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

<pre>Advisory Reports Areas of activity The Health Council’s task is to advise ministers and parliament on issues in the field of public health. Most of the advisory reports that the Council produces every year are prepared at the request of one of the ministers. In addition, the Health Council issues unsolicited advice that has an ‘alerting’ function. In some cases, such an alerting report leads to a minister requesting further advice on the subject. Health Council of the Netherlands www.healthcouncil.nl Optimum healthcare What is the optimum result of cure and care in view of the risks and opportunities? Environmental health Which environmental influences could have a positive or negative effect on health? Prevention Which forms of prevention can help realise significant health benefits? Healthy working conditions How can employees be protected against working conditions that could harm their health? Healthy nutrition Which foods promote good health and which carry certain health risks? Innovation and the knowledge infrastructure Before we can harvest knowledge in the field of healthcare, we first need to ensure that the right seeds are sown. Health Council of the Netherlands Acetaldehyde Re-evaluation of the carcinogenicity and genotoxicity 2014/28 Acetaldehyde 2014/28 627350_V23_OM_Paars_ENG.indd Alle pagina's 24-10-14 11:02</pre>

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<pre>Acetaldehyde
    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 Acetaldehyde
Uw kenmerk            : DGV/BMO/U-932542
Ons kenmerk           : U-8234/JR/cn/246-W19
Bijlagen              :1
Datum                 : 13 november 2014
Geachte minister,
Graag bied ik u hierbij het advies aan over de gevolgen van beroepsmatige blootstelling aan
aceetaldehyde.
Dit advies is een herevaluatie van een eerder door de Gezondheidsraad uitgebracht advies
voor een classificatie als kankerverwekkende stof. De raad is gevraagd om deze herevalua-
tie 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.
      De conclusie van het advies is opgesteld door een vaste subcommissie van de Commis-
sie Gezondheid en beroepsmatige blootstelling aan stoffen (GBBS) van de Gezondheids-
raad. De subcommissie heeft daarbij gebruik gemaakt van commentaren die zijn ontvangen
op een openbaar concept van dit advies en van de oordelen die intern zijn ingewonnen bij
de Beraadsgroep Gezondheid en omgeving.
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
Rijnstraat 50                                                     Postbus 16052
2515 XP Den Haag                                                  2500 BB Den Haag
E - m a il : jo l a n d a . r i jn k e l s @g r. n l              w w w. g r. n l
Te l e f o o n ( 0 7 0 ) 3 4 0 6 6 3 1
</pre>

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<pre></pre>

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<pre>Acetaldehyde
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. 2014/28, The Hague, Noverber 13, 2014
</pre>

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<pre>The Health Council of the Netherlands, established in 1902, is an independent
scientific advisory body. Its remit is “to advise the government and Parliament on
the current level of knowledge with respect to public health issues and health
(services) research...” (Section 22, Health Act).
     The Health Council receives most requests for advice from the Ministers of
Health, Welfare & Sport, Infrastructure & the Environment, Social Affairs &
Employment, Economic Affairs, and Education, Culture & Science. The Council
can publish advisory reports on its own initiative. It usually does this in order to
ask attention for developments or trends that are thought to be relevant to
government policy.
     Most Health Council reports are prepared by multidisciplinary committees of
Dutch or, sometimes, foreign experts, appointed in a personal capacity. The
reports are available to the public.
                 The Health Council of the Netherlands is a member of the European
                 Science Advisory Network for Health (EuSANH), a network of science
                 advisory bodies in Europe.
This report can be downloaded from www.healthcouncil.nl.
Preferred citation:
Health Council of the Netherlands. Acetaldehyde - Re-evaluation of the
carcinogenicity and genotoxicity. The Hague: Health Council of the Netherlands,
2014; publication no. 2014/28.
all rights reserved
ISBN: 978-94-6281-016-7
</pre>

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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
   Summary of toxicokinetics 21
.1 Absorption, distribution and elimination 21
.2 Metabolism 22
   Contents                                       7
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<pre>    Genotoxicity 23
 .1 Non-human information 23
 .2 Human information 31
 .3 Other relevant information 31
 .4 Summary and discussion of mutagenicity 36
 .5 Comparison with criteria 37
 .6 Conclusions on classification and labelling 38
    Carcinogenicity 39
 .1 Non-human information 39
 .2 Human information 44
 .3 Other relevant information 45
 .4 Summary and discussion of carcinogenicity 48
 .5 Comparison with criteria 48
 .6 Conclusions on classification and labelling 49
    References 51
    Annexes 59
A   Request for advice 61
B   The Committee 63
C   The submission letter (in English) 65
D   Comments on the public review draft 67
E   IARC evaluation and conclusion 69
F   Classification on carcinogenicity 73
G   Classification on mutagenicity 75
    Acetaldehyde
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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 Gezondheidsraad, hierna kortweg aange-
duid 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 aceetaldehyde. Aceetaldehyde wordt vooral
gebruikt als intermediair bij de synthese van diverse producten, waaronder de
synthese van azijnzuur. Het wordt verder onder meer gebruikt als oplosmiddel bij
de productie van diverse chemische stoffen en als conserveringsmiddel voor bij-
voorbeeld vis en fruit.
De commissie concludeert dat aceetaldehyde 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 aan
om aceetaldehyde te classificeren als mutageen voor geslachtscellen in categorie
1B (stof die beschouwd moet worden als een stof die erfelijke mutaties
 Zie bijlage F (carcinogeniteit) en G (mutageniteit) voor classificatiesysteem.
Samenvatting                                                                     9
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<pre>  veroorzaakt in de geslachtscellen van mensen).* Aceetaldehyde heeft een
  stochastisch genotoxisch werkingsmechanisme.
  Zie Annex F (carcinogeniteit) en G (mutageniteit) voor classificatiesysteem.
0 Acetaldehyde
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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 acetaldehyde. Acetaldehyde is mainly used as
intermediate, for instance in the production of acetic acid. It, furthermore, is used
for instance as a solvent in the production of various chemical substances, and as
a fish and fruit preservative.
The committee concludes that acetaldehyde is presumed to be carcinogenic to
man, and recommends classifying the substance in category 1B.* Based on the
available data, the committee furthermore recommends classifying acetaldehyde
as a germ cell mutagen in category 1B (substance to be regarded as if it induces
heritable mutations in the germ cells of humans).* The substance acts by a
stochastic genotoxic mechanism.
 See Annex F (carcinogenicity) and G (mutagenicity) for the classification system.
Executive summary                                                                     11
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<pre>2 Acetaldehyde</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, the ministry of Social Affairs and
        Employment asked the Health Council to update the evaluations and
        classification 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 acetaldehyde. An earlier
        evaluation of this substance was published in 2012.1 The re-evaluation now
        includes a proposal for classification on germ cell mutagenicity.
            The Committee is aware that acetaldehyde is an intermediate substance in the
        metabolism of ethanol, and that it has been suggested that acetaldehyde accounts
        for a great part of the toxic effects of ethanol. However, the Committee
        Scope                                                                              13
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<pre>    emphasizes that this report focuses on acetaldehyde alone and does not consider
    combined exposure with ethanol and ethanol-related adverse health effects.
1.2 Committee and procedures
    The re-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
    Minister can be found in Annex C.
         In 2014 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
    acetaldehyde, such an IARC-monograph is available, of which the summary and
    conclusion of IARC (1999) is inserted in Annex E.
         Furthermore, relevant data from 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 September 2014, using
    acetaldehyde and CAS no 75-07-0 as key words in combination with key words
    representative for carcinogenesis and mutagenesis.
 4  Acetaldehyde
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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                      : 200-836-8
        EC name                        : Acetaldehyde, ethanal
        CAS number (EC inventory)      : 75-07-0
        CAS number                     : 75-07-0
        CAS name                       : Acetaldehyde
        IUPAC name                     : Acetaldehyde
        CLP Annex VI Index number      : 605-003-00-6
        Molecular formula              : C2 H 4 O
        Molecular weight range         : 44.05 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
 able 2 Summary of physico-chemical properties.
 roperties                              Value                                    Reference    Comment
 tate of the substance                : Liquid at 20 °C and 101.3 kPa            IUCLID 2000
Melting/freezing point                : -123.5 °C                                SCCNFP 20042
 oiling point                         : 20.4 °C                                  SCCNFP 20042
 elative density                      : 0.78 g/cm3 at 20 °C                      IUCLID 2000
 apour pressure                       : 98 kPa at 20 °C                          SCCNFP 20042
 urface tension                       :-                                         IUCLID 2000
Water solubility                      : Miscible at 20 °C                        IUCLID 2000
 artition coefficient n-octanol/water : log P, 0.43                              IARC 19993
 lash point                           : -40 °C (open cup), -38 °C (closed cup) IARC 19993
 lammability                          : Extremely flammable                      IUCLID 2000
 xplosive properties                  :-                                         IUCLID 2000
 elf-ignition temperature             :-
Oxidising properties                  :-
Granulometry                          :-
 tability in organic solvents         : - (and identity of relevant degradation products)
Dissociation constant (pKa)           : 13.6 at 25 °C                            NTP 2010
 iscosity                             : 0.2456 mPa x sec at 15 °C                SCCS 2012
2.4            International classifications
2.4.1          European Commission
               Acetaldehyde is classified for carcinogenicity in Annex VI of regulation (EC) No
               1272/2008 as follows: Carc 2 (suspected human carcinogen; H351: suspected of
               causing cancer). The substance is not classified for mutagenic activity. The
               classification by the European Commission dates from 1991.
2.4.2          IARC
               In 1999, IARC concluded that there was inadequate evidence in humans for the
               carcinogenicity of acetaldehyde, and that there was sufficient evidence in
               experimental animals (see Annex E).3 Therefore, IARC classified the substance
               in Group 2B (‘possibly carcinogenic to humans’).
                   In 2010, IARC evaluated the risk of cancer due to alcohol consumption,
               including acetaldehyde. It confirmed that there was sufficient evidence in animal
               experiments for the carcinogenicity of acetaldehyde.4 Moreover, in 2012 IARC
 6             Acetaldehyde
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<pre>concluded that ‘acetal-dehyde associated with alcohol consumption’ is
carcinogenic to humans (Group 1).5
Identity of the substance                                             17
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<pre>8 Acetaldehyde</pre>

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<pre> hapter 3
        Manufacture and uses
3.1     Manufacture
        Not relevant for classification.
3.2     Identified uses
        Acetaldehyde is an aldehyde, occurring widely in nature. For instance, it occurs
        naturally in coffee, bread, and ripe fruit, and is produced by plants as part of their
        normal metabolism. Acetaldehyde is also formed endogenously in humans in
        small amounts, for instance during the breakdown of ethanol in the body. It is,
        furthermore, present in tobacco smoke.
            Acetaldehyde is produced on a large industrial scale for many purposes and
        uses.6 For instance, it is used as an intermediate in the production of acetic acid;
        in the production of cellulose acetate, pyridine derivates, perfumes, paints
        (aniline dyes), plastics and synthetic rubber; in leather tanning and silvering
        mirrors; as a denaturant for alcohol; in fuel mixtures; as a hardener for gelatine
        fibres; in glue and casein products; as a preservative for fish and fruit; in the
        paper industry; and, as a flavouring agent.
        Manufacture and uses                                                                   19
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<pre>0 Acetaldehyde</pre>

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<pre> hapter 4
        Summary of toxicokinetics
        The data presented below is a summary from evaluations and reviews by others,
        such as IARC,3-5 IPCS,7 DFG,8 and SCCNFP.2
4.1     Absorption, distribution and elimination
        In human volunteers, a significant uptake (45-70%) by the respiratory tract of
        inhaled acetaldehyde was observed after a very short exposure duration of 45 to
        75 seconds. In various tissues of rats, acetaldehyde was found to be increased
        after a single exposure by inhalation, compared to unexposed control animals.
        Limited data obtained from animal experiments suggest that acetaldehyde
        (administered by intraperitoneal injection) may be partially transferred from
        maternal to foetal blood. It is also found in foetal liver. In a few studies
        acetaldehyde was detected in the blood and brain of animals, which were given
        the substance by intragastric administration or intraperitoneal injections. No data
        are available on dermal or percutaneous absorption.
            Data on elimination are very limited. In one study using dogs, a single
        administration of acetaldehyde via a stomach tube revealed the presence of the
        substance in urine in minor quantities, but in most dogs no urinary acetaldehyde
        could be detected at all. Most likely this is due to the rapid metabolism of the
        substance in the liver.
        Summary of toxicokinetics                                                           21
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<pre>4.2 Metabolism
    Quantitative data on metabolism of acetaldehyde are based on animal
    experiments. Acetaldehyde is rapidly oxidized into acetate by NAD+-dependent
    acetaldehyde dehydrogenases. These enzymes are located in the cells of most
    tissues, including the liver, mucosal tissue of the respiratory tract, and the testes
    of mice. Acetaldehyde dehydrogenases show genetic polymorphism that gives
    rise to differences in vulnerability in humans concerning toxicity. To a minor
    part, the substance is probably oxidized by cytochrome P450 2E1, and by
    different aldehyde oxidases. Acetate is further metabolised into carbon dioxide
    and water by the citric acid cycle. There is no reason to believe that metabolism
    of acetaldehyde in rodents is significantly different from that of humans.
        In general, data indicate a highly effective metabolism, in that half-time
    values in the blood for acetaldehyde were found to be three minutes in rats (after
    repeated exposure by inhalation) and mice (single intraperitoneal injection). For
    humans, no reliable data on half-times are available.
        Acetaldehyde is a highly reactive electrophile, which reacts with nucleophilic
    groups of cellular macromolecules, such as proteins and DNA, to form adducts.
 2  Acetaldehyde
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<pre>  hapter     5
             Genotoxicity
             Numerous studies have been performed on the genotoxic properties of
             acetaldehyde (see Tables 3 through 11).
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          Klimisch9 References
                                          Rangea                 - negative       scoreb
                                                                 + positive
Micro-organisms
  everse mutation;   S. typhimurium       0 - 10,000 µg/plate    - (tested in two 2         Mortelmans et al.
multi-substance      TA98, TA100,                                laboratories)              198610
 tudy                TA1535, TA1537
  everse mutation    S. typhimurium       0.005, 0.01, 0.1, 1.0, -                2         ECHA
                     TA98, TA100,         5.0, and 10 µg/plate:                             registration data,
                     TA1535, TA1537,      + and - S9                                        vitro.001, study
                     TA1538                                                                 report 1979
                                                                                            (echa.europe.eu;)
  everse mutation    S. typhimurium       0.1 - 1.0 ml/chamber, -                 2         Dillon et al.
                     TA100, TA102,        vapour; - and + S9                                199811
                     TA104
             Genotoxicity                                                                                   23
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<pre>  everse mutation   S. typhimurium       Max. non-toxic dose:    -                       3; only one strain  Marnett et al.
                    TA104                2,515 µg/ml; -S9                                tested              198512
  everse mutation   S. typhimurium       0 - 3 µg/plate;         -                       3; only one strain  Chang et al.
                    TA102                cytotoxic over 5,000                            tested, no positive 199713
                                         µg/plate                                        control
  everse mutation   S. typhimurium       10 µg/plate (exact      -                       3; one dose tested  Rosenkranz
                    TA1535, TA1537       dose not given)                                 only                197714
  everse mutation   S. typhimurium       0.5% in air (highest    -                       4; from secondary   JETOC 199715
                    TA98, TA100,         dose; - and + S9)                               source
                    TA1535, TA1537
  everse mutation   S. typhimurium       No exposure             -                       4; abstract only    Sasaki and Endo
                    TA98 and TA100       concentration given;                                                197816
                                         +/- S9
  everse mutation   E. coli WP2 uvrA     Six different           -                       2                   Hemminki et al.
                                         concentrations in the   (also alkylation rate                       198017
                                         range of 0.02 to 10     did not increase)
                                         mM for 18 hours (-
                                         S9)
  everse mutation   E. coli WP2 uvrA     0.5% in air (highest    -                       4; from secondary   JETOC 199715
                                         dose; - and + S9)                               source
  everse mutation   E. coli WP2 uvrA     0.1%                    +                       4; abstract only;   Igali and Gaszó
                                                                                         no data on          198018
                                                                                         controls; no data
                                                                                         on viability
  hromosomal        Aspergillus          Up to 300 µg/ml; -S9 + (chromosomal             3                   Crebelli et al.
 berration          nidulans                                     malsegregation);                            198919
                                                                 percentage survivors
                                                                 decreases from 100
                                                                 µg/ml onwards
Mammalian cells
Gene mutation       Human TK6 cells;     0.001, 0.005, 0.01,     - hprt locus;           1                   Budinsky et al.
                    mutants determ-      0.05, 0.25, 0.5, 1.0, 2 + tk locus (dose-                           201320
                    ined at the hprt and and 4 mM for 24         dependent increase)
                    tk locus             hours
Gene mutation       Human lympho-        0 - 2.4 mM (24 hr-      + (dose-related         2                   He and Lambert
                    cytes, hprt locus    treatment, 0-0.6 mM     increase in number of                       199021
                                         (48-hr treatment);      mutants)
                                         doses selected were
                                         based on low-
                                         cytotoxicity); -S9
Gene mutation       Human                2.4 mM for 22 hours;    + (mutation spectrum    2                   Noori and Hou
 pectrum            lymphocytes, hprt    cloning efficiency      of acetaldehyde                             200122
                    locus                was 50% at 1.2 mM       induced mutations
                                         compared to control     was different from
                                                                 control)
Gene mutation       Human                1.2 to 2.4 mM for 24 + (dose-dependent          3; no positive      Lambert et al.
                    lymphocytes from     hours;                  increase in number of   control; no data    199423
                    donors, hprt locus   0.2 to 0.6 mM for 48 mutants); large            on cytotoxicity
                                         hours                   genomic deletions;
                                                                 most lesions are likely
                                                                 point mutations
 4           Acetaldehyde
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<pre>Gene mutation;     Mouse lymphoma 176 - 352 µg/ml; -S9            +; growth reduces       2                 Wangenheim and
multi-substance    L5178T cells, tk                               with increasing                           Bolcsfoldi 198824
 tudy              locus                                          exposure
Gene mutation      Human fibroblast 0, 0.25, 0.5, 1.0 and         + (after replication).  2                 Matsuda et al.
                   cell line with shuttle 2.0 M                   Mutations were                            199825
                   vector plasmid                                 specified as tandem
                   containing supF                                based substitutions
                   suppressor tRNA                                (GGTT); single-
                   gene                                           strand and double
                                                                  strand DNA mutations
                                                                  increased with
                                                                  increasing dose
Gene mutation      Normal human           Concentrations up to    + (bell-shaped dose-    2                 Grafström et al.
6-TG resistant     fibroblasts            10 mM for 5 hours;      response relationship);                   199426
mutations)                                positive and negative   survival at 5 mM was
                                          control included; cell  50%; cells treated
                                          viability tests         with 8 and 10 mM
                                          performed               showed delayed
                                                                  recovery of the growth
                                                                  rate.
  hromosome        Different DNA-         0.3, 0.6, 1.0, 1.8, 2.5 CA: + (concentration-   2; no positive    Mechilli et al.
 berrations        repair deficient       and 3.6 mM for 2        related increase)       control           200827
                   Chinese hamster        hours; 100
                   ovary cells            metaphases scored/
                                          group
  hromosome        Primary rat skin       0.1 - 10 mM for 12      12 hours: -             3; no positive    Bird et al. 198228
 berration         fibroblasts            and 24 hours; 50        24 hours: + (p<0.05),   controls; no data
                                          metaphases analysed/    except lowest dose,     on cytotoxicity
                                          dose                    concentration-related
                                                                  increase in aneuploidy
  hromosome        Chinese hamster        0, 20, 40 and 60 µg/    +                       3; no data on     Dulout and
 berration         embryonic diploid      ml; -S9                                         cytotoxicity; no  Furnus 198829
                   fibroblasts                                                            positive control
  hromosome        Human peripheral       0, 0.001 and 0.002 %    -                       3; no positive    Obe et al. 197930
 berration         lymphocytes (from      (v/v); 100 or 200                               control; no data
                   3 healthy              mitoses scored/                                 on cytotoxicity
                   volunteers)            sample
  hromosome        Human peripheral       0.02 and 0.04 mg/mL     +                       4; abstract only  Badr and Hussain
 berration         blood lymphocytes      culture medium; no                                                197731
                                          positive control
Micronuclei        Human                  0.005, 0.01, 0.05,      + (dose-related         1                 Budinsky et al.
                   lymphoblastoid         0.25, 0.5, 1.0, and 2   increase); with                           201320
                   TK6 cells              mM;                     increasing exposure
                                          plates sealed due to    also the number of
                                          volatility substances   apoptotic cells
                                                                  increased
             Genotoxicity                                                                                                   25
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<pre>Micronuclei            Human                  8 different            + (0.25, 0.5 and 1.0   2                   ECHA
                       lymphoblastoid         concentrations tested, mM)                                        registration data,
                       TK6 cells              between 0.005 and 4                                               vitro.002, study
                                              mM; negative and                                                  report 1979
                                              positive controls                                                  (echa.europe.eu)
                                              included; only data
                                              analysed when
                                              cytotoxicity was
                                              below 55%
Micronuclei; multi-    Human lympho-          0, 0.6, 0.8 and 1.0    + (dose-related        2; optimal doses    Migliore et al.
 ubstance study        cytes isolated from mM                        increase, p<0.05);     were assessed       199632
                       peripheral blood                              - (after hybridization determining
                       from one healthy                              with a centromeric     degree of
                       non-smoking donor                             DNA probe)             decrease in bi-/
                                                                                            mononucleated
                                                                                            ratio
Micronuclei; multi-    HepG2 and Hep3B 0, 0.9 and 9 mM for           + (concentrations-     2; no data on       Majer et al.
 ubstance study        cells                  24 hours; per          related increase)      cytotoxicity        200433
                                              experimental point
                                              1,500 cells evaluated.
Micronuclei            MCL-5 human            0 - 2 % (v/v; a range  + (from 0.4 %          2; no positive      Kayani and Parry
                       lymphoblastoid cell of 6 differrent           onwards, p<0.05),      control included    201034
                       line                   concentrations) for    dose-dependent
                                              22 hours; > 4,000      increase
                                              cells per dose         -: aneuploidy
                                              examined
Micronuclei            Primary rat skin       0.1 - 10 mM for 12,    + (p<0.05; except      3; no positive      Bird et al. 198228
                       fibroblasts            24 or 48 hours; >      lowest dose tested)    controls; no data
                                              1,000 cells analysed/                         on cytotoxicity
                                              dose
Micronuclei            V79 Chinese            0.5 - 10 mM (MN);      + (dose-dependent      2; No positive      Speit et al. 200835
                       hamster cells                                 increase)              control
    + or - S9, with or without metabolic activation system.
    Klimisch score is expressed in reliability levels (cited from original publication):
    •    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.
    •    Reliability 4 (not assignable). For example, only short abstract available; only secondary literature (review, tables,
         books, etc.).
5.1.2         In vivo data
              A summary on the in vivo mutagenicity of acetaldehyde is shown in Table 4.
 6            Acetaldehyde
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<pre> able 4 Summary of in vivo mutagenicity studies (animal studies).
Method              Animal                 Exposure conditions        Results                    Klimisch9    References
                                                                                                 scorea
 omatic cell mutagencicity
Gene mutation and Wildtype and knock-      Inhalation, 125 and 500    Micronuclei:               2            Kunugita et al.
micronuclei         out mice with inactive ppm vapour,                + in knock-out mice                     200836
                    ALDH2b gene; micro-    continuously for two       (p<0.05);
                    nuclei determined in   weeks; negative control    - in wild-type mice.
                    reticulocytes;         was inhalation of clean    Mutation (TCR mutant
                    mutations were         air                        frequency):
                    determined by T-cell                              + in knock-out mice
                    receptor (TCR) gene                               (p<0.05);
                    mutation assay                                    - in wild-type mice.
Gene mutation and Wildtype and knock-      Oral administration, 0     Micronuclei:               2            Kunugita et al.
micronuclei         out mice with inactive and 100 mg/kg bw, daily, + in knock-out mice                       200836
                    ALDH2 gene;            once a day for two         (p<0.05);
                    micronuclei            weeks; 5 - 10 animals/ - in wild-type mice.
                    determined in          group                      Mutation (TCR mutant
                    reticulocytes;                                    frequency):
                    mutations were deter-                             + in knock-out mice
                    mined by TCR gene                                 (p<0.05);
                    mutation assay                                    - in wild-type mice
Micronuclei; multi- Male SD and F344       Highest dose tested was + (250 mg/kg bw,              2; only      Wakata et al.
 ubstance study     rats, bone marrow      maximum tolerated          intraperitoneal injection, highest dose 199837
                    erythrocytes and       dose; at least four        both cell types)           tested
                    peripheral blood       animals/group
                    erythrocytes
Micronuclei         5 male CD-1 mice       0 - 400 mg/kg bw,          + (dose-related increase) 2             Morita et al.
                                           Intraperitoneal injection,                                         199738
                                           three dose levels; tests
                                           on acute toxicity
                                           performed
Micronuclei         Male Han rats, 5       Single intraperitoneal     + (at 24 and 48 hours), 2               Hynes et al.
                    animals/group          injection of 125 or 250    dose-related increase; no               200239
                                           mg/kg bw; blood            data at 72 hours due to
                                           samples collected after    toxicity
                                           0, 24, 48 and 72 hours
  hromosomal        Rat embryos            Single intra-amniotic      +                          4; original  Bariliak and
 berrations                                injection of 7,800 mg/kg                              publication Kozachuk
                                           bw                                                    available in 198340
                                                                                                 Russian only
Germ cell mutagenicity
Meiotic micronuclei C57BL/6J x C3H/He 125, 250, 375 and 500           - ; survival rate was      2            Lähdetie 198841
n spermatids        mouse early            mg/kg bw per day, single   significantly decreased
                    spermatids             dose, intraperitoneal      in highest exposure
                                           injection; 4 animals/      group
                                           group
             Genotoxicity                                                                                                   27
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<pre> ex-linked           Drosophila             1) Single injection of  + (injection)          2    Woodruff et al.
ecessive lethal      melanogaster           22,500 ppm; 2) 25,000   - (feed)                    198542
mutations; multi-                           ppm in feed; data
 ubstance study                             presented on mortality
                                            and sterility
    See footnote in Table 3 for explanation of the Klimisch-scores.
    ALDH2, aldehyde dehydrogenase 2 family (mitochondrial), converts acetaldehyde into acetate.
             Germ cells
             Lähdetie (1988) studied the induction of meiotic micronuclei in spermatids of
             mice.41 Mice (4 animals per group) were given a single intraperitoneal injection
             of acetaldehyde at a concentration of 0 (control vehicle), 125, 250, 375 and 500
             mg/kg bw. A group of mice served as positive control (cyclophosphamide
             injection). Thirteen days after treatment the mice were killed to examine the
             presence of meiotic micronuclei in early spermatids (1,000 spermatids scored per
             mouse). Compared to the vehicle control, the number of spermatids with
             micronuclei did not increase after acetaldehyde treatment, whereas in the
             positive control it did. The author reported that at a dose of 500 mg/kg bw all
             animals died due to acute toxicity, whereas all survived at lower doses. In a
             separate experiment, the author also investigated the sperm morphology in mice
             treated with acetaldehyde for a short period (up to 250 mg/kg bw; 5-day
             exposure regimen). However, acetaldehyde did not decrease sperm count, testis
             weight or seminal vesicle weight, nor did it induce abnormal sperm at the doses.
             The highest administered dose was lethal to half of the animals in the group.
                  The Committee noted that in a sex-linked recessive lethal mutation assay,
             acetaldehyde was positive after injection (Woodruff et al. 1985).42 This shows
             that the substance induces mutations in germ lines of the insect.
             Somatic cells
             Kunugita et al. (2008) studied the induction of gene mutations and micronuclei in
             knock-out mice having an inactive acetaldehyde dehydrogenase (Aldh2, converts
             acetaldehyde into acetate) gene.36 Both wildtype and the knockout mice inhaled
             acetaldehyde at concentrations of 0, 225 or 900 mg/m3, continuously for two
             weeks. In addition, groups of mice (5-10 animals per group) were given
             acetaldehyde orally at doses of 0 or 100 mg/kg bw, once a day for two weeks.
             Two weeks after the last exposure, all animals were killed and the number of
             reticulocytes with micronuclei was determined. Also the mutations in the TCR
             gene of T-lymphocytes was measured. Irrespective the route of exposure, in
 8           Acetaldehyde
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<pre>knockout mice, the number of micronuclei positive cells, and the frequency of
TCR gene mutations in lymphocytes was statistically significantly increased
compared to the respective controls. In wildtype animals, acetaldehyde did not
cause any effects on these endpoints. See Table 5 for a summary of the results.
     In a well-performed study, Wakata et al. (1998) showed that in bone marrow
polychromatic and peripheral blood erythrocytes of SD and F344 rats,
micronuclei were induced after exposure to acetaldehyde by a single
intraperitoneal injection of 250 mg/kg bw.37 Bone marrow and blood cells were
harvested 24 hours after the treatment. The study included concurrent negative
(solvent/vehicle) and positive (cyclophosphamide) controls.
     In addition, Morita et al. (1997) reported on acetaldehyde-induced
micronuclei in bone marrow polychromatic erythrocytes of male CD-1 mice.38
Five/six mice received the substance by a single intraperitoneal injection. Dose
levels were based on acute toxicity test results. Two different lots were used,
because the experiment was performed in two different laboratories. Twenty four
hours after injections, bone marrow cells were harvested for the micronucleus
assay. In Table 6 a summary of the results is shown.
Table 5 Induction of micronuclei and TCR gene mutations in knockout mice (Kunugita et al 2008).36
Exposure route                 Exposure level          Micronuclei in       Mutant frequency in
                                                       reticulocytes        T-cell receptor gene
Knock-out mice (Aldh2 -/-)
  Inhalation                      0 (control)          -                    -
                               225 mg/m3               +a                   Not determined
                               900 mg/m3               + b/c                +b
  Oral administration             0 (control)          -                    -
                               100 mg/kg bw            + b/c                + b/ c
Wildtype mice (Aldh2 +/+)
  Inhalation                      0 (control)          -                    -
                               225 mg/m3               -                    -
                               900 mg/m3               -                    -
  Oral administration             0 (control)          -                    -
                               100 mg/kg bw            -                    -
a    Compared to Aldh2 +/+ control mice (p<0.05).
b    Compared to Aldh2 +/+ control mice (p<0.01).
c    Compared to Aldh2 -/- control mice (p<0.05).
Genotoxicity                                                                                      29
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<pre>             Table 6 Induction of micronuclei in male CD mice (Morita et al. 1997).38
             Manufact. lot LD50                 Dose             Percentage of micronuclei in bone marrow cells
                                mg/kg bw        mg/kg bw         mean            SD               p-valuea
             Wako               470               0              0.12            0.08             -
                                                 95              0.22            0.15             0.132
                                                190              0.33            0.10             0.010
                                                380              0.85            0.21             0.000
             Merck              338               0              0.12            0.08             -
                                                100              0.10            0.07             0.726
                                                200              0.44            0.11             0.002
                                                300              0.62            0.16             0.000
                                                400              1.10            0.25             0.000
             a    P-value of pairwise comparisons.
 able 7 Induction of micronuclei in blood cells of rats treated with acetaldehyde (Hynes et al. 2002).39
Dose              Time (h)           Laboratorya      Mean RETb ± SD           Mean MNRETb per           Mean MNNCEb
mg/kg bw)                                                                      20,000 RET ± SD           ± SD
                    0                GW               1.29 ± 0.29              0.13 ± 0.06               0.01 ± 0.00
                                     LL               1.47                     0.14                      0.01
 25               24                 GW               0.80 ± 0.12              0.21 ± 0.07               0.01 ± 0.00
                                     LL               0.91                     0.19                      0.01
                  48                 GW               1.32 ± 0.21              0.30 ± 0.09               0.01 ± 0.00
                                     LL               1.37                     0.19                      0.01
                  72                 GW               1.82 ± 0.18              0.14 ± 0.05               0.01 ± 0.00
                                     LL               1.65                     0.18                      0.01
 50               24                 GW               1.00 ± 0.42              0.28 ± 0.07               0.02 ± 0.01
                                     LL               0.99                     0.32                      0.01
                  48                 GW               1.31 ± 0.25              0.33 ± 0.11               0.02 ± 0.01
                                     LL               1.14                     0.39                      0.01
                  72                 GW               1.90 ± 0.42              0.14 ± 0.05               0.01 ± 0.01
                                     LL               1.42                     0.16                      0.01
    GW, GlaxoWellcome; LL, Litron Laboratories.
    RET, reticulocytes; MNRET, micronucleated reticulocytes; MNNCE, micronucleated monochromatic erythrocytes. No
    data on statistical significance presented.
                  Hynes et al. (2002) exposed male Wistar Han rats (5 animals per group) to
             acetaldehyde by a single intraperitoneal injection of 125 or 250 mg/kg bw.39 For
             micronuclei testing, peripheral blood cells were harvested 0, 24, 48 and 72 hours
             after the injection. Micronuclei were scored by flow cytometric analysis. The
             study included negative (vehicle) and positive (cyclophosphamide) controls.
             Acetaldehyde at a dose of 250 mg/kg bw induced micronuclei, with maximum
             increases at 48 hours (see Table 7).
 0           Acetaldehyde
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<pre>5.2          Human information
             Table 8 summarizes a few studies performed on humans, in which effects were
             related to acetaldehyde. All volunteers were alcohol abusers or smokers.
 able 8 Summary of human studies.
Method            Population                Cells          Results and remarks             Quality and/or          References
                                                                                           reliability of study
DNA-adducts       Alcohol abusers (n=24) Peripheral        + in alcohol abusers            Reliability low in that Fang and
32P-              and controls (n=12)       white blood    compared to controls            subjects in the         Vaca 199743
 ostlabelling)                              cells          (p<0.001). Average adduct       alcoholic group were
                                            (granulo-      levels (adducts /107            heavy smokers; in
                                            cytes and      nucleotides):                   control group one
                                            lymphocytes)   - abusers: 3.4 ± 3.8            moderate smoker.
                                                           - controls: 2.1 ± 0.8
DNA-adducts       Cancer-free male          Peripheral     +, adduct level was             Past exposure to        Matsuda et
                  Japanese alcoholic        white blood    significantly higher in         ethanol; no non-        al. 200644
                  patients with different   cells          alcoholics with ALDH2*1*2       alcoholic healthy
                  acetaldehyde                             genotype compared to            controls included
                  dehydrogenase (ALDH)                     alcoholics with ALDH2*1*1
                  genotypes                                genotype.
Acetaldehyde      Smokers, before and       Leucocytes     Decrease in number of           Reliability low,        Chen et al.
 pecific          after smoking cessation                  adducts after cessation. Note:  because of smoking      200745
DNA-adducts                                                cigarette smoke contains        history participants
N2-ethylidene-                                             acetalde-hyde, but also other   and co-exposure
 eoxiguanosine)                                            potential carcinogens.
5.3          Other relevant information
             In the Tables 9 and 10 data are shown on the DNA damaging and genotoxic
             (other than mutagenicity) properties of acetaldehyde.
 able 9 Summary of other information on DNA damage.
Method           Cell type                Concentration                Results                          Klimisch9   References
                                                                                                        scorea
n vivo studies
DNA-protein      Male Fischer-344 rats;   1) Inhalation; 100, 300,     1) + (respiratory mucosa;        2           Lam et al.
 rosslinks       DNA-protein cross-       1,000 and 3,000 ppm;         dose-dependent increase,                     198646
                 links studied in nasal   single 6-hour exposure       p<0.05);
                 respiratory mucosa and   2) inhalation; 1,000 ppm;    - (olfactory mucosa)
                 olfactory cells          6-hours/day, daily, 5-days   2) + (respiratory mucosa); +
                                          samples of three rats were   (olfactory mucosa, p<0.05)
                                          combined
             Genotoxicity                                                                                                     31
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<pre>n vitro tests using human cells
DNA single and Human lymphocytes            0, 1.56, 6.25, 25 and 100  + (single strand breaks at all  2; no         Singh and
 ouble strand      from two healthy         mM for one hour; for each  exposures)                      positive      Khan
 reaks             donors                   dose 50 cells were         + (double strand breaks at      control       199547
                                            analysed from each         100mM only)
                                            subject                    Authors reported that > 80%
                                                                       of cells were not viable after
                                                                       exposure to 100 mM for 2
                                                                       hours
  omet assayb      Human peripheral         3, 10, 30 and 100 mM for + (dose-dependent)                2             Blasiak et
                   blood lymphocytes        one hour; doses were                                                     al. 199948
                                            based on cytotoxicity data
  omet assaya      Human lymphocytes,       3 mM (lympho-cytes),       + No differences were noted     2; one dose Blasiak et
                   gastric and colonic      100 mM (gastric and        among the different cell types; tested only al. 200049
                   mucosa cells             colonic mucosa cells)      viability was over 70% at the
                                                                       tested doses
  omet assaya      Human bronchial          Exposure to 3, 10, 30 and +, dose-dependent effects        2             Grafström
                   epithelial cells         100 mM for 1 hour in       - for single strand breaks                    et al. 199426
                                            thiol free medium
DNA-adducts        DNA form primary         Incubation of cells with + (N2-ethyl-deoxiguanosine        3             Wang et al.
                   human liver cells,       5.7 mM                     adducts)                                      200650
                   samples from normal      [13C2]acetaldehyde; 12
                   liver                    liver samples analysed
Alkaline elution Human lymphocytes          10 - 20 mM for 4 hours     +, DNA cross-links              3; No data    Lambert et
 ssaya                                                                 - ,DNA strand-breaks            on            al. 198551
                                                                                                       cytotoxicity;
                                                                                                       no positive
                                                                                                       controls
Alkaline elution Normal human               1 mM for 1 hour            - (without metabolic            3; only one   Saladino et
 ssaya; multi-     bronchial epithelial                                activation); at 1 mM no         concentratio  al. 198552
 ubstance study cells and humane                                       significant growth reduction    n used
                   leucocytes                                          noted
Alkaline elution Human bronchial            10 mM for 1 hour           -                               3; only one Grafström
 ssaya             epithelial cells                                                                    dose tested; et al. 198653
                                                                                                       no data on
                                                                                                       con-trols; 10
                                                                                                       mM
                                                                                                       acetaldehyd
                                                                                                       e induced
                                                                                                       50%
                                                                                                       cytotoxicity
DNA-protein        EBV-transformed          0.035, 0.175, 0.875, 3.5 + (> 5 mM, p<0.05)                2             Costa et al.
 rosslinks         human Burkitt’s          and 17.5 mM for 2 hours;                                                 199754
                   lymphoma cells (EBV,     Maximum tolerated dose
                   Epstein Barr virus)      was 17.5 mM
DNA-adducts        normal epithelial cells, 1-100 mM for one hour; + (N2-ethyl-3’-dG-                  2             Vaca et al.
                   and SV40T antigen-       32P-postlabeling assay     monophosphate adducts,                        199855
                   immortalized human                                  dose-dependent
                   buccal epithelial
                   cells
  2           Acetaldehyde
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<pre>n vitro tests using rodent cells
  omet assaya      V79 Chinese hamster      0.2 - 20 mM               -; authors reported more than   2; no       Speit et al.
                   cells                                              50% reduction of cell viability positive    200835
                                                                      at 20 mM                        control
Alkaline elution Chinese hamster ovary 0.5, 1.5 and 4.5 mM for - (strand breaks);                     2; no       Marinari et
 ssaya             cells (K1 cells)         90 minutes                + (crosslinks);                 positive    al. 198456
                                                                      cell viability > 80%            control
Alkaline elution Primary rat hepatocytes 0.03, 0.3 and 3 mM for 3 -                                   3           Sina et al.
 ssaya; multi-                              hours; cytotoxicity < 55%                                             198357
 ubstance study
Other test systems
DNA-adducts        Calf thymus DNA          1 M for 30 minutes at 37 + (without metabolic             3; only one Ristow and
                                            °C; negative control      activation)                     concentra-  Obe 197858
                                            included                                                  tion tested
DNA-adducts        Calf thymus DNA          0.01-40 mM for 20 to 96 + (mainly N2-ethylidene-          2           Wang et al.
                                            hours                     deoxi-guanosine DNA-                        200059
                                                                      adducts, but also (< 10%)
                                                                      1,N-propano-deoxi-
                                                                      guanosine, N2-
                                                                      dimethyldioxane-
                                                                      deoxiguanosine, and a cross-
                                                                      link adduct detected).
DNA-adducts        Calf thymus DNA          1.8 mM for 92 hours; 32P- + (N2-ethyl-3’-dG-              3           Fang and
                                            postlabeling assay        monophosphate adducts)                      Vaca 199560
DNA-adducts        Calf thymus DNA in       Up to 79,000 µg/ml        +                               3           Fang and
                   2’-deoxy-guanosine-3’-                                                                         Vaca 199743
                   monophosphate
DNA-protein        Calf thymus DNA in       100, 300 and 1,000 mM +                                   3           Lam et al.
 rosslinks         2’-deoxy-guanosine-3’- for one hour                                                            198646
                   monophosphate
Alkaline elution Saccharomyces              0.85 M for 2 or 4 hours   +                               3; no       Ristow et
 ssaya             cerevisiae (yeast)                                                                 positive    al. 199561
                                                                                                      control; no
                                                                                                      data on
                                                                                                      statistical
                                                                                                      analysis
DNA repair         repair-deficient E.coli  Highest tested            - (- and + S9)                  3; method   Hellmer
 ost-mediated      K-12 uvrB/recA; tests    concentration 370 mM/L;                                   not         and
 ssay, in vivo;    performed in mice        - and + S9                                                validated   Bolcsfoldi
multi-substance                                                                                                   199262
 tudy
     See footnote in Table 3 for explanation of the Klimisch-scores.
     Comet assay and alkaline elution assay: DNA single and double strand breaks, DNA cross-links.
              Genotoxicity                                                                                                  33
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<pre> able 10 Summary of genotoxicity studies.
Method           Cell type            Concentration               Results and remarks Klimisch9            References
                                                                                      Scorea
Mammalian cells (in vitro tests)
 ister chromatid Different DNA-repair 0.3, 0.6, 1.0, 1.8, 2.5 and +                   2; no positive       Mechilli et al.
 xchange         deficient Chinese     3.6 mM for 2 hours; 250                        control              200827
                 hamster ovary cells metaphases scored/
                                       group
 ister chromatid Chinese hamster      0, 30, 100 and 300 µM; -    + (dose-dependent   2                    Brambilla et
 xchange         ovary cells           S9                         increase                                 al. 198663
 ister chromatid V79 Chinese hamster 0.2 - 5 mM                   + (dose-dependent   2; No positive       Speit et al.
 xchange         cells                                            increase)           control              200835
 ister chromatid Chinese hamster      0, 0.8, 2, 4, 7.8, 39.4 and +, dose-related     3; no data on        de Raat et al.
 xchange         ovary cells          78 µg/ml; + and - S9; 20    response            cytotoxicity; no     198364
                                       metaphases/sample                              positive control
                                       scored
 ister chromatid Chinese hamster      0.25x10-3, 0.5x10-3,        +                   3; no positive       Obe et al.
 xchange         ovary cells           1x10-3, and 1.5x10-3 %                         controls, no data on 197965
                                       (v/v); - S9; 100 mitoses                       cytotoxicity
                                       scored/ sample
 ister chromatid Human peripheral     0 - 1,080 µM; -S9;          +, dose-related     2; no positive       Böhlke et al.
 xchange         lymphocytes           reduction of cell growth   response            controls             198366
                                       noted above 720 µM
 ister chromatid Human peripheral     1 - 100 µM                  +                   2; no positive       Knadle 198567
 xchange         lymphocytes                                                          controls
 ister chromatid Human lymphocytes 40, 400 and 800 µM;            +                   3; limited           Véghelyi and
 xchange         and fibroblast of                                                    information on test  Osztovics
                 normal subjects                                                      protocol             197868
 ister chromatid Human lymphocytes 0, 63, 125, 250 500 and        + (dose-dependent   3; no positive       Norppa et al.
 xchange                              2,000 µM; -S9               increase)           controls; no data on 198569
                                                                                      cytotoxicity
 ister chromatid Human lymphocytes 0, 0.0005, 0.001, and          +, dose-related     3; no positive       Ristow and
 xchange                              0.002 % (v/v);              response            controls; no data on Obe 197858
                                      -S9                                             cytotoxicity
 ister chromatid Human lymphocytes 0 - 500 µM; - S9               +, dose-related     3; no data on        Sipi et al.
 xchange                                                          response            cytotoxicity; no     199270
                                                                                      positive controls
 ister chromatid Human peripheral     100 - 400 µM; - S9;         + (dose-dependent   3; no positive       Helander and
 xchange         lymphocytes           exposure performed in      increase)           controls; no data on Lindahl-
                                       capped bottles                                 cytotoxicity         Kiessling
                                                                                                           199171
 ister chromatid Human peripheral     2x10-3 % (v/v);             +                   3; no positive       Obe et al.
 xchange         lymphocytes           + or - acetaldehyde                            controls, no data on 198672
                                       metabolizing enzyme                            cytotoxicity
                                       ALDH
 ister chromatid Human lymphocytes 100 - 2,400 µM;                + (dose-dependent   3; no positive       He and
 xchange                               - S9                       increase            controls used, no    Lambert
                                                                                      data on cytotoxicity 198573
 4           Acetaldehyde
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<pre>ister chromatid Human peripheral         0 - 0.001% (v/v); -S9      + (dose-dependent       3; limited           Jansson
xchange           lymphocytes                                       increase)               information on test 198274
                                                                                            protocol
odents (in vivo somatic cell tests)
ister chromatid Bone-marrow cells of Single intra-peritoneal        + at the highest        2                    Korte et al.
xchange           Chinese hamsters       injection of 0.01, 0.1 and exposure level only; at                      198175
                  (strain not specified) 0.5 mg/kg bw; 6-7          this level signs of
                                         animals/ dose; negative    intoxica-tion were
                                         and positive control       noted; no signs of
                                         included                   intoxication at 0.1 and
                                                                    0.01 mg/kg bw
ister chromatid Male mouse (NIH)         0.4, 4.0, 40 and 400 mg/ + (40 and 400 mg/kg       3; number of mice Torres-
xchange           bone marrow cells      kg bw, single              bw, p<0.05)             per group not given; Bezauri et al.
                                         intraperitoneal injection Mitotic index and        no positive control 200276
                                                                    average generation
                                                                    time did not differ
                                                                    from control
ister chromatid Male CBA mouse           Single intraperi-toneal +                          3; low number of     Obe et al.
xchange                                  injection of 1 or 0.5 mL                           animals in study, no 197930
                                         of a                                               positive controls
                                         10-4 % (v/v) solution;
                                         one animal/ dose
odents (in vivo germ cell tests)
ister chromatid Mouse                    Single intraperitoneal     + (all doses applied,   2; authors did test Madrigal-
xchange           spermatogonial cells injection; 0.4, 4.0, 40      p<0.05); no clear       for intoxication;    Bujaidar et al.
                                         and 400 mg/kg bw; 4 - 5    exposure-response       concentrations used 200277
                                         animals/concentration;     relationship observed   were considered
                                         cells were isolated, 53 h                          non-toxic/-lethal
                                         after injection.
    See footnote in Table 3 for explanation of the Klimisch-scores.
            Germ cells
            Madrigal-Bujaidar et al. (2002) injected NIH mice (4-5 mice per group) with
            acetaldehyde at concentrations of 0 (vehicle control), 0.4, 4, 40 and 400 mg/kg
            bw (single treatment), or cyclophosphamide (positive control).77 Fifty-three
            hours later, the animals were killed, and the tunica albuginea was removed from
            each testes to obtain spermatogonial cells in the seminiferous tubules. A
            statistically significant increase in the number of cells with sister chromatid
            exchange was reported (30 metaphases per mouse scored; see Table 11). The
            authors determined a LD50-dose of 560 mg/kg bw.
            Somatic cells
            Lam et al. (1986) reported on the formation of DNA-protein crosslinks in the
            nose tissue of male Fischer-344 rats after inhalation exposure.46 The animals
            Genotoxicity                                                                                                      35
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<pre>    were exposed to acetaldehyde at concentrations of 0,180, 540, 1,800 and 5,400
    mg/m3 for a single six hours, or to 5,400 mg/m3, 6 hours a day for 5 consecutive
    days. Immediately after the final exposure the animals were killed, and nasal
    respiratory mucosa was obtained for further examination. After a single
    inhalation, a dose dependent increase in DNA-protein crosslinks was observed in
    the respiratory mucosa, but not in the olfactory mucosa. Short-term repeated
    inhalation induced DNA-protein crosslinks in the respiratory and the olfactory
    mucosa.
    In bone marrow cells of Chinese hamsters (6-7 animals per group), a single
    intraperitoneal injection of acetaldehyde increased the number of sister
    chromatid exchanges at the two highest doses applied (0.1 and 0.5 mg/kg bw;
    Korte et al., 1981).75 The authors reported that exposure to concentrations of 0.6
    mg/kg bw and higher was lethal.
    Table 11 Sister chromatid exchanges in spermatogonial cells of mice treated with acetaldehyde
    (Madrigal-Bujaidar et al. 2002).77
    Dose (mg/kg bw)                      SCE/cell ± SDa                   SCE increase
       0                                 1.9 ± 0.16
       0.4                               2.9 ± 0.33b                      1.1
       4                                 4.1 ± 0.34b                      2.2
      40                                 4.6 ± 0.51b                      2.7
    400                                  5.1 ± 0.8b                       3.2
      50 (cyclophosphamide)              6.0 ± 0.1b                       4.1
    a    SCE, sister chromatid exchange.
    b    Statistically significant different compared to control, p< 0.05.
5.4 Summary and discussion of mutagenicity
    Below, only data are summarized of reliable (with or without restrictions)
    experimental design (according to the Klimisch criteria (1997)).9
    Germ cell genotoxicity
    The Committee found two animal studies on germ cell genotoxicity by
    acetaldehyde. The first is the study by Lähdetie et al. (1988), in which a single
    intraperitoneal injection of acetaldehyde did not induce meiotic micronuclei in
    early spermatids nor sperm abnormalities.41 The second study is published by
    Mardigal-Bujaidar et al. (2002), and considers the induction of sister chromatid
    exchanges in mouse spermatogonial cells.77 Although no clear dose-response
 6  Acetaldehyde
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<pre>    relationship could be assessed, the authors reported that acetaldehyde induced
    sister chromatid exchanges (see Table 11). However, based on this endpoint
    alone, the Committee cannot conclude that acetaldehyde is genotoxic in germ
    cells.
    Mutagenicity in bacteria and mammalian cells
    Numerous data have been presented on the mutagenic properties of acetaldehyde
    in bacteria, mammalian cells (other than germ cells) and rodents (see Tables 3
    and 4). Overall, negative outcomes were found in bacteria using the reverse
    mutation assay, whereas positive outcomes (gene mutations, chromosome
    aberrations) were reported in mammalian cells in vitro, and in rodents in vivo
    (gene mutation and micronuclei in blood cells). In part of these positive studies
    also a dose-related response was found. Based on these findings, the Committee
    concludes that acetaldehyde has mutagenic properties in at least somatic
    mammalian cells in vitro and in vivo.
    DNA damage and cytogenicity
    In addition to mutagenicity testing, various studies have been performed showing
    that acetaldehyde induced DNA damage (DNA-crosslinks, DNA-adducts, and
    DNA strand breaks) (see Table 9) in vivo and in vitro. Together with data on
    mutagenicity, these data indicate that acetaldehyde may damage DNA directly.
    Therefore, the Committee is of the opinion that acetaldehyde acts by a stochastic
    genotoxic mechanism. Data on human volunteers are limited, since factors like
    alcohol (ab)use and smoking may have influenced the outcomes (see Table 8).
        Numerous data have been presented on the induction of sister chromatid
    exchanges by acetaldehyde using in vitro, and to a lesser extent, in vivo test
    systems. In most of these studies acetaldehyde scored positive, and in some of
    these studies also a dose-related response was found. Based on these findings,
    the Committee concludes that acetaldehyde induces cytogenetic effects.
5.5 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 germ
    cell mutagenicity, and the only animal germ cell mutagenicity study did not show
    Genotoxicity                                                                      37
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<pre>    mutagenic activity (Lähdetie et al., 1988).41 Overall, due to a lack of data the
    Committee concludes that there is no positive direct evidence for in vivo
    heritable germ cell mutagenicity of acetaldehyde.
         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 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). Sufficient evidence has been found for in vivo mutagenicity
    testing in somatic cells of mammals. Regarding the second part of the criterion,
    there is limited evidence that acetaldehyde is genotoxic (sister chromatid
    exchanges) in germ cells of mice (Madrigal-Bujaidar et al. 2002), when the
    substance was given by intraperitoneal injection.77 These findings indicate that
    acetaldehyde is able to reach the germ cells, and interacts with the genetic
    material, which would be in line with the findings on absorption and distribution
    kinetics (see Chapter 4). However, in another animal study no abnormal sperm
    cells, and no meiotic micronuclei in spermatids were observed at dose levels
    inducing acute toxicity (Lähdetie et al. 1988).41
         Overall, the Committee is of the opinion that some evidence exists that
    acetaldehyde has potential to cause mutations in germ cells. Therefore, it
    recommends classifying the substance in category 1B.
5.6 Conclusions on classification and labelling
    Based on the available data, the Committee recommends classifying
    acetaldehyde as a germ cell mutagen in category 1B (substance to be regarded as
    if they induce heritable mutations in the germ cells of humans).
    The Committee is furthermore of the opinion that acetaldehyde acts by a
    stochastic genotoxic mechanism.
 8  Acetaldehyde
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<pre> hapter     6
            Carcinogenicity
6.1         Non-human information
            Data on animal carcinogenicity studies are summarized in Table 12.
 able 12 Summary of animal carcinogenicity studies on acetaldehyde exposure.
 pecies        Design                  Exposure levels            Observations and remark                      References
Oral administration
 ats, Sprague 50 animals/sex/group;    0 - 50 - 250 - 500 - 1,500 Klimisch-score: 2.                           Soffritti et
Dawley         animals kept in         - 2,500 mg acetaldehyde/ General: No difference between control and     al., 200278
               observation until       L drinking water (ad       exposed animals on consumption, body
               spontaneous death (last libitum; dose in kg/kg bw weight and survival.
               animal died in week     not given).                Lesions: Number of malignant tumour-
               161); gross necroscopy                             bearing animals did not differ significantly
               and histopathological                              from controls; Number of tumours per 100
               examinations.                                      animals was statistically significantly
                                                                  increased at 50 (females only), and at 2,500
                                                                  mg/L (males – female – both sexes,
                                                                  *p<0.05):
                                                                  - 0 mg/L: 34% - 46% - 40%
                                                                  - 50 mg/L: 52% - 82%* - 67%
                                                                  - 2,500 mg/L: 66%*- 78%*- 72%.
                                                                  Remark: The EFSA noted that the animals
                                                                  may have been infected with mycoplasma
                                                                  pulmonis. Therefore, DECOS considers the
                                                                  study of questionable relevance.
            Carcinogenicity                                                                                              39
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<pre> ats, Wistar  10 male animals/ group; 0 or 120 mM in drinking     Klimisch-score: 3 (only one dose used, short Homann et
              study duration 8 months; water (ad libitum; dose in exposure period, limited examination of       al., 199779
              immuno-histochemistry kg/kg bw not given).          tissues).
              and histopathological                               General: No difference between control and
              examination of the                                  exposed animals on consumption, body
              tongue, epiglottis, and                             weight and survival.
              forestomach; no other                               Lesions: No cancerous or dysplastic lesions
              tissue examined.                                    observed. Microscopic examination revealed
                                                                  hyperplasia in basal layers of squamous
                                                                  epithelia in the examined tissues of exposed
                                                                  animals.
nhalation
 ats, Wistar  105 animals/sex/ group;   0 - 1,350 - 2,700 - 5,400 Klimisch-score: 2.                            Woutersen
              six hours/day, five days/ mg/m3; due to toxicity,   General: lower survival and body weights      et al.,
              week for 28 months;       the highest exposure      were observed in exposed animals compared     198680
              gross necroscopy and      level was reduced to      to controls.
              histopathological         1,800 mg/m3 over a        Lesions: exposure induced malignant tumour
              examination.              period of 11 months.      in the respiratory tract. See main text and
                                                                  Table 13.
                                                                  Note: only the respiratory tract was examined
                                                                  for the presence of abnormalities.
Hamster,      36 animals/sex/group;     4,500 mg/m3 (week 1-9), Klimisch-score: 2 (no standard procedure of     Feron et al.,
 yrian golden seven hours/day, five     4,050 mg/m3 (week 10- doses applied).                                   198281
              days/week for 52 weeks, 20), 3,600 mg/m3 (week General: from week 4 onwards, exposed
              week 53-81, post-         21-29), 3,240 mg/m3       animals showed significant reduced body
              exposure period; gross    (week 30-44) and 2,970 weight compared to controls; reduction
              necroscopy and            mg/m3 (week 45-52); due diminished partly in the post-exposure
              histopathological         to considerable growth period.
              examination; 6 animals/ retardation and to avoid Lesions: exposure induced rhinitis,
              sex were killed for       early death, exposures    hyperplasia and metaplasia in the nasal,
              interim examination.      were reduced gradually laryngeal and tracheal epithelium. Also
                                        during experiment.        laryngeal and nasal carcinomas and polyps
                                                                  were observed; respiratory tract tumours:
                                                                  0/30 - 8/29 (male, control-exposed)
                                                                  0/28 - 5/29 (female, control-exposed)
Hamster,      35 animals/group (males 0 or 2,700 mg/m3            Klimisch-score: 2 (only one sex used, only    Feron et al.,
 yrian golden only); 7 hours/day, five                            one dose applied).                            197982
              days/week for 52 weeks,                             General: in exposed animals, body weights
              animals killed after 78                             were slightly lower than in controls. In the
              weeks; at week 52, 5                                last part of the exposure period mortality
              animals were killed for                             increased more rapidly in exposed animals
              interim examination;                                than in controls.
              gross necroscopy and                                Lesions: no substance-related tumours found.
              histopathological                                   Acetaldehyde induced hyperplastic,
              examination.                                        metaplastic and inflammatory changes.
                                                                  Note: exposure level may have been too low
                                                                  to induce adverse health effects.
 0           Acetaldehyde
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<pre>Dermal exposure
 ats           14 to 20 animals;       (Total) dose not known;   Klimisch-score: 4 (data from secondary          Watanabe
               subcutaneous injection. repeated injections.      source; original study in Japanese; no abstract and
                                                                 available))                                     Sugimoto
                                                                 General: no data.                               195683
                                                                 Lesions: spindle-cell sarcomas at site of
                                                                 injections (in four animals that survived the
                                                                 period up to 554 days).
ntratracheal installation
Hamsters,      35 animals/sex/group;   0 or 2% acetaldehyde      Klimisch-score: 3 (only one dose applied;       Feron et al.,
 yrian golden weekly installations for (installation volume, 0.2 experiment not performed according to           197982
               52 weeks, experiment    mL).                      today’s standard methods).
               was terminated at week                            General: no clear effects on body weight or
               104.                                              mortality.
                                                                 Lesions: No substance-related tumours found.
                                                                 Hyperplastic and inflammatory changes
                                                                 observed in the bronchioalveolar region of
                                                                 exposed animals.
6.1.1       Carcinogenicity: oral administration
            Male and female Sprague-Dawley rats (50 animals/sex/group) were exposed to
            0, 50, 250, 500, 1,500 and 2,500 mg/L acetaldehyde in drinking water (dose in kg
            bw not given), beginning at six weeks of age (Soffritti et al., 2002).78 Animals
            were kept under observation until spontaneous death. In various organs and
            tissues neoplastic lesions were observed. However, no clear increase in number
            of tumour-bearing animals was found in any of the exposed groups compared to
            the control group. The investigators reported a significantly increased total
            number of tumours (per 100 animals) in groups exposed to 50 mg/L (females
            only), and 2,500 mg/L (males; females). The Committee noted the lack of
            statistical analysis, and the limited examination of non-neoplastic end-points.
            Furthermore, the European Food Safety Authority (EFSA) has evaluated the
            studies performed by the European Ramazzi Foundation of Oncology and
            Environmental Sciences, who performed this study, and noted that the animals
            used by this foundation, may have been infected with Mycoplasma pulmonis.
            This may have resulted in chronic inflammatory changes.84 For these reasons, the
            Committee considers the findings of the study of questionable relevance.
                 Homann et al. (1997) have given male Wistar rats (N=10/group) either water
            containing acetaldehyde (120 mM) or tap water to drink for eight months.79
            Animals were then sacrificed, and of each animal tissue samples were taken from
            the tongue, epiglottis, and forestomach. No tumours were observed. However, in
            these organs, microscopic examination revealed statistically significant
            Carcinogenicity                                                                                               41
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<pre>      hyperplasia of the basal layers of squamous epithelia in rats receiving
      acetaldehyde (compared to controls). Furthermore, in the three organs of the
      treated animals, cell proliferation was significantly increased, and the epithelia
      were significantly more hyperplastic, than in control animals.
6.1.2 Carcinogenicity: inhalation
      In a carcinogenicity study by Woutersen et al. (1986), Wistar rats (105 animals/
      sex/group) inhaled acetaldehyde at a concentration of 0, 750, 1,500 or 3,000 ppm
      (0, 1,350, 2,700 or 5,400 mg/m3) for six hours a day, five days per week for a
      maximum of 28 months.80 The highest exposure level was reduced progressively
      over a period of eleven months to 1,000 ppm (1,800 mg/m3) due to toxicity. The
      study focussed on lesions in the respiratory tract.
          In general, animals exposed to acetaldehyde showed lower survival rates and
      body weights compared to controls. This was most pronounced in males exposed
      to the highest concentration of acetaldehyde. Gross examination at autopsy did
      not reveal acetaldehyde-related lesions, except for decolourisation of the fur and
      nasal swellings in all exposed groups. Microscopic examination revealed several
      non-neoplastic lesions in the respiratory tract of males and females, such as:
      hyperplasia in the respiratory nasal and olfactory epithelium; squamous
      metaplasia in the respiratory nasal epithelium; and, squamous metaplasia/
      hyperplasia in the larynx. These lesions were mainly noted in the mid and/or
      high exposure groups, and were statistically significantly increased compared to
      controls. No lesions were found in the lungs.
          In the nose, also exposure-related neoplastic lesions were observed (see
      Table 13). It concerned squamous cell carcinoma in the respiratory epithelium of
      the nose, and adenocarcinomas in the olfactory epithelium. The relative lower
      tumour incidences in the high exposure groups were explained by the
      investigators by early mortality due to other causes than cancer. According to the
      authors, the observations support the hypothesis that nasal tumours arise from
      degeneration of the nasal epithelium. The same research group reported earlier
      on degeneration of the olfactory epithelium in rats inhaling acetaldehyde for four
      weeks, under comparable experimental conditions (Appelman et al., 1986).85
          In a separate publication, the same authors reported on the interim results
      obtained in the first 15 month of the study (Woutersen et al. 1984).86 In short,
      nasal lesion were reported in exposed animals, indicating chronic and permanent
      inflammation.
          In a study by Feron et al. (1982), Syrian golden hamsters (n=36/sex/group)
      inhaled decreasing concentrations of acetaldehyde (from 2,500 ppm to 1,650
 2    Acetaldehyde
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<pre>ppm (equal to 4,500 to 2,970 mg/m3)) or clean room air, for seven hours a day,
five days per week for 52 weeks.81 The concentrations were reduced during the
study because of considerable growth retardation and to avoid early death.
Acetaldehyde induced rhinitis, hyperplasia and metaplasia of the nasal, laryngeal
and tracheal epithelium. The exposed animals also developed laryngeal
carcinomas with a few laryngeal polyps, and nasal polyps and carcinomas. The
incidences of respiratory tract tumours were 0/30 (males, control), 8/29 (males,
exposed), 0/28 (females, control) and 5/29 (females, exposed) (see Table 14).
According to the Committee, the study by Feron et al. supports the findings of
the carcinogenicity study by Woutersen et al. (1986) with rats.
     Male Syrian golden hamsters (n=35/group) were exposed to 1,500 ppm
(2,700 mg/m3) acetaldehyde combined with weekly intratracheal instillations of
benzo[a]pyrene (0.0625, 0.125, 0.25, 0.5 or 1 mg/kg bw) (Feron et al., 1979).82
The exposure was for seven hours a day, five days per week for 52 weeks. No
tumours were found in hamsters exposed to acetaldehyde alone, whereas in
animals treated with benzo[a]pyrene alone, or with a combination of
acetaldehyde and benzo[a]pyrene, a dose-related increase in respiratory-tract
tumours were found.
Table 13 Respiratory tract tumour incidences in rats, which were exposed by inhalation to
acetaldehyde for 28 months.80
Exposure level (ppm)                               0           750        1,500        3,000-1,000
Male animals
Nose:
   Papilloma                                         0/49       0/52        0/53         0/49
   Squamous cell carcinoma                           1/49       1/52      10/53a       15/49b
   Carcinoma in situ                                 0/49       0/52        0/53         1/49
   Adenocarcinoma                                    0/49      16/52b     31/53b       21/49b
Larynx: carcinoma in situ                            0/50       0/50        0/51         0/47
Lungs: poorly differentiated adenocarcinoma          0/55       0/54        0/55         0/52
Female animals
Nose:
   Papilloma                                         0/50       1/48        0/53         0/53
   Squamous cell carcinoma                           0/50       0/48        5/53       17/53b
   Carcinoma in situ                                 0/50       0/48        3/53         5/53
   Adenocarcinoma                                    0/50       6/48a     26/53b       21/53b
Larynx: carcinoma in situ                            0/51       0/46        1/47         0/49
Lungs: poorly differentiated adenocarcinoma          0/53       1/52        0/54         0/54
a    Fischer exact test: p<0.05.
b    Fischer exact test: p<0.001.
Carcinogenicity                                                                                    43
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<pre>      Table 14 Respiratory tract tumour incidences in hamsters, which were exposed by inhalation to
      acetaldehyde for 52 weeks (Feron et al., 1982).81
                                                 Incidence of tumours:      Incidence of tumours:
                                                 males                      females
                                                 Control       Acetaldehyde Control       Acetaldehyde
      Nose
        Adenoma                                  0/24          1/27         0/23          0/26
        Adenocarcinoma                           0/24          0/27         0/23          1/26
        Anaplastic carcinoma                     0/24          1/27         -             -
      Larynx
        Polyp/papilloma                          0/20          1/23         0/22          1/20
        Carcinoma in situ                        0/20          3/23         0/22          0/20
        Squamous cell carcinoma                  0/20          2/23         0/22          1/20
        Adeno-squamous cell carcinoma            -             -            0/22          2/20
      Total                                      0/30          8/29a        0/28          5/29
      a    Statistical significance (Fisher’s exacttest).
6.1.3 Carcinogenicity: dermal exposure
      Watanabe et al. (1956) reported on the induction of sarcomas in rats given
      acetaldehyde by subcutaneous injections.83 The Committee noted the limited
      study design, such as the small number of animals and the lack of a control
      group.
6.1.4 Carcinogenicity: other routes of exposure
      No tumours were found in Syrian golden hamsters (n=35/sex/dose), which were
      given acetaldehyde by intratracheal installations, weekly or biweekly, for 52
      weeks, followed by a recovery period for another 52 weeks (Feron et al., 1979).82
      Doses applied were 0.2 mL of 2% or 4% solutions. In positive controls, which
      were given benzo[a]pyrene and N-nitrosodiethylamine, a variety of tumours in
      the respiratory tract were found.
6.2   Human information
      No human studies addressing the carcinogenicity of acetaldehyde alone have
      been retrieved from public literature.
 4    Acetaldehyde
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<pre>      In East-Germany, nine cancer cases were found in a factory where the main
      process was dimerization of acetaldehyde, and where the main exposures were to
      acetaldol, acetaldehyde, butyraldehyde, crotonaldehyde and other higher,
      condensed aldehydes, as well as to traces of acrolein.87,88 Of these cancer cases,
      five were bronchial tumours and two were carcinomas of the oral cavity. All nine
      patients were smokers. The relative frequencies of these tumours were reported
      to be higher than those observed in the population of East-Germany. A matched
      control group was not included. The Committee noted the combined exposure
      with other potential carcinogenic substances, the small number of cases, and the
      poorly defined exposed population.
6.3   Other relevant information
6.3.1 Alcohol consumption
      Regarding the general population, some investigators suggest a role for
      acetaldehyde in cancer development (and other disorders) in humans after
      alcohol consumption, in particular in people with a genetic predisposition of one
      of the enzymes that are involved in ethanol metabolism.3,4,89-95 Acetaldehyde is
      the major metabolite of ethanol (ethyl alcohol).3,92,96-98 First, ethanol is oxidized
      by alcohol dehydrogenase (ADH) to acetaldehyde, and subsequently
      acetaldehyde is converted by aldehyde dehydrogenase (ALDH2) to acetate. Both
      enzymes show genetic polymorphisms. This means that depending on the
      genotype, the enzymes may lead to a faster breakdown of ethanol to
      acetaldehyde, and/or to a slower breakdown of acetaldehyde to acetate. Thus,
      people having unfavourable genotypes of these enzymes are likely to be exposed
      internally to higher levels of acetaldehyde after alcohol consumption than would
      be the case when not having one of these isoenzymes. This would increase the
      susceptibility to cancer development after alcohol consumption, since it is
      suggested that acetaldehyde possesses carcinogenic properties.
          Several studies reported on the association between genetic polymorphism
      and ethanol-related cancer development, all suggesting a role for acetaldehyde.
      As a result, a few meta-analyses have been performed to get more clarity. For
      instance, Chang et al. (2012) performed a meta-analysis to study the association
      between ADH1B* and ADH1C genotypes in head and neck cancer risk.99 The
      analysis included twenty-nine studies. According to the authors, having at least
      one of the fast alleles ADH1B*2 or ADH1C*1 reduced the risk for head and
      neck cancer (odds ratios: 0.50 (95% confidence interval (CI), 0.37-0.68) for
      ADH1B*2; 0.87 (95%CI, 0.76-0.99).
      Carcinogenicity                                                                       45
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<pre>       Wang et al. (2012) performed a meta-analysis to derive a more precise
  estimate of the relationship between ADH1C genotypes, and breast cancer
  risk.100 Twelve case-control studies were included in the analysis, covering 6,159
  cases and 5,732 controls (all Caucasians). The investigators did not find any
  significantly increased breast cancer risk that could be related to any ADH1C
  genotype.
       Boccia et al. (2009) reported on a meta-analysis to study the relationship
  between ALDH2 homozygous and heterozygous genotypes, alcohol
  consumption, and head and neck cancer.101 The analysis included six case-
  control studies, covering 945 Japanese cases and 2,917 controls. For the analysis,
  the investigators used a Mendelian randomization approach. The homozygous
  genotype ALDH2*2*2 (unable to metabolize acetaldehyde) reduced the risk of
  head and neck cancer, whereas the heterozygous genotype ALDH2*1*2 (partly
  able to metabolize acetaldehyde) did significantly increase the risk compared to
  the homozygous ALDH2*1*1 genotype (able to metabolize acetaldehyde).
  According to the authors, the reduction of cancer risk in ALDH2*2*2 was most
  likely explained by the fact that people having this genotype consumed markedly
  lower levels of alcohol compared to the other genotypes, probably due to
  discomfort. Therefore, the authors conclude that their study supports the
  hypothesis that alcohol increases head and neck cancer risk through the
  carcinogenic action of acetaldehyde.
       The same results were obtained by Fang et al. (2011), who carried out a meta-
  analysis of ALDH2 genotypes and esophageal cancer development.102 Data from
  sixteen studies (hospital- or population-based, one multicenter study) were
  analysed, covering 2,697 Asian cases and 6,344 controls. The analysis showed
  that the heterozygous ALDH2*1*2 genotype increased the risk of esophageal
  cancer, whereas the homozygous ALDH2*2*2 genotype reduced the risk.
       Yokoyama and Omori (2005) reviewed a number of case-control studies
  (including those performed by themselves) on the relationship of genetic
  polymorphism of ADH1B, ADH1C and ALDH2 genotypes and esophageal, and
  head and neck cancer risk.103 They found positive associations between the less-
  active ADH1B*1 genotype and inactive heterozygous ALDH2*1*2 genotype,
  ADH has seven isoenzymes, which are divided into five classes. Most relevant for alcohol
  metabolism in the liver of adults are the class one isoenzymes ADH1B and ADH1C (formerly known
  as ADH2 and ADH3 isoenzymes).99 For each isoenzyme two or three different alleles are known,
  leading to different genotypes and thus to functional polymorphism. The genotypes of the isoenzyme
  ADH1B are expressed as ADH1B*1, ADH1B*2 and ADH1B*3; those for the isoenzyme ADH1C
  are expressed as ADH1C*1 and ADH1C*2. The metabolic speed is highest for homozygote
  genotypes ADH1B*2, ADH1B*3 and ADH1C*1. ADH1B*1 and ADH1C*2 are considered slow
  metabolisers.
6 Acetaldehyde
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<pre>      and the risk for esophageal cancer in East Asian heavy drinkers. Light-to-
      moderate drinkers showed a higher vulnerability. According to the authors, some
      studies suggest similar associations for the risk for head and neck cancer in
      moderate-to-heavy-drinking Japanese. Data on ADH1C genotype were
      controversial.
          The Committee emphasizes that in none of the studies on genetic
      polymorphism and alcohol-related cancer risk, a direct association was found
      between acetaldehyde and cancer, although the indirect data are suggestive for
      this.
6.3.2 Cell transformation tests
      Koivisto and Salaspuro (1998) reported on a transformation test in which human
      colon adenocarcinoma cell line Caco-2 were used to study changes in cell
      proliferation, cell differentiation, and adhesion due to exposure to
      acetaldehyde.104 In the absence of cell cytotoxicity, on acute exposure (for 72
      hours), acetaldehyde (0.5 or 1 mM) inhibited the cell proliferation rate, but on
      chronic exposure (for five weeks) it stimulated cell proliferation. Furthermore,
      acetaldehyde clearly disturbed the cell differentiation (concentration applied was
      1 mM for 7, 14 or 21 days); and, a clear decrease of adhesion of Caco-2 cells to
      collagens was observed when acetaldehyde was applied to the cells at a
      concentration of 0.5 or 1 mM for four days. According to the authors, the
      increased proliferation rate, disturbed differentiation, and reduced adhesion,
      would in vivo predict more aggressive and invasive tumour behaviour.
          Eker and Sanner (1986) used a rat kidney cell line in a two-stage cell
      transformation assay.105 Acetaldehyde (up to 3 mM) did not affect cytotoxicity
      nor did it induce colony formation of the cells. When acetaldehyde treatment (3
      mM) was followed by a tumour promoter 12-O-tetradecanoylphorbol-13-acetate
      (TPA), the ability of the cells to form colonies was increased.
          In a poorly reported study by Abernathy et al. (1982), acetaldehyde (10-100
      µl/ml (LC50, 25 µg/ml)) induced cell transformation in C3H/10T½ cells, in the
      presence of TPA.106 Treatment with acetaldehyde alone did induce transformed
      foci.
          The Committee emphasizes that the value of transformation test in assessing
      carcinogenic potential is under debate. Therefore, it attaches little value to the
      outcomes of these tests.
      Carcinogenicity                                                                    47
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<pre>6.4 Summary and discussion of carcinogenicity
    Epidemiological studies are not available. In the literature, it is suggested that
    acetaldehyde may play a role in cancer development in humans after alcohol
    consumption, in particular in combination with a genetic predisposition for
    enzymes that convert ethanol in acetaldehyde, and for enzymes that convert
    acetaldehyde in acetate. The Committee emphasizes that in none of the studies
    on genetic polymorphism and alcohol-related cancer risk, a direct association
    was found between acetaldehyde and cancer, although the indirect data are
    suggestive for this.
        Regarding animal carcinogenicity studies, chronic inhalation of acetaldehyde
    induced squamous cell carcinomas and adenocarcinomas in the nose of male and
    female rats. In hamsters, inhaling the substance, one study showed the presence
    of laryngeal and nasal tumours, whereas in another study – using a lower
    exposure concentration – no tumours were observed at all.
6.5 Comparison with criteria
    For epidemiological data there is little or no data to support statements
    concerning an association between exposure to acetaldehyde and cancer.
    Therefore, the Committee is of the opinion that human data are insufficient to
    make a final conclusion on the carcinogenic potential of acetaldehyde in humans.
    For animal data, the Committee found sufficient evidence of carcinogenicity,
    since a causal relationship was established between malignant tumours in
    animals and chronic inhalation to acetaldehyde in two studies (Woutersen et al.,
    1986, Feron et al., 1982), the main route of exposure in an occupational
    environment.80,81 According to the CLP classification criteria, acetaldehyde
    should, therefore, be classified as “presumed to have carcinogenic potential for
    humans”, which corresponds to classification in category 1B. Supporting
    evidence for its carcinogenic potential is that the substance has mutagenic
    properties, and acts by a stochastic genotoxic mechanism.
        The Committee noticed that in 1991, the European Commission classified the
    substance as a carcinogen in category 2 (according to the current CLP-system).
    The classification was based on the same carcinogenicity studies as described in
    the present report. Most likely the difference in outcome is explained by
    differences in criteria used presently (criteria laid down in Regulation No. 1272/
    2008) and used in the late eighties of the twentieth century (criteria laid down in
    Annex VI of Directive 67/548/EEC).
 8  Acetaldehyde
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<pre>6.6 Conclusions on classification and labelling
    The Committee concludes that acetaldehyde is presumed to be carcinogenic to
    man, and recommends classifying the substance in category 1B*.
    See for classification system Annex F.
    Carcinogenicity                                                             49
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<pre>0 Acetaldehyde</pre>

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<pre>hapter 7
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   esophageal and head and neck cancers. Alcohol 2005; 35(3): 175-185.
04 Koivisto T, Salaspuro M. Acetaldehyde alters proliferation, differentiation and adhesion properties of
   human colon adenocarcinoma cell line Caco-2. Carcinogenesis 1998; 19(11): 2031-6.
05 Eker P, Sanner T. Initiation of in vitro cell transformation by formaldehyde and acetaldehyde as
   measured by attachment-independent survival of cells in aggregates. Eur J Cancer Clin Oncol 1986;
   22(6): 671-676.
06 Abernathy D, Frazelle J, Boreiko C. Effects of ethanol, acetaldehyde and acetic acid in the C3H/
   10T1/2 CI 8 cell transformation assay. Environ Mutagen 1982; 4: 331.
07 The Health Council. Guideline to the classification of carcinogenic compounds. Guide for classifying
   compounds in terms of their carcinogenic properties and for assessing their genotoxicity. The Hague:
   Report no. A10/07E; 2010: publication no. A10/07E.
8  Acetaldehyde
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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
  Annexes
                                      59
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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                                                                                        61
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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.
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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
     •  J.M. Rijnkels, scientific secretary
        Health Council of the Netherlands, The Hague
     The Committee                                                                63
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<pre>  With respect to the data presentation and interpretation, the Committee consulted
  an additional expert, Mr. 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.
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<pre>nnex C
     The submission letter (in English)
     Subject          : Submission of the advisory report Acetaldehyde
     Your Reference: DGV/MBO/U-932342
     Our reference : U-8234/JR/cn/246-W19
     Enclosed         :1
     Date             : November 13, 2014
     Dear State Secretary,
     I hereby submit the advisory report on the effects of occupational exposure to
     acetaldehyde.
     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
     included the opinions by the Health Council's Standing Committee on Health and
     the Environment.
     The submission letter (in English)                                                   65
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<pre>  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
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<pre>nnex D
     Comments on the public review draft
     A draft of the present report was released in 2014 for public review. The
     following organisations and persons have commented on the draft document:
     • D. Coggon, University of Southampton, UK
     • T.J. Lentz and Q. Ma, National Institute for Occupational Safety and Health
         (NIOSH), Cincinnati OH, USA.
     Comments on the public review draft                                           67
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<pre>nnex E
     IARC evaluation and conclusion
     Acetaldehyde (Group 2B), Volume 71 (1999) (p. 319)
     Summary of Data Reported and Evaluation
     Exposure data
     Exposure to acetaldehyde may occur in its production, and in the production of
     acetic acid and various other chemical agents. It is a metabolite of sugars and
     ethanol in humans and has been detected in plant extracts, tobacco smoke, engine
     exhaust, ambient and indoor air, and in water.
     Human carcinogenicity data
     An increased relative frequency of bronchial and oral cavity tumours was found
     among nine cancer cases in one study of chemical workers exposed to various
     aldehydes. Oesophageal tumours have been associated with genetically
     determined, high metabolic levels of acetaldehyde after drinking alcohol.
         Three case-control studies assessed the risk of oral, pharyngeal, laryngeal
     and oesophageal cancer following heavy alcohol intake, according to genetic
     polymorphism of enzymes involved in the metabolism of ethanol to
     acetaldehyde (alcohol dehydrogenase 3) and in the further metabolism of
     acetaldehyde (aldehyde dehydrogenase 2 and glutathione S-transferase M1).
     IARC evaluation and conclusion                                                   69
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<pre>  Despite limitations in the study design and the small size of most of the studies,
  these studies consistently showed an increased risk of alcohol-related cancers
  among subjects with the genetic polymorphisms leading to higher internal doses
  of acetaldehyde following heavy alcohol intake as compared to subjects with
  other genetic polymorphisms.
  Animal carcinogenicity data
  Acetaldehyde was tested for carcinogenicity in rats by inhalation exposure and in
  hamsters by inhalation exposure and by intratracheal instillation. It produced
  tumours of the respiratory tract following inhalation, particularly
  adenocarcinomas and squamous-cell carcinomas of the nasal mucosa in rats and
  laryngeal carcinomas in hamsters. In hamsters, it did not cause an increased
  incidence of tumours following intratracheal instillation. Inhalation of
  acetaldehyde enhanced the incidence of respiratory-tract tumours produced by
  intratracheal instillation of benzo[a]pyrene.
  Other relevant data
  Acetaldehyde is metabolized to acetic acid. During inhalation exposure of rats,
  degeneration of nasal epithelium occurs and leads to hyperplasia and
  proliferation.
      Acetaldehyde causes gene mutations in bacteria and gene mutations, sister
  chromatid exchanges, micronuclei and aneuploidy in cultured mammalian cells,
  without metabolic activation. In vivo, it causes mutations in Drosophila
  melanogaster but not micronuclei in mouse germ cells. It causes DNA damage in
  cultured mammalian cells and in mice in vivo. Acetaldehyde-DNA adducts have
  been found in white blood cells from human alcohol abusers.
  Evaluation
  There is inadequate evidence in humans for the carcinogenicity of acetaldehyde.
  There is sufficient evidence in experimental animals for the carcinogenicity of
  acetaldehyde.
  Overall evaluation
  Acetaldehyde is possibly carcinogenic to humans (Group 2B).
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<pre>Previous evaluations: Vol. 36 (1985); Suppl. 7 (1987).
Synonyms: Acetic aldehyde; ‘Aldehyde’; Ethanal; Ethylaldehyde.
IARC evaluation and conclusion                                 71
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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
                                                                                67/548/EEC            EC No 1272/2008
                                                                                before                as from
                                                                                12/16/2008            12/16/2008
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: Health Council of the Netherlands, 2010; publication no. A10/07E.107
            Classification on carcinogenicity                                                                         73
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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
     3.5.1.1        A 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.
     3.5.1.2        The 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
     3.5.2.1        This 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
     Classification on mutagenicity                                                                            75
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<pre>            mutagenicity or 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.
            3.5.2.2      For the purpose of classification for germ cell mutagenicity, substances are allocated to
            one of two categories as shown in Table 3.5.1.
            3.5.2.3      Specific considerations for classification of substances as germ cell mutagens
            3.5.2.3.1    To 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.
            3.5.2.3.2    The 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.
 able 3.5.1 Hazard categories for germ cell mutagens.
 ategories                      Criteria
 ATEGORY 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.
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.
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<pre>3.5.2.3.3     Classification 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.
3.5.2.3.4     In vivo heritable germ cell mutagenicity tests, such as:
•    rodent dominant lethal mutation test;
•    mouse heritable translocation assay.
3.5.2.3.5     In vivo somatic cell mutagenicity tests, such as:
•    mammalian bone marrow chromosome aberration test;
•    mouse spot test;
•    mammalian erythrocyte micronucleus test.
3.5.2.3.6     Mutagenicity/genotoxicity tests in germ cells, such as:
(a) mutagenicity tests:
•    mammalian spermatogonial chromosome aberration test;
•    spermatid micronucleus assay;
(b) Genotoxicity tests:
•    sister chromatid exchange analysis in spermatogonia;
•    unscheduled DNA synthesis test (UDS) in testicular cells.
3.5.2.3.7     Genotoxicity tests in somatic cells such as:
•    liver Unscheduled synthesis test (UDS) in vivo;
•    mammalian bone marrow Sister Chromatid Exchanges (SCE);
3.5.2.3.8     In vitro mutagenicity tests such as:
•    in vitro mammalian chromosome aberration test;
•    in vitro mammalian cell gene mutation test;
•    bacterial reverse mutation tests.
3.5.2.3.9     The 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.
Classification on mutagenicity                                                                           77
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<pre>  3.5.3          Classification criteria for mixtures
  3.5.3.1        Classification of mixtures when data are available for all ingredients or only for some
  ingredients of the mixture
  3.5.3.1.1      The 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).
  3.5.3.2        Classification of mixtures when data are available for the complete mixture
  3.5.3.2.1      Classification of mixtures will be based on the available test data for the individual
  ingredients of the mixture using concentration limits for the ingredients 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
  analysis of germ cell mutagenicity test systems. Adequate documentation supporting the
  classification shall be retained and made available for review upon request.
  3.5.3.3        Classification of mixtures when data are not available for the complete mixture:
  bridging principles
  3.5.3.3.1      Where 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.
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<pre>3.5.4          Hazard communication
3.5.4.1        Label elements shall be used in accordance with Table 3.5.3, for substances or mixtures
meeting the criteria for classification in this hazard class.
Table 3.5.3 Label elements of germ cell mutagenicity.
Classification                            Category 1A or Category 1B     Category 2
GHS Pictograms
Signal word                               Danger                         Warning
Hazard Statement                          H340: May cause genetic        H341: Suspected of causing
                                          defects (state route of        genetic defects (state route of
                                          exposure if it is conclusively exposure if it is conclusively
                                          proven that no other routes of proven that no other routes of
                                          exposure cause the hazard)     exposure cause the hazard)
Precautionary Statement Prevention        P201, P202, P281               P201, P202, P281
Precautionary Statement Response          P308 + P313                    P308 + P313
Precautionary Statement Storage           P405                           P405
Precautionary Statement Disposal          P501                           P501
3.5.5          Additional classification considerations
It is increasingly accepted that the process of chemical-induced tumorigenesis 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                                                                           79
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<pre>Health Council of the Netherlands
Advisory Reports
The Health Council’s task is to       In addition, the Health Council
advise ministers and parliament on    issues unsolicited advice that
issues in the field of public health. has an ‘alerting’ function. In some
Most of the advisory reports that     cases, such an alerting report
the Council produces every year       leads to a minister requesting
are prepared at the request of one    further advice on the subject.
of the ministers.
Areas of activity
Optimum healthcare                    Prevention                          Healthy nutrition
What is the optimum                   Which forms of                      Which foods promote
result of cure and care               prevention can help                 good health and
in view of the risks and              realise significant                 which carry certain
opportunities?                        health benefits?                    health risks?
Environmental health                  Healthy working                     Innovation and
Which environmental                   conditions                          the knowledge
influences could have                 How can employees                   infrastructure
a positive or negative                be protected against                Before we can harvest
effect on health?                     working conditions                  knowledge in the
                                      that could harm their               field of healthcare,
                                      health?                             we first need to
                                                                          ensure that the right
                                                                          seeds are sown.
www.healthcouncil.nl
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<br><br>