<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>Nitrous oxide
Evaluation of the effects on reproduction, recommendation for classification
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<pre>Aan de Staatssecretaris van Sociale Zaken en Werkgelegenheid
Onderwerp       :  aanbieding advies
Uw kenmerk      :  DGV/BMO-U-932542
Ons kenmerk     :  U 948/AB/jt/543-Y2
Bijlagen        :  1
Datum           :  1 mei 2000
Mijnheer de Staatssecretaris,
Bij brief van 3 december 1993, nr. DGV/BMO-U-932542, verzocht de Staatssecretaris
van Welzijn, Volksgezondheid en Cultuur namens de Minister van Sociale Zaken en
Werkgelegenheid om naast het afleiden van gezondheidskundige advieswaarden ook te
adviseren over andere onderwerpen ten behoeve van de bescherming van beroepsmatig
aan stoffen blootgestelde personen. In 1995 heeft de Staatssecretaris van Sociale Zaken
en Werkgelegenheid besloten tot het opstellen van een zogenaamde niet-limitatieve repro-
tox-lijst. Op deze lijst komen stoffen die volgens de richtlijnen van de Europese Unie in-
gedeeld moeten worden in categorie 1 of 2 wat betreft effecten op de voortplanting. De
Gezondheidsraad is verzocht om voor stoffen een classificatie volgens de EU-criteria
voor te stellen.
In 1996 heb ik hiervoor de Commissie Reproductietoxische stoffen ingesteld.
Hierbij bied ik u - gehoord de Beraadsgroep Gezondheid en Omgeving - de publikatie
van de commissie aan over lachgas. Deze publikatie heb ik heden ter kennisname aan de
Minister van Volksgezondheid Welzijn en Sport en aan de Minister van Volkshuisves-
ting, Ruimtelijke Ordening en Milieubeheer gestuurd.
Hoogachtend,
w.g.
prof. dr JJ Sixma
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<pre>Nitrous oxide
Evaluation of the effects on reproduction, recommendation for classification
Committee for Compounds toxic to reproduction,
a committee of the Health Council of the Netherlands
to
the Minister and State Secretary of Social Affairs and Employment
No. 2000/03OSH, The Hague, 1 May 2000
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<pre>Preferred citation:
Health Council of the Netherlands: Committee for Compounds toxic to reproduction. Ni-
trous oxide; Evaluation of the effects on reproduction, recommendation for classifica-
tion. The Hague: Health Council of the Netherlands, 2000; publication no.
2000/03OSH.
all rights reserved
ISBN: 90-5549-316-3
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<pre>    Contents
    Samenvatting 7
    Executive summary 8
1   Scope 9
1.1 Background 9
1.2 Committee and procedure 9
1.3 Additional considerations 10
1.4 Labelling for lactation 11
1.5 Data 12
1.6 Presentation of conclusions 12
1.7 Final remark 12
2   Nitrous oxide 13
2.1 Introduction 13
2.2 Human studies 13
2.3 Animal studies 15
2.4 Conclusion 20
    References 22
    Contents                       5
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<pre>  Annexes 27
A The committee 28
B Comments on the public draft 30
C Directive (93/21/EEC) of the European Community 31
D Fertility and developmental toxicity studies 37
E Abbreviations and conversion factors 51
  Contents                                           6
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<pre>Samenvatting
Op verzoek van de Minister van Sociale Zaken en Werkgelegenheid beoordeelt de Ge-
zondheidsraad de effecten op de reproductie van stoffen waaraan mensen tijdens de be-
roepsuitoefening kunnen worden blootgesteld. De Commissie Reproductietoxische
stoffen, een commissie van de Raad, adviseert een classificatie van reproductietoxische
stoffen volgens Richtlijn 93/21/EEC van de Europese Unie. In het voorliggende rapport
heeft de commissie lachgas onder de loep genomen.
De aanbevelingen van de commissie zijn:
    Voor effecten op de fertiliteit adviseert de commissie lachgas in categorie 3 (stoffen
    die in verband met hun mogelijke voor de vruchtbaarheid van de mens schadelijke
    effecten reden geven tot bezorgdheid) te classificeren en met R62 (mogelijk gevaar
    voor verminderde vruchtbaarheid) te kenmerken.
    Voor effecten op de ontwikkeling adviseert de commissie lachgas in categorie 3
    (stoffen die in verband met hun mogelijke voor de ontwikkeling schadelijke effecten
    reden geven tot bezorgdheid voor de mens) te classificeren en met R63 (mogelijk
    gevaar voor beschadiging van het ongeboren kind) te kenmerken.
    Voor effecten tijdens lactatie adviseert de commissie om lachgas niet te kenmerken
    wegens onvoldoende gegevens.
Samenvatting                                                                               7
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<pre>Executive summary
On request of the Minister of Social Affairs and Employment, the Health Council of the
Netherlands evaluates the effects on the reproduction of substances at the workplace.
The Health Council’s Committee for Compounds Toxic to Reproduction recommends to
classify compounds toxic to reproduction according to the Directive 93/21/EEC of the
European Union. In the present report the committee has reviewed nitrous oxide.
The committee’s recommendations are:
    For effects on fertility, the committee recommends to classify nitrous oxide in cate-
    gory 3 (substances which cause concern for human fertility) and to label nitrous
    oxide with R62 (possible risk for impaired fertility).
    For developmental toxicity, the committee recommends to classify nitrous oxide in
    category 3 (substances which cause concern for humans owing to possible develop-
    mental toxic effects) and to label nitrous oxide with R63 (possible risk of harm to
    the unborn child).
    For effects during lactation, the committee is of the opinion that due to a lack of ap-
    propriate data nitrous oxide should not be labelled with R64.
Executive summary                                                                           8
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<pre>Chapter 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 re-
        quested the Health Council of the Netherlands to classify compounds toxic to reproduc-
        tion. The classification is performed according to the guidelines of the European Union
        (Directive 93/21/EEC) by the Health Council’s Committee for Compounds Toxic to Re-
        production. The committee’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
        toxic to reproduction (class 1 and 2) of the European Union.
1.2     Committee and procedure
        The present document contains the classification of nitrous oxide by the Health Coun-
        cil’s Committee for Compounds Toxic to Reproduction. The members of the committee
        are listed in Annex A. The first draft of this report was prepared by Mrs ir IDH Waal-
        kens-Berendsen at the Department of Neurotoxicology and Reproduction Toxicology of
        the TNO Nutrition and Food Research Institute, Zeist, The Netherlands, by contract
        with the Ministry of Social Affairs and Employment. The classification is based on the
        evaluation of published human and animal studies concerning adverse effects with res-
        pect to fertility and development and lactation of the above mentioned compound.
        Scope                                                                                    9
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<pre>    Classification and labelling was performed according to the guidelines of the European
    Union listed in Annex C.
     Classification for fertility and development:
     Category 1      Substances known to impair fertility in humans (R60)
                     Substances known to cause developmental toxicity in humans (R61)
     Category 2      Substances which should be regarded as if they impair fertility in humans (R60)
                     Substances which should be regarded as if they cause developmental toxicity in humans
                     (R61)
     Category 3      Substances which cause concern for human fertility (R62)
                     Substances which cause concern for humans owing to possible developmental toxic ef-
                     fects (R63)
     No classification for effects on fertility or development
     Labelling for lactation:
                     May cause harm to breastfed babies (R64)
                     No labelling for lactation
    In November 1999, the President of the Health Council released a draft of the report for
    public review. The individuals and organisations that commented on the draft report are
    listed in Annex B. The committee has taken these comments into account in deciding on
    the final version of the report.
1.3 Additional considerations
    The classification of compounds toxic to reproduction on the basis of the Directive
    93/21/EEC is ultimately dependent on an integrated assessment of the nature of all pa-
    rental and developmental effects observed, their specificity and adversity, and the dosa-
    ges at which the various effects occur. The directive necessarily leaves room for
    interpretation, dependent on the specific data set under consideration. In the process of
    using the directive, the committee has agreed upon a number of additional considera-
    tions.
         If there is sufficient evidence to establish a causal relationship between human expo-
         sure to the substance and impaired fertility or subsequent developmental toxic ef-
         fects in the progeny, the compound will be classified in category 1, irrespective the
         general toxic effects (see Annex C, 4.2.3.1 category 1).
         Adverse effects in a reproductive or developmental study, in the absence of data on
         parental toxicity, occurring at dose levels which cause severe toxicity in other stu-
         dies, need not necessarily lead to a category 2 classification.
    Scope                                                                                                  10
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<pre>         If, after prenatal exposure, small reversible changes in foetal growth and in skeletal
         development (e.g. wavy ribs, short rib XIII, incomplete ossification) in offspring oc-
         cur in a higher incidence than in the control group in the absence of maternal effects,
         the substance will be classified in category 3 for developmental toxicity. If these ef-
         fects occur in the presence of maternal toxicity, they will be considered as a conse-
         quence of this and therefore the substance will not be classified for developmental
         toxicity (see Annex C, 4.2.3.3 developmental toxicity final paragraph).
         Clear adverse reproductive effects will not be disregarded on the basis of reversibili-
         ty per se.
         Effects on sex organs in a general toxicity study (e.g. in a subchronic or chronic
         toxicity study) may warrant classification for fertility.
         The committee not only uses guideline studies (studies performed according to
         OECD standard protocols*) for the classification of compounds, but non-guideline
         studies are taken into consideration as well.
1.4 Labelling for lactation
    The recommendation for labelling substances for effects during lactation is also based on
    Directive 93/21/EEC. The Directive defines that substances which are absorbed by wo-
    men and may 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,
    should be labelled with R64. Unlike the classification of substances for fertility and de-
    velopmental effects, which is based on a hazard identification only (largely independent
    of dosage), the labelling for effects during lactation is based on a risk characterisation
    and therefore also includes consideration of the level of exposure of the breastfed child.
         Consequently, a substance should be labelled for effects during lactation when it is
    likely that the substance would be present in breast milk in potentially toxic levels. The
    committee considers a compound as potentially toxic to the breastfed child when exposu-
    re to this compound via the milk results in an intake exceeding an exposure limit for the
    general population, e.g. the acceptable daily intake (ADI).
*   Organisation for Economic Cooperation and Development
    Scope                                                                                        11
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<pre>1.5 Data
    Literature searches were conducted in the on-line databases Toxline and Medline, star-
    ting from 1966 up 1998. 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. References are divided in literature cited and lite-
    rature consulted but not cited. Before finalising the public draft the committee performed
    an additional literature search in Medline and Toxline for the period 1998 to 1999. The
    results of this search were no reason for the committee to adjust the recommendations.
         The committee chose to describe human studies in the text, starting with review arti-
    cles. Of each study the quality of the study design (performed according to international-
    ly acknowledged guidelines) and the quality of documentation are considered. The
    human studies are summarised in Annex D.
         Animal data are described in the text and summarised in Annex D.
1.6 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.
1.7 Final remark
    The classification of compounds is based on hazard evaluation* only, which is one of a
    series of elements guiding the risk evaluation process. The committee emphasises 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 characterisation, human
    exposure assessment and recommendations of other organisations.
*   for definitions see Tox95
    Scope                                                                                       12
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<pre>Chapter 2
        Nitrous oxide
2.1     Introduction
         Name                :  nitrous oxide
         CAS No.             :  10024-97-2
         Use                 :  anaesthetic gas
         Mol weight          :  44.01
         Chem formula        :  N2O
         Conversion factors  :  1% = 10.000 ppm = 18 g/m3
                                1 ppm = 1.8 mg/m3
2.2     Human studies
        Human studies are described in more detail in Tables 1 and 2 (Annex D).
        Fertility
        Wyrobeck et al. (1981) collected semen samples from 46 anaesthesiologists and 26 be-
        ginning residents in anaesthesiology and detected no differences in sperm concentration
        and number of abnormal sperm (Wyr81).
        Nitrous oxide                                                                           13
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<pre>     Anderson et al. (1992) found no differences in the quality of sperm of surgical pa-
tients before and after exposure to N2O in combination with halothane (And92).
     Knill-Jones et al. (1972) reported unexplained infertility amongst female and male
anaesthetists (Kni72). In 1975, Knill-Jones et al detected no effects of paternal exposure
to a mixture of anaesthetics on involuntary infertility in a case-control study (Kni75).
     Rowland et al. (1992) reported effects on time to pregnancy and spontaneous abor-
tion in female dental assistants after unscavenged exposure to N2O (Row92).
     Ahlborg et al. (1996) reported an effect of N2O on time to pregnancy in a small
group of midwives who assisted in more than 30 deliveries (N2O exposed) per month
(Ahl96).
     In a recent study, Peelen et al. (1999) reported that the time to pregnancy was not
affected in operation chamber assistants (Pee99). In this study the concentrations of se-
veral of anaesthetic gasses were measured; the maximal N2O concentration measured
was 318 mg/m3 ( 0.02%).
     In these human studies, age differences occurred between the exposed and control
group and no consideration was given to social factors, medication, illnesses and possi-
ble stress. Furthermore, the composition of the anaesthetic gas mixtures, in which N2O
was one component, the duration of exposure and its timing in pregnancy and exposure
to other chemicals such as inorganic mercury (amalgam), were often not reported.
     For these reasons, the committee considered the quality of these studies to be insuk-
ficient for classification.
Development
Several epidemiological studies were performed in which female anaesthetists, dental as-
sistants, operating nurses and wives of male anaesthetists were inquired about the course
and outcome of their pregnancies, with specific attention for miscarriages and congenital
anomalies (Ask70, Coh71, Kni72, Ros73 Ame74, Cor74a, Coh75, Pha77, Ros78,
Eri79, Coh80, Lau81, Hei84, Hem85, Joh87, Row92). In all studies, except Eri79,
Lau81, Hei84 and Hem 85, some effects on these parameters were suggested. However,
the studies were criticised by several authors (Wal75, Fer78, Ves78, Dud81) for the fol-
lowing reasons: studies were retrospective and often loaded questionnaires were used.
Age differences occurred between the exposed and control groups and no consideration
was given to social factors, medication, illnesses and possible stress.
     Furthermore, the composition of the anaesthetic gas mixtures, in which N2O was one
component, the duration of exposure and its timing in pregnancy and other exposure
such as inorganic mercury (amalgam), were often not reported.
     For these reasons, the committee considered the quality of these studies to be insuk-
ficient for classification.
Nitrous oxide                                                                              14
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<pre>    In a recent study Peelen et al. (1999) studied the effects of exposure to anaesthetic gas-
    ses on time to pregnancy, spontaneous abortions, preterm birth, low birth weight and
    congenital anomalies in operation personnel (Pee 99). An increased risk for spontaneous
    abortion (OR, odds ratio =1.3 (CI 95%: 0.8-2.1)), preterm birth (OR=1.9 (CI 95%:
    1.2-3.0)) and congenital abnormalities (CI 95%: OR=1.6 (0.9-2.9)) was observed. After
    correction for alcohol use, work circumstances and other environmental exposure on the
    working place, the OR for preterm birth was 1.4 and the OR for congenital abnormali-
    ties was 1.8. In this study, the concentration of anaesthetic gasses was measured; the
    maximal N2O concentration measured was 318 mg/m3. However, operation personnel
    was exposed to a mixture of anaesthetic gasses. For that reason it was not clear if N2O
    caused the slight increase in reproductive effects.
    Lactation
    No publications were found concerning the excretion of nitrous oxide in human breast
    milk.
2.3 Animal studies
    Fertility and developmental toxicity studies with N2O in experimental animals are sum-
    marised in annex D (table 3 and 4), respectively.
    Fertility
    Kripke et al. (1976) reported a significant reduction in mean testicular weight and dama-
    ge to dividing spermatogenic cells in the seminiferous tubules in all rats (LEW/f Mai)
    exposed to a mixture of 20% N2O (360 g/m3), 20 % oxygen and 60% nitrogen for vari-
    ous time periods up to 35 days (Kri76). Evidence of injury of the seminiferous tubules
    was found in some animals after exposure for 2 days; by 14 days such damage was
    found in all animals. Recovery of spermatogenesis was observed after removing the ani-
    mals to room air for 6 days. However, repair of the histological architecture of affected
    tubules was observed in only 1 of 5 animals sacrificed after 6 days in room air. Other
    animals had still severely affected tubules 10 days after removal from N2O-exposure.
    General toxicity was not described in this study.
         Male (C57B1/C3H)F1 mice were exposed by inhalation to air, containing 8 or 80 %
    N2O (144 or 1440 g/m3) for 4 h/day for 5 days (Lan81a). After 28 days, epididymal
    sperm was evaluated for morphological changes. The percentage abnormal sperm in
    both N2O-exposed groups was comparable to the control group (1.42 + 0.08 (8%
    Nitrous oxide                                                                              15
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<pre>group), 1.64 + 0.15 (80% group) versus 1.44 + 0.19 in the control). General toxicity
was not described in this study.
     Male mice (Swiss/ICR) were exposed to 0, 0.5, 5 or 50% N2O (0, 9, 90 or 900
     3
g/m ) for 4 h/day, 5 days/week for 9 weeks and then mated overnight for 1 week to un-
treated, virgin females (Maz82). No effects on fertility were observed. General toxicity
was not described in this study.
     Male and female Swiss Webster mice were exposed to 0, 0.5, 5 or 50% N2O (0, 9,
90 or 900 g/m3) by inhalation for 4 h/day, 5 days/week for 14 weeks (Maz83). After ex-
posure, germ cells were examined for evidence of injury. There were no significant diffe-
rences among the 4 inhalation exposure groups in testes weight, percentage of
abnormally shaped sperm, sperm count or histological appearance of the testes; the mean
percentage (+ SE) of abnormal sperm cells ranged from 8.9 + 2.4 (5% N2O) to 13.5 +
0.5 (50% N2O) with a concurrent control value of 10.4 + 2.3%. There was no significant
difference between the mean number of oocytes in mice treated with 50% N2O and in
control mice. The number of abnormal sperm cells in all inhalation (air and N2O ex-
posed) groups was rather high. The number of abnormal sperm cells in the animals trea-
ted with saline (intraperitoneally) was 2.5 + 0.3. N2O exposure caused no excitement or
general anaesthesia.
     Viera et al. (1983) exposed male Wistar rats by inhalation to 0 or 0.5% N2O/air
mixtures (v/v) (0 or 9 g/m3) for 30 days (Vie83). Immediately thereafter each male was
mated with three nulliparous female rats and mated again with three more nulliparous
rats after a 6-month recovery period. There was a significant reduction in mean litter
size of the females (7 for the exposed group versus 12 in the control group) mated with
N2O-exposed male rats directly after the N2O-exposure period and these offspring were
smaller in size (length and body weight). Mating after a 6 months recovery period of ex-
posed males with nulliparous females did not show these effects. General toxicity was
not described in this study.
     Kugel et al. (1990) exposed virgin female rats (Sprague Dawley rats) by inhalation
for 8 h per day during 4 days (one oestrus cycle) to 30% N2O (540 g/m3). All
N2O-exposed rats exhibited disrupted cycles following the first day of exposure; 11 out
of 12 animals went into constant proestrus for up to 3 weeks (Kug90). Control rats
cycled normally. Following exposure 8 control and 8 N2O-exposed rats were perfused,
brains sectioned, and LHRH (luteinising hormone releasing hormone) cells identified by
immunochemistry. A 33-fold increase in LHRH cells was noted in N2O treated rats. In
addition, 12 N2O-exposed female rats were mated with proven male breeders. Six of the
12 N2O-exposed rats and 12 of 12 control rats gave birth. No differences were noted in
litter size or weight. General toxicity was not described in this study.
     Holson et al. (1995) exposed adult male or female rats (Crl:COBS CD(SD) BR out-
bred albino) to trace concentrations of N2O (0, 0.1, 0.5 or 1.0% in air; 0, 1.8, 9 or 18
Nitrous oxide                                                                             16
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<pre>g/m3) for 6h/day either throughout gestation (females) of for 9 weeks (males) (Hol95).
They studied effects of N2O on male fertility by mating treated males with untreated fe-
males by examining uterine contents. There was no evidence for a substantial decline in
fertility by mating exposed males. Litter size was not significantly reduced after mater-
nal exposure, although there was a small dose-related trend for increased resorptions and
decreased live births with increasing paternal N2O exposure. Maternal and offspring
weights were normal from conception through adulthood. The offspring of the treated
adults was subjected to an extensive behavioural battery of tests.
Developmental toxicity
Fink et al. (1967) and Shepard et al. (1968) found defects of vertebral and rib ossifica-
tion in 100% of the rat foetuses from dams (Sprague Dawley) exposed to a mixture of
45-50 % N2O (810- 900 g/m3), 21-25 % oxygen, and nitrogen for 2 to 6 days beginning
on day 8 of gestation (Fin67, She68). A dose response relationship was observed bet-
ween an increased proportion of foetal resorptions and foetal weight decrement with an
increase in the number of days of exposure. Foetal length of the exposed group was sig-
nificantly reduced. The sex ratio (number of males compared to the number of females)
was reduced in surviving foetuses of the exposed group. Maternal toxicity was not
described in this study.
     Corbett et al. (1973) studied the effects in rats after inhalatory exposure to low con-
centrations of N2O: (I) 1.5% N2O (~27 g/m3) for 24 h/day from gestation days 8-13, (II)
0.1% N2O (~1.8g/m3) for 24 h/day from gestation days 12-19, (III) 0.1% or 0.01%
(~1.8 or 0.18 g/m3) for 8 h/day from gestation day 10-13, 14-19 or 10-19 (Cob73). The
number of implantations per dam was reduced and the foetal death rate increased in the
dams exposed for 24 h/day to N2O. Furthermore, the foetal death rate was increased in
the dams exposed to 0.01% and 0.1% for 8 h/day during gestation days 10-13, and 0.1%
group for 8 h/day for gestation days 14-19. Maternal toxicity was not described in this
study.
     Bussard et al. (1974) studied exposure to 60% N2O (1080 g/m3) and 0.6% halot-
hane by inhalation for 3 h/day during gestation days 9, 10 or 11 of pregnant hamsters
(Mesocricetus auratus) (Bus74). Exposure on day 11 resulted in an increased number of
resorptions. Mean foetal weight and crown-rump length were decreased when females
were exposed on gestation day 10 or 11.
     Pope et al. (Pop78) studied the effects in pregnant Sprague Dawley rats during ge-
station (up to day 21) after exposure by inhalation to 0, 1, 10 and 50% N2O (0, 18, 180
or 900 g/m3) for 8 h/day. Foetal loss and skeletal and gross anomalies were not affected;
in the 10 and 50% N2O-exposed groups foetal body weight was reduced. Maternal toxi-
city was not described in this study.
Nitrous oxide                                                                                17
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<pre>     Shah et al. (1979) exposed pregnant Golden Syrian hamsters during organogenesis
(gestation days 7, 8, 9, 10 or 11) by inhalation (during 24 h) to different concentrations
of N2O (70-95%; 1260-1620 g/m3) (Sha79). A significant increase in the number of re-
sorptions was observed. Developmental effects (cleft palate, limb defects, gut herniation
and foetal oedema) were observed in a small number of foetuses. A dose relationship
was not observed. Maternal toxicity was not described in this study.
     Lane et al. (1980) exposed Sprague Dawley rats for 24 h to 70-75% N2O
(1260-1350 g/m3) on gestation day 9 and found an increased number of resorptions and
malformed foetuses (gastroschisis, micro/anophthalmia, cleft lip, hydrocephaly)
(Lan80). Effects were linked to vitamin B12 deficiency. Maternal toxicity was not
described in this study.
     Vieira et al. (1980) studied the effects in Wistar rats exposed to low concentrations
N2O (0. 025, 0.05, 0,1%; 0.45, 0. 9, 1.8 g/m3) by inhalation during 24 h/day during the
entire gestation period (Vie80). In the 0.1% exposure-group, a significant increase in the
number of resorptions and a significant decrease in the number of live foetuses and in
foetal crown-rump length was observed. Maternal toxicity was not described in this stu-
dy.
     Female mice (Swiss/ICR) were exposed by inhalation for 4 h/day during days 6-15
of gestation to 0, 0.5, 5 or 50% N2O (0, 9, 90 or 900 g/m3) (Maz 82). No adverse deve-
lopmental effects were observed after treatment. Maternal toxicity was not described in
this study.
     Mazze et al. (Maz84) studied the effects of inhalatory exposure to 0, 0.75, 7.2, 25
and 75% N2O (0, 13.5, 130, 450 or 1350 g/m3) during 24 h on gestation day 9 in Spra-
gue Dawley rats. After exposure to 75% N2O, a significant increase in early and late re-
sorptions and consistent developmental effects (e.g. runts, ocular malformations and
limb deformities) were observed. During exposure to 75% N2O, rats appeared drowsy
and their motor co-ordination was impaired, food and water intake was decreased.
     Koëter and Rodier (1986) studied the effects of pre-and postnatal exposure to 75%
N2O (1350 g/m3) in DUB/ICR mice and observed retarded physical development, retar-
ded surface and air righting (pre- and postnatal exposure) and affected total activity
(postnatal exposure) (Koe86).
     Tassinari et al. (1986) studied the effects of 75% N2O (1350 g/m3) by inhalation in
Sprague Dawley rats after exposure during several periods of gestation (24 h/day during
gestation days 11-15 or 16-20 or 8 h/day during gestation days 9-13, 11-15, 14-15 or 15
only) (Tas86). Both 24 hours exposure-periods gave a significant reduction in foetal and
maternal body weight, an effect not observed after the 8 hour exposures. No gross
morphological or skeletal changes were observed.
     Mazze et al. (1986) found developmental effects (increased number of resorptions
per dam, decreased foetal weight and decreased ossification) in the presence of light ma-
Nitrous oxide                                                                              18
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<pre>ternal anaesthesia after exposure of Sprague Dawley rats to 75% N2O (1350 g/m3) for 6
h/day by inhalation when exposed from gestation days 14-16 of gestation (Maz86).
     Mazze et al. (1988) exposed Sprague Dawley rats on day 8 of gestation to 50-75%
N2O (900-1350 g/m3) for 24 h alone or in combination with halothane and folate
(Maz88). Exposure to N2O alone resulted in a significantly increased number of resorp-
tions and in major and minor skeletal abnormalities. Halothane administered in combina-
tion with N2O protected against these effects; folinic acid (5 mg/kg body weight/day
gestation days 5-13) did not.
     Fujinaga et al. (1989) exposed Sprague Dawley rats by inhalation to 60% N2O
(1080 g/m3) to study the susceptible period (gestation days 6, 7, 8, 9, 10, 11 or 12) for
developmental effects (Fuj89). There were no differences among the groups in number of
implantations and live foetuses, mean foetal weight and sex ratio. The number of resorp-
tions was higher in the N2O-treated groups exposed on days 8 and 11 of gestation than in
the corresponding control groups. Skeletal malformations of the ribs and vertebrae were
increased following exposure on day 9 of gestation. On day 8 of exposure the incidence
of right-sided aortic arch and left-sided umbilical artery, abnormalities indicative of alte-
red laterality, were increased. Female rats were mildly sedated during exposure; 15 out
of 140 female rats died during the exposure period. Maternal weight was decreased
among all N2O-exposed female rats.
     Rice (1990) studied behavioural effects in offspring of Swiss mice (Hla:[SW]Br) ex-
posed by inhalation to 0, 5, 15 or 35% N2O (0, 90, 270 or 630 g/m3) for 4 h/day on days
6 through 15 of gestation (Ric90). Exposures did not affect reproduction indices and
survival or physical milestones of development. Body weights showed significant expo-
sure effects that could be isolated to specific exposure groups; however, N2O-exposed
mice tended to weigh more than control animals. On postnatal days (PN) 126 or 127 no
effect on brain weights were observed. Ability to stay on a rotarod was not affected by
prenatal N2O exposure. Prenatal exposure to N2O resulted in hypo-reactivity of the
startle reflex on PN95 for all N2O-exposed groups. Maternal toxicity was not described
in this study.
     Holson et al. (1995) exposed adult male or female rats (Crl:COBS CD(SD) BR out-
bred albino) to trace concentrations of N2O (0, 0.1, 0.5 or 1.0% in air; 0, 1.8, 9 or 18
g/m3) for 6 h/day either throughout gestation (females) of for 9 weeks (males) (Hol95).
Litter size was not significantly reduced after maternal exposure although there was a
small dose-related trend for resorptions to increase and live births to decrease with incre-
asing paternal N2O exposure. Maternal and offspring weights were normal from concep-
tion through adulthood. The offspring of the treated adults were subjected to an
extensive behavioural battery (negative geotaxis, 23-h activity, complex maze, passive
avoidance response, developmental activity, amphetamine challenge, auditory startle,
Nitrous oxide                                                                                 19
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<pre>    barbiturate anaesthesia). There were no significant long-term behavioural alterations in
    offspring exposed to trace levels of N2O via dam or sire.
    Lactation
    No publications were available.
2.4 Conclusion
    In human studies, some effects of N2O were observed on fertility (Kni72, Row92, Ahl96
    ). In other studies in man (Wyr81, And92, Kni75 and Pee 99) no effects of N2O exposu-
    re were observed. The committee is of the opinion that the studies in man were insuffici-
    ent for classification because confounding factors such as mixed gas exposure to
    anaesthetic, other exposures (e.g.. amalgam), differences in age between the control and
    the exposed groups and stress may have played a role.
          In inhalatory studies in rats the following effects were observed: decreased testis
    weight and injury of the seminiferous tubules (20% N2O, Kri76), reduced litter size and
    smaller offspring in females mated with exposed males (0.5% N2O Vie83), disrupted
    cycles after exposure of females, increase in LHRH cells in the brain and decreased fer-
    tility (30% N2O, Kug90). However, in these studies the general toxicity was not descri-
    bed. In other studies in mice (concentrations up to 80%) and rats (concentrations up to
    80%) no effects on fertility were observed.
          Therefore, based on the animal studies the committee recommends to classify N2O in
    category 3 (‘substances which cause concern for human fertility’) and to label the com-
    pound with R62 (‘Possible risk of impaired fertility’).
    Epidemiological studies (Ask70, Coh71, Kni72, Ros73, Ame74, Cor74a, Coh75, Pha77,
    Ros78, Eri79, Coh80, Lau81, Hei84, Hem85, Joh87, Row92 and Pee99) gave rise to
    concern about the effect of anaesthetic gas mixtures containing N2O on abortions, foetal
    development, preterm birth and congenital anomalies. However, the committee is of the
    opinion that it is not clear whether the effects described were caused by N2O. Confoun-
    ding factors such as exposure to gas mixtures, other exposures (amalgam, radiation),
    differences in age between the control and the exposed groups and stress may have play-
    ed a role. For that reason the quality of the studies was considered insufficient.
          In several studies in rats, hamsters and mice, effects of N2O exposure on resorp-
    tions, foetal weight and developmental anomalies were observed at concentrations bet-
    ween 0.01 and 95% N2O (1.8-1620 g/m3). In several of the studies maternal toxicity was
    not described. In the other studies (concentrations of up to 75% N2O) effects on body
    Nitrous oxide                                                                             20
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<pre>weight gain and mild sedation were reported. In one study (concentration N2O 60%) ma-
ternal mortality rate of about 10% was described (Fuj89).
    Therefore, in view of the animal studies with respect to the effects on development,
the committee recommends to classify N2O in category 3 (’substances which cause con-
cern for humans owing to possible developmental toxic effects’) and label the compound
with R63 (’Possible risk of harm to the unborn child’).
No data concerning the excretion of N2O in human or animal milk were available.
    Therefore, a lack of appropriate data precludes the assessment of N2O for labelling
for effects during lactation.
Proposed classification for fertility
Category 3, R62.
Proposed classification for developmental toxicity
Category 3, R63.
Proposed labelling for effects during lactation
Lack of appropriate data precludes assessment of N2O for labelling for effects during
lactation.
For the committee,
The Hague, 1 May 2000
dr ASAM van der Burght,                  dr BJ Blaauboer,
scientific secretary                     chairman
Nitrous oxide                                                                            21
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<pre>       References
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Ame74  American Society of Anesthesiologists, Ad Hoc Committee. Occupational disease among operating room
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Ask70  Askrog V, Harvald B. Teratogen effekt inhalationsanaestetika. Nord Medicin 1970; 16:498-500.
Bus74  Bussard DA, Stoelting RK, Peterson R, Ishaq M. Fetal changes in hamsters anesthetized with nitrous
       oxide and halothane. Anesthesiology 1974; 41: 275-278.
Coh71  Cohen EN, Bellville JW, Brown BW. Anesthesia, pregnancy and miscarriage: a study of operating nurses
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Coh75  Cohen EN, Brown BW, Bruce DL et al. A survey of anesthetic Health hazards among dentists. J Am
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Coh80  Cohen EN, Brown BW, Wu ML et al. Occupational disease in dentistry and chronic exposure to trace
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Cor73  Corbett TH, Cornell RG, Endres JL, Millard RI. Effects of low concentrations of nitrous oxide on rat
       pregnancy. Anesthesiology 1973; 39: 299-301.
Cor74a Corbett TH, Cornell RG, Endres JL, Lieding K. Birth defects among children of nurse-anesthetists.
       Anesthesiology 1974; 41:341-344.
Dud81  Dudziak R. Nebenwirkungen von flüchtigen Anästhetika auf das Anästhesiepersonal unter besonderer
       Berücksichtigung des Mutterschutzgesetzes. Anästh Intensiv Med 1981; 22:81-92.
       References                                                                                           22
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<pre>Eri79  Ericson A, Källèn B Survey of infants born in 1973 or 1975 to Swedish women working in operating
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Fer78  Ferstandig LL. Trace Concentrations of anesthetic gasses: a critical review of their disease potential.
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Fin67  Fink BR, Shepard TH , Blandau RJ. Teratogenic activity of nitrous oxide. Nature 1967; 214: 146-148.
Fuj89  Fujinaga M, Baden JM, Mazze RI. Susceptible period of nitrous oxide teratogenicity in Spraque-Dawley
       rats. Teratology 1989; 40: 439-444.
Hei84  Heidam LZ. Spontaneous abortions among dental assistants, factory workers, painters, and gardening
       workers: a follow up study. J Epidemiol Comm Health 1984; 38: 149-155.
Hem85  Hemminki K, Kyronen P, Lindbohm ML. Spontaneous abortions and malformations in the offspring of
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       registered information of outcome. J Epidemiol Community Health 1985; 39: 141-147.
Hol95  Holson RR, Bates HK, LaBorde JB, Hansen DK. Behavioral teratology and dominant lethal evaluation of
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Joh87  Johnson JA, Buchan RM, Reif JS. Effect of waste anesthetic gas and vapor exposure on reproductive out-
       come in veterinary personnel. Am Ind Hyg Ass J 1987; 48: 62-66.
Kni72  Knill-Jones RP, Rodrigues LV, Moir DD, Spence AA. Anaesthetic practice and pregnancy: controlled
       survey of women anaesthetists in the United Kingdom. Lancet 1972; 1:1326-1328.
Kni75  Knill-Jones RP, Newman BJ, Spence AA. Anaesthetic practice and pregnancy: controlled survey of male
       anaesthetists in the United Kingdom. Lancet 1975; 2:807-809.
Koe86  Koëter HBWM, Rodier PM. Behavioral effects in mice exposed to nitrous oxide or halothane; prenatal
       vs. postnatal exposure. Neurobehav Toxicol Teratol 1986; 8:189-194.
Kri76  Kripke BJ, Kelman AD, Shah NK, Balogh K, Handler AH. Testicular reaction to prolonged exposure to
       nitrous oxide. Anesthesiology 1976; 44: 104-113.
Kug90  Kugel G, Letelier C, Zive MA, King JC. Nitrous oxide and infertility. Anesth Prog 1990; 37: 176-180.
Lan81a Land PC, Owen El, Linde HW. Morphologic changes in mouse spermatozoa after exposure to inhalatio-
       nal anesthetics during early spermatogenesis. Anesthesiology 1981; 54: 53-56.
Lan80  Lane GA, Nahrwold ML, Tait AR, Taylor-Busch M, Cohen PJ. Anesthetics as teratogens: nitrous oxide is
       fetotoxic, xenon is not. Science 1980; 210: 899-901.
Lau81  Lauwerys R, Siddons M, Misson CB et al. Anaestetic health hazards among Belgian nurses and physi-
       cians. Int Arch Occup Environ Health 1981: 48:195-203.
Maz82  Mazze RI, Wilson AI, Rice SA, Baden JM. Reproduction and fetal development in mice chronically ex-
       posed to nitrous oxide. Teratology 1982; 26: 11-16.
Maz83  Mazze RI, Rice SA, Wyrobek AJ, Felton JS, Brodsky JB, Baden JM. Germ cell studies in mice after pro-
       longed exposure to nitrous oxide. Toxicol Appl Pharm 1983; 67: 370-375
Maz84  Mazze RI, Wilson AI, Rice Sa, Baden JM. Reproduction and fetal development in rats exposed to nitrous
       oxide. Teratology 1984; 30: 259-265.
Maz86  Mazze RI, Fujinaga M, Rice S, Harris SB, Baden JM. Reproductive and teratogenic effects of nitrous
       oxide, halothane, isoflurane, and endoflurane in Sprague-Dawley rats. Anesthiology 1986; 64:339-344.
       References                                                                                                23
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<pre>Maz88 Mazze RI, Fujinaga M, Baden JM. Halothane prevents nitrous oxide teratogenicity inSprague-Dawley
      Rats: Folinic acid does not. Teratology 1988; 38: 121-127.
Pee99 Peelen S, Roeleveld N, Heederik D, Kromhout H, de Kort W. Reproductie-toxische effecten bij zieken-
      huispersoneel. Ministerie van Sociale Zaken en Werkgelegenheid 1999.
Pha77 Pharoah POD, Alberman E, Doyle P. Outcome of pregnancy among women in anestetic practice. Lancet
      1977; 1:34-36.
Pop78 PopeWDB, Halsey MJ, Phil D, Lansdown BG, Simmonds A, Bateman PE. Fetotoxicity in rats following
      chronic exposure to halothane, nitrous oxide, or methoxyflurane. Anesthesiology 1978; 48: 11-16.
Ric90 Rice SA. Effects of prenatal N2O exposure on startle reflex reactivity. Teratology 1990; 42: 373-381.
Ros73 Rosenberg P, Kirves A. Miscarriages among operating theatre staff. Acta Anaeast Scand 1973; 53: 37-42.
Ros78 Rosenberg PH, Väntinnen H. Occupational hazards to reproduction and health in anaestetists and paedia-
      tricians. Acta Anaesthesiol Scand 1978; 22:202-207.
Row92 Rowland AS, Day-Baird D. Reduced fertility among women employed as dental assistants exposed to
      high levels of nitrous oxide. New Eng J Med 1992; 327: 993-997.
Sha79 Shah RM, Burdett DN, Donaldson D. The effects of nitrous oxide on the developing hamster embryos.
      Can J Physiol Pharmacol 1979; 57: 1229-1232.
She68 Shepard TH, Fink BR. Teratogenic activity of nitrous oxide in rats. Toxic Anesth Proc Res Symp 1967.
      1968: 308-323.
Tas86 Tassinari MS, Mullenix PJ, Moore PA. The effects of nitrous oxide after exposure during middle and la-
      te gestation. Toxicol Ind Health 1986; 2: 261-271.
Tox95 Niesink RJM, de Vries J, Hollinger MA, eds. Toxicology, Principles and Applications, Boca Raton: CRC
      Press, 1995:385
Ves78 Vessey MP. Epidemiological studies of the occupational hazards of anaesthesia- a review. Anaesth 1978;
      33:430-438.
Vie80 Vieira E, Cleaton-Jones P, Austin JC, Moyes DG, Shaw R. Effects of low concentrations of nitrous oxide
      on rat fetuses. Anesth Analg 1980; 59: 175-177.
Vie83 Vieira E, Cleaton-Jones P, Moyes D. Effects of intermittent 0.5% nitrous oxide/air (v/v) on the fertility of
      male rats and the post-natal growth of their offspring. Anaesthesia 1983; 38: 319-323.
Wal75 Walts LF, Forsythe AB, Moore JG. Critique: Occupational disease among operating room personnel.
      Anesthesiology 1975; 42:608-611.
Wyr81 Wyrobek AJ, Brodsky J, Gordon L, Moore DH, Watchmaker G, Cohen EN. Sperm studied in anesthesio-
      logists. Anesthesiology 1981; 55: 527-532.
      Literature consulted but not referred to in the text of the report
Ald86 Aldrige LM, Tunstall ME. Nitrous oxide and the fetus: a review and the results of a retrospective study
      of 175 cases of anesthesia for insertion of shirodkar suture. Br J Anaesth 1986; 58: 1348-1356.
Bai92 Baird PA. Occupational exposure to nitrous oxide-not a laughing matter. N Eng J Med 1992; 327:
      1026-1027.
      References                                                                                                   24
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<pre>Bro83  Brodsky JB. Anesthesia and surgery during early pregnancy and fetal outcome. Clin Obst Gyn 1983;
       26,2:449-457.
Bro84  Brodsky JB, Baden JM, Serra M, Kundomal Y. Nitrous oxide inactivates methionine synthetase activity
       in rat testis. Anesthesiology 1984; 61: 66-68.
Bro93  Brodsky JB. Nitrous oxide and fertility. N Eng J Med 1993; 328: 284-285
Buc87  Buckley DN, Brodsky JB. Nitrous oxide and male fertility. Reprod Toxicol 1987; 1: 93-97.
Bur85  Buring JE, Hennekens CH, Mayrent SL, Rosner B, Greenberg ER, Colton T. Health experiences of ope-
       rating room personnel. Anesthesiology 1985; 62: 325-330.
Bus76  Bussard DA. Congenital anomalies and inhalation anesthetics. JADA 1976; 93: 606-609.
Coa79a Coate WB, Kapp RW, Ulland BM, Lewis TR. Toxicity of low concentration long-term exposure to an
       airborne mixture of nitrous oxide and halothane. J Environ Pathol Toxicol 1979; 2: 209-231.
Coa79b Coate WB, Kapp RW, Lewis TR. Chronic exposure to low concentrations of halothane-nitrous oxide: re-
       productive and cytogenetic effects in the rat. Anesthesiology 1979; 50: 310-318.
Cor74b Corbett TH. Inhalation anesthesia: an occupational hazard. Hospital Practice 1974; 9: 81-88.
Don95  Donaldson D, Meechan JG. The hazards of chronic exposure to nitrous oxide: an update. Br Dent J 1995;
       178: 95-100.
Ebi94  Ebi KL, Rice SA. Reproductive and developmental toxicity of anesthetics in humans. Anesth Toxicity
       1994; 175-198.
Fri88  Friedman JM. Teratogen update: anesthetic agents. Teratology 1988; 37: 69-77.
Fri96  Friedler G. Paternal exposures: impact on reproductive and developmental outcome. An overview. Phar-
       macology Biochemistry and Behavior 1996; 55: 691-700.
Fuj94  Fujinaga M, Baden JM. Methionine prevents nitrous oxide-induced teratogenicity in rat embryos grown
       in culture. Anesthesiology 1994: 81: 184-189.
Gre68  Green CD. Strain sensitivity of rats to nitrous oxide. Anesth Analg 1968; 47: 509-514.
Har81  Hardin BD, Bond GP, Sikov MR, Andrew FD, Beliles RP, Niemeier RW. Testing ofselected workplace
       chemicals for teratogenic potential. Scand J Work Environ Health 1981; 7: 66-75.
Hem85  Hemminki K, Vineis P. Extrapolation of the evidence on teratogenicity of chemicals between humans
       and experimental animals: chemicals other than drugs. Teratogenesis, Carcinog Mutagen 1985;
       5:251-358.
Inf85  Infante PF, Tsongas TA. Anesthetic gases and pregnancy: a review of evidence for an occupational ha-
       zard. Occupational Hazards and Reproduction 1985: 287-294.
Jon98  Jones HE, Balster RL. Inhalant abuse in pregnancy. Obstet Gynecol Clin North Am 1998; 25: 153-167.
Kee86  Keeling PA, Rocke DA, Nunn JF, Monk SJ, Lumb MJ, Halsey MJ. Folinic acid protection against nitrous
       oxide teratogenicity in the rat. Br J Anaesth 1986; 58: 528-534.
Lan79  Land PC, Owen EL, Linde HW. Mouse sperm morphology following exposure to anesthetics during early
       spermatogenesis. Anesthesiology 1979; 51: S259.
Lan80  Land PC, Owen EL, Murphy NL. Nitrous oxide does not alter spermatogenesis in the mouse. Anesthesio-
       logy 1980; 53: S255.
       References                                                                                            25
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<pre>Lan81b Lane GA, DuBoulay PM, Tait AR, Taylor-Busch M, Cohen PJ. Nitrous oxide is teratogenic: halothane is
       not. Anesthesiology 1981; 55:A252.
Mar97  Marx T. Belastung des Arbeitsplatzes mit volatilen Anasthetika und Lachgas. Anasthesiol Intensivmed
       Notfallmed Schmerzther 1997; 32: 532-540.
Maz89  Mazze RI, Källén B. Reproductive outcome after anesthesia and operation during pregnancy: a registry
       study of 5405 cases. Am J Obstet Gynecol 1989; 161: 1178-1185.
Ram80  Ramazzotto L. Carlin R, Warchalowski G. Effects of chronic exposure to nitrous oxide on gestation in the
       rat. Fed Proc Am Soc Exp Biol 1980; 39: 506.
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Rod86  Rodier PM, Aschner M, Lewis LS, Koëter HBWM. Cell proliferation in developing brain after brief ex-
       posure to nitrous oxide or halothane. Anesthesiology 1986; 64: 680-687.
Tra94  Tran N, Elias J, Rosenberg T, Wylie D, Gaborieau D, Yassi A. Evaluation of waste anesthetic gases, mo-
       nitoring strategies, and correlations between nitrous oxide levels and health symptoms. Am Ind Hyg As-
       soc J 55; 1994: 36-41.
Vil90  Vilar C. Link to fertility problems puts N2O under scrutiny. Dentistry 1990; 10: 13-15.
Web80  Webman MS. The effects of intermittent chronic exposure of nitrous oxide on rat fertility and pregnancy.
       Pediatr Dent 1980; 2: 208-216.
Wyn93a Wynn RL. Nitrous oxide and fertility, part I. Gen Dent 1993; 41: 122-123.
Wyn93b Wynn RL. Nitrous oxide and fertility, part II. Gen Dent 1993; 41: 212-214.
Yag91  Yagiela JA. Health hazards and nitrous oxide: a time for reappraisal. Anesth Prog 1991; 38: 1-11.
       References                                                                                                 26
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<pre>A The committee
B Comments on the public draft
C Directive (93/21/EEG) of the European Community
D Fertility and developmental toxicity studies
E Abbreviations and conversion factors
  Annexes
                                                  27
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<pre>Annex A
      The committee
         BJ Blaauboer, chairman
         Toxicologist; Research Institute of Toxicology, Utrecht
         JN van den Anker
         Professor of pediatrics and Neonatology; Erasmus University, Rotterdam
         AM Bongers, advisor
         Ministry of Social Affairs and Employment, The Hague
         HFP Joosten
         Toxicologist; NV Organon, Department of Toxicology and Drug Disposition, Oss
         D Lindhout
         Professor of Clinical Genetics/Teratology; Erasmus University, Rotterdam
         JHJ Copius Peereboom-Stegeman
         Toxicologist; Catholic University Nijmegen, Nijmegen
         AH Piersma
         Reproductive toxicologist; National Institute of Public Health and the Environment,
         Bilthoven
         A Stijkel
         Toxicologist; Environmental Awareness Foundation, ‘s-Graveland
         PJJM Weterings
         Toxicologist; Weterings Consultancy BV, Rosmalen
         ASAM van der Burght, scientific secretary
         Health Council of the Netherlands, The Hague
      The committee                                                                          28
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<pre>The first draft of the present document was prepared by IDH Waalkens-Berendsen, from
the TNO Nutrition and Food Research Institute in Zeist, by contract with the Ministry of
Social Affairs and Employment.
Secretarial assistance: E Vandenbussche-Parméus.
Lay-out: J van Kan.
The committee                                                                            29
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<pre>Annex B
      Comments on the public draft
      A draft of the present report was released in 1999 for public review. No persons or orga-
      nisations have commented on the draft report.
      Comments on the public draft                                                              30
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<pre>Annex C
      Directive (93/21/EEC) of the European
      Community
      4.2.3         Substances toxic to reproduction
      4.2.3.1       For the purposes of classification and labelling and having regard to the present
                    state of knowledge, such substances are divided into 3 categories:
      Category 1:
      Substances known to impair fertility in humans
      There is sufficient evidence to establish a causal relationship between human exposure to the substance
      and impaired fertility.
      Substances known to cause developmental toxicity in humans
      There is sufficient evidence to establish a causal relationship between human exposure to the substance
      and subsequent developmental toxic effects in the progeny.
      Category 2:
      Substances which should be regarded as if they impair fertility in humans:
      Directive (93/21/EEC) of the European Community                                                         31
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<pre>There is sufficient evidence to provide a strong presumption that human exposure to the substance may
result in impaired fertility on the basis of:
     Clear evidence in animal studies of impaired fertility in the absence of toxic effects, or, evidence of
     impaired fertility occurring at around the same dose levels as other toxic effects but which is not a
     secondary non-specific consequence of the other toxic effects.
     Other relevant information.
Substances which should be regarded if they cause developmental toxicity to humans:
There is sufficient evidence to provide a strong presumption that human exposure to the substance may
result in developmental toxicity, generally on the basis of:
     Clear resuts in appropriate animal studies where effects have been observed in the absence of signs
     of marked maternal toxicity, or at around the same dose levels as other toxic effects but which are
     not a secondary non-specific consequence of the other toxic effects.
     Other relevant information.
Category 3:
Substances which cause concern for human fertility:
Generally on the basis of:
     Results in appropriate animal studies which provide sufficient evidence to cause a strong suspicion
     of impaired fertility in the absence of toxic effects, or evidence of impaired fertility occurring at
     around the same dose levels as other toxic effects, but which is not a secondary non-specific conse-
     quence of the other toxic effects, but where the evidence is insufficient to place the substance in Ca-
     tegory 2.
     Other relevant information.
Substances which cause concern for humans owing to possible developmental toxic effects:
Generally on the basis of:
     Results in appropriate animal studies which provide sufficient evidence to cause a strong suspicion
     of developmental toxicity in the absence of signs of marked maternal toxicity, or at around the same
     dose levels as other toxic effects but which are not a secondary non-specific consequence of the
     other toxic effects, but where the evidence is insufficient to place the substance in Category 2.
     Other relevant information.
Directive (93/21/EEC) of the European Community                                                              32
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<pre>4.2.3.2       The following symbols and specific risk phrases apply:
Category 1:
For substances that impair fertility in humans:
T; R60: May impair fertility
For substances that cause developmental toxicity:
T; R61: May cause harm to the unborn child
Category 2:
For substances that should be regarded as if they impair fertility in humans:
T; R60: May impair fertility
For substances that should be regarded as if they cause developmental toxicity in humans:
T; R61: May cause harm to the unborn child.
Category 3:
For substances which cause concern for human fertility:
Xn; R62: Possible risk of impaired fertility
For substances which cause concern for humans owing to possible developmental toxic effects:
Xn; R63: Possible risk of harm to the unborn child.
4.2.3.3       Comments regarding the categorisation of substances toxic to reproduction
Reproductive toxicity includes impairment of male and female reproductive functions or capacity and the
induction of non-inheritable harmful effects on the progeny. This may be classified under two main head-
ings of 1) Effects on male or female fertility, 2) Developmental toxicity.
1    Effects on male or female fertility, includes adverse effects on libido, sexual behaviour, any aspect
     of spermatogenesis or oogenesis, or on hormonal activity or physiological response which would in-
Directive (93/21/EEC) of the European Community                                                            33
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<pre>      terfere with the capacity to fertilise, fertilisation itself or the development of the fertilised ovum up
      to and including implantation.
2     Developmental toxicity, is taken in its widest sense to include any effect interfering with normal de-
      velopment, both before and after birth. It includes effects induced or manifested prenatally as well
      as those manifested postnatally. This includes embrytoxic/fetotoxic effects such as reduced body
      weight, growth and developmental retardation, organ toxicity, death, abortion, structural defects (te-
      ratogenic effects), functional defects, peri-postnatal defects, and impaired postnatal mental or physi-
      cal development up to and including normal pubertal development.
Classification of chemicals as toxic to reproduction is intended to be used for chemicals which have an
intrinsic or specific property to produce such toxic effects. Chemicals should not be classified as toxic to
reproduction where such effects are solely produced as a non-specific secondary consequence of other
toxic effects. Chemicals of most concern are those which are toxic to reproduction at exposure levels
which do not produce other signs of toxicity.
The placing of a compound in Category 1 for effects on Fertility and/or Developmental Toxicity is done
on the basis of epidemiological data. Placing into Categories 2 or 3 is done primarily on the basis of ani-
mal data. Data from in vitro studies, or studies on avian eggs, are regarded as ‘supportive evidence’ and
would only exceptionally lead to classification in the absence of in vivo data.
In common with most other types of toxic effect, substances demonstrating reproductive toxicity will be
expected to have a threshold below which adverse effects would not be demonstrated. Even when clear
effects have been demonstrated in animal studies the relevance for humans may be doubtful because of
the doses administrated, for example, where effects have been demonstrated only at high doses, or where
marked toxicokinetic differences exist, or the route of administration is inappropriate. For these or simi-
lar reasons it may be that classification in Category 3, or even no classification, will be warranted.
Annex V of the Directive specifies a limit test in the case of substances of low toxicity. If a dose level of
at least 1000 mg/kg orally produces no evidence of effects toxic to reproduction, studies at other dose le-
vels may not be considered necessary. If data are available from studies carried out with doses higher
than the above limit dose, this data must be evaluated together with other relevant data. Under normal
circumstances it is considered that effects seen only at doses in excess of the limit dose would not neces-
sarily lead to classification as Toxic to Reproduction.
Effects on fertility
For the classification of a substance into Category 2 for impaired fertility, there should normally be clear
evidence in one animal species, with supporting evidence on mechanism of action or site of action, or
chemical relationship to other known antifertility agents or other information from humans which would
Directive (93/21/EEC) of the European Community                                                                 34
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<pre>lead to the conclusion that effects would be likely to be seen in humans. Where there are studies in only
one species without other relevant supporting evidence then classification in Category 3 may be appropri-
ate.
Since impaired fertility may occur as a non-specific accompaniment to severe generalised toxicity or
where there is severe inanition, classification into Category 2 should only be made where there is eviden-
ce that there is some degree of specificity of toxicity for the reproductive system. If it was demonstrated
that impaired fertility in animal studies was due to failure to mate, then for classification into Category 2,
it would normally be necessary to have evidence on the mechanism of action in order to interpret whether
any adverse effect such as alteration in pattern of hormonal release would be likely to occur in humans.
Developmental toxicity
For classification into Category 2 there should be clear evidence of adverse effects in well conducted stu-
dies in one or more species. Since adverse effects in pregnancy or postnatally may result as a secondary
consequence of maternal toxicity, reduced food or water intake, maternal stress, lack of maternal care,
specific dietary deficiencies, poor animal husbandry, intercurrent infections, and so on, it is important
that the effects observed should occur in well conducted studies and at dose levels which are not associa-
ted with marked maternal toxicity. The route of exposure is also important. In particular, the injection of
irritant material intraperitoneally may result in local damage to the uterus and its contents, and the re-
sults of such studies must be interpreted with caution and on their own would not normally lead to classi-
fication.
Classification into Category 3 is based on similar criteria as for Category 2 but may be used where the
experimental design has deficiencies which make the conclusions less convincing, or where the possibili-
ty that the effects may have been due to non-specific influences such as generalised toxicity cannot be ex-
cluded.
In general, classification in category 3 or no category would be assigned on an ad hoc basis where the
only effects recorded are small changes in the incidences of spontaneous defects, small changes in the
proportions of common variants such as are observed in skeletal examinations, or small differences in
postnatal developmental assessments.
Effects during Lactation
Substances which are classified as toxic to reproduction and which also cause concern due to their effects
on lactation should in addition be labelled with R64 (see criteria in section 3.2.8).
Directive (93/21/EEC) of the European Community                                                                35
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<pre>For the purpose of classification, toxic effects on offspring resulting only from exposure via the breast
milk, or toxic effects resulting from direct exposure of children will not be regarded as ‘Toxic to Repro-
duction’, unless such effects result in impaired development of the offspring.
Substances which are not classified as toxic to reproduction but which cause concern due to toxicity
when transferred to the baby during the period of lactation should be labelled with R64 (see criteria in
section 3.2.8). This R-phrase may also be appropriate for substances which affect the quantity or quality
of the milk.
R64 would normally be assigned on the basis of:
a    toxicokinetic studies that would indicate the likelihood that the substance would be present in po-
     tentially toxic levels in breast milk, and/or
b    on the basis of results of one or two generation studies in animals which indicate the presence of ad-
     verse effects on the offspring due to transfer in the milk, and/or
c    on the basis of evidence in humans indicating a risk to babies during the lactational period.
     Substances which are known to accumulate in the body and which subsequently may be released in-
     to milk during lactation may be labelled with R33 and R64.
Directive (93/21/EEC) of the European Community                                                             36
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<pre>Annex D
      Fertility and developmental toxicity
      studies
      Fertility and developmental toxicity studies 37
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<pre>Table 1.1 Fertility studies with NO2 in man.
authors   exposure                study type/       study/ comparison population          investigated effects remarks
                                  data collection                                         and results
Wyr81     Mixture: waste anaes- Cohort study;       46 anaesthesiologist.                 No effect on sperm   Mixed exposure
          thetics                 semen sampling 26 beginning residents in anaesthesi-    concentration and    Controlled for
          Exposure for at least                     ology                                 sperm abnormali-     smoking, medical
          one year                                                                        ties                 history, sauna use
And92     Mixture: anaesthesia Cohort study; se- 17 patients;                             No effect sperm      Mixed exposure
          during surgery dura- men sampling         preoperative sample served as control volume, count mo- No covariates con-
          tion about 55 minutes                                                           tility and sperm     sidered
                                                                                          abnormalities
Kni72     Mixture: waste anaes- Retrospective       893 pregnancies in 563 female         Increased infertili- Mixed exposure
          thetics                 survey UK; pos-   anaesthesists;                        ty due to unknown No covariates con-
          Employment in           tal questionnaire 1,835 pregnancies in 828 female phy- causes                sidered
          anaesthesia during 1st                    sicians
          or 2nd trimester of
          pregnancy
Kni75     Mixture: waste anaes-   Retrospective     Married male anaesthesists reporting After paternal ex- Mixed exposure
          thetics                 survey UK;        at least 1 pregnancy for their wives; posure: involuntary No covariates con-
          Employment in           postal question-  5,891 pregnancies with only paternal infertility negative sidered
          anaesthesia during 1st  naire             exposure and 166 pregnancies with
          or 2nd trimester                          only maternal exposure.
                                                    Male doctors registered in 1972;
                                                    7,296 pregnancies without paternal
                                                    or maternal exposure
Row92     N2O at least 5 hours    Retrospective     Female dental assistants pregnant     Mean time to con-    Controlled for age,
          scavenged or unsca-     survey USA        within previous 4 years; 127 exposed  ception was increa-  race, family inco-
          venged exposure in      1987/1988         to scavenged N2O and 63 exposed to    sed in the           me, exercise, li-
          dentistry               telephone inter-  unscavenged N2O.                      unscavenged group    festyle habits and
                                  views             215 unexposed female dental assis-                         other occupational
                                                    tants who were pregnant within pre-                        exposure.
                                                    vious 4 years.
             Fertility and developmental toxicity studies                                                                        38
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<pre>Table 1.2 Fertility studies with NO2 in man.
authors   exposure           study type/         study/                    investigated effects and results   remarks
                             data collection     comparison population
Ahl96     N2O                Retrospective sur- 972 midwives               47% became pregnant in the first   Controlled for re-
          Employment         vey Sweden;                                   cycle in the day-time group com-   productive histo-
          midwives who       postal questionnai-                           pared to 49.0%, 36.1%, and         ry, age
          worked only        re                                            31.9% in the permanent night,      employment, oc-
          day-or night                                                     two-shift, and three-shift groups, cupational expo-
          time, in two-                                                    respectively.                      sures, health
          shift or three-                                                  A decreased fecundability was      problems, medi-
          shift rotas                                                      observed for shift work and >30    cation and li-
                                                                           N2O exposed deliveries per         festyle habits
                                                                           month
Pee99     Mixture: waste     Retrospective sur-  427 pregnant females (age No effect on time to pregnancy     Mixed exposure
          anaesthetics       vey                 22-37 years) employed in                                     Controlled for
          N2O concentra-     The Netherlands     anaesthesia.                                                 age, education,
          tion: maximum      1990-1997;          1,010 pregnant females                                       menstrual cycle,
          318 mg/m3          postal questionnai- (age 22-37 years) nurses                                     life style and cir-
          Employment in      re                  employed in department of                                    cumstances du-
          anaesthesia                            orthopaedics, gynaecology                                    ring work.
                                                 or surgery
             Fertility and developmental toxicity studies                                                                      39
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<pre>Table 2.1 Developmental studies in man.
authors exposure              study type/          study/ comparison population     investigated effects and     remarks
                              data collection                                       results
Ask70    Mixture: waste       Retrospective survey Pregnancies conceived during     Spontaneous abortions po- Mixed exposure
         anaesthetics         Denmark;             employment: in 229 nurse         sitive in wives of male      No covariates
                              postal questionnaire anaesthetists, 26 female anaes-  anaesthesiologists           considered
                                                   thesiologists, 137 wives of male Premature delivery posi-
                                                   anaesthesiologists/ pregnancies  tive in wives of male
                                                   conceived before employment:     anaesthesiologists
                                                   in 85 nurse anaesthetists, 8 fe- Sex ratio: increased num-
                                                   male anaesthesiologists and 119  ber of females in female
                                                   in wives of male anaesthesiolo-  anaesthesiologists
                                                   gists
Coh71    Mixture: waste       Retrospective survey 1. 67 OT nurses/ 921 general     Spontaneous abortions po- Mixed exposure
         anaesthetics         USA                  duty nurses                      sitive                       No covariates
         Any exposure in      1. 1966-1970;        2. 50 female anaesthesiologists; Congenital abnormalities considered
         operating room       personal interview   81 female physicians             negative
         (OT)                 2. 1965-1970;
                              postal questionnaire
Kni72    Mixture: waste       Retrospective survey 893 pregnancies in 563 female    Spontaneous abortions,       Mixed exposure
         anaesthetics         UK;                  anaesthesists;                   stillbirth and sex ratio ne- No covariates
         Employment in        postal questionnaire 1,835 pregnancies in 828 female  gative                       considered
         anaesthesia du-                           physicians                       Congenital abnormalities
         ring 1st or 2nd tri-                                                       positive
         mester of
         pregnancy
Ros73    Mixture: waste       Retrospective survey 257 pregnancies in 58 anaesthe-  Spontaneous abortions po- Mixed exposure
         anaesthetics         Finland 1965-1972;   sia and 124 scrub nurses;        sitive                       No covariates
         Employment in        postal questionnaire 150 pregnancies in 75 casualty   Low birth weight and con- considered
         anaesthesia or                            department and 43 intensive      genital abnormalities ne-
         scrub nurse                               care unit nurses                 gative
            Fertility and developmental toxicity studies                                                                       40
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<pre>Table 2.2 Developmental studies in man.
authors   exposure               study type/        study/ comparison population     investigated effects and remarks
                                 data collection                                     results
Ame74     Mixture: waste anaes-  Retrospective sur- 18,568 pregnancies in 29,810     Spontaneous abortions Mixed exposure
          thetics                vey USA            exposed OT personnel from 4      positive in exposed fe- Maternal age
          Females exposure du-   1972-1974;         societies.                       males                    and smoking
          ring 1st trimester of  postal question-   5,620 pregnancies in 10,420      Congenital abnormali-
          pregnancy and work in  naire              unexposed physicians, nurses     ties positive in exposed
          OT during previous ca-                    and their wives from 2 societies females and wives of ex-
          lendar year. Males,                       plus unexposed individuals and   posed males
          work in OT during ye-                     their wives from study popula-
          ar prior ro pregnancy                     tion
Cor74a    Mixture: waste anaes-  Retrospective sur- 434 births to 268 nurses who     Congenital abnormali-    Mixed exposure
          thetics                vey USA;           practised anaesthesia during     ties positive            Age
          OT employment during   postal question-   pregnancy.
          pregnancy              naire and telepho- 261 births to nurses who did
                                 ne interview       not practice anaesthesia during
                                                    pregnancy and published inci-
                                                    dence rates
Coh75     Mixture: waste anaes-  Retrospective sur- 1,668 male dentists and oral     Spontaneous abortion     Mixed exposure
          thetics                vey USA;           surgeons exposed to anaesthe-    positive                 age and smoking
          Exposure to anaesthe-  postal question-   tics.                            Congenital abnormali-    habit of wife at
          tics at least 3 hours  naire              1,560 male dentists and oral     ties negative            time of pregnan-
          weekly during calendar                    surgeons not exposed to anaes-                            cy
          year preceding wife’s                     thetics.
          pregnancy
Kni75     Mixture: waste anaes-  Retrospective sur- Married male anaesthesists re-   Spontaneous abortion     Mixed exposure
          thetics                vey UK;            porting at least 1 pregnancy for positive after maternal No covariates
                                 postal question-   their wives; 5,891 pregnancies   exposure; negative after considered
                                 naire              with only paternal exposure      paternal exposure
                                                    and 166 pregnancies with only    Congenital malforma-
                                                    maternal exposure.               tions positive after pa-
                                                    Male doctors registered in       ternal exposure and
                                                    1972; 7,296 pregnancies wit-     negative after maternal
                                                    hout paternal or maternal expo-  exposure
                                                    sure
             Fertility and developmental toxicity studies                                                                    41
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<pre>Table 2.3 Developmental studies in man.
authors   exposure              study type/      study/ comparison population     investigated effects and re- remarks
                                data collection                                   sults
Kni75     Mixture: waste        Case-control     1) 4,074 pregnancies with pater- 1) Spontaneous abortions     Mixed exposure
          anaesthetics          study UK;        nal exposure;                    and still birth negative;    Matched on ma-
          OT employment du-     postal question- 4,074 pregnancies without pater- Congenital abnormalities     ternal smoking
          ring 1st trimester of naire            nal exposure.                    positive                     habits, birth or-
          pregnancy                              2a) 435 pregnancies with mater-  2 a and b) Spontaneous       der, maternal
                                                 nal exposure;                    abortions positive and       and paternal age
                                                 435 pregnancies without pater-   stillbirth negative
                                                 nal or maternal exposure.        2a) Congenital malforma-
                                                 2b) 368 pregnancies with mater-  tions positive
                                                 nal exposure;                    2b) Congenital malforma-
                                                 772 pregnancies without pater-   tions negative
                                                 nal or maternal exposure.
Pha77     Mixture: waste        Retrospective    670 pregnancies while employed   Spontaneous abortion, low    Mixed exposure
          anaesthetics          survey UK;       as anaesthesiologist;            birth weight, and congeni-   Maternal age,
          Appointment as        postal question- 1,977 pregnancies while women    tal abnormalities (cardio-   smoking habits
          anaesthesiologist at  naire            had no medical appointments      vascular) positive           and parity
          time of conception                                                      Stillbirth increased
Ros78     Mixture: waste        Retrospective    248 pregnancies in anaesthesio-  Spontaneous abortions ne- Mixed exposure
          anaesthetics          survey Finland   logists families/                gative                       Smoking habits
          Member Finnish So-    1961-1976/       266 pregnancies in pediatricians Low birth weight positive
          ciety of Anaesthesio- postal question- families (no OT exposure)        Congenital abnormalities
          logists               naire                                             (musculoskeletal) increa-
                                                                                  sed
Eri79     Mixture: waste        Cohort Sweden    494 women who worked throug-     Birth weight, perinatal de- Mixed exposure
          anaesthetics          1973 and 1975;   hout pregnancy, 37 women who     ath rate, congenital abnor- Maternal age
          women working in Registry data         worked more than half of their   malities negative            and parity
          OT during pregnan-                     pregnancies and 10 women who     Pregnancy duration in
          cy who gave birth in                   worked less than half of their   weeks decreased
          1973 and 1975                          pregancies;
                                                 19,127 women employed in me-
                                                 dical work who delivered in
                                                 1973 or 1975
             Fertility and developmental toxicity studies                                                                      42
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<pre>Table 2.4 Developmental studies in man.
authors   exposure           study type/       study/                            investigated effects and re-    remarks
                             data collection   comparison population             sults
Coh80     N2O exposure       Retrospective     Wives of 21,634 male dentists     Spontaneous abortion positive   Exposure to in-
          Exposure in year   survey USA;       and 21,202 female chairside as-   in wives of male dentists and   organic mercury
          preceding preg-    postal question-  sistants/                         female chairside assistants     Maternal age
          nancy; light expo- naire             Those not exposed in any years    also when they are exposed to   and smoking his-
          sure was 1- 8                        before conception, including ye-  N2O only;                       tory and history
          hours weekly and                     ar of conception                  Congenital abnormalities ne-    of previous
          heavy exposure                                                         gative in wives of male den-    spontaneous
          was more than 8                                                        tists and positive in female    abortion or con-
          hours weekly                                                           chairside assistants also when  genital abnorma-
                                                                                 they are exposed to N2O only.   lities
Lau81     Mixture: waste     Retrospective     Pregnancies in 149 anaesthesio-   Spontaneous abortions, sum Mixed exposure
          anaesthetics       survey            logists and their wives and 240   of all abnormal pregnancies, Maternal smo-
          Exposure anaes-    Belgium/          OT nurses and their wives;        premature birth, stillbirth and king habits
          thetic gasses by   postal question-  pregnancies in 531 occupational   congenital abnormalities ne-
          one or both pa-    naire             physicians, dermatologists, in-   gative.
          rents during or in                   tensive care, and other nurses    Sex ratio increased number of
          the year before                      and their wives                   males
          pregnancy
Hei84     N2O exposure       Historical pro-   352 pregnancies in dental assis- Spontaneous abortions nega-      Controlled for
          Dental assistants  spective study    tants; 255 N2O-exposed and 97 tive                                gravidity, preg-
          in poorly ventila- Denmark; women    not N2O- exposed; control                                         nancy order and
          ted clinics        entire reproduc-  group comparable with respect                                     age.
                             tive life before  to work exposures and move-                                       Amalgam expo-
                             1980              ments                                                             sure
Hem85     Mixture: waste     Case-control Fin- 1) 169 employed nurses who        1) Spontaneous abortion ne-     1 and 2. Mixed
          anaesthetics       land 1973-1979;   had spontaneous abortion/ 469     gative                          exposure
          Postal question-   registry data for employed nurses who gave          2) Congenital abnormalities     Matched on ma-
          naire concerning   outcomes          birth to a healthy infant         negative                        ternal age and
          exposure during                      2) 38 employed nurses who ga-                                     other potential
          1st trimester                        ve birth to an infant with conge-                                 exposures
                                               nital abnormalities/ 99
                                               employed nurses who gave
                                               birth to a healthy infant.
                                               1 and 2: cases excluded from
                                               controls
              Fertility and developmental toxicity studies                                                                       43
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<pre>Table 2.5 Developmental studies in man.
authors   exposure         study type          study/                        investigated effects and results  remarks
                           /data collection    comparison population
Joh87     Mixture: waste Case-control USA;     278 spontaneous abortions     Spontaneous abortion increased
          anaesthetics and postal questionnai- and stillbirths and 98 live   in female veterinarian assistants
          N2O              re (additional      birth with congenital abnor-  and positive in wives of female
                           questionnaire sent  malities that occurred to fe- veterinarians exposed to N2O and
                           to senior female    male veterinarians and        negative in female veterinarians.
                           veterinary assis-   veterinarian assistants and   Spontaneous abortion increased
                           tants)              wives of male veterinarians/  in female veterinarians and vete-
                                               642 normal pregnancies cho-   rinarian assistants exposed to
                                               sen on a stratified random    waste anaesthetics and negative
                                               basis                         in wives of male veterinarians.
                                                                             Congenital abnormalities nega-
                                                                             tive in female veterinarians
Row92     N2O at least 5   Retrospective sur-  Female dental assistants      Spontaneous abortion increased    Controlled for
          hours scavenged  vey                 pregnant within previous 4    in the unscavenged group          age, race, family
          or unscavenged   USA 1987/1988;      years; 127 exposed to sca-                                      income, exerci-
          exposure in      telephone inter-    venged N2O and 63 exposed                                       se, lifestyle ha-
          dentistry        views               to unscavenged N2O;                                             bits and other
                                               215 unexpected female den-                                      occupational ex-
                                               tal assistants who were preg-                                   posure.
                                               nant within previous 4 years.
Pee99     Mixture: waste   Retrospective sur-  427 pregnant females (age     Increased risk for abortion, pre- Mixed exposure
          anaesthetics     vey                 22-37 years) employed in      term birth and congenital abnor-  Controlled for
          N2O concentra-   The Netherlands     anaesthesia;                  malities                          age, education,
          tion: maximum    1990-1997/postal    1,010 pregnant females (age                                     menstrual cycle,
          318 mg/m3        questionnaire       22-37 years) nurses employ-                                     life style and
          Employment in                        ed in department of ortho-                                      circumstances
          anaesthesia                          paedics, gynaecology or                                         during work.
                                               surgery
             Fertility and developmental toxicity studies                                                                      44
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<pre>Table 3.1 Fertility studies in animals with N2O.
authors   species             experimental peri-      dose and route   general toxicity effects on reproductive    remarks
                              od/design                                                 organs/effects on repro-
                                                                                        duction
Kri76     LEW/f Mai rats 1,2,3,4,5,7,10,14,21,        0 and 20% N2O Not described       Decreased testis weight
          4-6 males/ group 28,32,35 days              (inh)                             after 28 days exposure in
                              8 h/day                                                   both exposure groups
                              or continuously for                                       (8/24h).
                              24h                                                       After 14 days injury to
                                                                                        seminiferous tubules in
                                                                                        both exposure groups.
                                                                                        No change in plasma tes-
                                                                                        tosterone level
Lan81a    (C57B1/C3H)F1 4h/day during 5 days 0, 8 or 80%               Not described    No change in abnormal
          mice                Sacrifice 28 days after N2O (inh)                         spermatozoa
          5 males/group       exposure
Maz82     Swiss /ICR mice     4h/day, 5 days/week     0, 0.5, 5 or 50% Not described    No effects on fertility
          18-20 ma-           for 9 weeks followed    N2O (inh)
          les/group           by a 1 week mating
                              period with 2 untrea-
                              ted females/male
Maz83     Swiss Webster       4h/day,                 0, 0.5, 5 or 50% No excitement No differences in testis      The percentage
          mice                5 days/week for 14      N2O (inh)        or general       weight, abnormal shaped    of abnormal
          14-15 males/        weeks                                    anaesthesia      sperm, sperm count or      sperm in the sa-
          group                                                                         histological appearance    line control was
          9 saline controls                                                             of the testis.             much lower
          15 females/ group                                                             No significant difference  than in the air
                                                                                        between the number of      control 2.5 + 0.3
                                                                                        oocytes of the group trea- versus 10.4 +
                                                                                        ted with 50% N2O and       2.3
                                                                                        the control group
             Fertility and developmental toxicity studies                                                                          45
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<pre>Table 3.2 Fertility studies in animals with N2O.
authors species               experimental period/design    dose and route general   effects on reproductive or-        re-
                                                                           toxicity  gans/effects on reproduction       marks
Vie83    Wistar rats          6h/day,                       0 or 0.5% N2O  Not       In the females mated immedia-
         12 males/ group      5 days/week for 30 days im- (inh)            described tely after exposure of the ma-
                              mediately thereafter each ma-                          les: reduced litter size (7 versus
                              le was mated with 3                                    12 pups in the controls) and
                              nulliparous females. Males                             smaller offspring.
                              were mated again after a 6                             No effects after recovery peri-
                              months recovery period                                 od.
Kug90    Sprague Dawley       8h/day during 4 days          0 or 30% N2O   Not       All N2O-exposed females exhi-
         rat 32 females/                                    (inh)          described bited disrupted cycles.
         group;                                                                      33-fold increase in LHRH cells
         10 N2O females                                                              in brains of N2O-exposed fema-
         exposed in proe-                                                            les
         strus                                                                       Decreased fertility
Hol95    Crl:COBS             paternal study 6h/day 5       0, 0.1, 0.5 or No effect No effect on conception rate,
         CD(SD) BR (out-      days/week for 9 weeks         1% N2O         on body   total number of implants/litter,
         bred albino) rat     Males were mated with 2 un- (inh)            weight    live foetuses/litter or resorp-
         10-12 ma-            treated females                                        tions/litter
         les/group
             Fertility and developmental toxicity studies                                                                    46
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<pre>Table 4.1 Developmental toxicity studies in animals with N2O.
authors  species                  experimental   dose and route      maternal toxicity developmental toxicity    remarks
                                  period/design
Fin67,   Sprague Dawley rat       2, 4 or 6 days 0 or 45-50% N2O     Not described     Increased number of re-   Very small
She68    6 control females        exposure be- (inh)                                   sorptions, decreased foe- number
         3, 7, 2 females after 2, ginning on day                                       tal weight with increase  (3 and 2)
         4 or 6 days exposure,    8 of gestation                                       in number of days of ex-  of animals
         respectively                                                                  posure                    after 2 and
                                                                                                                 6 days ex-
                                                                                                                 posure
Cor73    rats (strain not descri- 1. 24 h/day    1. 0 or 1.5% N2O    Not described     1. Decreased number of
         bed)                     GD 8-13        2. 0 or 0.1% N2O                      implantations
         7-12 pregnant fema-      2. 24h/day     3. 0.01 or 0.1% N2O                   Increased foetal death
         les/group                GD 12-19       4. 0.01 or 0.1% N2O                   rate
                                  3. 8h/day      5. 0, 0.01 or 0.1%                    2. Decreased number of
                                   GD 10-13      N2O                                   implantations
                                  4. 8h/day      (inh)                                 Increased foetal death
                                  GD 14-19                                             rate
                                  5. 8h/day                                            3. Increased foetal death
                                  GD 10-19                                             rate
                                                                                       4. Increased foetal death
                                                                                       rate
Bus74    Hamster (Mesocrite-      3h/day during 0 or 60% N2O and     females became    Exposure GD 10: decrea- Mixed ex-
         tus auratus)             GD 9, 10 or 11 0.6% halothane      immobile and      sed foetal weight and     posure
         8-9 pregnant fema-                      (inh)               asleep after      crown-rump length.
         les/group                                                   15-30 min in      Exposure GD 11: Increa-
                                                                     chamber; when     sed number of resorptions
                                                                     removed from      and decreased foetal
                                                                     chamber females   weight and crown-rump
                                                                     became mobile     length.
                                                                     after 5 minutes
Pop78    Sprague Dawley rats      8h/day during 0, 1, 10 or 50%      Maternal toxicity 10 and 50% group decre-
         7-10 pregnant fema-      the gestation N2O (inh)            not described     ased foetal weight
         les/group                period (21
                                  days)
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<pre>Table 4.2 Developmental toxicity studies in animals with N2O.
authors species                experimental      dose and route      maternal toxi-  developmental toxicity             remarks
                               period/design                         city
Sha79    Golden Syrian Ham-    24h during GD 0, 70, 80, 90 or        Not described   Increased (NS) number of
         sters                 7, 8, 9, 10 or 11 95% N2O                             malformations no relation to do-
         5 females/group                         (inh)                               se or day of exposure
                                                                                     GD 7 95%: Increased number of
                                                                                     resorptions
                                                                                     GD 10 90%: Increased number
                                                                                     of resorptions
                                                                                     GD 11 90%: Increased number
                                                                                     of resorptions
Lan80    Sprague Dawley        24h on GD 9       0 and 75% N2O       Not described   Increased number of resorptions
          rats                                   (inh)                               and malformed foetuses
         30- 31 pregnant fema-
         les/group
Vie80    Wistar rats           24h during the    0, 0.025, 0.05 or   Not described   0.1% group increased number of
         12 females/group      entire gestation  0.1% N2O                            resorptions; decreased number
                               period            (inh)                               of live foetuses and foetal
                                                                                     crown-rump length
Maz82 Swiss ICR mice           4h/day GD 6-15 0, 0.5, 5 or 50%       Not described   No adverse developmental ef-
         24-34 females/group                     N2O (inh)                           fects
Maz84 Sprague Dawley rats      1. 24 h on GD 9 1. 0 or 75%           0.75, 7.5 and   Combined data of the 4 experi-
         Groups 1, 2 and 4 ti- 2. 24 h on GD 9 2. 0, 0.75, 7.5 or    25 % no effect  ments showed :
         me-mated females ob-                    75%                 75% group:      7.5-75% group increasing fre-
         tained from breeder,  3. 24 h on GD 9 3. 0 and 75%          rats appeared   quency with increasing concen-
         group 3 was mated in- 4. 24 h on GD 9 4. 0 and 25% and      drowsy, impai-  tration of extra lumbar rib
         house.                                  food deprivation on red motor coor- 75%group: increased number of
         About 26 females/                       GD 9                dination and    early and late resorptions; incre-
         group                                   N2O (inh)           decreased food  ased number of runts, ocular
                                                                     and water in-   malformations, limb deformities
                                                                     take
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<pre>Table 4.3 Developmental toxicity studies in animals with N2O.
authors  species          experimental peri-    dose and route maternal toxicity developmental toxicity         remarks
                          od/design
Koe86    DUB/ICR mice Females GD 14             0 and 75% N2O  After exposure on  no effect on pup body
         (n=5-6/group;    6h/day                (inh)          GD 14 females re-  weight; except for an increa-
         dams or litters) Litters PN 2 exposed                 sumed activity     sed pup weight on PN 2
                          4h/day                               within a few mi-   ear unfolding retarded after
                          Females exposed on                   nutes.             pre- and postnatal exposure
                          GD 14 were allowed                                      air and surface righting were
                          to litter.                                              retarded during test period
                          Physical landmarks:                                     after pre- and postnatal ex-
                           ear opening                                            posure
                           eye opening                                            locomotion was affected af-
                          Behavioural measu-                                      ter pre- and postnatal expo-
                          res:                                                    sure
                           surface righting                                       total activity was affected
                           locomotion                                             postnatal exposure
                           air righting
                           general activity
Tas86    Sprague Dawley 24 h/day GD 11-15       0 or 75% N2O   24h GD 11-15 and  24h GD 11-15 and 16-20: re-    Small
         rats             or 16-20 or 8h/day    (inh)          16-20: reduced    duced foetal weight; no other  number of
         3-7 exposed fe- GD 9-13, 11-15                        maternal body     developmental effects          exposed
         males                                                 weight            8h                             females
                                                               8h: no effect
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<pre>Table 4.4 Developmental toxicity studies in animals with N2O.
authors species             experimental pe- dose and route   maternal toxicity    developmental toxicity             remarks
                            riod/design
Maz86    Sprague Dawley     GD 8-10, 11-13     0 or 75% N2O   Light general        GD 14-16: increased number re-
         rats               or 14-16           (inh)          anaesthesia; decrea- sorptions and decreased foetal
         19-25 females/                                       sed weight gain      weight
         group
Maz88    Sprague Dawley     24 h on GD 8       0 or 50-75%    decreased weight     increased number of resorptions
         rats                                  N2O (inh)      gain                 and increased number of
         20-30 females/                                                            malformations (right-sided aor-
         group                                                                     tic arch) and minor skeletal ano-
                                                                                   malies (vertebral and rib
                                                                                   anomalies)
Fuj89    Sprague Dawley     24h on GD 6, 7, 0 or 60% N2O      Females mildly se-   GD 8: increase in right-sided
         rats               8, 9, 10, 11 or 12 (inh)          dated                aortic arch and left-sided umbi-
         20 females/group                                     15 out of 140 fema-  lical artery
                                                              les died during the  GD 8 and 11: increased number
                                                              exposure             of resorptions
                                                              Maternal weight      GD 9: skeletal malformations
                                                              was decreased        increased
Ric90    Swiss              4h/day GD 6-15 0, 5, 15 or 35%    Not described        No reproductive effects.
         (Hla:[SW]Br)       Females were al- N2O (inh)                             N2O-exposed F1- mice tend to
         mice               lowed to litter                                        weight more than air exposed
         10 litters/group                                                          mice.
         were studied                                                              No effect on brain weight.
                                                                                   No effect on rotarod perform-
                                                                                   ance.
                                                                                   Hyporeactivity in startle reflex
                                                                                   on PN 95 in all N2O-exposed
                                                                                   animals
Hol95    Crl:COBS           males 6h/day 5 0, 0.1, 0.5 or 1% No effect on body     No effect on live births after
         CD(SD) BR out-     days/week for 9 N2O (inh)         weight males and     maternal exposure.
         bred albino rat    weeks                             females              Small (NS) on live births with
         10-12 males and    Females 6h/day                                         increasing paternal exposure.
         females/group      during gestation                                       No effect on long-term behavi-
                            Behavioural me-                                        oural alterations in the offspring
                            asures in                                              after maternal or paternal N2O
                            F1-pups                                                exposure
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<pre>Annex E
      Abbreviations and conversion factors
      Abbreviations used:
      bw          body weight
      d           day
      F           female(s)
      GD          Gestation day
      i.p.        intraperitoneal
      i.v.        intravenous
      M           male(s)
      n           number of animals
      no          number
      ns          not significant
      NOAEL       no adverse effect level
      OECD        Organisation for Economic Cooperation and Development
      OT          Operating room
      PN          postnatal
      w           week
      Conversion factors
      1%          10.000 ppm
      1 ppm       1.8 mg/m3
      Abbreviations and conversion factors                              51
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<pre>Abbreviations and conversion factors 52</pre>

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<br><br>