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

<pre>www.healthcouncil.nl Innovation and the knowledge infrastructure Before we can harvest knowledge in the field of healthcare, we first need to ensure that the right seeds are sown. Healthy working conditions How can employees be protected against working conditions that could harm their health? Environmental health Which environmental influences could have a positive or negative effect on health? Healthy nutrition Which foods promote good health and which carry certain health risks? Prevention Which forms of prevention can help realise significant health benefits? Optimum healthcare What is the optimum result of cure and care in view of the risks and opportunities? Areas of activity Advisory Reports The Health Council’s task is to advise ministers and parliament on issues in the field of public health. Most of the advisory opinions that the Council produces every year are prepared at the request of one of the ministers. In addition, the Health Council issues unsolicited advice that has an ‘alerting’ function. In some cases, such an alerting report leads to a minister requesting further advice on the subject. Health Council of the Netherlands 1,4-Dioxane 2015/26 2015/26 Re-evaluation of the carcinogenicity and genotoxicity 1,4-Dioxane Health Council of the Netherlands</pre>

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<pre>1,4-Dioxane
     Re-evaluation of the carcinogenicity and genotoxicity
</pre>

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<pre></pre>

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<pre>Aan de minister van Sociale Zaken en Werkgelegenheid
Onderwerp              : aanbieding advies 1,4-Dioxane
Uw kenmerk             : DGV/BMO/U-932542
Ons kenmerk            : U- 866033/DC/fs/246-W20
Bijlagen               :2
Datum                  : 13 november 2015
Geachte minister,
Graag bied ik u hierbij het advies 1,4-Dioxane aan.
      Dit advies is een herevaluatie van een eerder door de Gezondheidsraad uitgebracht
advies voor classificatie als kankerverwekkende stof. De raad is gevraagd om deze hereva-
luatie omdat de voorgestelde classificatie uit het eerdere advies afwijkt van de classificatie
die op dit moment in de Europese Unie wordt gehanteerd. Tevens is de raad gevraagd de
stof te classificeren voor mutageniteit. De classificaties in het voorliggende advies zijn
gebaseerd op het Europese classificatiesysteem.
Dit advies is opgesteld door een vaste subcommissie van de Commissie Gezondheid en
beroepsmatige blootstelling aan stoffen (GBBS), de Subcommissie Classificatie van carci-
nogene stoffen. De subcommissie heeft daarbij gebruik gemaakt van commentaren die zijn
ontvangen op het openbare concept van dit advies. Het advies is getoetst door de Beraads-
groep Gezondheid en omgeving van de Gezondheidsraad.
Ik heb dit advies vandaag ter kennisname toegezonden aan de staatssecretaris van Infra-
structuur en Milieu en aan de minister van Volksgezondheid, Welzijn en Sport.
Met vriendelijke groet,
prof. dr. J.L. Severens,
vicevoorzitter
Bezoekadres                                                         Postadres
Parnassusplein 5                                                    Postbus 16052
2 5 11 V X       Den Haag                                           2500 BB         Den Haag
E - m a i l : d . c o e n e n @ g r. n l                            w w w. g r. n l
Te l e f o o n ( 0 7 0 ) 3 4 0 7 4 7 3
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<pre></pre>

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<pre>1,4-Dioxane
Re-evaluation of the carcinogenicity and genotoxicity
Subcommittee on the Classification of Carcinogenic Substances of the
Dutch Expert Committee on Occupational Safety,
a Committee of the Health Council of the Netherlands
to:
the Minister of Social Affairs and Employment
No. 2015/26, The Hague, November 13, 2015
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<pre>The Health Council of the Netherlands, established in 1902, is an independent
scientific advisory body. Its remit is “to advise the government and Parliament on
the current level of knowledge with respect to public health issues and health
(services) research...” (Section 22, Health Act).
     The Health Council receives most requests for advice from the Ministers of
Health, Welfare and Sport, Infrastructure and the Environment, Social Affairs
and Employment, and Economic Affairs. The Council can publish advisory
reports on its own initiative. It usually does this in order to ask attention for
developments or trends that are thought to be relevant to government policy.
     Most Health Council reports are prepared by multidisciplinary committees of
Dutch or, sometimes, foreign experts, appointed in a personal capacity. The
reports are available to the public.
                 The Health Council of the Netherlands is a member of the European
                 Science Advisory Network for Health (EuSANH), a network of science
                 advisory bodies in Europe.
This report can be downloaded from www.healthcouncil.nl.
Preferred citation:
Health Council of the Netherlands. 1,4-Dioxane - Re-evaluation of the
carcinogenicity and genotoxicity. The Hague: Health Council of the Netherlands,
2015; publication no. 2015/26.
all rights reserved
ISBN: 978-94-6281-060-0
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<pre>   Contents
   Samenvatting 9
   Executive summary 11
   Scope 13
.1 Background 13
.2 Committee and procedures 14
.3 Data 14
   Identity of the substance 15
.1 Name and other identifiers of the substance 15
.2 Composition of the substance 15
.3 Physico-chemical properties 16
.4 International classifications 16
   Manufacture and uses 19
.1 Manufacture 19
.2 Identified uses 19
   Contents                                       7
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<pre>    Summary of toxicokinetics 21
 .1 Absorption, distribution and elimination 21
 .2 Metabolism 23
    Genotoxicity 25
 .1 Non-human information 25
 .2 Human information 34
 .3 Summary and discussion of mutagenicity 34
 .4 Comparison with criteria 35
 .5 Conclusions on classification and labelling 36
    Carcinogenicity 37
 .1 Non-human information 37
 .2 Human information 44
 .3 Other relevant information 45
 .4 Summary and discussion of carcinogenicity 47
 .5 Comparison with criteria 48
 .6 Conclusions on classification and labelling 48
    References 49
    Annexes 55
A   Request for advice 57
B   The Committee 59
C   The submission letter (in English) 61
D   Comments on the public review draft 63
E   IARC evaluation and conclusion 65
F   Classification on carcinogenicity 67
G   Classification on mutagenicity 69
H   Criteria for testing reliability of animal and in vitro studies 75
    1,4-Dioxane
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<pre>Samenvatting
Op verzoek van de minister van Sociale Zaken en Werkgelegenheid evalueert en
beoordeelt de Gezondheidsraad de kankerverwekkende eigenschappen van stof-
fen waaraan mensen tijdens het uitoefenen van hun beroep kunnen worden bloot-
gesteld. De evaluatie en beoordeling worden verricht door de Subcommissie
Classificatie van carcinogene stoffen van de Commissie Gezondheid en beroeps-
matige blootstelling aan stoffen van de Raad, hierna kortweg aangeduid als de
commissie. Verder heeft het ministerie aan de Gezondheidsraad gevraagd om een
aantal stoffen te herevalueren en daarbij ook een voorstel voor classificatie voor
mutageniteit in geslachtscellen te doen. In het voorliggende advies herevalueert
de commissie 1,4-dioxaan. De stof wordt vooral gebruikt als oplosmiddel in de
papier-, katoen- en textielindustrie, in koelvloeistof voor auto's, als uitgangsstof
voor de synthese van andere stoffen, als schuimmiddel in de polymeerindustrie
en bij de productie van cosmetische stoffen en shampoos.
De commissie concludeert dat 1,4-dioxaan beschouwd moet worden als
kankerverwekkend voor de mens, en beveelt aan de stof in categorie 1B te
classificeren.* Op basis van de beschikbare gegevens beveelt de commissie
verder aan om 1,4-dioxaan te classificeren als mutageen voor geslachtscellen in
categorie 2 (stof die reden geeft tot bezorgdheid voor de mens omdat zij mogelijk
Zie Annex F (carcinogeniteit) en G (mutageniteit) voor classificatiesysteem.
Samenvatting                                                                         9
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<pre>  erfelijke mutaties in de geslachtscellen van mensen veroorzaakt). De stof kan
  kanker veroorzaken via een niet-stochastisch genotoxisch werkingsmechanisme.
0 1,4-Dioxane
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<pre>Executive summary
At request of the Minister of Social Affairs and Employment, the Health Council
of the Netherlands evaluates and judges the carcinogenic properties of
substances to which workers are occupationally exposed. The evaluation is
performed by the Subcommittee on Classifying carcinogenic substances of the
Dutch Expert Committee on Occupational Safety of the Health Council,
hereafter called the Committee. In addition, the ministry asked the Health
Council to re-evaluate a series of substances, and to include in the re-evaluation a
proposal for classification on germ cell mutagenicity. In this report, such a re-
evaluation was made for 1,4-dioxane. 1,4-Dioxane is mainly used as solvent in
the paper, cotton and textile industry; in coolant for cars, and as base component
for the synthesis of other substances, such as foaming agents in the polymer
industry, production of cosmetics, and shampoos.
The Committee concludes that 1,4-dioxane is presumed to be carcinogenic to
man, and recommends classifying the compound in category 1B.*
    Based on the available data, the Committee recommends classifying
1,4-dioxane as a germ cell mutagen in category 2 (Substances which cause
concern for humans owing to the possibility that they may induce heritable
mutations in the germ cells of humans). The substance acts via a non-stochastic
genotoxic mechanism.
See Annex F (carcinogenicity) and G (mutagenicity) for the classification system.
Executive summary                                                                    11
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<pre>2 1,4-Dioxane</pre>

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<pre> hapter 1
        Scope
1.1     Background
        In the Netherlands a special policy is in force with respect to occupational use
        and exposure to carcinogenic substances. Regarding this policy, the Minister of
        Social Affairs and Employment has asked the Health Council of the Netherlands
        to evaluate the carcinogenic properties of substances, and to propose a
        classification (see Annex A). The assessment and the proposal for a classification
        are expressed in the form of standard sentences (see Annex F). In addition to
        classifying substances on carcinogenicity, the Health Council also assesses the
        genotoxic properties of the substance in question.
            Recently, with reference to the EU Regulation 1272/2008 on classification,
        labelling and packaging of substances (see Annex G), the ministry of Social
        Affairs and Employment asked the Health Council to update the evaluations and
        classifications on carcinogenicity of a series of substances, and to propose for
        these substances a classification on germ cell mutagenicity as well.
        In this report, such an update was performed for 1,4-dioxane. An earlier
        evaluation of this substance was published in 2011.1 The re-evaluation now
        includes a proposal for classification on germ cell mutagenicity.
        Scope                                                                              13
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<pre>1.2 Committee and procedures
    The evaluation is performed by the Subcommittee on Classifying carcinogenic
    substances of the Dutch Expert Committee on Occupational Safety of the Health
    Council, hereafter called the Committee. The members of the Committee are
    listed in Annex B. The submission letter (in English) to the State Secretary can
    be found in Annex C.
         In 2015 the President of the Health Council released a draft of the report for
    public review. The individuals and organisations that commented on the draft are
    listed in Annex D. The Committee has taken these comments into account in
    deciding on the final version of the report. The received comments, and the
    replies by the Committee, can be found on the website of the Health Council.
1.3 Data
    The evaluation and recommendation of the Committee is standardly based on
    scientific data, which are publicly available. The starting points of the
    Committees’ reports are, if possible, the monographs of the International Agency
    for Research on Cancer (IARC). This means that the original sources of the
    studies, which are mentioned in the IARC-monograph, are reviewed only by the
    Committee when these are considered most relevant in assessing the
    carcinogenicity and genotoxicity of the substance in question. In the case of 1,4-
    dioxane, such an IARC-monograph is available, of which the summary and
    conclusion of IARC (1999) is inserted in Annex E.
         Furthermore, relevant data of the European Chemicals Agency (ECHA) were
    retrieved and included in this advisory report.
         Additional data were obtained from the online databases Toxline, Medline
    and Chemical Abstracts, covering the period up to October 2015, using 1,4-
    dioxane and CAS no 123-91-1 as key words in combination with key words
    representative for carcinogenesis and mutagenesis.
 4  1,4-Dioxane
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<pre> hapter 2
        Identity of the substance
2.1     Name and other identifiers of the substance
        Table 1 Substance identity.
        EC number                          : 204-661-8
        EC name                            : 1,4-dioxane
        CAS number (EC inventory)          : 123-91-1
        CAS number                         : 123-91-1
        CAS name                           : 1,4-dioxane
        IUPAC name                         : 1,4-dioxane
        CLP Annex IV Index number          : 603-024-00-5
        Molecular formula                  : C4 H 8 O 2
        Molecular weight range             : 88.12 g/mol
        Structural formula                 :
2.2     Composition of the substance
        Not applicable.
        Identity of the substance                         15
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<pre>2.3   Physico-chemical properties
      Table 2 Summary of physico-chemical properties
      Properties                      Value                                    Reference   Comment
      State of the substance        : Colourless liquid                        ATSDR 20122
      Melting/freezing point        : 11.8 °C                                  ATSDR 20122
      Boiling point                 : 101.1 °C                                 ATSDR 20122
      Relative density              : 1.0329                                   ATSDR 20122
      Vapour pressure               : 38.1 mm Hg at 25 °C                      ATSDR 20122
      Surface tension               :-
      Water solubility              : Miscible                                 ATSDR 20122
      Partition coefficient         : Log Kow -0.27                            ATSDR 20122
      (n-octanol/water)
      Flash point                   : 5-18 °C                                  ATSDR 20122
      Flammability                  : Limits at 25 °C lower: 2.0%; upper: 22%  ATSDR 20122
      Explosive properties          : Vapour forms explosive mixture with air  ATSDR 20122
                                      over wide range
      Self-ignition temperature : 180 °C                                       ATSDR 20122
      Oxidising properties          : none                                     ECHA3
      Granulometry                  :-
      Stability in organic solvents : Yes                                      ECHA3
      Dissociation constant (pKa): No dissociating properties                  ECHA3
      Viscosity                     : 1.27 mm2/s at 20 °C; 0.93 mm2/s at 40 °C ECHA3
2.4   International classifications
2.4.1 European Commission
      1,4-Dioxane is classified for carcinogenicity in Annex VI of regulation (EC) No
      1272/2008 of the European Parliament as follows: Carc 2 (suspected human
      carcinogen; H351: suspected of causing cancer). The substance is not classified
      for germ cell mutagenicity. The classification by the European Commission dates
      from January 2000.
2.4.2 Health Council of the Netherlands
      In 2011, the Dutch Expert Committee on Occupational Standards, a Committee
      of the Health Council of the Netherlands concluded that 1,4-dioxane should be
      regarded as carcinogenic to humans (comparable with EU category 1B) and
      considered the substance as a non genotoxic carcinogen.1,4 Furthermore, the
 6    1,4-Dioxane
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<pre>      Committee recommended an HBROEL TWA 8 hours for 1,4-dioxane of 20
      mg/m3 (6 ppm). This was based on the lowest observed adverse exposure limit
      (LOAEL) of 180 mg/m3 (50 ppm) for nasal lesions in rats after lifetime exposure
      to 1,4-dioxane.1
2.4.3 IARC
      In 1999, IARC concluded that there was inadequate evidence in humans for the
      carcinogenicity of 1,4-dioxane, and that there was sufficient evidence in
      experimental animals (see Annex E). Therefore, IARC classified the compound
      in Group 2B (‘possibly carcinogenic to humans’).5
      Identity of the substance                                                       17
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<pre>8 1,4-Dioxane</pre>

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<pre> hapter 3
        Manufacture and uses
3.1     Manufacture
        Not relevant for classification.
3.2     Identified uses
        1,4-Dioxane is used as a solvent in the production of lacquers, varnishes,
        cleaning and detergent preparations, adhesives, cosmetics, deodorant fumigants,
        emulsions and polishing compositions, pulping of wood, extraction medium for
        animal and vegetable oils, laboratory chemical (eluent in chromatography),
        cassettes, plastic and rubber, and insecticides and herbicides (BASF information;
        HSDB 1996; Grant Chemicals 1977). Furthermore, it is used as a stabilizer for
        1,1,1-trichloroethane. However, this use is diminished considerably as a result of
        the restriction of the use of substances depleting the ozone layer (Grant
        Chemicals 1977).6
        Manufacture and uses                                                               19
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<pre>0 1,4-Dioxane</pre>

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<pre> hapter 4
        Summary of toxicokinetics
        The data presented below is a summary based on evaluations and reviews by
        others, such as DECOS, IARC, ATSDR, DFG, and EPA.1,2,4,5,7,8
4.1     Absorption, distribution and elimination
4.1.1   Absorption
        Inhalation and oral
        Four healthy volunteers inhaled 50 ppm 1,4-dioxane (180 mg/m3) for 6 hours,
        after which the blood and the urine was examined (Young et al., 1977).9 The
        substance was rapidly and extensively absorbed as evidenced by a rapid
        accumulation in plasma. Limited human data are available to evaluate the oral or
        inhalatory absorption of 1,4-dioxane.
            1,4-Dioxane was rapidly and almost completely absorbed after oral and
        inhalation exposure of mice (Sweeney et al., 2008).10
        Dermal
        Dermal absorption occurs, but it is low, probably due to evaporation of the
        material. In experiments with Rhesus monkeys, 2.3 and 3.4% of the dioxane,
        which was applied non occlusively as a methanol solution or as lotion on the
        Summary of toxicokinetics                                                        21
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<pre>      forearm skin, was excreted in the urine (Marzulli et al., 1981).11 In vitro studies
      show that 12% of an applied dose passes through excised skin under occlusion,
      and only 0.3% when not occluded (ECETOC 1983).12
4.1.2 Distribution
      No data are available for the distribution of 1,4-dioxane in human tissues. In
      addition, no data are available for the distribution of 1,4-dioxane in animals
      following oral or inhalation exposure. After intraperitoneal administration of 3H-
      labelled dioxane to rats, 3H label was found in all tissues investigated at
      comparable levels (Woo et al., 1977) between 1 and 16 hours after
      administration. Mikheleev et al., (1990) report similar findings.13,19,20
4.1.3 Elimination and pharmacokinetics
      In humans exposed for 6 hours to 180 mg 1,4-dioxane/m3 (in a chamber under
      dynamic airflow conditions) dioxane in plasma rapidly accumulated to nearly
      steady state after 4 hours of exposure. It was excreted in urine as its metabolite
      ß-hydroxyethoxyacetic acid (HEAA) over the next 24 hours of which approx.
      50% during the first 6 hour period. In humans exposed for 6 hours to 180 mg
      1,4-dioxane/m3 (50 ppm) 99.3% of the absorbed dose (assuming that urinary
      excretion was the only excretory route) was eliminated via the urine as
      ß-hydroxyethoxyacetic acid (HEAA); the remainder was unchanged dioxane
      (Young et al., 1977).9 After the 6 hr exposure period the plasma 1,4-dioxane
      concentration decreased exponentially, indicating that the elimination was not
      saturated. The plasma elimination T½ was 59 minutes (Young et al., 1977).9
          Physiologically-based pharmacokinetic (PB-PK) models were developed by
      Reitz et al., (1990) and Leung and Paustenbach (1980), which were further
      improved by Sweeney et al., (2008).10,14,15 The plasma concentrations as well as
      HEAA urinary excretion after exposure to dioxane by inhalation or gavage in
      mice and rats could reasonably well be predicted, but the human volunteer data
      of Young et al., (1977) did not fit adequately in the model.9 Only the urinary
      excretion data of Young et al., (1978) were well predicted by the model.16 A
      physiologically based pharmacokinetic modelling study indicates that 1,4-
      dioxane may also be excreted into human milk (Fisher et al., 1997).17
          1,4 -Dioxane is rapidly excreted in rats via the urine. The major metabolite is
      2-hydroxyethoxyacetic acid (HEAA) (Woo et al., 1977a,b).18,19 At low pH,
      HEAA is rearranged (reversibly) to 1,4-dioxan-2-one.
 2    1,4-Dioxane
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<pre>4.2 Metabolism
    1,4-Dioxane is metabolized by cytochrome P-450’s, possibly of the 2A and 2D
    family (Sweeney et al., 2008).11 Induction of the cytochrome P-450 enzymes
    increases the rate of HEAA formation, whereas inhibition decreases HEAA
    formation (Woo 1977b, Woo 1978).19,20
        Repeated oral administration of 1,000 mg/kg of 1,4-dioxane induced dioxane
    metabolism in rats, but at doses of 10 mg/kg no such effect was observed (Young
    et al., 1978).16
        At a single oral dose of 20 mg/kg in mice the metabolism was so rapid that
    1,4-dioxane could hardly be detected in blood; saturation of metabolism seemed
    to occur above 200 mg/kg (Sweeney et al., 2008).10
        In rats the capacity to metabolise 1,4-dioxane to HEAA is also limited.
    A single oral dose of 10 mg/kg bw was rapidly metabolised and excreted (as
    HEAA) via the urine, while a single oral dose of 100 1,000 mg/kg bw, saturated
    the metabolism, resulting in a decreased proportion of urinary excretion of
    HEAA, and increased excretion of 1,4-dioxane in urine and the expired air (Dietz
    et al., 1982, Reitz et al., 1990, Young et al., 1978).15,16,21 Young et al., (1978)
    observed a statistically significant increase of 14CO2 excretion at multiple oral
    doses of 14C-labelled dioxane compared to the control; it is unclear as yet how
    this mechanistically reflects metabolism of dioxane.16 It has been suggested by
    SCOEL that at high dose another, presumably reactive metabolite of
    1,4-dioxane, β-hydroxyethoxyacetaldehyde (HEA) might be responsible for
    toxicity: in the toxicity studies, morphological and biochemical changes were
    observed at exposure concentrations which lead to saturation of the
    metabolism.22 SCOEL postulated, without further evidence that HEA may be
    assumed to be the reactive metabolite that is responsible for some of the toxicity
    seen with 1,4-dioxane, including carcinogenicity in experimental animals.22
    Summary of toxicokinetics                                                           23
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<pre>Figure 1 Suggested metabolic pathways of 1,4-dioxane in the rat (Woo et al. 1977a in EPA 2013).8,18 [I], 1,4-dioxane; [II],
 iethylene glycol; [III], β-hydroxyethoxy acetic acid (HEAA); [IV], 1,4-dioxane-2-one; [V], 1,4-dioxane-2-ol;
 VI] β-hydroxyethoxy acetaldehyde (HEA). Note: Metabolite [V] is a likely intermediate in pathway b as well as pathway c.
The proposed pathways are based on the metabolites identified; the enzymes responsible for each reaction have not been
 etermined. The proposed pathways do not account for metabolite degradation to the labelled carbon dioxide identified in
 xpired air after labelled 1,4-dioxane exposure.
 4            1,4-Dioxane
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<pre> hapter     5
            Genotoxicity
5.1         Non-human information
5.1.1       In vitro data
            Data on in vitro mutagenicity testing are presented in Table 3.
 able 3 Summary of in vitro mutagenicity studies.
Method              Cell type                 Concentration         Results          Klimisch             References
                                              Range*                - negative       Score**
                                                                    + positive
Micro-organisms
 everse mutation    S. typhimurium            0, 156, 313, 625,     -                2                    Morita et al.,
                    TA98, TA100, TA1535,      1,250, 2,500, and                                           199823
                    TA1537                    5,000 µg/plate
                    E. coli WP2uvrA and       +/- preincubation
                    WP2
 everse mutation    S. typhimurium            0, 5.17, 15.5, 31.0,  - (highest dose  2                    Stott et al., 198124
                    TA98, TA100, TA1535,      62.0 and 103 mg/plate bacteriostatic -
                    TA1537, TA1538                                  S9)
 everse mutation    S. typhimurium            0,100, 133, 1,000,    -                2                    Haworth et al.,
                    TA98, TA100, TA1535,      1,333, and 10,000                                           198325
                    TA1537                    µg/plate
 everse mutation    S. typhimurium            0, 10, 31, 103        -                3 (only two strains; Nestmann et al.,
                    TA100, TA1535             mg/plate                               methodological       198426
                                              preincubation                          deficiencies)
            Genotoxicity                                                                                                   25
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<pre> everse mutation      S. typhimurium         Dose levels not           -               3 (dose levels not    Khudoley et al.,
                      TA98, TA100, TA1530,   provided                                  provided)             198727
                      TA1535, TA1537
 everse mutation      S. typhimurium         4, 20, 100, 500, 2,500 -                  2                     Echa registration
                      TA98, TA100, TA1535,   µg/plate                                                        data, vitro 001
                      TA1537, TA1538                                                                         study report
                                                                                                             1979-04-02
                                                                                                             (echa.europe.eu)3
Mammalian cells
Gene mutation         Mouse lymphoma         0, 1,250, 2,500 and       - (slight       2                     Morita and
                      L5178Y cells, tk locus 5,000 µg/ml:              decrease in                           Hayashi 199823
                                             3 and 24 hr exposure      relative
                                                                       survival at
                                                                       5,000 µg/ml
                                                                       +S9)
Gene mutation         Mouse lymphoma         0, 312.5, 625, 1,250,     -               2                     McGregor et al.,
                      L5178Y cells, tk locus 2,500, 5,000 µg/ml                                              199128
                                             (-S9)
                                             0, 1,000, 2,000, 3,000,
                                             4,000, 5,000 µg/ml
                                             (+S9)
Gene mutation         Chinese hamster ovary, 0.05, 0.1, 0.5, 1.0, 5.0, -               2                     Echa registration
                      K1 cells               10.0 mg/ml                                                      data, vitro 003
                                                                                                             study report
                                                                                                             1991-8-9
                                                                                                             (echa.europe.eu)3
Micronucleus          Chinese hamster ovary, 0, 1,250, 2,500 and       -               2                     Morita and
                      K1 cells               5,000 µg/ml:                                                    Hayashi 199823
                                             5 and 44 hr exposure
                                             (+/-S9)
 hromosome            Chinese hamster ovary, 0, 1,250, 2,500 and       -               2                     Morita and
 berration            K1 cells               5,000 µg/ml                                                     Hayashi 19823
                                             (+/-S9)
 hromosome            Chinese hamster ovary 1,050, 3,500, 10,520       -               3 (no data on purity; Galloway et al.,
 berration            cells                  µg/ml                                     no data on negative 198729
                                             (+/-S9)                                   control or
                                                                                       cytotoxicity)
Other supporting studies
 ister chromatid      CHO-K1 cells           0, 1250, 2,500 and        - (dose-related 2                     Morita and
 xchange                                     5,000 µg/ml (+/- S9)      cytotoxicity                          Hayashi 199823
                                             3 and 26 hr exposure      observed)
 ister chromatid      CHO cells              1,050, 3,500, 10,520      + (-S9 at       3 (no data on purity, Galloway et al.,
 xchange                                     µg/ml (+/-S9);            10,520          negative control or 198729
                                             positive and negative     µg/ml);         cytotoxicity)
                                             controls included         - (+S9)
 6           1,4-Dioxane
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<pre>UDS                     Rat primary hepatocytes Incubation with 0,       - (at 1mM       2                    Goldsworthy
                        F344                     0.001, 0.01, 0.1 or 1   signs of                             et al., 199130
                                                 mM; -S9 only            cytotoxicity)
UDS                     Rat primary hepatocytes 10-8 to 1 M              -               3 (methodological    Stott et al., 198124
                                                                                         deficiencies)
Comet assay’; DNA Rat primary hepatocytes        0.03, 0.3, 3.0, 10, 30  + (at cytotoxic 3 (methodological    Sina et al., 198331
 amage, single strand                            mM; positive and        concentrations deficiencies)
 reak measured by                                negative controls       of 0.3 and
 lkaline elution***                              included; -S9 only      higher)
DNA damage              Photobacterium           Not specified;          -               4 (no standard test, Kwan et al., 1990
Mutatox assay)          phosphoreum M169         -S9 only                                relevance unknown;   (results taken
                        (strain sensitive to DNA                                         concentrations not   from ATSDR
                        damaging agents, DNA-                                            specified)           2012)2
                        intercalating agents,
                        DNA-synthesis
                        inhibitors, and direct
                        mutagens.
Aneuploidy              S. cerevisiae D61M       1.48, 1.96, 2.44, 2.91, - (toxicity     3 (no metabolic      Zimmerman et
                                                 3.38, 4.31, 4.75%       observed; only  activation; no       al., 198532
                                                 (repeated plating after tested -S9)     validated method)
                                                 addition-nil
                                                 incubation of 5 hr at
                                                 3.85 and 4.31%);
                                                 positive and negative
                                                 controls included
  + or - S9, with or without metabolic activation system. ** See Annex H.
              Conclusion
              The in vitro studies summarised in Table 3 show no mutagenic activity of 1,4-
              dioxane when using bacteria or mammalian cells. Negative outcomes were also
              found in the unscheduled DNA synthesis and sister chromatide exchange assay.
              Genotoxicity                                                                                                     27
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<pre>5.1.2        In vivo data
             Data on the in vivo mutagenicity testing are presented in Table 4.
 able 4 Summary of in vivo mutagenicity studies (animal studies).
Method          Animal            Exposure conditions              Results                     Klimisch score*  References
 omatic cell mutagenicity
Micronuclei      CD-1 mice, male 0, 500, 1,000, 2,000 and           - (toxicity at 3,200 mg/kg 2                 Morita 199433
                 peripheral blood; 3,200 mg/kg bw (two              bw, 1/5 males died at this
                 5/group           intraperitoneal injections, 1/ dose), cytotoxicity not
                                   day); positive and negative      tested, but IP dosing
                                   control
Micronuclei      B6C3F1 mice,      0, 2,000, 3,000, 4,000 mg/kg - (decreased PCE/NCE 2                           McFee et al.,
                 male bone         bw (intraperitoneal injection) ratio)                                         199434
                 marrow; 5/group 0, 500, 1,000, 2,000 mg/kg
                                   bw
                                   (intraperitoneal injection, 3x); - (500 and 1,000 mg/kg
                                   two studies in two different bw were positive in one
                                   labs                             trial and one laboratory
                                                                    only; no dose-related
                                                                    increase). Decreased
                                                                    PCE/NCE ratio
Micronuclei      C57BL6 mice,      0, 900, 1,800, 3,600 mg/kg       + (dose-related increase) 2                  Mirkova
                 male bone         bw (oral gavage) for 24 hr,      no data on cytotoxicity                      199435
                 marrow:           3,600 mg/kg bw also for 48
                 10/group          hr sampling time
                 C57BL6 mice,      0, 900, 1,800, 3,600 mg/kg       + (dose-related increase) 2
                 male bone         bw (oral gavage) for 24 hr,      no data on cytotoxicity
                 marrow 4/group 3,600 mg/kg bw also for 48
                                   hr sampling time
                  C57BL6 mice, 0 and 3,600 mg/kg bw (oral + (no data on                         3
                 male bone         gavage) for 24 hr                cytotoxicity)               (methodological
                 marrow 10/group                                                                deficiencies)
                 C57BL6 mice       0 and 5,000 mg/kg bw (oral + (no data on                     3
                 female bone       gavage) for 24 hr or 48 hr       cytotoxicity)               (methodological
                 marrow:           sampling time                                                deficiencies)
                 5/group
                 BALB/c mice,      0 and 5,000 mg/kg bw (oral - (1/6 death occurred in 3
                 males bone        gavage) for 24 hr                5,000 mg/kg bw after 24 (methodological
                 marrow; 6/group                                    hr); irrelevant exposure deficiencies)
                                                                    levels. No data on
                                                                    cytotoxicity
 8           1,4-Dioxane
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<pre>Micronuclei      CD-1 mice, male  1,500, 2,500 and 3,500 mg/kg     + (dose-related increase 2                   Roy et al.,
  ollow-up study bone marrow; 5/  bw (oral gavage, 5 days); 24     in MN frequency and                          200536
 f Morita and    group            hr sampling time;                decrease in PCE/NCE
Hayashi 1998                      CRESH and FISH staining          ratio; >90% micronuclei
                                  used to demonstrate              caused by chromosome
                                  aneuploidy; implantation of      breakage; induction of
                                  BrdU releasing osmotic           cell proliferation
                                  pumps used to demonstrate
                                  cell proliferation in liver and
                                  to increase sensitivity of the
                                  test
                 CD-1 mice, male 1,500, 2,500 and 3,500 mg/kg      + (from 2,500 mg/kg bw 2
                 hepatocytes; 5/ bw (oral gavage, 5 days)          dose-related increase in
                 group            24 hr sampling time; CRESH       MN in proliferating cells
                                  and FISH staining used to        only; caused by
                                  demonstrate aneuploidy;          chromosome breakage;
                                  implantation of BrdU             induction of cell
                                  releasing osmotic pumps used     proliferation
                                  to demonstrate cell
                                  proliferation in liver and to
                                  increase sensitivity of the test
Micronuclei      CBA mice, male 1,800 mg/kg bw (oral,              - (decreased PCE/NCE       2                 Tinwell and
  ollow-up of    bone marrow; 4 gavage);                           ratio)                                       Ashby 199437
 tudy Mirkova    animals          Giemsa staining**
 994
                 CBA mice, male 1,800 mg/kg bw (oral,              -                          3 (one dose only;
                 bone marrow; 8 gavage); Acridine orange                                      no data
                 animals          staining                                                    cytotoxicity;
                                                                                              acridine orange
                                                                                              staining**)
                 C57BL6 mice,     3,600 mg/kg bw (oral,            -                          3 (max. dose
                 male bone        gavage); acridine orange                                    level; no data on
                 marrow; 4        staining                                                    cytotoxicity
                 animals                                                                      methodological
                                                                                              deficiencies;
                                                                                              acridine orange
                                                                                              staining**)
Micronuclei      CD-1 mice, male 1,000, 2,000 and 3,000 mg/kg - (in peripheral blood)         3 (method not     Morita and
  ollow-up of    peripheral blood bw (oral gavage); partial        + (in hepatocytes; from validated:           Hayashi 199823
 tudy Mirkova    and hepatocytes; hepatoectomy 24 hr after         2,000 mg/kg bw; dose- partial
 994, same dose 5/group           dosing; peripheral blood         related increase);         hepatectomy to
evels                             obtained from tail vein 24       intraspecies differences stimulate
                                  hours after hepatectomy;         at 2,000, but not at 3,000 mitosis)
                                  hepatocytes analysed 5 days mg/kg bw; valid positive
                                  after hepatectomy                and negative controls
              Genotoxicity                                                                                                  29
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<pre> ransgenic        Gpt delta        0, 200, 1,000 or 5,000 ppm in  - (0 to 1,000 ppm)         4 (poster abstract Fukushima
odent gene        transgenic male  drinking water for up to 16    + (5,000 ppm), for         only; no details et al., 200938
mutation          rats; 30 animals weeks; at the end of treatment increased mutation         on methods or
Analysis of       divided in four  all animals were killed, and   frequency of gpt           outcomes
GST-P positive    groups (number   livers excised for further     transgenes (p<0.001),      reported)
oci and PCNA-     of animals per   analyses                       GST-P-positive foci
 ositive cell     group not given)                                (p<0.001), and PCNA-
ndex                                                              positive cell index
                                                                  (p<0.001)
Germ cell mutagenicity
 ex-linked        Drosophila       35,000 ppm in feed for 7       3 (classification based on                    Yoon et al.,
ecessive lethal melanogaster       days, or 50,000 ppm by         studies in mammalians;                        198539
mutations                          injection; negative controls   no OECD guideline
                                   included                       anymore)
Meiotic non-      Drosophila       1, 1.5, 2, 3 and 3.5%          + (not dose related,       3 (less relevant   Munõz and
 isjunction       melanogaster     (feeding);                     cytotoxic doses)           test system;       Barnett 200240
                                   negative controls included;                               unusual strains)
                                   oocytes were obtained for
                                   evaluation 24 and 48 hr after
                                   mating
Dominant lethal Mouse, male        2,550 mg/kg bw (single         -                          3 (no positive     BASF 197741
est               NMRI, 20/sex     intraperitoneal injection)                                control; no        (results taken
                                                                                             toxicity observed  from ECHA
                                                                                             in highest dose;   registration
                                                                                             methodological     data, Ex Key
                                                                                             deficiencies)      Genetic
                                                                                                                toxicity in
                                                                                                                vivo.001)3
Other supporting studies
UDS               Male rat liver   1% (1,500 mg/kg bw/day) in     - (at 1 mM signs of        2                  Goldsworthy
                  F344 and         drinking water for 1 week      cytotoxicity)                                 et al., 199130
                  primary          (pretreatment rats) followed
                  hepatocytes      by hepatocyte incubation
                                   with 0, 0.001, 0.01, 0.1 or 1
                                   mM; -S9 only
UDS               Male rat liver   1,000 mg/kg bw (oral,          - (cytotoxicity not        2
                  F344; 3/group    gavage),                       observed)
                                   2 hr and 12 hr sampling time
UDS               Male rat liver   1% (1,500 mg/kg bw/day) in     - (no increase in NG; no 2
                  F344; 3/group    drinking water for 2 weeks or  cytotoxicity observed)
                                   2% (3,000 mg/kg bw/day) in     - Two-fold hepatocytes
                                   drinking water for 1 week      proliferation observed at
                                                                  1%
UDS               Male F344 rats;  1% (1,500 mg/kg bw/day) in - (at highest dose signs of 2
                  3/group; nasal   drinking water for 8 days      toxicity were observed);
                  epithelial cells (pre-treatment), followed by only morphologically
                  and hepatocytes  0, 10, 100 or 1,000 mg/kg bw normal cells were scored
                  examined         (single gavage dose)
 0            1,4-Dioxane
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<pre>UDS              SD rat liver; 4   1,000 mg/kg bw (14C oral        -                         3 (no positive   Stott et al.,
                 rats/group        gavage)                                                   control;         198124
                                                                                             (methodological
                                                                                             deficiencies)
UDS              SD rat liver; 6   0, 10, 1,000 mg/kg bw/day       + (1.5 fold increase at   3 (no positive
                 males/group       (drinking water for 11 wks) 1,000 mg/kg, a cytotoxic control;
                                                                   concentration)            (methodological
                                                                                             deficiencies)
Comet assay’;    Female SD rats, 0, 168, 840, 2,550, 4,200 mg/ + (from 2,550 mg/kg bw, 2                      Kitchin and
DNA damage,      3-5/group;        kg bw (oral gavage twice) for dose-related increase; but                   Brown 199042
 ingle strand    histopathological 21 and 4 h before sacrifice     irrelevant dose levels)
 reak measured   examination of
 y alkaline      liver                                             Histopathology liver: 3/5
 lution assay***                                                   rat of 2,550 mg/kg
                                                                   showed mild to minimal
                                                                   periportal vacuolar
                                                                   degenerations in liver
                                                                   samples in the absence of
                                                                   hepatic necrosis or
                                                                   substantial cellular
                                                                   toxicity. No
                                                                   histopathological lesions
                                                                   found in other dose
                                                                   groups.
  eplicative     Male F344 rats; Gavage; 1,000, 1,500, 2,000 + (24 hr-response time: 2                        Miyagawa
DNA synthesis    4/group;          and 4,000 mg/kg bw; 24 hr       dose-related increase                      et al., 199943
marker for cell  hepatocytes       and 48 hr response time;        from 1,000 mg/kg bw,
 roliferation)   isolated after    thymidine and BrdU              but no increase at 4,000
                 exposure for      incorporation                   mg/kg bw; relationship
                 testing                                           was bell shaped; no
                                                                   hepatotoxicity at any
                                                                   dose level)
                                                                   (48 hr-response time; no
                                                                   hepatocytotoxicity)
  eplicative     Rat hepatocytes 0, 1,000, 2,000 mg/kg bw,         + at 2,000 mg/kg bw       3 (no validated Uno et al.,
DNA synthesis                      oral gavage; positive and       (signs cytotoxicity at    test method)     199444
 ssay                              negative controls included      1,000 and 2,000 mg/kg
                                                                   bw)
DNA alkylation   SD rat liver; 4-6 1,000 mg/kg bw 14C              -                         3 (positive      Stott et al.,
                 males/group       (gavage); DNA isolation                                   control missing; 198124
                                   from hepatocytes and HPLC                                 (methodological
                                   analysis                                                  deficiencies;
                                                                                             limited study)
  NA synthesis;  Male SD rat;      Intravenous injection; activity +                         3 (no positive   Kurl et al.,
nhibition of     numbers not       measured in isolated                                      control; no      198145
  NA             reported          hepatocytes; 10 and 100 mg/                               validate method)
 olymerase A                       rat (2 and 20 mg/kg bw)
 nd B
              Genotoxicity                                                                                                  31
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<pre>DNA repair, host Repair-deficient Highest tested concentration -                               3 (method not   Hellmer and
mediated assay, E coli K-12          1150 mM; + and – S9;                                      validated)      Bolcsfoldi
n vivo            uvrB/recA; tests positive and negative controls                                              199246
                  performed in       included
                  mice
  See Annex H. ** According to OECD guideline, the Giemsa stain is preferred for detection of micronuclei; the acridine orange
 tain is a DNA stain that can eliminate artefacts. *** Comet assay and alkaline elution assay: DNA single and double strand
 reaks, DNA cross-links.
             Conclusion
             Germ cells
             No animal studies are available on the mutagenicity of 1,4-dioxane in germ cells.
             The outcome of a sex-linked recessive lethal mutagenicity test using Drosophila
             melanogaster, was negative (Yoon et al., 1985).39 However, the Committee
             considers this test not relevant for humans.
             Somatic cells
             As summarised in Table 4, a number of studies using mice have been performed
             on the mutagenic properties of 1,4-dioxane. The induction of micronuclei was
             mainly investigated in bone marrow cells, but also in peripheral blood cells and
             in hepatocytes. Furthermore, the Committee noted that dose levels over the limit
             dose of 2,000 mg/kg bw have been used. The Committee does not consider these
             higher dose levels relevant for evaluation of the genotoxicity.
                  1,4-Dioxane did not induce an increase in bone marrow cells with
             micronuclei in animals which were given the substance by intraperitoneal
             injection. In one study a decreased ratio of PCE/NCE was reported, which is an
             indirect measure of bone marrow toxicity (McFee et al., 1994).34 This indicates
             that 1,4-dioxane at least reached the bone marrow.
                  In studies in which mice were given the substance orally positive results were
             observed in dose level above the limit dose of 2,000 mg/kg bw up to 5,000 mg
             1,4-dioxane/kg bw. However, in a few studies a dose-related statistically
             significant increase in number of cells with micronuclei already started at doses
             below this limit dose. For instance, Mirkova et al., (1994) reported a statistically
             significant dose-related increase in bone marrow cells with micronuclei from 900
             mg/kg bw/day and Roy et al., (2005) from 1,500 mg/kg bw which paralleled with
             a dose-related decrease in the PCE/NCE ratio, a measure for cytotoxicity in bone
             marrow cells and thus bioavailability in bone marrow cells.35,36 Decreases in
  2          1,4-Dioxane
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<pre>bone marrow cell proliferation were also observed. Roy et al., (2005) also
observed that the induced micronuclei are formed primarily from chromosomal
breakage.36
    In other studies, no induction of cells with micronuclei by 1,4-dioxane was
observed below the limit dose of 2,000 mg/kg bw although in one study a
decreased ratio of PCE/NCE was reported (Tinwell and Ashby 1994).37
Overall, the Committee noted that in the majority of the animal studies no data
on cytotoxicity were reported, which makes it difficult to interpret the outcomes
correctly. However, in most studies dose levels were used exceeding the limit
dose, making them less relevant. Secondly, the differences in outcomes among
the studies could also be partially explained by the use of a small number of
animals, different dose regimen and testing methods. However, the Committee
cannot ignore the dose-related positive findings of the micronuclei studies of
Roy et al., (2005) and Mirkova et al., (1994) in bone marrow in which at doses
below the limit dose of 2,000 mg/kg bw statistically significant increases in cells
with micronuclei were found. Based on these results, the Committee considers
that 1,4-dioxane may have genotoxic potential.
Other in vivo studies have also been summarised in Table 4. Kitchen and Brown
1990 found a dose-related increase in DNA single-strand breaks at 2,500 and
5,000 mg/kg bw 1,4-dioxane (oral administration by gavage) in the liver of
rats.42 At these relatively high dose levels no significant cytotoxicity was
observed. In another study, 1,4-dioxane did not induce DNA-alkylation in
hepatocytes of rats, which were given the substance by gavage at a concentration
of 1,000 mg/kg bw (Stott et al., 1981).24 No other reliable data on DNA damage
due to exposure to 1,4-dioxane are available.
    In vivo data on unscheduled DNA synthesis showed negative outcomes.
Miyagawa et al., (1999) showed that cell proliferation (measured as replicative
DNA synthesis) could occur without signs of hepatotoxicity.43 In their study, rats
were exposed to 1,4-dioxane to up to 4,000 mg/kg bw (single administration by
gavage). Tests for cell proliferation were performed 24 or 48 hours after
administration. After 24 hours a clear bell-shaped relationship was found with no
significant increase in proliferation at the highest concentration tested. However,
data obtained after 48 hours did not show indications of cell proliferation at any
concentration level.
The majority of these studies support the conclusion that 1,4-dioxane may have
genotoxic potential.
Genotoxicity                                                                        33
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<pre>5.2         Human information
            In Table 5 data are shown on 1,4-dioxane exposure in humans.
 able 5 Summary of human studies.
Method              Population         Cells           Results and remarks Quality/reliability References
                                                                           of study
 hromosomal         6 German workers; Human peripheral Negative (compared 4 (Data from         Thiess et al., 197647
 berrations         6-15 year exposure lymphocytes     to controls)        secondary sources;  (results taken from
                    to unspecified                                         no study details    EU Risk
                    airborne levels                                        given)              Assessment Report
                                                                                               2002)
5.3         Summary and discussion of mutagenicity
            Below, only data are summarised of reliable experimental design according to
            the Klimisch criteria 1 and 2 (see Annex H).
            Germ cell genotoxicity
            As no genotoxicity studies of 1,4-dioxane in germ cells were found, the
            Committee is not able to make a conclusion whether 1,4-dioxane is mutagenic in
            germ cells.
            Somatic cell genotoxicity
            1,4-Dioxane was investigated in genotoxicity tests for the 3 endpoints of
            genotoxicity: gene mutations, structural and numerical chromosome aberrations.
            The Committee noted that in the majority of the animal studies no data on
            cytotoxicity were reported, which makes it difficult to interpret the outcomes.
            Also in most studies dose levels were used exceeding the limit dose, making
            them less relevant to determine the genotoxicity of 1,4-dioxane. Furthermore, the
            differences in outcomes among the studies could also be partially explained by
            the use of a small number of animals, different dose regimen and testing
            methods.
            1,4 Dioxane did not induce gene mutations in bacteria nor in mammalian cells in
            vitro. Exposure to 1,4-dioxane did not result in an increase in cells with
 4          1,4-Dioxane
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<pre>    chromosome aberrations or micronuclei. The majority of the supporting
    genotoxicity tests (Table 3) confirmed the negative findings in in vitro tests.
    Unexpectedly, the in vivo genotoxicity studies gave contradictory results.
    Exposure to high doses of 1,4-dioxane, above the limit dose of 2,000 mg/kg bw,
    resulted in an increase of cells with micronuclei indicating to a cytotoxic rather
    than a genotoxic effect. Occasionally positive results were also found in
    micronucleus tests with doses below the limit dose of 2,000 mg/kg bw. The
    Committee cannot ignore these positive findings and considers that 1,4-dioxane
    also has a genotoxic potential. Aneuploidy was not observed. The majority of the
    supportive in vivo genotoxicity tests (Table 4) confirmed the in vivo results.
    As the important in vitro tests are negative but part of the in vivo tests
    unexpectedly positive predominantly at doses above the limit dose, it can be
    concluded that 1,4-dioxane has to be considered as a non-stochastic genotoxic
    substance and that the positive results may be due to cytotoxicity and thus
    proliferation induction. The positive results found in the tests measuring
    replicative DNA synthesis as a marker for cell proliferation confirm this mode of
    action. Since occasionally positive results in the micronucleus tests were found at
    doses below the limit dose of 2,000 mg/kg bw a stochastic genotoxic mechanism
    as secondary mode of action cannot be excluded.
    Overall, the Committee concludes that 1,4-dioxane is mutagenic in vivo in
    mammalian cells and acts predominantly by a non-stochastic genotoxic
    mechanism.
5.4 Comparison with criteria
    According to the criteria in Annex VI of the European regulation No. 1272/2008
    (see Annex G), classification as a mutagen in category 1 is warranted when
    positive evidence for in vivo heritable germ cell mutagenicity in humans (1A) or
    mammals (1B) has been reported. No data have been presented on human or
    animal germ cell mutagenicity. Overall, due to a lack of data the Committee
    concludes that there is no positive evidence for in vivo heritable germ cell
    mutagenicity of 1,4-dioxane.
        In addition, substances may be categorized in 1B if there are “positive results
    from in vivo somatic cell mutagenicity tests in mammals, in combination with
    some evidence that the substance has potential to cause mutations to germ cells”.
    The latter may be based on a) “supporting evidence from mutagenicity/
    Genotoxicity                                                                        35
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<pre>    genotoxicity tests in germ cells in vivo”, or b) “by demonstrating the ability of
    the substance or its metabolites to interact with the genetic material of germ
    cells” (see Annex G). In case of 1,4-dioxane no supporting evidence is available
    that suggests that the substance has potential to cause mutations in germ cells.
    A substance may be classified as a germ cell mutagen in category 2 if there is
    positive evidence from animal studies and/or from in vitro studies obtained from:
    somatic cell mutagenicity tests in vivo, or other in vivo somatic cell genotoxicity
    tests, which are supported by positive results from in vitro mutagenicity assays.
    1,4-Dioxane did not show genotoxicity in vitro. In vivo data show an increase in
    micronuclei formation in several studies. Therefore, the Committee concludes
    that 1,4-dioxane should be classified in category 2.
5.5 Conclusions on classification and labelling
    Based on the available data, the Committee recommends classifying 1,4-dioxane
    as a germ cell mutagen in category 2 (Substances which cause concern for
    humans owing to the possibility that they may induce heritable mutations in the
    germ cells of humans). The substance may cause cancer via a non-stochastic
    genotoxic mechanism.
 6  1,4-Dioxane
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<pre> hapter    6
           Carcinogenicity
6.1        Non-human information
           Data on animal carcinogenicity studies are summarized in Table 6.
 able 6 Summary of animal carcinogenicity studies on 1,4-dioxane exposure.
 pecies    Design               Exposure levels      Observations and remarks (Klimisch score)*           References
nhalation
 at        50 males*/group;     0, 50, 250, 1,250    Klimisch-score: 1                                      Kasai et al.,
 344/      study duration: 6 h/ ppm (v/v)                                                                   200949
DuCrj      day, 5 days/wk for (calculated as 180, Neoplastic lesions: +
           104 weeks;           900 and 4,500 mg/ Significant induction of nasal squamous cell
           hematology, clinical m3) by inhalation    carcinomas, hepatocellular adenomas, peritoneal
           biochemistry, gross (whole body           mesotheliomas and subcutis fibroma (see Table 7).
           necropsy and         vaporisation         General: Decreased survival rate at 250 and 1,250
           histopathological    technique);          ppm towards end of 2-yr exposure period. At 1,250
           examination                               ppm terminal body weights decreased, relative liver
                                Actual exposure      weight increased and plasma ALT, AST and gamma-
           *Reason for          levels were:         GTP enzyme activities increased.
           selecting male       50.2 + 1.4           Non-neoplastic lesions: Increased incidences of
           animals was the      250.9 + 3.2          nuclear enlargement in respiratory and olfactory
           absence of           1,247.5 + 18.6 ppm epithelia in all exposed. Increased incidences of
           mesotheliomas in                          nuclear enlargement in liver of 1,250 ppm and in
           females in a                              kidney of 250 and 1,250 ppm exposed groups.
           previous 2-year oral                      Statistically significant inflammation and necrosis,
           study with 1,4-                           recurrent cell death and repair in respiratory and
           dioxane (Kano                             olfactory epithelia and atrophy in olfactory
           et al., 2009)48                           epithelium, hydropic change and sclerosis of lamina
           Carcinogenicity                                                                                             37
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<pre>                                                         propria and proliferation nasal gland within exposed
                                                         groups.
                                                         At 1,250 ppm necrosis of hepatocytes and hydropic
                                                         changes in renal proximal tubule were observed as
                                                         well as squamous cell hyperplasia in nasal cavity and
                                                         altered cell foci in liver. At 250 ppm and above
                                                         squamous cell metaplasia was observed
 at          288 rats/sex for      111 ppm (400 mg/      Klimisch-score: 3 (methodological deficiency as no       Torkelson et al.,
Wistar       dose group; 192       m3) by inhalation     MTD was used at selecting concentration levels)          197450
             rats/sex for control; (whole body)
             study duration 7 hr/                        Neoplastic lesions: -
             day, 5 days/wk,                             No substance-related tumours found.
             during 2 years;                             General: no observable substance-related effects with
             haematology,                                respect to behaviour, growth, or mortality rate. no
             clinical                                    differences between control and exposed animals on
             biochemistry, Gross                         haematology and clinical chemical, all were within
             necropsy and                                the physiological limits; no substance-related gross
             histopathological                           and microscopic findings
             examinations
Oral administration
 at          50 animals/sex/       0, 0.02, 0.1, 0.5%    Klimisch-score: 2                                          Yamazaki
 344/        group;                (w/w) in drinking                                                                et al., (1994),
DuCrj        study duration 104    water (ad libitum)    Neoplastic lesions: +                                      Japan
             weeks;                                      Significant induction of nasal squamous cell carcinomas Bioassay
             haematology,          Actual dose levels: in females and hepatocellular adenomas and carcinomas Research
             clinical              m: 0, 11, 55, 274     in males and females, peritoneal mesotheliomas in          Center (1998)
             biochemistry, gross   mg/kg bw/day;         males, and mammary gland adenomas in females (see Summarised
             necropsy and          f: 0, 18, 83, 429 mg/ Table 8).                                                  by Kano
             histopathological     kg bw/day             General: Significantly decreased survival rates at 0.5%; et al., 200948
             examination                                 retarded growth rates and decreased terminal body
                                                         weights; relative liver weights significantly increased in
                                                         0.1 and 0.5% dosed males and 0.5% dosed females; no
                                                         effect on food nor water consumption
Mouse        50 animals/sex/       0, 0.05, 0.2, 0.8% Klimisch-score: 2                                             Yamazaki
 rj:BDF1     group;                w/w) in drinking                                                                 et al., (1994),
             study duration 104 water (ad libitum). Neoplastic lesions: +                                           Japan
             weeks;                                      Significant induction of hepatocellular tumours in both Bioassay
             haematology,          Actual dose levels: sexes. Two nasal tumours in the highest dose groups for Research
             clinical              m: 0, 49, 191, 677 tumour incidences (see Table 9).                              Center
             biochemistry, gross mg/kg bw/day;           General: Significantly decreased survival rates at 0.2     (1998),
             necropsy and          f: 0, 66, 278, 964    and 0.8% females. Significantly retarded growth rates Summarised
             histopathological     mg/kg bw/day          and terminal body weights in 0.2 and 0.8% males and        by Kano et
             examination                                 females. Relative liver weight significantly increased in al., 200948
                                                         0.8% males and females and in 0.2% males;
                                                         significantly decreased food and water consumption in
                                                         0.8% males and females
 8          1,4-Dioxane
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<pre> at      60 animals/sex/    0, 0.01, 0.1, 1% in  Klimisch-score: 2                                          Kociba et al.,
 herman  group;             drinking water (ad                                                              197451
         study duration 716 libitum)             Neoplastic lesions: +
         days; haematology,                      Treatment related hepatocellular carcinomas and nasal
         gross necropsy and Actual dose levels squamous cell carcinomas (see Table 10).
         histopathological  m: 0, 9.6, 94, 1,015 General: Body weights were significantly lower in
         examination        mg/kg bw/day         animals exposed to 1% than controls. water
                            f: 0, 19, 148, 1,599 consumption was slightly less in animals exposed to 1%
                            mg/kg bw/day         than controls; severe reduction in survival rate of
                                                 animals exposed to 1% during first 4 months of study (p
                                                 <0.05); after 4 month survival rate was the same for all
                                                 groups; a significantly increased liver weight and liver/
                                                 body weight ratio in rats exposed to 1% 1,4-dioxane;
                                                 gross and histopathological examination revealed
                                                 variable degrees of renal tubular epithelial and
                                                 hepatocellular degeneration and necrosis, accompanied
                                                 by regenerative activities in liver (hepatocellular
                                                 hyperplastic nodule formation) and renal tubuli in rats at
                                                 0.1 and 1.0%.
                                                 No difference between control and exposed animals on
                                                 haematology
 at      35 rats/sex/group; 0, 0.5, 1% (v/v) in Klimisch-score: 2                                           NCI 197852
Osborne- study duration 110 drinking water (ad
Mendel   weeks; gross       libitum).            Neoplastic lesions: +
         necropsy and                            Significant induction of nasal squamous cell carcinomas
         histopathological  Actual dose levels in males and females and hepatocellular adenomas in
         examination        m: 0, 240, 530 mg/ females (see Table 11).
                            kg bw                General: a significant positive dose-related trend in
                            f: 0, 350, 640 mg/kg mortality; no clinical signs other than fluctuations in
                            bw                   mean body weights of males probably due to mortality.
                                                 Histopathology:
                                                 Tubular degeneration in kidney
                                                 Liver cytomegaly
                                                 Gastric ulceration of stomach:
                                                 - m: 0/33, 5/28, 5/30
                                                 Pneumonia:
                                                 - m: 8/30, 15/31, 14/33
                                                 - f: 6/30, 5/34, 25/32
Mouse    50 mice/sex/group; 0, 0.5, 1% (v/v) in Klimisch-score: 2                                           NCI 197852
 6C3F1   study duration 90  drinking water (ad
         weeks; gross       libitum).            Neoplastic lesions: +
         necropsy and                            Significant induction of hepatocellular adenomas or
         histopathological  Actual dose levels carcinomas in females and males (see Table 12).
         examination        m: 0, 720, 830 mg/ General: A significant positive dose-related trend in
                            kg bw/day            mortality for females.
                            f: 0, 380, 860 mg/kg Pneumonia:
                            bw/day               - m: 1/49, 9/50, 17/47
                                                 - f: 2/50, 33/47, 32/36
                                                 Rhinitis:
                                                 - m: 0/49, 1/50, 1/49
                                                 - f: 0/50, 7/48, 8/39
                                                 No clinical signs other than altered body weights
         Carcinogenicity                                                                                               39
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<pre>  at        30 male/group;        0, 0.75, 1.0, 1.4,   Klimisch-score: 3 (only one sex; limited reporting of       Hoch-Ligeti
 D          study duration 13     1.8% drinking        results, no tables and graphs, limited duration)            et al., 197053
            months; necropsy at   water (ad libitum).
            16 months; gross      Total dose/rat based Neoplastic lesions: -
            necropsy;             on a daily fluid     Non-neoplastic lesions:
            histopathological     intake of 36 ml:     Nasal cavity, squamous cell carcinomas (0, 0.75, 1.0,
            examination only in   104, 142, 191, 198,  1.4, 1.8%):
            nasal cavity with     213 and 256 gram.    0/30, 1/30,1/30, 2/30, 2/30
            gross lesions         Using a ref. body
                                  weight of 0,523 kg
                                  chronic exposure
                                  male CD: 0, 430,
                                  574, 803, 1,032 mg/
                                  kg bw/day)
  at        26 exposed males, 9 0, 1% in drinking      Klimisch score: 3 (rats received 1 wk terramycin prior to   Argus et al.,
Wistar,     control males; study water (ad libitum)    start test; limited number of rats; one sex; only one dose, 196554
            duration 63 wk;       (using a ref. body   limited duration; Control group of 9 rats).
            gross necropsy and weight of 0,462 kg      Neoplastic lesions:
            histopathological     chronic exposure     (0 and 1%, respectively):
            examination           male Wistar: 640     - Lymphosarcoma: 1/9, 0/26
                                  mg/kg bw/day)        - Liver tumours: 0/9, 6/26
                                                       - Kidney cell carcinoma: 0/9, 1/26
                                                       Histological changes in liver
Osborne rat 35/sex/group; study 0,.5 and 1.0 % in      Klimisch score: 3 (minimal reported; purity not             King et al.,
 nd         duration 42 weeks. drinking water          specified)                                                  197355
  6C3F1     Control group 34      0.5 and 1.0% in diet
mice        weeks                                      Neoplastic lesions: -
                                                       General: Mortality only in rats; increased weight gain in
                                                       male rat and mice; histopathological lesions of lung and
                                                       liver in rats only
Guinea pig  24 Guinea pigs;       0.5-2% in drinking Klimisch score 4                                              Hoch-Ligeti
            study duration 23     water                                                                            and Argus
            months                                     Neoplastic lesions: 2 gallbladder carcinomas; 3 early       (1970)53
                                                       hepatomas; 1 kidney adenoma
ntraperitoneal injection
Mice A/J    16/sex/group; study   Intraperitoneal:     Klimisch score: 3 (Limited gross necropsy and               Stoner
 ulmonary duration 24 weeks;      0, 4,800, 12,000,    histopathology; short duration)                             et al., 198656
umour       gross necropsy of     and 24,000 mg/kg     Neoplastic lesions:
 ssay       limited organs (liver bw                   Intraperitoneal, lung tumours
            kidney, spleen                              (0, 4,800, 12,000, 24,000, respectively):
            intestines, stomach,  Oral: 0 and 24,000 - m: 1/14, 1/16, 6/16, 2/11
            thymus and salivary   mg/kg bw             - f: 7/15, 3/16, 5/16, 3/13
            and endocrine                              Oral, lung tumours (0 and 24,000, respectively):
            glands);              3 applications/wk - m: 51/135 and 4/15
            histopathological     for 8 weeks,         - f: 32/131 and 5/14
            examination of        followed by 16 wks
            gross lesions; lungs  observation
            and livers examined
            on tumours
 0          1,4-Dioxane
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<pre>Mice A/J    30 males/group;     0, 400, 1,000 and    Klimisch score: 3 (only lung tumours studied, short         Maronpot
 ulmonary   study duration 16   2,000 mg/kg bw;      duration)                                                   et al., 198657
umour       weeks; removal of   3 applications/wk
 ssay       lungs and           for 8 weeks,         Neoplastic lesions:
            histopathological   followed by 8 wks    Lung tumours in %( 0, 400, 1,000, and 2,000
            examination         observation          respectively): 33, 17, 48, and 62
Dermal administration
Mice,       30/sex/group; study 3 applications/wk    Klimisch score: 3 (minimal reported; purity not             King et al.,
 wiss-      duration 78 weeks.  of 0.2 mM 1,4-       specified)                                                  197355
Webster     gross necropsy and  dioxane solution in
            histopathological   acetone on shaved    Neoplastic lesions: no papilloma, one malignant
            examination.        back for 78 wks.     lymphoma. One suspected carcinoma (f) and one
                                Acetone as negative  subcutaneous tumour (m)
                                control              General: increase in male body weight
Osborne rat 35/sex/group; study 0,.5 and 1.0 % in    Klimisch score: 3 (limited test design no haematology King et al.,
 nd         duration 42 weeks. drinking water;       clinical biochemistry; minimal reported; purity not         197355
  6C3F1     Control group 34    0.5 and 1.0% in diet specified)
mice        weeks
                                                     General: Mortality only in rats; increased weight gain in
                                                     male rat and mice.
                                                     Histopathologic lesions in the lung and liver in rats only.
  See Annex H.
6.1.1       Carcinogenicity: inhalation
            Male F344/DuCrj rats (50/group) were whole-body exposed to 0, 180, 900 and
            4,500 mg 1,4-dioxane/m3, for 6 hours a day, 5 days/week for 104 weeks (Kasai
            et al., 2009).49 Details on tumour incidences are shown in Table 7. In summary,
            1,4-dioxane induced a statistically significant increase in hepatocellular
            adenomas (highest exposure group only), peritoneal mesothelioma (two highest
            exposure groups), and in nasal squamous cell carcinoma (highest exposure group
            only). The investigators also reported on pre-neoplastic lesions, such as
            squamous cell metaplasia, characterized by replacement of transitional and
            respiratory epithelia by squamous epithelium with or without keratinisation
            occurred in rats exposed to 900 mg/m3 and higher. In addition, increased
            incidences of nuclear enlargement in the respiratory and olfactory epithelia, and
            atrophy and respiratory metaplasia in the olfactory epithelium, were noted in the
            nasal cavity of male rats exposed at 180 mg 1,4-dioxane/m3 and higher.
            Torkelson exposed Wistar rats to 400 mg 1,4-dioxane/m3 for 7 hours a day, five
            days a week for a total of 2 years.50 The substance did not induce neoplastic
            lesions.
            Carcinogenicity                                                                                                  41
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<pre>      Table 7 Tumour incidences in male rats exposed to 1,4-dioxane for 2 years (Kasai et al., 2009).49
      Exposure level (ppm, by inhalation)                    0          50         250          1,250
      • Nose cavity: squamous cell carcinoma                 0           0          1            6*
      • Liver: heptocellular adenoma                         1           2          3           21**
      • Liver: hepatocellular carcinoma                      0           0          1            2
      • Kidney: renal cell carcinoma                         0           0          0            4
      • Peritoneum: mesothelioma                             2           4         14**         41**
      • Mammary gland: fibroadenoma                          1           2          3            5
      • Mammary gland: adenoma                               0           0          0            1
      • Zymbal gland: adenoma                                0           0          0            4
      • Subcutis: fibroma                                    1           4          9**          5
      Fischer exact test: * p<0.05, ** p<0.01
6.1.2 Carcinogenicity: oral administration
      A number of animal carcinogenicity studies have been performed in which
      animals received 1,4-dioxane orally in drinking water (see Table 6). Regarding
      the well-performed studies, all showed that 1,4-dioxane induced tumours in for
      instance the nasal cavity and the liver of rats and mice. Details on tumour
      incidences for the distinctive studies are shown in the Tables 8 to 12. In addition,
      Table 8 Tumour incidences in rats exposed to 1,4-dioxane for 2 years (Kano et al., 2009).48
      Exposure level (%, w/w, in drinking water)             0           0.02      0.1          0.5
      Male rats (mg/kg bw/day)                               0          11         55           274
      • Nose cavity: squamous cell carcinoma                 0           0          0             3
      • Liver: hepatocellular adenoma                        3           4          7            32**
      • Liver: hepatocellular carcinoma                      0           0          0            14**
      • Liver: combined hepatocellular adenoma or            3           4          7            39**
         carcinoma
      • Peritoneum: mesothelioma                             2           2          5            28**
      • Mammary gland: fibroadenoma or adenoma               1           2          2             6
      • Subcutis: fibroma                                    5           3          5            12
      Female rats (mg/kg bw/day)                             0          18         83           429
      • Nose cavity: squamous cell carcinoma                 0           0          0             7**
      • Liver: hepatocellular adenoma                        3           1          6            48**
      • Liver: hepatocellular carcinoma                      0           0          0            10**
      • Liver: combined hepatocellular adenoma or            3           1          6            48**
         carcinoma
      • Peritoneum: mesothelioma                             1           0          0             0
      • Mammary gland: fibroadenoma or adenoma               8           8         11            18*
      • Subcutis: fibroma                                    0           2          1             0
      Fischer exact test: * p<0.05, ** p<0.01
 2    1,4-Dioxane
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<pre>Table 9 Tumour incidences in mice exposed to 1,4-dioxane for 2 years (Kano et al., 2009).48
Exposure level (%, w/w, in drinking water)                0         0.05       0.2     0.8
Male mice (mg/kg bw/day)                                  0       49        191        677
• Nose cavity: adenocarcinoma                             0         0          0           0
• Nose cavity: esthesioneuroepithelioma                   0         0          0           1
• Liver: hepatocellular adenoma                           9       17          23**       11
• Liver: hepatocellular carcinoma                       15        20          23         36**
• Liver: combined hepatocellular adenoma or             23        31          37**       40**
   carcinoma
Female mice (mg/kg bw/day)                                0       66        278        964
• Nose cavity: adenocarcinoma                             0         0          0           1
• Nose cavity: esthesioneuroepithelioma                   -         -          -           -
• Liver: hepatocellular adenoma                           5       31**        20**         3
• Liver: hepatocellular carcinoma                         0         6*        30**       45**
• Liver: combined hepatocellular adenoma or               5       35**        41**       46**
   carcinoma
Fischer exact test: * p<0.05, ** p<0.01.
Table 10 Tumour incidences in male and female rats (combined) exposed to 1,4-dioxane for 2 years
(Kociba et al., 1974).51
Exposure level (%, in drinking water)                   0         0.01      0.1          1
• Nose cavity: squamous cell carcinoma                  0         0         0            3***
• Liver: hepatocellular carcinoma                       1         0         1          10**
• Liver: hepatic tumours all types                      2         0         1          12*
Fisher exact probability test: *p=0.00022, **p=0.00033, ***p=0.05491.
Table 11 Tumour incidences in rats exposed to 1,4-dioxane for 2 years (NCI 1978).52
Exposure level (%, v/v, in drinking water)              0         0.5       1.0
Male rats
• Nose cavity: adenocarcinoma                           0/33        1/35      3/34
• Nose cavity: squamous cell carcinoma                  0/33      12/33     16/34***
• Nose cavity: rhabdomyoma                              0/33        1/33      0/34
• Liver: hepatocellular adenoma                         2/31        2/31      1/33
• Liver: hepatocellular carcinomas                      0/31        1/31      0/33
• Testis/epididymis: mesothelioma                       2/33        4/33      5/34
Female rats
• Nose cavity: adenocarcinoma                           0/33        0/35      1/35
• Nose cavity: squamous cell carcinoma                  0/34      10/35*** 8/35****
• Nose cavity: rhabdomyoma                              -         -         -
• Liver: hepatocellular adenoma                         0/31      10/33     11/32**
Fischer exact test: * p<0.05, ** p<0.01, *** p<0.001, **** p=0.003.
Carcinogenicity                                                                                  43
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<pre>              Table 12 Tumour incidences in mice exposed 1,4-dioxane for 2 years (NCI 1987).52
              Exposure level (%, v/v, in drinking water)              0            0.5          1.0
              Male mice
              • Nose cavity: adenocarcinoma                           0/49           0/50         1/47
              • Liver: heptocellular adenoma or carcinoma             8/49          19/50**** 28/47***
              Female mice
              • Nose cavity: adenocarcinoma                           0/50           1/48         0/37
              • Liver: heptocellular adenoma or carcinoma             0/50          21/48       35/37***
              Fischer exact test: * p<0.05, ** p<0.01, *** p<0.001, **** p=0.014.
              the tumour development was preceded by the induction of non-neoplastic
              lesions, which progressed to hepatocellular adenoma and carcinoma in rats and
              mice and to nasal squamous cell carcinoma in rats at higher dosages. Liver
              tumours were observed at higher tumour incidences in rats and mice from a
              concentration of approximately 0.05% 1,4-dioxane and higher, whereas
              neoplastic lesions in the nose were observed in rats at a concentration of 0.5%
              1,4-dioxane and higher.
6.1.3         Carcinogenicity: dermal exposure and other routes of exposure
              The Committee noted the low quality of the animal carcinogenicity studies on
              dermal exposure and administration of 1,4-dioxane by intraperitoneal injection.
              For this reason, the Committee considers these studies not relevant in assessing
              the carcinogenic properties of the substance.
6.2           Human information
 able 13 Summary of human studies.
Method            Population          Exposure level    Results and remarks             Quality and/or   References
                                                                                        reliability of
                                                                                        study
  ross sectional 74 workers           Concentrations    No evidence of liver of kidney Low (secondary    Thiess et al., 1976
 tudy; Germany exposed to             up to 54 mg/m3    cancer no higher cancer deaths source, no other  (source EU risk
                  unspecified                           than population at large. Two study details      assessment report
                  airborne levels                       pensioned employees died and given)              2002)47
                  for 3-41 years                        were diagnosed cancer:
                                                        squamous epithelial carcinoma
                                                        and myelofibrosis leukaemia
  4           1,4-Dioxane
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<pre>Mortality          165 employees      < 25 ppm (~         Manufacturing department:         Low              Buffler et al., 197858
ollow-up study;    exposed to 1,4-    90 mg/m3),          seven deaths, two from cancer
USA, chemical      dioxane since      during 28-89        (expected 4.9 and 0.9);
 ompany plant      1954               months              processing department: five
                                                          deaths of which one from
                                                          cancer (expected 4.9 and 0.8)
  etrospective     80 men             0.18-184 mg/m3      No signs of exposure related Low (secondary Barber, 1934 (source
 tudy                                 for some years      health effects                    source, no other EU risk assessment
                                                                                            study details    report 2002)47
                                                                                            given)
              Data on human carcinogenicity are shown in Table 13. The Committee noted the
              low quality of study reporting, in that data were obtained from secondary
              sources, and that study details were missing. Also, the size of the cohorts, and
              thus the power of the studies, were low. In none of the studies evidence for
              carcinogenicity due to occupational exposure to 1,4-dioxane could be assessed.
6.3           Other relevant information
 able 14 Initiation/promotion and cell transformation studies.
Method            Cell type              Concentration                 Results and remarks          Klimisch          References
                                                                                                    Score*
nitiation/promotion studies
Mice, SENCAR 25-40 females/dose;         1,000 mg/kg bw oral,          -                            2                 Bull et al.,
                  early papilloma        subcutaneous, or dermal                                                      198659
                  development as         for 2 wks, followed by 1
                  potential predictor    µg TPA dermal 3x/wk for
                  of carcinoma yields    20 wks. A single dose of
                                         1,000 mg/kg bw in a
                                         satellite group followed by
                                         acetone dermal served as
                                         negative control. TPA is a
                                         tumour promotor
  at              8-9 male/group         Partial hepatectomy of rats   + (Increase in number and 2                    Lundberg
  D               GGT-enzyme             was followed by 30 mg         total volume of foci only                      et al., 198760
                  altered foci of        intraperitoneal treatment     at toxic doses of 1,000
                  hepatocytes            with diethynitrosamine        mg/kg bw)
                  determined             DENA/kg (initiator).
                  10 days after last     Thereafter treatment with
                  treatment sacrifice    0, 100 and 1,000 mg 1,4-
                  and staining liver     dioxane/kg bw (gavage 1/
                  sections for GGT       d, 5 times/wk for 7 weeks.
                                         Controls with and without
                                         DENA initiation included
              Carcinogenicity                                                                                                    45
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<pre>Mice, Swiss-       30/sex/group; study  50 µg DMBA (dimethyl-        + Neoplastic lesions of     3 (limited test    King et al.,
Webster            duration 78 weeks.   benzanthracene)              skin, lung and kidney in    design no          197355
                   Gross necropsy and   for 1 wk, as initiator,      survivors: 4 papillomas     haematology
                   histopathology       followed by 3                (2m, 2f); 6 suspected       clinical
                                        applications/wk of 0.2 mM    carcinomas (3m, 3f); 2(m)   biochemistry;
                                        1,4-dioxane solution on      subcutaneous tumours.       minimal reported;
                                        shaved back for 78 wks.                                  purity not
                                        Acetone was the negative     Skin tumours increased      specified)
                                        control and croton oil the   sharply after 10 weeks. No
                                        positive control             skin tumours observed
                                                                     after dermal application in
                                                                     absence of DMBA
                                                                     initiation (Table 8).
                                                                     General: mortality up to
                                                                     25/36 after 60 weeks
  ell transformation
                   Balb/3T3 cells       0, 0.25, 0.5, 1.0, 2.0, 3.0, + (at cytotoxic             2                  Sheu et al.,
                                        4.0 mg/ml; 48 hr and 13      concentrations of 2 mg/                        198861
                                        days treatment; positive     ml)
                                        and negative controls
                                        included
                   Balb/3T3 cells       + and -S9                    - (with and without S9)     4                  Microbial
                                                                                                                    Associates
                                                                                                                    1980 (source
                                                                                                                    EU-risk
                                                                                                                    assessment
                                                                                                                    report)47
 iver pre-         Gpt delta transgenic 0, 200, 1,000 or 5,000 ppm - (0 to 1,000 ppm)            4 (poster abstract Fukushima
 eoplastic         rats, males; 30      in drinking water for up to + (5,000 ppm) for GST-P- only; no details on et al., 200938
marker             animals divided in 16 weeks; at the end of        positive foci (p<0.001), methods or
glutathione S- four groups (no. of treatment all animals were and PCNA-positive cell outcomes
ransferase,        animals per group    killed, and livers excised index (p<0.001)               reported)
 lacental form); not given)             for further analyses
 ell proliferation
PCNA-positive
ndex); see
 able 4.
  See Annex H.
                   As summarized in Table 14, 1,4-dioxane was clearly positive in a liver foci
               assay, (Lundberg et al., 1987), while a mouse skin papilloma test with a single
               dose of 1,4-dioxane was negative (Bull et al., 1986).59,60 No peroxisomal
               proliferation activity was observed after oral dosing with 1,4-dioxane (1% and
               2% in drinking water for 5 days in two studies; Goldsworthy et al., 1991, see
               Table 4).30
 6             1,4-Dioxane
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<pre>6.4 Summary and discussion of carcinogenicity
    Only a few epidemiological studies are available concerning the carcinogenic
    properties of 1,4-dioxane; they show no indications for carcinogenicity.
    However, as these studies have limited power, the Committee is of the opinion
    that the human data are insufficient for conclusions.
        Two carcinogenicity studies have been conducted, in which rats were
    exposed by inhalation to 1,4-dioxane vapour. In a recent study (Kasai et al.,
    2009), male F344/DuCrj rats were exposed to 1,4-dioxane concentrations of 180,
    900 and 4,500 mg/m3 (50, 250 and 1,250 ppm) for 2 years, 6 h/day, 5 days/wk.49
    In this study, an increased incidence of squamous cell carcinoma in the nasal
    cavity and hepatocellular adenoma in the liver was observed after exposure to
    4,500 mg/m3. Moreover, the incidence of peritoneal mesothelioma was
    statistically significant increased (dose dependently) after exposure to 900 and
    4,500 mg/m3 as well. Non-neoplastic and pre-neoplastic changes in the nasal
    cavity (nuclear enlargement of the olfactory and respiratory epithelium, and
    atrophy and metaplasia of the olfactory epithelium) were observed at the lowest
    exposure level, 180 mg/m3, and above. In the inhalation study of Torkelson,
    Wistar rats were exposed to 400 mg 1,4-dioxane/m3 for 7 hours a day, five days a
    week for a total of 2 years (Torkelson et al., 1974).50 The substance did not
    induce neoplastic lesions, probably because the exposure was too low. Moreover,
    the nasal cavity was not examined. Therefore, the Committee decided that this
    study cannot be used to indicate a lack of carcinogenic potential of 1,4-dioxane.
    1,4-Dioxane has been shown to be carcinogenic in several drinking water studies
    in rats, mice and guinea pigs (Kano et al., 2008, 2009).48,62 The target organs
    were the liver, and nasal cavities, while also peritoneal mesothelioma were
    induced. The relevance of the effects on the nasal cavity for humans after
    exposure via drinking water was questioned by Stickney et al., (2003).63
    Although the nasal lesions and nasal tumours were consistently seen after
    exposure to 1,4-dioxane through the drinking water, such lesions could result
    from water entering the nasal cavity when the animals drink from sipper bottles
    (Sweeney et al., 2008).10 However, because nasal tumours were also observed
    after inhalatory exposure in rats, these are considered relevant for humans by the
    Committee.
    Carcinogenicity                                                                    47
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<pre>6.5 Comparison with criteria
    According to the criteria in Annex VI of the European regulation No. 1272/2008
    substance, classification as a known or presumed human carcinogen is warranted
    when positive evidence for carcinogenicity is obtained in humans (category 1A),
    or rodents (category 1B). In humans, no evidence for carcinogenicity is found.
    However, the Committee is of the opinion that the studies of Kasai et al., 2009
    and Kano et al., 2008, 2009 show consistent carcinogenic effects (hepatocellular
    adenoma, squamous cell carcinoma in the nasal cavity and peritoneal
    mesothelioma) after exposure to dioxane by inhalation and via drinking water
    respectively.48,49,62 Because of these sound positive studies of Kasai et al., 2009
    and Kano et al., 2008, 2009, the Committee recommends classifying 1,4-dioxane
    as a substance that is presumed to have carcinogenic potential for humans. This
    corresponds with a classification in category 1B.
         The Committee noticed that from 2000, the European Commission classified
    the substance as a carcinogen in category 2 (according to the current CLP-
    system). The classification was based on other carcinogenicity studies as
    described in the present report.
6.6 Conclusions on classification and labelling
    Based on the available data, the Committee concludes that 1,4-dioxane is
    presumed to be carcinogenic to man, and recommends classifying the substance
    for carcinogenicity in category 1B.*
    See for classification system Annex F.
 8  1,4-Dioxane
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<pre>  References
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  ECHA. 1,4-Dioxane. ECHA data base. www.echa.europa.eu Consulted: 28-10-2015
  Health Council of the Netherlands. Rapport inzake grenswaarde 1,4-Dioxaan. Gezondheidskundig
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0 Sweeney L, Thrall K, Poet T, et al. Physiologically based pharmacokinetic modeling of 1,4-Dioxane
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1 Marzulli F, Anjo D, Maibach H. In vivo skin penetration studies of 2,4-toluenediamine,
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  References                                                                                        49
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<pre>2 ECETOC. 1,4-dioxane. European Environment Agency; 1983: JACC 002.
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8 Woo Y, Areas I, Argus M, Griffin G, Nishiyama Ka. Structural identification of p-dioxane-2-one as
  the major metabolite of p-dioxane. Naunyn-Schmiedeberg's Arch Pharmacol 1977; 299: 283-87.
9 Woo Y, Arcos J, Argus M. Metabolism in vivo of dioxane: identification of p-dioxane-2-one as the
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0 Woo YT, Argus MF, Arcos JC. Effect of mixed-function oxidase modifiers on metabolism and
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1 Dietz FK, Stott WT, Ramsey JC. Nonlinear pharmacokinetics and their impact on toxicology:
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2 SCOEL. Recommendation from Scientific Committee on Occupational Exposure Limits for
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3 Morita T, Hayashi M. 1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral
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4 Stott W, Quast J, Watanabe PG. Differentiation of the mechanism of carcinogenicity of 1,4-dioxane
  and 1,3 hexachlorobutadiene in the rat. Toxicol Appl Pharmacol 1981; 60: 287-300.
5 Haworth S, Lawlor T, Mortelmans K, Speck W, Zeiger E. Salmonella mutagenicity test results for
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6 Nestmann ER, Otson R, Kowbel DJ, Bothwell PD, Harrington TR. Mutagenicity in a modified
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7 Khudolei VV, Mizgirev IV, Pliss GB. [Mutagenic activity of carcinogens and other chemical agents
  in Salmonella typhimurium tests]. Vopr Onkol 1986; 32(3): 73-80.
8 McGregor DB, Brown AG, Howgate S, McBride D, Riach C, Caspary WJ. Responses of the L5178Y
  mouse Lymphoma cell forward mutation assay. V: 27 coded chemicals. Environ Mol Mutagen 1991;
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9 Galloway S, Armstrong MJ, Reuben C, Colman S. Chromosome aberrations and sister chromatid
  exchanges in Chinese hamster ovary cells; evaluations of 108 chemicals. Environ Mol Mutag 1987;
  10: 1-175.
0 1,4-Dioxane
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<pre>0 Goldsworthy TL, Monticello TM, Morgan KT, Bermudez E, Wilson DM, Jäckh R. et al. Examination
  of potential mechanisms of carcinogenicity of 1,4-dioxane in rat nasal epithelial cells and
  hepatocytes. Arch Toxicol 1991; 65(1): 1-9.
1 Sina J, Bean C, Dysart G, et al. Evaluation of the alkaline elution/rat hepatocyte assay as a predictor
  of carcinogenic/mutagenic potential. Mutation Res 1983; 113: 357-91.
2 Zimmermann FK, Mayer V, Scheel I, Resnick MA. Acetone, methyl ethyl ketone, ethyl acetate,
  acetonitrile and other polar aprotic solvents are strong inducers of aneuploidy in Saccharomyces
  cerevisiae. Mutation Res 1985; 149: 339-51.
3 Morita T. No clastogenicity of 1,4-dioxane as examined in the mouse peripheral blood micronucleus
  test. MMS Communications 1994; 2: 7-8.
4 McFee A, Abbott M, Gulati D, et al. Results of mouse bone marrow micronucleus studies on
  1,4-dioxane. Mutation Res 1994; 322: 145-8.
5 Mirkova E. Activity of the rodent carcinogen 1,4-dioxane in the mouse bone marrow micronucleus
  assay. Mutation Res 1994; 322: 142-4.
6 Roy S, Thilagar A, Eastmond D. Chromosome breakage is primarily responsible for the micronuclei
  induced by 1,4-dioxane in the bone marrow and liver of young CD-1 mice. Mutat Res 2005; 586(1):
  28-37.
7 Tinwell H, Ashby J. Activity of 1,4-dioxane in mouse bone marrow micronucleus assays. Mutation
  Research 1994; 332: 141-150.
8 Fukushima S, Wei M, Omori M, Morimura K, Kinoshita A, Musumura K ea. Carcinogenicity and in
  vivo mutagenicity of 1,4-dioxane in gpt delta rats. Experimental and Toxicologic Pathology 2009;
  61(poster abstract): 282.
9 Yoon J, Mason J, Valencia R, Woodruff R, Zimmering S. Chemical mutagenesis testing in
  Drosophila. IV Results of 45 coded compounds tested for the National Toxicology program. Environ
  Mutagen 1985; 7: 349-67.
0 Muñoz E, Mazar B. The rodent carcinogens 1,4-dioxane and thiourea induce meiotic non-disjunction
  in Drosophila melanogaster females. Mutat Res 2002; 517(1-2): 231-8.
1 BASF. Unveröffentlichte Untersuchungen. XXV/431. 21-3-1977. Abteilung Toxikologie.
2 Kitchin K, Brown J. Is 1,4-dioxane a genotoxic carcinogen? Cancer Lett 1990; 53: 67-71.
3 Miyagawa M, Shirotori T, Tsuchitani M, Yoshikawa K. Repeat-assessment of 1,4-dioxane in a rat-
  hepatocyte replicative DNA synthesis (RDS) test: evidence for stimulus of hepatocyte proliferation.
  Exp Toxicol Pathol 1999; 51(6): 555-558.
4 Uno Y, Takasawa H, Miyagawa M, Inoue Y, Yoshikawa K. An in vivo-in vitro replicative DNA
  synthesis (RDS) test using rat hepatocytes as an early prediction assay for nongenotox
  hepatocarcinogens screening of 22 known positives and 25 noncarcinogens. Mutation Research
  1994; 320: 189-205.
5 Kurl RN, Poellinger L, Lund J, Gustafsson JA. Effects of dioxane on RNA synthesis in the rat liver.
  Arch Toxicol 1981; 49(1): 29-33.
  References                                                                                              51
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<pre>6 Hellmer L, Bolcsfoldi G. An evaluation of the E. coli K-12 uvrB/recA DNA repair host-mediated
  assay. I. In vitro sensitivity of the bacteria to 61 compounds. Mutat Res 1992; 272(2): 145-160.
7 European Union risk assesment report: 1,4-Dioxane. 2002: EUR 19833 EN, 1-129.
8 Kano H, Umeda Y, Kasai T, Sasaki T, Matsumoto M, Yamazaki K et al. Carcinogenicity studies of
  1,4-dioxane administered in drinking-water to rats and mice for 2 years. Food Chem Toxicol 2009;
  47(11): 2776-2784.
9 Kasai T, Kano H, Umeda Y, Sasaki T, Ikawa N, Nishizawa T et al. Two-year inhalation study of
  carcinogenicity and chronic toxicity of 1,4-dioxane in male rats. Inhal Toxicol 2009; 21(11):
  889-897.
0 Torkelson T, Leong B, Kociba R, Richter WA, Gehring PA, . 1,4-Dioxane. II. Results of a 2-year
  inhalation study in rats. Toxicol Appl Pharmacol 1974; 30: 287-98.
1 Kociba R, McCollister S, Park C, et al. Results of a 2-year ingestion study in rats. Toxicol Appl
  Pharmacol 1974; 30: 275-286.
2 National Cancer Institute. Bioassay of 1,4-dioxane for possible carcinogenicity. 1978: 80. Internet:
  PM:12830218.
3 Hoch-Ligeti C, Argus MF, Arcos JC. Induction of carcinomas in the nasal cavity of rats by dioxane.
  Br J Cancer 1970; 24(1): 164-167.
4 Argus MF, Arcos JC, Hochligeti C. Studies on the carcinogenic activity of protein-denaturing agents:
  hepatocarcinogenicity of dioxane. J Natl Cancer Inst 1965; 35(6): 949-958.
5 King ME, Shefner AM, Bates RR. Carcinogenesis bioassay of chlorinated dibenzodioxines and
  related chemicals. Environm Health Perspectives 1973; 163-170.
6 Stoner GD, Conran PB, Greisiger EA, Stober J, Morgan M, Pereira MA. Comparison of two routes of
  chemical administration on the lung adenoma response in strain A/J mice. Toxicol Appl Pharmacol
  1986; 82(1): 19-31.
7 Maronpot RR, Shimkin MB, Witschi HP, Smith LH, Cline JM. Strain A mouse pulmonary tumor test
  results for chemicals previously tested in the National Cancer Institute carcinogenicity tests. J Natl
  Cancer Inst 1986; 76(6): 1101-1112.
8 Buffler P, Wood SM, Suarex L, Killian DJ. Mortality follow-up of workers exposed to 1,4-dioxane.
  J of Occup Med 1978; 20: 255-259.
9 Bull R, Robinson M, Lauri RD. Association of carcinoma yield with early papilloma development in
  SENCAR mice. Environ Health Persp 1986; 68: 11-17.
0 Lundberg I, Ekdahl M, Kronevi T, et al. Three industrial solvents investigated for tumor promoting
  activity in the rat liver. Cancer Letters 1987; 36: 29-33.
1 Sheu C, Moreland F, Kelm L, et al. In vitro BALB/3T3 cell transformation assay of nonoxynol-9 and
  1,4-dioxane. Environ Mol Mutag 1988; 11: 41-8.
2 Kano H, Umeda Y, Saito M, Senoh H, Ohbayashi H, Aiso S et al. Thirteen-week oral toxicity of
  1,4-dioxane in rats and mice. J Toxicol Sci 2008; 33(2): 141-53.
3 Stickney JA, Sager SL, Clarkson JR, Smith LA, Locey BJ, Bock MJ et al. An updated evaluation of
  the carcinogenic potential of 1,4-dioxane. Regul Toxicol Pharmacol 2003; 38(2): 183-195.
2 1,4-Dioxane
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<pre>4 Klimisch HJ, Andreae M, Tillmann U. A systematic approach for evaluating the quality of
  experimental toxicological and ecotoxicological data. Regul Toxicol Pharmacol 1997; 25(1): 1-5.
  References                                                                                      53
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<pre>4 1,4-Dioxane</pre>

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<pre>A Request for advice
B The Committee
C The submission letter (in English)
D Comments on the public review draft
E IARC evaluation and conclusion
F Classification on carcinogenicity
G Classification on mutagenicity
H Criteria for testing reliability of animal and in vitro studies
  Annexes
                                                                  55
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<pre>6 1,4-Dioxane</pre>

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<pre>nnex A
     Request for advice
     In a letter dated October 11, 1993, ref DGA/G/TOS/93/07732A, to, the State
     Secretary of Welfare, Health and Cultural Affairs, the Minister of Social Affairs
     and Employment wrote:
     Some time ago a policy proposal has been formulated, as part of the simplification of the
     governmental advisory structure, to improve the integration of the development of recommendations
     for health based occupation standards and the development of comparable standards for the general
     population. A consequence of this policy proposal is the initiative to transfer the activities of the
     Dutch Expert Committee on Occupational Standards (DECOS) to the Health Council. DECOS has
     been established by ministerial decree of 2 June 1976. Its primary task is to recommend health based
     occupational exposure limits as the first step in the process of establishing Maximal Accepted
     Concentrations (MAC-values) for substances at the work place.
     In an addendum, the Minister detailed his request to the Health Council as
     follows:
     The Health Council should advice the Minister of Social Affairs and Employment on the hygienic
     aspects of his policy to protect workers against exposure to chemicals. Primarily, the Council should
     report on health based recommended exposure limits as a basis for (regulatory) exposure limits for air
     quality at the work place. This implies:
     •    A scientific evaluation of all relevant data on the health effects of exposure to substances using a
          criteria-document that will be made available to the Health Council as part of a specific request
     Request for advice                                                                                        57
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<pre>      for advice. If possible this evaluation should lead to a health based recommended exposure limit,
      or, in the case of genotoxic carcinogens, a ‘exposure versus tumour incidence range’ and a
      calculated concentration in air corresponding with reference tumour incidences of 10-4 and 10-6
      per year.
  •   The evaluation of documents review the basis of occupational exposure limits that have been
      recently established in other countries.
  •   Recommending classifications for substances as part of the occupational hygiene policy of the
      government. In any case this regards the list of carcinogenic substances, for which the
      classification criteria of the Directive of the European Communities of 27 June 1967 (67/548/
      EEG) are used.
  •   Reporting on other subjects that will be specified at a later date.
  In his letter of 14 December 1993, ref U 6102/WP/MK/459, to the Minister of
  Social Affairs and Employment the President of the Health Council agreed to
  establish DECOS as a Committee of the Health Council. The membership of the
  Committee is given in Annex B.
8 1,4-Dioxane
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<pre>nnex B
     The Committee
     •  R.A. Woutersen, chairman
        toxicologic pathologist, TNO Quality of Life, Zeist; professor of translational
        toxicology, Wageningen University and Research Centre, Wageningen
     •  J. Van Benthem
        genetic toxicologist, National Health Institute for Public health and the
        Environment, Bilthoven
     •  P.J. Boogaard
        toxicologist, SHELL International BV, The Hague
     •  G.J. Mulder
        emeritus professor of toxicology, Leiden University, Leiden
     •  M.J.M. Nivard
        molecular biologist and genetic toxicologist, Leiden University Medical
        Center, Leiden
     •  G.M.H. Swaen
        epidemiologist, Maastricht University, Maastricht
     •  E.J.J. van Zoelen
        professor of cell biology, Radboud University Nijmegen, Nijmegen
     •  T.M.M. Coenen, scientific secretary
        Health Council of the Netherlands, The Hague
     The Committee                                                                      59
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<pre>  With respect to the data presentation and interpretation, the Committee consulted
  an additional expert, J.J.A. Muller, toxicologist from Bureau Reach, National
  Health Institute for Public Health and the Environment, Bilthoven.
  The Health Council and interests
  Members of Health Council Committees are appointed in a personal capacity
  because of their special expertise in the matters to be addressed. Nonetheless, it
  is precisely because of this expertise that they may also have interests. This in
  itself does not necessarily present an obstacle for membership of a Health
  Council Committee. Transparency regarding possible conflicts of interest is
  nonetheless important, both for the chairperson and members of a Committee
  and for the President of the Health Council. On being invited to join a
  Committee, members are asked to submit a form detailing the functions they
  hold and any other material and immaterial interests which could be relevant for
  the Committee’s work. It is the responsibility of the President of the Health
  Council to assess whether the interests indicated constitute grounds for non-
  appointment. An advisorship will then sometimes make it possible to exploit the
  expertise of the specialist involved. During the inaugural meeting the
  declarations issued are discussed, so that all members of the Committee are
  aware of each other’s possible interests.
0 1,4-Dioxane
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<pre>nnex C
     The submission letter (in English)
     Subject          : Submission of the advisory report 1,4-Dioxane
     Your Reference: DGV/BMO/U-932542
     Our reference : U- 866033/DC/fs/246-W20
     Enclosed         :2
     Date             : November 13, 2015
     Dear Minister,
     I hereby submit the advisory report on the effects of occupational exposure to
     1,4-dioxane.
     This advisory report is a re-evaluation of an advisory report on the classification
     as a carcinogenic substance that has earlier been published by the Health
     Council. The Council is asked for a re-evaluation because the proposed
     classification differs from the classification that applies in the European Union.
     In addition, the Council is asked to also propose a classification for mutagenicity.
     The classifications are based on the European classification system.
     The conclusions in the advisory report were drawn by a subcommittee of the
     Health Council’s Dutch Expert Committee on Occupational Safety (DECOS).
     The subcommittee has taken comments into account from a public review, and
     The submission letter (in English)                                                   61
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<pre>  included the opinions by the Health Council's Standing Committee on Health and
  the Environment.
  I have today sent copies of this advisory report to the State Secretary of
  Infrastructure and the Environment and to the Minister of Health, Welfare and
  Sport, for their consideration.
  Yours sincerely,
  (signed)
  Professor J.L. Severens,
  Vice President
2 1,4-Dioxane
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<pre>nnex D
     Comments on the public review draft
     A draft of the present report was released in 2015 for public review. The
     following organisations and persons have commented on the draft document:
     • H. Stengel, BASF SE, Ludwigshafen, Germany
     • T.J. Lentz, P. Joseph, National Institute for Occupational Safety and Health
         (NIOSH), USA.
     All comments received and the response of the Committee will be publicly
     available (www.gezondheidsraad.nl) from the moment of presentation of the
     final report.
     Comments on the public review draft                                            63
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<pre>4 1,4-Dioxane</pre>

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<pre>nnex E
     IARC evaluation and conclusion
     1,4-dioxane (Group 2B)
     VOL.: 71 (1999) (p. 589).5
     Summary of Data Reported and Evaluation.
     Exposure data
     Exposure to 1,4-dioxane may occur during its manufacture and its use as a
     solvent in a wide range of organic products. It has been detected in ambient air.
     Human carcinogenicity data
     Deaths from cancer were not elevated in a single, small prospective study of
     workers exposed to low concentrations of dioxane.
     Animal carcinogenicity data
     1,4-Dioxane was tested for carcinogenicity by oral administration in mice, rats
     and guinea-pigs. It produced an increased incidence of hepatocellular adenomas
     and carcinomas in mice, tumours of the nasal cavity, liver subcutaneous tissues,
     mammary gland and peritoneal mesotheliomas in rats and tumours of the liver
     IARC evaluation and conclusion                                                    65
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<pre>  and gall-bladder in guinea-pigs. No increase in tumours was seen in rats
  following inhalation exposure. In the mouse-lung adenoma assay, intraperitoneal
  injection of 1,4-dioxane increased the incidence of lung tumours in males; no
  such effect was seen following oral administration. In a two-stage liver foci assay
  in rats, 1,4-dioxane showed promoting activity.
  Other relevant data
  1,4-Dioxane is rapidly absorbed upon inhalation or after oral administration, but
  its penetration of skin is poor. The major metabolite is β-hydroxyethoxyacetic
  acid, which is rapidly excreted. In rats, the elimination of 1,4-dioxane and its
  metabolites is progressively delayed as doses are increased, indicating saturation
  of metabolism. No clinical signs or changes in mortality were found in a cohort
  of exposed workers. In rats, 1,4-dioxane produces degenerative and necrotic
  changes in liver and renal tubules. High doses can significantly increase the total
  hepatic cytochrome P450 content. No reproductive effects of 1,4-dioxane
  exposure of rats have been reported. Most of the broad of tests for genotoxic
  activity have produced negative results, but positive results were obtained in a
  cell transformation assay and conflicting results were obtained in mouse bone-
  marrow cell tests for micronucleus induction.
  Evaluation
  There is inadequate evidence in humans for the carcinogenicity of 1,4-dioxane.
  There is sufficient evidence in experimental animals for the carcinogenicity of
  1,4-dioxane.
  Overall evaluation
  1,4-dioxane is possibly carcinogenic to humans (Group 2B).
  Previous evaluations: Vol. 11 (1976); Suppl. 7 (1987).
  Synonyms: ‘1,4-diethylene dioxide’.
6 1,4-Dioxane
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<pre> nnex       F
            Classification on carcinogenicity
            The Committee expresses its conclusions in the form of standard phrases*:
 ategory   Judgement of the Committee (GRGHS)                                           Comparable with EU Categorya
                                                                                        (before 16        (as from 16
                                                                                        December 2008)    December 2008)
A          The compound is known to be carcinogenic to humans.                          1                 1A
           • It acts by a stochastic genotoxic mechanism.
           • It acts by a non-stochastic genotoxic mechanism.
           • It acts by a non-genotoxic mechanism.
           • Its potential genotoxicity has been insufficiently investigated.
               Therefore, it is unclear whether the compound is genotoxic.
B          The compound is presumed to be as carcinogenic to humans.                    2                 1B
           • It acts by a stochastic genotoxic mechanism.
           • It acts by a non-stochastic genotoxic mechanism.
           • It acts by a non-genotoxic mechanism.
           • Its potential genotoxicity has been insufficiently investigated.
               Therefore, it is unclear whether the compound is genotoxic.
           The compound is suspected to be carcinogenic to man.                         3                 2
3)         The available data are insufficient to evaluate the carcinogenic             not applicable    not applicable
           properties of the compound.
4)         The compound is probably not carcinogenic to man.                            not applicable    not applicable
    See Section 3.6 (Carcinogenicity) of Regulation No. 1272/2008 of the European Parliament and of the council of 16
    December 2008 on classification, labelling and packaging of substances.
            Health Council of the Netherlands. Guideline to the classification of carcinogenic compounds. The
            Hague: 2010; publication no. A10/07.
            Classification on carcinogenicity                                                                            67
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<pre>8 1,4-Dioxane</pre>

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<pre>nnex G
     Classification on mutagenicity
     Source: Section 3.5 (Germ cell mutagenicity) of Regulation No. 1272/2008 of the European
     Parliament and of the council of 16 December 2008 on classification, labelling and packaging of
     substances.
     3.5.1          Definitions and general considerations
     3.5.1.1A mutation means a permanent change in the amount or structure of the genetic material in a
     cell. The term ‘mutation’ applies both to heritable genetic changes that may be manifested at the
     phenotypic level and to the underlying DNA modifications when known (including specific base pair
     changes and chromosomal translocations). The term ‘mutagenic’ and ‘mutagen’ will be used for
     agents giving rise to an increased occurrence of mutations in populations of cells and/or organisms.
     3.5.1.2The more general terms ‘genotoxic’ and ‘genotoxicity’ apply to agents or processes which
     alter the structure, information content, or segregation of DNA, including those which cause DNA
     damage by interfering with normal replication processes, or which in a non-physiological manner
     (temporarily) alter its replication. Genotoxicity test results are usually taken as indicators for
     mutagenic effects.
     3.5.2          Classification criteria for substances
     3.5.2.1This hazard class is primarily concerned with substances that may cause mutations in the germ
     cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or
     Classification on mutagenicity                                                                       69
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<pre>  genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in
  classifying substances and mixtures within this hazard class.
  3.5.2.2For the purpose of classification for germ cell mutagenicity, substances are allocated to one of
  two categories as shown in Table 3.5.1.
  3.5.2         Specific considerations for classification of substances as germ cell mutagens
  3.5.2.3.1To arrive at a classification, test results are considered from experiments determining
  mutagenic and/or genotoxic effects in germ and/or somatic cells of exposed animals. Mutagenic and/
  or genotoxic effects determined in in vitro tests shall also be considered.
  3.5.2.3.2The system is hazard based, classifying substances on the basis of their intrinsic ability to
  induce mutations in germ cells. The scheme is, therefore, not meant for the (quantitative) risk
  assessment of substances.
  Table 3.5.1 Hazard categories for germ cell mutagens.
  Categories                           Criteria
  CATEGORY 1:                          Substances known to induce heritable mutations or to be regarded
                                       as if they induce heritable mutations in the germ cells of humans.
                                       Substances known to induce heritable mutations in the germ cells
                                       of humans.
                    Category 1A:       The classification in Category 1A is based on positive evidence
                                       from human epidemiological studies. Substances to be regarded
                                       as if they induce heritable mutations in the germ cells of humans.
                    Category 1B:       The classification in Category 1B is based on:
                                       • positive result(s) from in vivo heritable germ cell mutagenicity
                                           tests in mammals; or
                                       • positive result(s) from in vivo somatic cell mutagenicity tests
                                           in mammals, in combination with some evidence that the
                                           substance has potential to cause mutations to germ cells. It is
                                           possible to derive this supporting evidence from mutagenicity/
                                           genotoxicity tests in germ cells in vivo, or by demonstrating
                                           the ability of the substance or its metabolite(s) to interact with
                                           the genetic material of germ cells; or
                                       • positive results from tests showing mutagenic effects in the
                                           germ cells of humans, without demonstration of transmission
                                           to progeny; for example, an increase in the frequency of
                                           aneuploidy in sperm cells of exposed people.
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<pre>CATEGORY 2:                         Substances which cause concern for humans owing to the
                                    possibility that they may induce heritable mutations in the germ
                                    cells of humans. The classification in Category 2 is based on:
                                    • positive evidence obtained from experiments in mammals and/
                                        or in some cases from in vitro experiments, obtained from:
                                    • somatic cell mutagenicity tests in vivo, in mammals; or
                                    • other in vivo somatic cell genotoxicity tests which are
                                        supported by positive results from in vitro mutagenicity
                                        assays.
                                    Note: Substances which are positive in in vitro mammalian
                                    mutagenicity assays, and which also show chemical structure
                                    activity relationship to known germ cell mutagens, shall be
                                    considered for classification as Category 2 mutagens.
3.5.2.3.3Classification for heritable effects in human germ cells is made on the basis of well
conducted, sufficiently validated tests, preferably as described in Regulation (EC) No 440/2008
adopted in accordance with Article 13(3) of Regulation (EC) No 1907/2006 (‘Test Method
Regulation’) such as those listed in the following paragraphs. Evaluation of the test results shall be
done using expert judgement and all the available evidence shall be weighed in arriving at a
classification.
3.5.2.3.4In vivo heritable germ cell mutagenicity tests, such as:
•    rodent dominant lethal mutation test;
•    mouse heritable translocation assay.
3.5.2.3.5In vivo somatic cell mutagenicity tests, such as:
•    mammalian bone marrow chromosome aberration test;
•    mouse spot test;
•    mammalian erythrocyte micronucleus test.
3.5.2.3.6Mutagenicity/genotoxicity tests in germ cells, such as:
h    mutagenicity tests:
     •   mammalian spermatogonial chromosome aberration test;
     •   spermatid micronucleus assay;
i    genotoxicity tests:
     •   sister chromatid exchange analysis in spermatogonia;
     •   unscheduled DNA synthesis test (UDS) in testicular cells.
3.5.2.3.7Genotoxicity tests in somatic cells such as:
•    liver Unscheduled synthesis test (UDS) in vivo;
•    mammalian bone marrow Sister Chromatid Exchanges (SCE);
Classification on mutagenicity                                                                         71
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<pre>  3.5.2.3.8In vitro mutagenicity tests such as:
  •     in vitro mammalian chromosome aberration test;
  •     in vitro mammalian cell gene mutation test;
  •     bacterial reverse mutation tests.
  3.5.2.3.9The classification of individual substances shall be based on the total weight of evidence
  available, using expert judgement (See 1.1.1). In those instances where a single well-conducted test is
  used for classification, it shall provide clear and unambiguously positive results. If new, well
  validated, tests arise these may also be used in the total weight of evidence to be considered. The
  relevance of the route of exposure used in the study of the substance compared to the route of human
  exposure shall also be taken into account.
  3.5.3          Classification criteria for mixtures
  3.5.3.1Classification of mixtures when data are available for all ingredients or only for some
  ingredients of the mixture
  3.5.3.1.1The mixture shall be classified as a mutagen when at least one ingredient has been classified
  as a Category 1A, Category 1B or Category 2 mutagen and is present at or above the appropriate
  generic concentration limit as shown in Table 3.5.2 for Category 1A, Category 1B and Category 2
  respectively.
  Table 3.5.2 Generic concentration limits of ingredients of a mixture classified as germ cell mutagens
  that trigger classification of the mixture.
                               Concentration limits triggering classification of a mixture as:
  Ingredient classified as: Category 1A mutagen Category 1B mutagen Category 2 mutagen
  Category 1A mutagen          ≥ 0,1 %                   -                         -
  Category 1B mutagen          -                         ≥ 0,1 %                   -
  Category 2 mutagen           -                         -                         ≥ 1,0 %
  Note. The concentration limits in the table above apply to solids and liquids (w/w units) as well as
  gases (v/v units).
  3.5.3.2Classification of mixtures when data are available for the complete mixture.
  3.5.3.2.1Classification of mixtures will be based on the available test data for the individual
  ingredients of the mixture using concentration limits for the ingredients classified as germ cell
  mutagens. On a case-by-case basis, test data on mixtures may be used for classification when
  demonstrating effects that have not been established from the evaluation based on the individual
  ingredients. In such cases, the test results for the mixture as a whole must be shown to be conclusive
  taking into account dose and other factors such as duration, observations, sensitivity and statistical
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<pre>              analysis of germ cell mutagenicity test systems. Adequate documentation supporting the
              classification shall be retained and made available for review upon request.
              3.5.3.3Classification of mixtures when data are not available for the complete mixture: bridging
              principles.
              3.5.3.3.1Where the mixture itself has not been tested to determine its germ cell mutagenicity hazard,
              but there are sufficient data on the individual ingredients and similar tested mixtures (subject to
              paragraph 3.5.3.2.1), to adequately characterise the hazards of the mixture, these data shall be used in
              accordance with the applicable bridging rules set out in section 1.1.3.
              3.5.4          Hazard communication
              3.5.4.1Label elements shall be used in accordance with Table 3.5.3, for substances or mixtures
              meeting the criteria for classification in this hazard class.
 able 3.5.3 Label elements of germ cell mutagenicity.
 lassification                                Category 1A or Category 1B                  Category 2
GHS Pictograms
 ignal word                                   Danger                                      Warning
Hazard Statement                              H340: May cause genetic defects (state      H341: Suspected of causing genetic
                                              route of exposure if it is conclusively     defects (state route of exposure if it is
                                              proven that no other routes of exposure     conclusively proven that no other routes
                                              cause the hazard)                           of exposure cause the hazard)
 recautionary Statement Prevention            P201, P202, P281                            P201, P202, P281
 recautionary Statement Response              P308 + P313                                 P308 + P313
 recautionary Statement Storage               P405                                        P405
 recautionary Statement Disposal              P501                                        P501
              3.5.5          Additional classification considerations
              It is increasingly accepted that the process of chemical-induced tumourigenesis in humans and
              animals involves genetic changes for example in proto-oncogenes and/or tumour suppresser genes of
              somatic cells. Therefore, the demonstration of mutagenic properties of substances in somatic and/or
              germ cells of mammals in vivo may have implications for the potential classification of these
              substances as carcinogens (see also Carcinogenicity, section 3.6, paragraph 3.6.2.2.6).
              Classification on mutagenicity                                                                                        73
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<pre>4 1,4-Dioxane</pre>

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<pre>nnex H
     Criteria for testing reliability of animal
     and in vitro studies
     To assess the reliability of animal and in vitro studies, the Committee uses the
     criteria set by Klimisch et al., 1997.64 A summary of the criteria of the reliability
     scores is given below. Only studies with a reliability score of 1 or 2 are
     considered in assessing genotoxicity and carcinogenicity.
     Reliability 1 (reliably without restriction)
     For example, guideline study (OECD, etc.); comparable to guideline study; test
     procedure according to national standards (DIN, etc.).
     Reliability 2 (reliable with restrictions)
     For example, acceptable, well-documented publication/study report which meets
     basic scientific principles; basic data given: comparable to guidelines/standards;
     comparable to guideline study with acceptable restrictions.
     Reliability 3 (not reliable)
     For example, method not validated; documentation insufficient for assessment;
     does not meet important criteria of today standard methods; relevant
     methodological deficiencies; unsuitable test system.
     Criteria for testing reliability of animal and in vitro studies                       75
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<pre>  Reliability 4 (not assignable)
  For example, only short abstract available; only secondary literature (review,
  tables, books, etc.).
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<pre>Health Council of the Netherlands
Advisory Reports
The Health Council’s task is to       In addition, the Health Council
advise ministers and parliament on    issues unsolicited advice that
issues in the field of public health. has an ‘alerting’ function. In some
Most of the advisory opinions that    cases, such an alerting report
the Council produces every year       leads to a minister requesting
are prepared at the request of one    further advice on the subject.
of the ministers.
Areas of activity
Optimum healthcare                    Prevention                          Healthy nutrition
What is the optimum                   Which forms of                      Which foods promote
result of cure and care               prevention can help                 good health and
in view of the risks                  realise significant                 which carry certain
and opportunities?                    health benefits?                    health risks?
Environmental                         Healthy working                     Innovation and
health                                conditions                          the knowledge
Which environmental                   How can employees                   infrastructure
influences could have                 be protected against                Before we can harvest
a positive or negative                working conditions                  knowledge in the
effect on health?                     that could harm their               field of healthcare,
                                      health?                             we first need to
                                                                          ensure that the right
                                                                          seeds are sown.
www.healthcouncil.nl
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