OFA        Growth               subcutaneously    throughout       (p<0.001). Lower concentrations of
               rats              investigated at                        gestation and    several brain metabolites and lower
                                 PND1 and -21, and                      weight gain      hippocampal gene expression at PND7
                                 brain metabolism                       during the third (p<0.05)
                                 at PND7                                week decreased
                                                                        (p<0.001 and
                                                                        p<0.01,
                                                                        respectively)
Kutzler et al. Pregnant ewes     Ewes were treated    0 or 8            Not described    Decreased foetal body weight (p<0.05).
2004)          (n=9) and         on GD103-104,        mg per ewe                         Prolonged gestation length (p<0.05).
               control (n=11)    110-111 and          intramuscularly                    Normal birth weight. Decreased
               used for          117-118              (4 doses of 2                      newborn brain weight (p<0.05) and
               prenatal                               mg per ewe                         biparietal diameter (p<0.05). Various
               development                            within 48 hrs;                     other postnatal growth measures normal
               (cohort 1)                             a dose
               -----------------                      corresponds
               n= 8 (treated                          to 29-33
               and control                            µg/kg bw)
               cohort 2) or
               n=13 (treated
               and control
               cohort 3) used
               for postnatal
               development
Uno et al.     Rhesus            Monkeys were         Single; 0, 0.5, 5 Not described    Irreversible deficit and poorly
1990)          Monkeys           treated, Single;     or 10 mg/kg bw.                    differentiated hippocampal and cortal
               (n=unknown)       GD132 or             Multiple;                          neurons
                                 Multiple; GD132      0 (4x 0), 0.5
                                 and 133.             (4x 0.125),
                                 Caesarean section    5 (4x 1.25) or
                                 GD135 for acute      10 (4x 2.5
                                 effects or GD162     mg/kg).
                                 for chronic effects. Intramuscularly
                                 Brain investigated
Moritz et al.  Pregnant          Ewes were infused    0 or 0.020        Not described    No effects
2002)          merino ewes (route not                 mg/kg bw/day
               (n=18) and        mentioned) for 20
               control (n=15) days. Ewes were
                                 killed on GD45. A
                                 second cohort was
                                 killed on GD130.
                                 In a third cohort
                                 the lambs were
                                 studied at 2
                                 months of age
 2            Dexamethasone
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<pre>Uno et al.      Rhesus         Monkeys were         0 or 5          Not described Irreversible reduction of hippocampal
1994)           monkeys        treated on GD132     mg/kg bw/day                  size and high plasma cortisol and post
                (n=8) and      and 133. In infants  (route                        stress levels (statistics not reported)
                control (n=3)  cortisol levels and  unknown)
                               brain was
                               investigated at 20
                               months of age
  ffects on immune and endocrine system
Negić et al. Pregnant          Rats were treated    0, 1.0 or 0.5   Not described Marked reduction of morphometric
2007)           Wistar         on GD16-19.          mg/kg bw/day                  parameters of LH and FSH (p<0.05).
                females.       Autopsy at GD 19     subcutaneously                Size of LH and FSH cells on day 19 of
                Sacrifice on   or GD21. Foetal                                    pregnancy affected.
                GD19 (n=8) pituitary                                              Reversible decrease in cell volume
                and on GD21 investigated                                          (normalization of LH and FSH cell
                (n=8) and two                                                     function on day 21 of pregnancy).
                control groups                                                    No prolonged effects of dexamethasone
                (n=8/group)                                                       treatment during pregnancy on LH and
                                                                                  FSH cells
 tojanoski      Wistar rats    Rats were treated    1.0 (GD 16),    Not described Pituitary gland: smaller number and
 t al. (2006)   (n=unknown) on GD16, 17 and         0.5 (GD 17                    volume of ACTH cells (p<0.05).
                               18. Autopsy at        and 18)                      Adrenal organs: reduced proliferative
                               GD19 and             mg/kg bw/day                  activity of cortical cells of zona
                               pituitary and        subcutaneously;               glomerulosa (p<0.05)
                               adrenal glands       Controls
                               investigated         received
                                                    vehicle
 age et al.     Spraque        Rats were treated    0 or 0.1        Not described Immature male offspring showed
2001)           Dawley rats    on GD14-19.          mg/kg bw/day                  decreased serum testosterone (p<0.05)
                (n=9/group)    Allowed to litter    subcutaneously                and testosterone production in Leydig
                               and immature and                                   cells (p<0.001). Serum ACTH and
                               adult males                                        corticosterone were reduced (p<0.001).
                               investigated                                       Mature male offspring demonstrated
                                                                                  higher serum ACTH and corticosterone
                                                                                  (p<0.001), and higher serum
                                                                                  testosterone (p<0.05) and testosterone
                                                                                  production in Leydig cells (p<0.001)
Hristić et al.  Pregnant       Rats were treated 0 or 0.3           Not described Decreased volume of foetal adrenal
1997)           Wistar rats    during 5 days,       mg/kg bw/day                  gland (p<0.005), its zona glomerulosa
                (n=10/group)   started on GD16. subcutaneously                    plus capsula (p<0.005) and its inner
                               First cohort killed                                zone (p<0.005), atrophic changes in the
                               after 24 h after the                               gland, reduction of the volume and total
                               last injection.                                    number of adrenocortical cells (p<0.05
                               Second and third                                   and p<0.005, respectively). These
                               cohort killed on                                   morphometric changes were found in
                               PND3 and 14.                                       3- and 14-day-old pups
                               Structure and
                               function of adrenal
                               gland investigated
               Fertility and developmental toxicity studies                                                               83
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<pre>Holson et al. Spraque        Rats were treated    0.1              Considerable    Reduced pup birth weight (effect still
1995)         Dawley         on GD14 to 21.       mg/kg bw/day reduction in        seen at PND85) (p<0.01).
              derived CD     Sexual behaviour     subcutaneously maternal weight   Male adrenal weight at birth reduced
              rats (n=4/     offspring                             gain (p<0.0001) (p<0.01); male anogenital distance
              group)         measured                                              reduced (p<0.05).
                                                                                   Adult male sexual performance (number
                                                                                   of ejaculations per trial and latency to
                                                                                   first ejaculation after cohabitation with
                                                                                   oestrous females (p<0.05)) affected, and
                                                                                   lordosis quotient following exposure of
                                                                                   castrated hormone-primed males to stud
                                                                                   males affected (p<0.05)
 akker et al. Wistar rats    Rats were treated    0 or 400 µg of Not described     Reduced pup birth weight compared to
1995)         (n=7) and two  on GD 17 and 19.     dexamethasone                    either control group, on PND 1.
              control groups Effects on thymus,   -21-phosphate                    Decreased T cell numbers in thymus and
              (n=6)          spleen,              intra-                           spleen on PND 1 (p<0.05 or <0.005). T
                             hypothalamus and     peritoneally, or                 cell numbers in spleen were reduced at
                             blood plasma of      no injection                     all neonatal ages studied (PND 1, 7 and
                             offspring were                                        20; p<0.05 or <0.005). Altered pattern of
                             examined at PND                                       neonatal changes in peptide expresion in
                             1, 7 and 20                                           corticotropin-releasing hormone
                                                                                   neurons. No effects on
                                                                                   adrenocorticotrophic hormone and
                                                                                   corticosterone levels in plasma
 akker et al. Wistar rats    Rats were treated    0 or 1.2         Not described   No altered corticosteroid response to a
1998)         (3 animals per on GD 17 and 19.     mg/kg bw                         novel environment at 20 days of age, as
              group)         Effects on           dexamethasone                    compared to either control group. No
                             corticosterone       -21-phosphate                    effects on immunoglobulin-2a
                             response to a mild   intra-                           production after immunization with
                             stressor and on      peritoneally, or                 conjugated pneumococcal
                             humoral and          no injection                     polysaccharide at 6 wks of age.
                             cellular immune                                       Oxazolone-induced contact
                             responsiveness                                        hypersensitivity responses at 8 wks of
                             were measured                                         age, was lower compared to saline
                                                                                   controls (p<0.005), but not different
                                                                                   from untreated controls
 u et al.     Sprague-       Rat were treated at  0 or 0.1         Not described   Three out of five inflammation
2014)         Dawley rats    gestational days     mg/kg bw/day                     mediators decreased at day 7, one of
              (6-8 animals   14-20. Male          dexamethasone                    them, matrix metalloproteinase-9, still at
              per group)     offspring were       intra-                           day 120 (p<0.05). Upon concanavalin-A
                             killed at day 7 or   peritoneally                     stimulation prenatal dexamethasone
                             day 120 after birth.                                  treatment reduced tumour necrosis
                             Spleens were                                          factor-α production (p<0.05), but not
                             collected for                                         interferon-γ production in spleen cells at
                             immune analysis                                       day 120
 4           Dexamethasone
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<pre>Hristić et al.  Wistar rats    Rats were treated   Single dose of   Not described Decreased adrenal weight and volume,
1995)           (n=5/group)    on GD16.            0 or 1.5                       volume of the zona glomerulosa plus
                               Dams and foetuses   mg/kg bw                       capsule, and volume of the inner zone,
                               sacrificed on       (route                         both in foetuses and 3-day-old rats (p-
                               GD21 en some        unknown)                       values <0.05, <0.025 or <0.005).
                               neonatal animals                                   Decreased number, but not total volume
                               sacrificed at PND                                  of cells (p<0.05 or p<0.005).
                               4 or 15. Adrenal                                   Morphological signs of necrosis. In 14-
                               glands investigated                                day-old animals, degree of atrophic
                                                                                  changes in adrenal cortex reduced
 ffects on renal function
Dodic et al. Pregnant          Ewes were treated   0 or 0.002       Not described No evidence of altered renal function or
2003)           Mereno ewes    from GD25 to 45.    mg/kg bw/day                   predisposition to adult hypertension
                (n=11) and     Ewes allowed to     intravenously
                controls (n=9) lamb and blood
                               pressure and renal
                               function in
                               offspring
                               investigated
Ortiz et al.    Pregnant       Rats were treated   two daily        Not described Length of gestation, litter size, body
2001)           Sprague        on GD11 and 12,     intraperitoneal                weight and kidney weight at PND1 not
                Dawley rats    13 and 14, 15 and   injections of 0                affected. In the offspring of dams treated
                (n=unknown) 16, 17 and 18, 19      or 0.2                         at GD15 and 16 the glomerular number
                               and 20 or 20 and    mg/kg bw                       was 30% reduced (p<0.01) and the
                               21.                                                systolic pressure was higher (p<0.05).
                               Blood pressure,                                    After maternal treatment at GD17 and
                               glomerular number                                  18 a 20% reduction of glomeruli was
                               and inulin                                         measured (p<0.01). No effects in other
                               clearance were                                     treatment regimens
                               measured in
                               offspring at
                               PND60 or 90
Ortiz et al.    Pregnant rats Rats were treated    Twice daily 0 or Not described In the offspring of dams treated at GD15
2003)           (n=unknown) on GD11 and 12,        0.2 mg/kg bw                   and 16 the glomerular number was 20%
                               13 and 14, 15 and   intra-                         reduced at 6 to 9 months of age
                               16, 17 and 18, or   peritoneally                   (p<0.05). This was similar to the
                               19 and 20. Effects                                 reduction at 3 wks of age. After
                               on neonates and                                    treatment at GD17 and 18 the
                               longterm effects                                   glomerular number was 17% reduced at
                               were investigated                                  6 months of age (p<0.05). After
                                                                                  treatment at days 13 and 14, 15 and 16,
                                                                                  or 17 and 18 of gestation blood pressure
                                                                                  was elevated at 6 months of age
                                                                                  (p<0.05). The GD13-14 group did not
                                                                                  show a reduced glomerular number
 lotkin et al. Pregnant        Rats were treated 0, 0.2 or 0.8      Maternal body Body and kidney weights lower
1991)           Sprague        on GD 17, 18 and mg/kg bw/day        weight gain   (p<0.01). DNA content of the kidney
                Dawley rats    19. Foetal heart    subcutaneously   slower, but   diminished (p<0.01). Cell packing
                (n=unknown)    and kidney were                      specifics not density increased (p<0.01 or p<0.05,
                               investigated                         reported      depending on the dose of
                                                                                  dexamethasone) and cells enlarged, after
                                                                                  initial decrease in size (p<0.01)
               Fertility and developmental toxicity studies                                                              85
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<pre> ain et al.     Pregnant      Rats were treated    0 or 0.1        Not described     Hypertension and reduced nephron
2014)           Sprague       on GD 16-22.         mg/kg bw/day                      numbers (p<0.05). No changes in kidney
                Dawley rats   Offspring blood      subcutaneously                    expression of genes involved in
                (n=unknown)   pressure and                                           apoptosis or nephrogenesis, except for a
                              kidney were                                            gene coding for a class I histone
                              investigated at 16                                     deacetylase. Some genes of the renin-
                              wks of age                                             angiotensin system upregulated (p<0.05)
                              (n=12/group)
 ain et al.     Pregnant      Rats were treated    0 or 0.2        Not described     Hypertension (p<0.05). Various genes
2014a)          Sprague       on GD15-16.          mg/kg bw/day                      involved in apoptosis or nephrogenesis
                Dawley rats   Offspring blood      subcutaneously                    upregulated (p<0.05)
                (n=unknown) pressure and
                              kidney were
                              investigated at 16
                              wks of age
                              (n=8-10/group)
  ogers et al. Pregnant       Rats were treated    0 or 0.6        Maternal weight   Both male and female offspring had
2014)           Sprague       on GD16-20.          mg/kg bw/day gain during          reduced birth weights (p<0.05).
                Dawley rats   Offspring blood      subcutaneously pregnancy          Increased blood pressure at 7 wks (males
                (n=unknown) pressure and                           (GD19-21) was     only), 37 wks (females only, males not
                              kidney were                          reduced (p<0.05)  examined) and 65 wks of age (p<0.05).
                              investigated up                                        Males had reduced nephron counts and
                              until 65 wks of age                                    increased glucocorticoid receptor gene
                              (n=10-12/group)                                        expression in kidneys (p<0.05); female
                                                                                     kidneys not examined
  ffects on skeletal function
 wolin-Eide Pregnant          Rats were treated 0 or 0.1           Maternal toxicity Male pups showed transient increases in
 t al. (2002) Wistar rats     during GD9, 11       mg/kg bw/day was not observed     crown-rump length and tibia and femur
                (n=5/group)   and 13. Allowed to intramuscularly                     lengths at 3-6 wks of age (p<0.05 or
                              litter and 6 ♂ and 6                                   p<0.001). Cortical bone dimensions
                              ♀ killed at 6                                          altered in 12-week old females (p<0.05
                              weeks. Remaining                                       or p<0.001). Areal bone mineral
                              ♂♂ at 10 weeks,                                        densities of long bones and spine
                              remaining ♀♀ at                                        unchanged in both male and female
                              12 weeks of age                                        offspring
  ffects on hearing
Hougaard et Pregnant          Rats were treated 0 or 0.1           The body weight   The number of live pups and the pup
 l. (2007)      Wistar rats   during GD14-21, mg/kg/day            gain during       weight at PND3 (day 0 not reported)
                (n=20-22)     females allowed to subcutaneously    pregnancy of      were lower (p<0.05 and p<0.001,
                              litter and hearing                   dams was lower    respectively). At weaning they had
                              loss investigated                    from GD19         returned to normal (data not shown).
                                                                   onwards           No effect on hearing loss
                                                                   (p<0.001)
  anlon et al.                Rats were treated    0 or 0.1        Not described     Increased susceptibility of the inner ear
2003)                         from GD14 until      mg/kg bw intra-                   to acoustic noise trauma in adult life.
                              parturition.         peritoneally                      This acoustic trauma was significantly
                              Females allowed                                        reduced after treatment with the free
                              to litter and                                          radical scavenger
                              hearing loss
                              investigated
  6            Dexamethasone
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<pre>  ffects in nonhuman primates
 e Vries et al. African vervet Monkeys were      0, 0.05, 0.12 or No effect on        No effect on foetal birth weight. A
 2007)          monkeys        treated from mid- 0.2 mg/kg bw/d maternal              disordered glucose-insulin homeostasis
                (n=10/group) gestation (exact    by diet          parameters          (p<0.02 or p=0.051, depending on
                               day unknown) up                                        dexamethasone dose), reduced
                               to birth                                               pancreatic β cell mass (p<0.0001), and
                                                                                      elevated blood pressure (p<0.04) and
                                                                                      cortisol levels (p<0.05)
Coe et al.      Rhesus         Exposure varied: Intramuscular Reduced maternal Transient increase in foetal blood
 2005)          monkeys        initiation at     injections at    cortisol levels and pressure, post-treatment effects on foetal
                (Macaca        GD120 to 143,     different dose increased             adrenal glands (smaller) and cortisol
                nemestrina or duration 2 to 42   levels, varying maternal blood       levels in umbilical cord blood (reduced),
                Papio          days              from 0.1         pressure            reduced foetal body size and smaller
                monkeys)                         mg/kg bw/day                         head circumferences, increase in weight
                                                 to 15                                of foetal liver, higher glycogen content
                                                 mg/kg bw/day                         and elevated blood glucose increased
                                                                                      insulin levels, reduced total brain and
                                                                                      cerebellum weights at preterm delivery
                                                                                      and delivery at term, morphological
                                                                                      changes in the hippocampus, decreased
                                                                                      thymus and spleen weights and reduced
                                                                                      lymphocyte proliferative responses
Abbreviations: bw=body weight; GD=gestation day(s); hr(s)=hour(s); n=number; PND= postnatal day(s); PNW=postnatal
week(s); wk(s)=week(s).
               Fertility and developmental toxicity studies                                                                  87
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<pre>8 Dexamethasone</pre>

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