<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>Acetaldehyde
    Evaluation of the carcinogenicity and genotoxicity
</pre>

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<pre></pre>

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<pre>Aan de staatssecretaris van Sociale Zaken en Werkgelegenheid
Onderwerp             : aanbieding advies Acetaldehyde
Uw kenmerk            : DGV/MBO/U-932342
Ons kenmerk           : U-7438/JR/fs/246-H17
Bijlagen              :1
Datum                 : 23 november 2012
Geachte staatssecretaris,
Graag bied ik u hierbij het advies aan over de gevolgen van beroepsmatige blootstelling aan
aceetaldehyde.
Dit advies maakt deel uit van een uitgebreide reeks waarin kankerverwekkende stoffen wor-
den geclassificeerd volgens richtlijnen van de Europese Unie. Het gaat om stoffen waaraan
mensen tijdens de beroepsmatige uitoefening kunnen worden blootgesteld.
      Dit advies is opgesteld door een vaste subcommissie van de Commissie Gezondheid en
beroepsmatige blootstelling aan stoffen (GBBS), de Subcommissie Classificatie van carci-
nogene stoffen. Het advies is getoetst door de Beraadsgroep Gezondheid en omgeving van
de Gezondheidsraad.
Ik heb het 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. W.A. van Gool,
voorzitter
Bezoekadres                                                      Postadres
Parnassusplein 5                                                 Postbus 16052
2 5 11 V X D e n        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
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<pre></pre>

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<pre>Acetaldehyde
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 State Secretary of Social Affairs and Employment
No. 2012/22, The Hague, November 23, 2012
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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.
                 The Health Council of the Netherlands is a member of the International Network
                 of Agencies for Health Technology Assessment (INAHTA), an international
                 collaboration of organisations engaged with health technology assessment.
 I NA HTA
This report can be downloaded from www.healthcouncil.nl.
Preferred citation:
Health Council of the Netherlands. Acetaldehyde. Evaluation of the
carcinogenicity and genotoxicity. The Hague: Health Council of the Netherlands,
2012; publication no. 2012/22.
all rights reserved
ISBN: 978-90-5549-913-7
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<pre>   Contents
   Samenvatting 9
   Executive summary 11
   Scope 13
.1 Background 13
.2 Committee and procedures 13
.3 Data 14
   General information 15
.1 Identity, and physico-chemical properties 15
.2 IARC classification 16
   Carcinogenicity 17
.1 Observations in humans 17
.2 Carcinogenicity studies in animals 19
.3 Cell transformation tests 22
   Mode of action 25
.1 Genotoxic mode of action 25
.2 Non-genotoxic mode of action 28
   Contents                                     7
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<pre>    Classification 29
 .1 Evaluation of data on carcinogenicity and genotoxicity 29
 .2 Recommendation for classification 30
    References 31
    Annexes 37
A   Request for advice 39
B   The Committee 41
C   The submission letter 43
D   Comments on the public review draft 45
E   IARC evaluation and conclusion 47
F   Carcinogenic classification of substances by the Committee 51
    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 raad, hierna kortweg aangeduid als de
commissie. In het voorliggende advies neemt de commissie aceetaldehyde onder
de loep. 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 stof-
fen en als bewaarmiddel voor bijvoorbeeld vis en fruit.
De commissie concludeert dat aceetaldehyde beschouwd moet worden als kan-
kerverwekkend voor de mens, en beveelt aan de stof in categorie 1B te classifice-
ren.* Aceetaldehyde heeft een stochastisch genotoxisch werkingsmechanisme.
Volgens het classificatiesysteem van de Gezondheidsraad (zie bijlage F).
Samenvatting                                                                      9
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<pre>0 Acetaldehyde</pre>

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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 this report, the Committee evaluates
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 compounds, and as a fish and fruit preservative.
The Committee concludes that acetaldehyde is presumed to be carcinogenic to
man, and recommends classifying the compound in category 1B.* Based on the
available data, acetaldehyde acts by a stochastic genotoxic mechanism.
According to the classification system of the Health Council (see Annex F).
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). In addition to classifying substances, the Health
        Council also assesses the genotoxic properties of the substance in question. The
        assessment and the proposal for a classification are expressed in the form of
        standard sentences (see Annex F)
             This report contains the evaluation of the carcinogenicity and genotoxicity of
        acetaldehyde.
1.2     Committee and procedures
        The evaluation is performed by the Subcommittee on Classifying carcinogenic
        substances of the Dutch Expert Committee on Occupational Safety of the Health
        Council, hereafter called the Committee. The members of the Committee are
        listed in Annex B. The submission letter (in English) can be found in Annex C.
             In 2012 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
        Scope                                                                               13
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<pre>    listed in Annex D. The Committee has taken these comments into account in
    deciding on the final version of the report.
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 is inserted in annex E.
         Additional data were obtained from the online databases Toxline, Medline
    and Chemical Abstracts, covering the period 1997 to October 2012, 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
        General information
2.1     Identity, and physico-chemical properties
        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. It is also a metabolite during the breakdown of ethanol in the
        body, and is present in tobacco smoke. Acetaldehyde is produced on a large
        industrial scale for many purposes and uses.1 For instance, it is used as an
        intermediate in the production of acetic acid, but also in the production to for
        instance cellulose acetate, and pyridine derivates. It is furthermore used: in the
        production of 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.
        The identity, and its properties are shown below. 1-4
        CAS registry number    :   75-07-0
        EINECS number          :   200-836-8
        Synonyms               :   Ethanal, acetic aldehyde, ethylaldehyde, acetic aldehyde
        Appearance             :   Colourless volatile liquid
        General information                                                                    15
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<pre>    Chemical and structure :  C 2H 4O
    formula
    Molecular weight       :  44.05
    Boiling/melting point  :  29°C and -123.5°C
    Vapour pressure        :  2.5 kPa at -50°C; 44.0 kPa at 0°C; 101.3 kPa at 20.16°C
    Vapour density         :  1.52 (air=1)
    Solubility             :  Miscible in water and most common solvents
    Conversion factor      :  1 ppm = 1.8 mg/m³; 1mg/m³ = 0.56 ppm (at 25°C, 101.3 kPa)
    EU Classification      :  Carc. 2; H224 (extremely flammable liquid and vapour),
                              H319 (causes serious eye irritation), H335 (may cause respiratory
                              irritation), and H351 (suspected of causing cancer).
2.2 IARC classification
    In 1999, IARC concluded that there was inadequate evidence in humans for the
    carci-nogenicity of acetaldehyde, and that there was sufficient evidence in
    experimental animals.5 Therefore, IARC classified the compound in Group 2B
    (‘possible 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.6 More importantly, in 2012 IARC concluded that ‘acetaldehyde
    associated with alcohol consumption’ is carcinogenic to humans.7
 6  Acetaldehyde
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<pre> hapter 3
        Carcinogenicity
3.1     Observations in humans
        No human studies addressing the carcinogenicity of acetaldehyde alone have
        been retrieved from public literature.
        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.8,9 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 compounds, the small number of cases, and the
        poorly defined exposed population.
        Regarding the general population, some investigators suggest a role for acetalde-
        hyde 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.5,6,10-16 Acetaldehyde is the
        major metabolite of ethanol (ethyl alcohol).5,13,17-19 First, ethanol is oxidized by
        alcohol dehydrogenase (ADH) to acetaldehyde, and subsequently acetaldehyde
        Carcinogenicity                                                                      17
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<pre>  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 (see also Chapter 4).
       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-analysis 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.20 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).
       Wang et al. (2012) performed a meta-analysis to derive a more precise
  estimate of the relationship between ADH1C genotypes, and breast cancer risk.21
  Twelve case-control studies were included in the analysis, covering 6,159 cases
  and 5,732 controls (all Caucasians). The investigators did not found 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.22 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
  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).20 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.
8 Acetaldehyde
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<pre>      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.23 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.24 They found positive associations between the less-
      active ADH1B*1 genotype and inactive heterozygous ALDH2*1*2 genotype,
      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, direct evidence was found that
      acetaldehyde had caused cancer, although the data indirectly are suggestive for
      this.
3.2   Carcinogenicity studies in animals
3.2.1 Inhalation
      In a Dutch carcinogenicity study, 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.25 The highest exposure level was reduced progressively over a period of
      eleven months to 1,000 ppm (1,800 mg/m3) due to toxicity.
          In general, animals exposed to acetaldehyde showed lower survival rates and
      body weights compared to controls. This was most pronounced in males exposed
      Carcinogenicity                                                                   19
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<pre>             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 1). 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.26
 able 1 Tumour incidences in rats, which were exposed by inhalation to acetaldehyde for 28 months.25
 xposure 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           10/53*     15/49***
   Carcinoma in situ                                        0/49           0/52            0/53       1/49
   Adenocarcinoma                                           0/49          16/52***        31/53***   21/49***
 arynx: carcinoma in situ                                   0/50           0/50            0/51       0/47
 ungs: poorly differentiated adenocarcinoma                 0/55           0/54            0/55       0/52
 emale animals
Nose:
   Papilloma                                                0/50           1/48            0/53       0/53
   Squamous cell carcinoma                                  0/50           0/48            5/53      17/53***
   Carcinoma in situ                                        0/50           0/48            3/53       5/53
   Adenocarcinoma                                           0/50           6/48*          26/53***   21/53***
 arynx: carcinoma in situ                                   0/51           0/46            1/47       0/49
 ungs: poorly differentiated adenocarcinoma                 0/53           1/52            0/54       0/54
 ischer exact test: * p<0.05, ** p<0.01, *** p<0.001.
 0           Acetaldehyde
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<pre>          In another study, Syrian golden hamsters (n=36/sex/group) inhaled
      decreasing concentrations of acetaldehyde (2,500 ppm to 1,650 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.27 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), 0/29
      (females, control) and 5/29 (females, exposed).
          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).28 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.
3.2.2 Oral intake
      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.29 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.30 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.
      Animals were then sacrificed, and of each animal tissue samples were taken from
      Carcinogenicity                                                                   21
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<pre>      the tongue, epiglottis, and forestomach. No tumours were observed. However, in
      these organs, microscopic examination revealed statistically significant hyper-
      plasia 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.31
3.2.3 Dermal exposure
      Watanabe et al. (1956) reported on the induction of sarcomas in rats given
      acetaldehyde by subcutaneous injections.32 The Committee noted the limited
      study design, such as the small number of animals and the lack of a control
      group.
3.2.4 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.28 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.
3.3   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 acetal-
      dehyde.33 In the absence of cell cytotoxicity, on acute exposure (for 72 hours),
      acetaldehyde (500 or 1,000 µM) 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
      1,000 µM 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 500 or 1,000 µM 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.34 Acetaldehyde (up to 3,000 µM) did not affect cytotoxicity
 2    Acetaldehyde
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<pre>nor did it induce colony formation of the cells. When acetaldehyde treatment
(3,000 µM) was followed by a tumour promoter 12-O-tetradecanoylphorbol-
13-acetate, the ability of the cells to form colonies was increased.
Carcinogenicity                                                              23
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<pre>4 Acetaldehyde</pre>

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<pre> hapter 4
        Mode of action
4.1     Genotoxic mode of action
4.1.1   Gene mutation tests
        In vitro
        Vapours of acetaldehyde were not mutagenic to S. typhimurium or E.coli WP2
        uvrA, with or without metabolic activation.35-38 In addition, it did not induce
        mutations (dose range 0.1 -1.0 mL) in S. typhimurium tester strains TA97a,
        TA100, TA102 and TA104, in the presence and absence of a exogenous
        metabolic activation system, although the results on strain TA102 were
        equivocal.39
             Without an exogenous metabolic activation system, acetaldehyde induced
        gene mutations in mouse lymphoma L5178T cells.40 Also in human lymphocytes
        it induced mutations.41
             Using a shuttle vector plasmid, acetaldehyde (doses applied: 0.25, 0.5, 1.0,
        and 2.0 M) increased the frequency of mutations on the supF gene. Furthermore,
        after the plasmid was replicated in human fibrobast cell lines, it was observed
        that the majority of the mutations were specific tandem base substitutions (GG to
        TT).42
             In another study, acetaldehyde induced 1,N2-propano-dG adducts in a DNA
        vector that next was introduced into human xeroderma pigmentosum A (XPA)
        Mode of action                                                                    25
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<pre>      cells to allow replication.43 Analysis of the DNA of these cells showed major
      miscoding events, such as G->T and G->C transversions.
4.1.2 Cytogenetic tests
      In vitro
      Acetaldehyde increased the frequency of sister chromatid exchanges in Chinese
      Hamster Ovary cells (without an exogenous metabolic activation system), and in
      human lymphocytes.41,44-48,48-56
          It furthermore induced chromosomal aberrations in human lymphocytes;46,48,57
      positive- and negative-centromere-staining micronuclei in human lymphocytes;58
      aneuploidy in embryonic Chinese hamster diploid fibroblasts (without exogenous
      metabolic activation);59 chromosomal malsegregation in Aspergillus nidulans.60
      In vivo
      Acetaldehyde, when given to animals, increased the frequency of sister
      chromatid exchanges in Chinese hamster bone-marrow cells;61 chromosomal
      aberrations in rat embryos;62 and, chromosomal aberrations in mouse bone-
      marrow cells.46,62
          Furthermore, sister-chromatid exchanges in spermatogonial mouse cells were
      determined after intraperitoneal injection with acetaldehyde (0.4, 4.0, 40.0, and
      400 mg/kg bw). All four doses tested produced a positive response, although no
      clear exposure-response relationship was found. The lowest dose had an increase
      of sister chromatid exchange of a factor of 1.1 compared to the background
      value; the highest dose had an increase of a factor of 3.2.63
          In male mice, which were given an intraperitoneal injection of acetaldehyde,
      no abnormal sperm morphology or spermocyte micronuclei were observed.64
          Mice with an inactive ALDH2 gene were generated by gene targeting
      knockout as a model of ALDH2-deficient humans.65 The mice and a control
      group of wild-type ALDH2 mice (able to metabolize acetaldehyde), were
      continuously exposed to 125 and 500 ppm of acetaldehyde vapour for two
      weeks. Another group (knock-out and wild-type mice) was orally administered
      100 mg acetaldehyde/kg bw, daily, once a day for two weeks. The animals were
      killed at the end of the exposure period. The frequency of micronucleated
      reticulocytes induced by acetaldehyde was significantly increased in mice having
      the inactive ALDH2 gene, but not in the wild-type mice. The T-cell receptor
      (TCR) mutant frequency was also associated with the acetaldehyde exposure in
 6    Acetaldehyde
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<pre>      mice having an inactive ALDH2 gene, especially after oral administration;
      however, it was not associated with acetaldehyde exposure in wild-type mice.65
4.1.3 DNA-adducts
      In vitro
      Acetaldehyde-DNA adducts have been found in calf thymus DNA, in 2’-
      deoxyguanosine-3’-monophosphate.54,66,67 In another study, also using calf
      thymus DNA, mainly N2-ethylidene-deoxiguanosine DNA-adducts were
      found.68 In that study, three more stable adducts were detected, namely 1,N-
      propano-deoxiguanosine, N2-dimethyldioxane-deoxiguanosine, and a cross-link
      adduct. These three adducts were formed in substantially lower yield (less than
      10%) than the major adduct, but they were stable at the nucleoside level, and so
      may be more stable in DNA.
          Acetaldehyde-specific DNA adducts were also found in the DNA of: primary
      human liver cells, isolated from normal liver tissue (N2-ethyl-deoxiguanosine
      adducts);69 normal and SV40T antigen-immortalized human buccal epithelial
      cells (N2-ethyl-3’-dG-monophosphate adducts, dose-dependent, and at relatively
      non-toxic concentrations);70 and, in human embryonic kidney cell line 293 (N2-
      ethyl-deoxiguanosine adducts).71
      In vivo
      In humans, acetaldehyde induced statistically significantly higher levels of
      DNA-adducts in granulocytes and lymphocytes of twenty four alcohol abusers
      (p<0.001) compared to controls.66 The average adduct levels were 3.4±3.8 and
      2.1±0.8 adducts/107 nucleotides, respectively. In another study, investigators
      reported on a decrease in the number of acetaldehyde-specific DNA adducts (N2-
      ethylidene-deoxiguanosine) in leucocytes after smoking cessation.72 It is well
      known that cigarette smoke contains acetaldehyde (but also other potential
      carcinogens).
          Acetaldehyde-derived DNA-adducts were also found in blood samples taken
      from 44 cancer-free male Japanese alcoholic patients. The levels of these DNA-
      adducts were significantly higher in alcoholics with the ALDH2*1*2 genotype
      than in alcoholics with the ALDH2*1*1 genotype.73
      Mode of action                                                                   27
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<pre>4.1.4 Miscellaneous
      Acetaldehyde did not cause differential killing of repair-deficient Escherichia
      coli K-12 uvrB/recA cells.74
          Acetaldehyde induced DNA strand breaks and cross-links in human
      lymphocytes (without metabolic activation).75,76 However, acetaldehyde did not
      induce DNA strand breaks and cross-links in primary human bronchial epithelial
      cells or human leukocytes.75,76
4.2   Non-genotoxic mode of action
      In animal carcinogenicity studies using rats and hamsters, exposed animals
      showed signs of inflammation in the respiratory and olfactory epithelium of the
      nose.25,27 When inflammation becomes chronic and permanent, this can end in
      the development of cancer.
 8    Acetaldehyde
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<pre> hapter 5
        Classification
5.1     Evaluation of data on carcinogenicity and genotoxicity
        No epidemiological studies have been performed investigating cancer
        development due to exposure to acetaldehyde alone. 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 predis-
        position 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, direct evidence
        was found that acetaldehyde had caused cancer, although the data indirectly are
        suggestive for this. Overall, the Committee is of the opinion that human data are
        insufficient to make a final conclusion on the carcinogenic potential of
        acetaldehyde in humans.
            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 compound, 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. Based on these
        findings, the Committee concludes that there is sufficient evidence of
        carcinogenicity from animal experiments.
            Acetaldehyde is a reactive compound with stochastic genotoxic properties
        that induces stable DNA-adducts (mainly N2-ethylidene-dG), mutations, genome
        Classification                                                                    29
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<pre>    and chromosome aberrations in cultured mammalian (and human) cells. Genome
    and chromosomal aberrations, and DNA-adducts were also induced by
    acetaldehyde in vivo.
5.2 Recommendation for classification
    The Committee concludes that acetaldehyde is presumed to be carcinogenic to
    man, and recommends classifying the compound in category 1B.* Based on the
    available data, acetaldehyde acts by a stochastic genotoxic mechanism.
    According to the classification system of the Health Council (see Annex F).
 0  Acetaldehyde
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0 Wangenheim J, Bolcsfoldi G. Mouse lymphoma L5178Y thymidine kinase locus assay of 50
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5 Lambert B, Chen Y, He SM, Sten M. DNA cross-links in human leucocytes treated with vinyl acetate
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  References                                                                                           35
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<pre>6 Acetaldehyde</pre>

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<pre>A Request for advice
B The Committee
C The submission letter
D Comments on the public review draft
E IARC evaluation and conclusion
F Carcinogenic classification of substances by the Committee
  Annexes
                                                             37
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<pre>8 Acetaldehyde</pre>

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<pre>nnex A
     Request for advice
     In a letter dated October 11, 1993, ref DGA/G/TOS/93/07732A, to, the State
     Secretary of Welfare, Health and Cultural Affairs, the Minister of Social Affairs
     and Employment wrote:
     Some time ago a policy proposal has been formulated, as part of the simplification of the
     governmental advisory structure, to improve the integration of the development of recommendations
     for health based occupation standards and the development of comparable standards for the general
     population. A consequence of this policy proposal is the initiative to transfer the activities of the
     Dutch Expert Committee on Occupational Standards (DECOS) to the Health Council. DECOS has
     been established by ministerial decree of 2 June 1976. Its primary task is to recommend health based
     occupational exposure limits as the first step in the process of establishing Maximal Accepted
     Concentrations (MAC-values) for substances at the work place.
     In an addendum, the Minister detailed his request to the Health Council as
     follows:
     The Health Council should advice the Minister of Social Affairs and Employment on the hygienic
     aspects of his policy to protect workers against exposure to chemicals. Primarily, the Council should
     report on health based recommended exposure limits as a basis for (regulatory) exposure limits for air
     quality at the work place. This implies:
     •    A scientific evaluation of all relevant data on the health effects of exposure to
          substances using a criteria-document that will be made available to the Health
     Request for advice                                                                                     39
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<pre>      Council as part of a specific request 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.
0 Acetaldehyde
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<pre>nnex B
     The Committee
     •  R.A. Woutersen, chairman
        Toxicologic Pathologist, TNO Innovation for Life, Zeist; Professor of
        Translational Toxicology, Wageningen University and Research Centre,
        Wageningen
     •  J. van Benthem
        Genetic Toxicologist, National 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
     •  Ms M.J.M. Nivard
        Molecular Biologist and Genetic Toxicologist, Leiden University Medical
        Center, Leiden
     •  G.M.H. Swaen
        Epidemiologist, Dow Chemicals NV, Terneuzen
     •  E.J.J. van Zoelen
        Professor of Cell Biology, Radboud University Nijmegen, Nijmegen
     •  J.M. Rijnkels, scientific secretary
        Health Council, The Hague
     The Committee                                                              41
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<pre>  The Health Council and interests
  Members of Health Council Committees are appointed in a personal capacity
  because of their special expertise in the matters to be addressed. Nonetheless, it
  is precisely because of this expertise that they may also have interests. This in
  itself does not necessarily present an obstacle for membership of a Health
  Council Committee. Transparency regarding possible conflicts of interest is
  nonetheless important, both for the chairperson and members of a Committee
  and for the President of the Health Council. On being invited to join a
  Committee, members are asked to submit a form detailing the functions they
  hold and any other material and immaterial interests which could be relevant for
  the Committee’s work. It is the responsibility of the President of the Health
  Council to assess whether the interests indicated constitute grounds for non-
  appointment. An advisorship will then sometimes make it possible to exploit the
  expertise of the specialist involved. During the inaugural meeting the
  declarations issued are discussed, so that all members of the Committee are
  aware of each other’s possible interests.
2 Acetaldehyde
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<pre>nnex C
     The submission letter
     Subject             : Submission of the advisory report Acetaldehyde
     Your Reference      : DGV/MBO/U-932342
     Our reference       : U-7438/JR/fs/246-H17
     Enclosed            :1
     Date                : November 23, 2012
     Dear State Secretary,
     I hereby submit the advisory report on the effects of occupational exposure to
     Acetaldehyde.
     This advisory report is part of an extensive series in which carcinogenic
     substances are classified in accordance with European Union guidelines. This
     involves substances to which people can be exposed while pursuing their
     occupation.
         The advisory report was prepared by the Subcommittee on the Classification
     of Carcinogenic Substances, a permanent subcommittee of the Health Council’s
     Dutch Expert Committee on Occupational Safety. The advisory report has been
     assessed by the Health Council’s Standing Committee on Health and the
     Environment.
     The submission letter                                                          43
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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 W.A. van Gool,
  President
4 Acetaldehyde
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<pre>nnex D
     Comments on the public review draft
     A draft of the present report was released in 2012 for public review. The
     following organisations and persons have commented on the draft document:
     • Mr. T.J.Lentz, National Institute for Occupational Safety and Health, USA.
     Comments on the public review draft                                          45
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<pre>6 Acetaldehyde</pre>

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<pre>nnex E
     IARC evaluation and conclusion
     Acetaldehyde (Group 2B)
     VOL.: 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
     IARC evaluation and conclusion                                                   47
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<pre>  acetaldehyde (aldehyde dehydrogenase 2 and glutathione S-transferase M1).
  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).
8 Acetaldehyde
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<pre>Previous evaluations: Vol. 36 (1985); Suppl. 7 (1987)
Synonyms: Acetic aldehyde; ‘Aldehyde’; Ethanal; Ethylaldehyde
IARC evaluation and conclusion                                49
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<pre>0 Acetaldehyde</pre>

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<pre> nnex       F
            Carcinogenic classification of
            substances by the Committee
            The Committee expresses its conclusions in the form of standard phrases:
 ategory    Judgement of the Committee (GRGHS)                                  Comparable with EU Category
                                                                                67/584/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
ource: Health Council of the Netherlands. Guideline to the classification of carcinogenic compounds. The Hague: 2010;
ublication no. A10/07E.77
            Carcinogenic classification of substances by the Committee                                                51
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<pre>2 Acetaldehyde</pre>

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<br><br>