<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>Trichloroacetic acid
     Evaluation of the carcinogenicity and genotoxicity
</pre>

====================================================================== Einde pagina 1 =================================================================

<br><br>====================================================================== Pagina 2 ======================================================================

<pre>Aan de staatssecretaris van Sociale Zaken en Werkgelegenheid
Onderwerp              : aanbieding advies Trichloroacetic acid
Uw kenmerk             : DGV/MBO/U-932342
Ons kenmerk            : U-7411/BvdV/fs/246-B17
Bijlagen               :1
Datum                  : 13 november 2012
Geachte staatssecretaris,
Graag bied ik u hierbij het advies aan over de gevolgen van beroepsmatige blootstelling aan
trichloorazijnzuur.
Dit advies maakt deel uit van een uitgebreide reeks waarin kankerverwekkende stoffen
worden 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
carcinogene 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
Infrastructuur 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 i l : b . v. d . v o e t @ g r. n l                      w w w. g r. n l
Te l e f o o n ( 0 7 0 ) 3 4 0 7 4 4 7
</pre>

====================================================================== Einde pagina 2 =================================================================

<br><br>====================================================================== Pagina 3 ======================================================================

<pre>Trichloroacetic acid
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. 2012/20, The Hague, November 13, 2012
</pre>

====================================================================== Einde pagina 3 =================================================================

<br><br>====================================================================== Pagina 4 ======================================================================

<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, Agriculture & Innovation, 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. Trichloroacetic acid. Evaluation of the
carcinogenicity and genotoxicity. The Hague: Health Council of the Netherlands,
2012; publication no. 2012/20.
all rights reserved
ISBN: 978-90-5549-919-9
</pre>

====================================================================== Einde pagina 4 =================================================================

<br><br>====================================================================== Pagina 5 ======================================================================

<pre>   Contents
   Samenvatting 7
   Executive summary 8
   Scope 9
.1 Background 9
.2 Committee and procedures 9
.3 Data 10
   General information 11
.1 Identity and physico-chemical properties 11
.2 IARC classification 12
   Carcinogenicity 13
.1 Observations in humans 13
.2 Carcinogenicity studies in animals 13
   Mode of action 18
.1 Genotoxic mode of action 18
.2 Non-genotoxic mode of action 20
   Contents                                    5
</pre>

====================================================================== Einde pagina 5 =================================================================

<br><br>====================================================================== Pagina 6 ======================================================================

<pre>    Classification 23
 .1 Evaluation and conclusion 23
 .2 Recommendation for classification 25
    References 26
    Annexes 30
A   Request for advice 31
B   The Committee 33
C   The submission letter 35
D   Comments on the public review draft 37
E   IARC Monograph 38
F   Carcinogenic classification of substances by the Committee 41
    Contents                                                      6
</pre>

====================================================================== Einde pagina 6 =================================================================

<br><br>====================================================================== Pagina 7 ======================================================================

<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 de beroepsmatige uitoefening kunnen worden bloot-
gesteld. De evaluatie en beoordeling worden verricht door de subcommissie
Classificatie van Carcinogene Stoffen van de Commissie Gezondheid en
Beroepsmatige Blootstelling aan Stoffen van de raad, hierna kortweg aangeduid
als de commissie. In het voorliggende advies neemt de commissie trichloroacetic
acid onder de loep. Trichloorazijnzuur is een stof die voornamelijk wordt
gebruikt als selectieve herbicide. Daarnaast wordt het ook gebruikt in metaal-,
plastic-, en textiel industrie, als toevoeging in minerale smeerolie en als een ana-
lytisch reagens. Het wordt ook gebruikt voor de lokale behandeling van wratten,
beschadigingen van de huid van hals of nek en andere dermatologische situaties.
Trichloorazijnzuur is een belangrijke eind metaboliet van trichlooroethyleen en
tetrachloorethyleen. Trichloorazijnzuur ontstaat ook bij chlorering van drinkwa-
ter en zwembaden.
    Op basis van de beschikbare gegevens is de commissie van mening dat de
gegevens over trichloorazijnzuur niet voldoende zijn om de kankerverwekkende
eigenschappen te evalueren (categorie 3).*
Volgens het classificatiesysteem van de Gezondheidsraad (zie bijlage F).
Samenvatting                                                                         7
</pre>

====================================================================== Einde pagina 7 =================================================================

<br><br>====================================================================== Pagina 8 ======================================================================

<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 the Classification of Carcinogenic
Substances of the Dutch Expert Committee on Occupational Standards of the
Health Council, hereafter called the Committee. In this report, the Committee
evaluates trichloroacetic acid. Trichloroacetic acid is mainly used as a selective
herbicide. It also finds use in the metal, plastics and textile industries, as an
additive in mineral lubricating oils and as an analytical reagent. It is used in the
topical treatment of warts, cervical lesions and other dermatological conditions.
Trichloroacetic acid is a major end metabolite of trichloroethylene and
tetrachloroethylene. Trichloroacetic acid also arises as a result of chlorination or
chloramination.
    The Committee is of the opinion that the available data are insufficient to
evaluate the carcinogenic properties of trichloroacetic acid (category 3).*
According to the classification system of the Health Council (see Annex F).
Executive summary                                                                    8
</pre>

====================================================================== Einde pagina 8 =================================================================

<br><br>====================================================================== Pagina 9 ======================================================================

<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 of trichloroacetic
        acid.
1.2     Committee and procedures
        The evaluation is performed by the subcommittee on the Classification of
        Carcinogenic Substances of the Dutch Expert Committee on Occupational Safety
        of the Health Council, hereafter called the Committee. The members of the
        Committee are listed in Annex B. The submission letter (in English) to the State
        Secretary can be found in Annex C.
            In June 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
        Scope                                                                               9
</pre>

====================================================================== Einde pagina 9 =================================================================

<br><br>====================================================================== Pagina 10 ======================================================================

<pre>    are 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 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 trichloroacetic acid, such
    an IARC-monograph is available, of which the summary and conclusion of
    IARC is inserted in Annex E.
        More recently published data were retrieved from the online databases
    Medline, Toxline, and Chemical Abstracts using carcino*, cancer*, mutagen*,
    chromosom*, genotox* (*; wildcard character) and CAS no. 76-03-9 as key
    words. The last updated online search was in September 2012 and covered
    publications from 2004. The new relevant data were included in this report.
    Scope                                                                                10
</pre>

====================================================================== Einde pagina 10 =================================================================

<br><br>====================================================================== Pagina 11 ======================================================================

<pre> hapter 2
        General information
        The data have been retrieved from the IARC evaluation of trichloroacetic acid1
        and the European Substance Information System (ESIS* and the INCHEM
        database of the International Programme on Chemical Safety (IPCS), which can
        be accessed via the inchem-site**.
2.1     Identity and physico-chemical properties
        Chemical name              :  Trichloroacetic acid
        CAS registry number        :  76-03-9
        EINECS number              :  200-927-2
        Synonyms                   :  Trichloorazijnzuur, TCA, TCA (acid), trichloroethanoic acid, trichloro
                                      methane carboxylic acid
        Appearance                 : Colourless to white deliquescent crystals with characteristic odour
        Use                        : Trichloroacetic acid is mainly used as a selective herbicide. It also finds
                                      use in the metal, plastics and textile industries, as an additive in mineral
                                      lubricating oils and as an analytical reagent. It is used in the topical
                                      treatment of warts, cervical lesions and other dermatological conditions.
                                      Trichloroacetic acid is a major and final metabolite of trichloroethylene
                                      and tetrachloroethylene in humans and therefore used as a biomarker for
                                      exposure to these substances.
        Chemical formula           : C2HCl3O2
        http://esis.jrc.ec.europa.eu (accessed October 11, 2012).
 *      www.inchem.org (accessed October 11, 2012), International Chemical Safety Card of trichloroacetic
        acid (0586). November 1998.
        General information                                                                                        11
</pre>

====================================================================== Einde pagina 11 =================================================================

<br><br>====================================================================== Pagina 12 ======================================================================

<pre>    Structural formula       :       Cl  O
                               Cl    C   C    OH
                                     Cl
    Molecular weight         : 163.39
    Boiling point            : 197.5 °C
    Melting point            : 59 °C
    Vapour pressure          : 1 mm/Hg at 51 °C
    Vapour density (air = 1) : 5.6
    Solubility               : Very soluble in water (1,306 g/100 g at 25 °C) and most organic solvents,
                               including acetone, benzene ether, methanol and ortho-xylene
    Conversion factor        : 1 mg/m3 = 6.68 ppm
                               1 ppm = 0.1497 mg/m3
    EU Classification        : Skin Corr. 1A: H314 (Causes severe skin burns and eye damage)
    (100% solution)            STOT SE 3: H335 (May cause respiratory irritation); C ≥1%
2.2 IARC classification
    In 2004, IARC1 concluded that there is inadequate evidence in humans, and
    limited evidence in experimental animals for the carcinogenicity of
    trichloroacetic acid. Trichloroacetic acid was not classifiable as to its
    carcinogenicity to humans (group 3).1
    General information                                                                                  12
</pre>

====================================================================== Einde pagina 12 =================================================================

<br><br>====================================================================== Pagina 13 ======================================================================

<pre> hapter 3
        Carcinogenicity
3.1     Observations in humans
        In its monograph of 2004 IARC1 described quite a number of human studies
        (cohorts, as well as case-control studies) that analysed risk for cancer with
        respect to one or more measures of exposure to complex mixtures of disinfection
        by-products that are found in most chlorinated and chloraminated drinking-
        waters. Some of these studies show indications for increased relative risks for
        melanoma, and tumours of the urinary bladder, liver, colon and lung, i.e. for a
        diversity of tumours. However, no data specifically on trichloroacetic acid were
        available. Therefore, no conclusion could be drawn with respect to the
        carcinogenicity in humans by trichloroacetic acid specifically.
             No additional human data have become available after the IARC evaluation
        of 2004.
3.2     Carcinogenicity studies in animals
        Trichloroacetic acid has been evaluated previously (IARC 19952, 20041) and was
        found to induce hepatocellular adenomas and carcinomas in male B6C3F1 mice
        and to possess promoter activity. The previous evaluation of trichloroacetic acid
        indicated that there was limited evidence in experimental animals for its
        carcinogenicity.
        Carcinogenicity                                                                   13
</pre>

====================================================================== Einde pagina 13 =================================================================

<br><br>====================================================================== Pagina 14 ======================================================================

<pre>3.2.1 Carcinogenicity studies
      Oral exposure
      In a study by Pereira (1996)3 groups of 93, 46 and 38 female B6C3F1 mice, 7-8
      weeks of age, were administered trichloroacetic acid in the drinking-water at
      concentrations of 2.0, 6.67 and 20.0 mmol/L (324, 1,080 or 3,240 mg/L),
      adjusted to pH 6.5-7.5 with sodium hydroxide. A control group received 20.0
      mmol/L sodium chloride. Mice were killed after 360 or 576 days (when high-
      dose mice became moribund) of exposure and only livers were removed for
      histopathology. The livers were weighed and evaluated for foci of altered
      hepatocytes (basophillic and eosinophillic foci), adenomas and carcinomas. Data
      from mice administered trichloroacetic acid were compared with those from
      control mice using Fishers’s exact test with a p-value < 0.05.
          After 360 or 576 days of exposure, the liver-to-body weight ratio was
      increased dose-dependently following treatment with trichloroacetic acid. The
      high dose of trichloracetic acid (20.0 mmol/L(=3,240 mg/L)) increased, in
      comparison with controls, the incidence of foci (11/18 versus 10/90 at 576 days),
      adenomas (7/18 versus 2/90 at 576 days) and carcinomas (5/20 versus 0/40 at
      360 days and 5/18 versus 2/90 at 576 days). The mid dose of trichloroacetic acid
      (6.67 mmol/L) increased the incidence of foci (9/27) and hepatocellular
      carcinomas (5/27) at 576 days, while the low dose of 2.0 mmol/L (324 mg/L) did
      not alter the incidence of any liver lesion. In control mice, the incidence was 1/40
      adenoma (2.5%) at 360 days and 10/90 foci (11.1%), 2/90 adenomas (2.2%) and
      2/90 carcinomas (2.2%) at 576 days.
          In a study by DeAngelo et al. (1997)4 groups of 50 male Fisher 344/N rats,
      28-30 days of age, received 0.05, 0.5 and 5 g/L neutralized trichloroacetic acid,
      adjusted to pH 6.9-7.1 with sodium hydroxide, or 2 g/L sodium chloride in the
      drinking-water for a total 104 weeks. Interim sacrifices were made at 15, 30, 45
      and 60 weeks. A complete necropsy of the animals was performed. The liver,
      kidney, spleen, testes and gross lesions were examined microscopically. A
      complete pathological examination was carried out on all tissues from all
      animals in the high-dose group.
          The high dose of trichloroacetic acid but not the low or mid dose decreased
      body weight (~11%). Trichloroacetic acid did not affect the absolute or relative
      (to body weight) weights of the liver, kidneys, spleen or testes except for a
      decrease in the absolute liver weights in rats administered 5.0 g/L (p < 0.05). At
      104 weeks, the number of animals per treatment group ranged form 20 to 24
      Carcinogenicity                                                                      14
</pre>

====================================================================== Einde pagina 14 =================================================================

<br><br>====================================================================== Pagina 15 ======================================================================

<pre>including one rat that died after 76 weeks. The number of rats with hepatocellular
adenomas varied between one and three among the treatment groups (4.2-15%).
A single hepatocellular carcinoma (1/22, 4.6%) was found in the high-dose
trichloroacetic acid-treated group. None of the treatment groups had a significant
increase in the incidence of any tumour in other organs.
    The ability of mixtures of di- and trichloroacetic acid to induce liver tumours
was studied in 6-week-old B6C3F1 male mice (Bull et al., 20025; IARC 20041).
As part of this experiment treatments included 0.5 and 2.0 g/L trichloroacetic
acid. Twenty animals were assigned to each of the groups that received the above
concentrations in their drinking-water for 52 weeks. Control animals were given
the vehicle only.
     The incidence of liver tumours (adenomas and carcinomas combined) was
significantly increased (p< 0.05) in both treatment groups (11/20 in the 0.5 g/L
and 9/20 in the 2 g/L group) compared to the incidence in the control group
(1/20).
    DeAngelo et al.6 determined the prevalence and multiplicity (average
number of tumours per animal) of hepatocellular neoplasia in the male B6C3F1
mouse exposed to trichloroacetic acid in the drinking-water. Male mice were
exposed in study 1 to 0.05, 0.5, and 5 g/L trichloroacetic acid for 60 wk (50
animals/group, and in study 2 to 0.05 and 0.5 g/L trichloroacetic acid for 104 wk
(low and mid-dose 58 animals/ group), and in study 3 to 4.5 g/L trichloroacetic
acid for 104 wk (high dose 72 animals/group).
    Time-weighted mean daily doses measured for the low, medium, and high
dose groups were consistent over the three studies, 6-8, 58-68, and 572-602
mg/kg,day for the 0.05, 0.5, and the 4.5-5 g/L treatment groups, respectively. No
significant changes in animal survival were noted across the studies. A
significant increase in the prevalence and multiplicity of hepatocellular tumours
was found in the 58-68 and 572-602 mg/kg/d trichloroacetic acid dose groups.
The high dose group of the 60 wk study had a prevalence of 38% (versus 12% in
controls), and a significant increase (p < 0.03) of the multiplicity of
hepatocellular carcinoma from 0.07 + 0.05 (control) to 0.42 + 0.11. The 104
week study high dose group had a prevalence of 78% (versus 12% in controls),
and showed a significant increase (p < 0.03) of the multiplicity of hepatocellular
carcinoma from 0.20 + 0.12 (control) to 1.50 + 0.22.
Carcinogenicity                                                                     15
</pre>

====================================================================== Einde pagina 15 =================================================================

<br><br>====================================================================== Pagina 16 ======================================================================

<pre>3.2.2 Tumour-promotion studies
      A couple of experimental studies were performed to investigate tumour
      promoting activity of trichloroacetic acid, using N-methyl-N-nitrosourea (MNU)
      as tumour initiating substance.
          In one study (Pereira & Phelps, 19967) groups of 6-40 female B6C3F1 mice,
      15 days of age, were initiated with an intraperitoneal injection of 25 mg/kg
      MNU. At 49 days of age, the animals received 2.0, 6.67 or 20.0 mmol/L (=324,
      1080 or 3,240 mg/L) trichloroacetic acid, adjusted to pH 6.5-7.5 with sodium
      hydroxide, or 20.0 mmol/L sodium chloride as a control for the sodium salt in the
      drinking-water. Mice were killed after 31 or 52 weeks of exposure.
          At 31 weeks, the high dose of trichloroacetic acid increased the incidence of
      hepatocellular adenomas in MNU-initiated mice from 0/10 to 6/10 and the
      multiplicity (average number of tumours per animal) from 0.00 to 1.30 ± 0.045.
      At 52 weeks, the mid and high doses of trichloroacetic acid significantly
      (p < 0.01) increased the incidence of carcinomas in MNU-initiated mice from
      4/40 to 5/6 and 20/24, and multiplicity from 0.10 ± 0.05 to 1.33 ± 0.42 and 2.79
      ± 0.48, respectively. At 52 weeks, the mid and high doses of trichloroacetic acid
      increased the incidence of adenomas from 7/40 to 16/24 and 5/6, respectively,
      and the multiplicity from 0.28 ± 0.11 to 2.00 ± 0.82 and 1.29 ± 0.24, respectively.
      In mice that were not administered MNU, the high dose of trichloroacetic acid
      significantly increased the incidence of carcinomas from 0/40 to 5/20.
          In a second study by Pereira et al. (1997)8 combinations of dichloroacetic
      acid and trichloroacetic acid have been evaluated for tumour-promoting activity.
      Female B6C3F1 mice, 15 days of age, were initiated with MNU (25 mg/kg bw)
      followed by exposure to 0, 7.8, 15.6, and 25 mmol/L (=1,006, 2,012, 3,225
      mg/L) dichloroacetic acid with or without 6.0 mmol/L (=972 mg/L)
      trichloroacetic acid or 0, 6.0 and 25 mmol/L (=0, 972 and 4,050 mg/L)
      trichloroacetic acid with or without 15.6 mmol/L (=2,012 mg/L) dichloroacetic
      acid. The pH of the dose solutions was adjusted to 6.5-7.5 with sodium
      hydroxide. Exposure was from week 4 to 48 of age, at which time the mice were
      killed.
          The high dose of dichloroacetic acid (25 mmol/L(=3,225 mg/L)) and
      trichlorocacetic acid (25 mmol/L(=4,050 mg/L)) significantly increased (p <
      0.05) the multiplicity of hepatocellular adenomas from 0.07 + 0.05 (no
      dichloroacetic acid or trichloroacetic acid) to 1.79 + 0.29 and 0.52 + 0.11,
      respectively. The lower doses of dichloroacetic acid and trichloracetic acid did
      not significantly increase the incidence or multiplicity of adenomas).
      Carcinogenicity                                                                     16
</pre>

====================================================================== Einde pagina 16 =================================================================

<br><br>====================================================================== Pagina 17 ======================================================================

<pre>     In a third study (Pereira et al., 20019) the effect of chloroform on liver
tumours promotion by trichloroacetic acid has been investigated. Groups of male
and female B6C3F1 mice, 15 days of age, were initiated with 30 mg/kg MNU. At
5 weeks of age, the mice started to receive in the drinking-water 4.0 g/L
trichloroacetic acid neutralized with sodium hydroxide and 0, 800 or 1,600 mg/L
chloroform and were killed at 36 weeks of age. The results were analysed for
statistical significane by a one-way ANOVA followed by the Tukey test with p-
value < 0.05.
     In MNU-initiated mice that did not receive trichloroacetic acid,
hepatocellular adenomas were found in 2/29 females and 2/8 males, while no
hepatocellular carcinomas were found. Trichloroacetic acid increased the
incidence of liver carcinomas (10/16) and adenomas (12/16) in male mice. In
female mice administered trichloroacetic acid, the incidence of mice with
hepatocellular adenocarcinomas and adenomas was not significantly altered:
4/14 and 2/14. In male mice administered trichloroacetic acid plus 0, 800, and
1,600 mg/L chloroform, the incidence of hepatocellular adenocarcinomas was
10/16, 7/9 and 6/8 and that of hepatocellular adenomas was 12/16, 6/9 and 1/8,
respectively. The incidence of mice with hepatocellular adenomas was
significantly lower in mice administered trichloroacetic acid plus 1600 mg/L
chloroform than in mice administered trichloroacetic acid (< 0.05). No altered
hepacotcyte foci, adenomas or adenocarcinomas were found in six MNU-
initiated male mice that were administered 1600 mg/L chloroform. Multiplicity
of tumours (adenomas plus adenocarcinomas) was increased in male mice from
0.25 + 0.16 to 3.18 + 0.82 (p< 0.001), but not in female mice, with 0.07 + 0.04
and 0.64 + 0.22 for control and trichloroacetic acid-exposed mice, respectively.
Sixty per cent of the tumours were adenocarcinomas, indicating that multiplicity
of adenocarcinomas was significantly increased in male mice exposed to
trichloroacetic acid.
Carcinogenicity                                                                  17
</pre>

====================================================================== Einde pagina 17 =================================================================

<br><br>====================================================================== Pagina 18 ======================================================================

<pre> hapter 4
        Mode of action
4.1     Genotoxic mode of action
4.1.1   Gene mutation assays
        In vitro
        Trichloroacetic acid was repeatedly shown to be not mutagenic to Salmonella
        typhimurium in a couple of studies including strains TA98, TA100, TA1535, and
        TA1538, in the presence or absence of metabolic activation. Trichloroacetic acid
        was weakly mutagenic in mouse lymphoma cells at high dose (3,000 µg/mL)
        (Harrington-Brock et al., 199810).
        In vivo
        Point mutations in exons 1, 2 and 3 of K- and H-ras proto-oncogenes were
        studied in trichloroacetic acid-induced liver tumours of male B6C3F1 mice (104-
        week treatment with 4.5 g/L in drinking-water). Trichloroacetic did not modify
        the incidence of mutations in exon 2 of H-ras in carcinomas (45% versus 58%
        for control). Only four carcinomas showed mutations in the other exons of H-ras
        or in K-ras. In tumours with mutation in exon 2 of H-ras, treatment with
        trichloroacetic acid dit not modify the mutational spectrum compared with that
        of spontaneous liver tumours, that is to say 80% of the mutations in codon 61
        Mode of action                                                                   18
</pre>

====================================================================== Einde pagina 18 =================================================================

<br><br>====================================================================== Pagina 19 ======================================================================

<pre>      were CAA-> AAA, and 20% were CAA->CGA (Ferreira-Gonzalez et al.,
      199511).
4.1.2 Cytogenetic assays
      In vitro
      In human cells in vitro, trichloroacetic acid did not induce chromosomal
      aberrations (Mackay et al., 199512).
      In vivo
      In one study (Mackay et al., 199512), trichloroacetic acid induced micronuclei
      and chromosomal aberrations in bone-marrow cells and abnormal sperm
      morphology after injection into Swiss mice in vivo.13 In another study, in which a
      10-fold higher dose was injected to C57BL/6jfBL10/-Alpk mice, no
      micronucleus formation was observed. Trichloroacetic acid induced the
      formation of micronuclei in erythrocytes of newt larvae in vivo (Giller et al.,
      1997).14 It also induced chromosomal aberrations in vivo in the bone marrow of
      the chicken Gallus domesticus (Bhunya et al., 1996).15
4.1.3 Miscellaneous
      In vitro
      Trichloroacetic acid did not induce λ prophage or SOS repair in Escherichia coli
      (DeMarini et al., 199416; Giller et al., 199714).
          Trichloroacetic acid did not induce DNA strand breaks or DNA damage in
      mouse, rat or hamster cells in vitro (Chang et al., 199217; Plewa et al., 200218;
      Stauber et al., 199819).
          After treatment with trichloroacetic acid, the level of malondialdehyde-
      derived adducts was increased in vitro (Beland, 199920).
      In vivo
      The level of 8-hydroxyguanosine-DNA adducts in the liver of B6C3F1 mice was
      not modified after administration of trichloroacetic acid through drinking-water
      (Parrish et al., 199621), was slightly increased after administration by gavage
      (Austin et al., 199622) and was clearly increased after intraperitoneal injection
      Mode of action                                                                     19
</pre>

====================================================================== Einde pagina 19 =================================================================

<br><br>====================================================================== Pagina 20 ======================================================================

<pre>      (Von Tungeln et al., 200223). After treatment with trichloroacetic acid, the level
      of malondialdehyde-derived adducts was increased in vivo (Von Tungeln et al.,
      200223).
4.2   Non-genotoxic mode of action
4.2.1 Peroxisome proliferation
      In vitro
      An in-vitro study of COS-1 cells transiently co-transfected with a peroxisome
      proliferator-activated receptor (PPAR) expression plasmid, pCMV-mPPARalpha,
      together with a reporter plasmid containing a peroxisome proliferator response
      element, Pluc4A6-880, clearly demonstrated that trichloroacetate directly
      activates PPARalpha (Zhou & Waxman, 199824). In other studies, trichloroacetic
      acid induced peroxisome proliferation in primary cultures of hepatocytes from
      rats and mice but not in those from humans (Elcombe, 198525; Walgren et al.,
      2000a26). In addition, human hepatocytes that expressed endogenous human
      PPARalpha did not respond to trichloroacetic acid, whereas human cells co-
      transfected with mouse PPARalpha and mouse retinoid X receptor plasmids
      displayed increased activity of the peroxisome proliferator response element
      reporter after treatment with trichloroacetic acid and other peroxisome
      proliferators. Retinoid X receptor that forms a heterodimer with PPAR enhanced
      PPAR-DNA binding and transcriptional activation (retinoid X receptor is a
      common partner for many steroid receptors) (Walgren et al., 2000b27).
          Smith et al.28,29 examined the induction of DNA synthesis and apoptosis by
      trichloroacetic acid in hepatocytes from B6C3F1, PPARalpha knockout and
      129/Sv wildtype mouse strains. The carcinogenic effect of trichloroacetic acid in
      mice, both belonging to the peroxisome proliferator class, is believed to involve
      agonist binding to the peroxisome proliferator activated receptor alpha
      (PPARalpha).
          Trichloroacetic acid (0.1-5.0 mM (=16.2-810 mg/L) produced a
      concentration-related increased DNA synthesis in both B6C3F1 and 129/Sv
      hepatocytes at 24, 48 and 72 hrs. In hepatocytes from PPARalpha knock-out
      mice it failed to increase DNA synthesis at any time point examined. In human
      hepatocytes, trichloroacetic acid decreased DNA synthesis. Apoptosis was
      increased by 2.5 (=405 mg/L) and 5.0 mM (=810 mg/L) trichloroacetic acid (~2-
      fold) in B6C3F1 hepatocytes after 48 and 72 hrs and by 5.0 mM (=810 mg/L)
      trichloroacetic acid (1.5-2.5-fold) in 129/Sv hepatocytes at 48 and 72 hr
      Mode of action                                                                     20
</pre>

====================================================================== Einde pagina 20 =================================================================

<br><br>====================================================================== Pagina 21 ======================================================================

<pre>exposure. No changes in apoptosis were seen in PPARalpha null or human
hepatocytes. In addition, peroxisomal beta oxidation, a measure of peroxisome
proliferation, was increased by trichloroacetic acid in hepatocytes from B6C3F1
(~2-4-fold over control), and 129/Sv (~2-fold) mice, whereas no induction was
seen in hepatocytes from PPARalpha null mice or in human hepatocytes.
    The studies of Walgren et al. (2004)30 were undertaken to determine whether
a primary rat hepatocyte model system could be used to examine structure-
activity relationships of haloacetates for the induction of peroxisomal palmitoyl-
CoA oxidation. The haloacetates tested differed in both type (iodo, bromo,
chloro and fluoro) and extent (mono, di and tri) of substitution. Significant
differences were observed in both potency and efficacy. Potency varied over
about two orders of magnitude, in the order of mono > di = tri. Within the
monohalo-substituted series, the order of potency was iodo > bromo > chloro,
with the fluoro analog being essentially inactive.
In vivo
As reported in the monograph on trichloroacetic acid (IARC, 19952), short-term
treatment (< 14 days) resulted in increases in cell replication rates in the liver of
mice. The elevated rates of replication were not sustained and became
substantially reduced compared with controls with and without chronic
pretreatment (Pereira, 19963; Stauber & Bull, 199731). In an experiment in which
treatment of male B6C3F1 mice with 2 g/L trichloroacetic acid was terminated
after 1 year (50 weeks), cell replication rates within tumours were not dependent
upon continued treatment (for an additional 2 weeks). Trichloroacetic acid did
not stimulate replication of initiated cells. As only one time-point was measured,
the possibility that trichloroacetic acid affected replication rates of preneoplastic
lesions cannot be ruled out (Stauber & Bull, 199731).
    In a 7 day in vivo study performed by Smith et al.28,29, trichloroacetic acid
(0.5 and 2.0 g/L) increased DNA synthesis (2.0-5.7-fold) and peroxisomal beta
oxidation in the 129/Sv mouse, while no changes in these endpoints were seen in
the PPARalpha null mice. Trichloroacetic acid did not alter levels of apoptosis in
either strain of mice.
    Non-hepatoproliferative changes (cytoplasmic alterations, inflammation, and
necrosis) in mice treated with trichloroacetic acid were mild and dose related in
the earlier described carcinogenicity study of DeAngelo et al.32 A TCA-induced
increase in liver palmitoyl CoA oxidase activity, a marker of peroxisome
proliferation, correlated with tumor induction. A linear association (r2 = .984 and
Mode of action                                                                        21
</pre>

====================================================================== Einde pagina 21 =================================================================

<br><br>====================================================================== Pagina 22 ======================================================================

<pre>      r2 = .987 for 60 and 104 weeks respectively) was found between peroxisome
      proliferation and tumour induction.
4.2.2 Methylation
      In vitro
      No assays with trichloroacetic acid were available to the Committee.
      In vivo
      Short-term oral treatment (11 days) of mice with trichloroacetate (25 mmol/L)
      inhibited methylation of DNA in liver, an effect that was not observed with long-
      term treatment (44 weeks) (Tao et al., 199833). However, methylation of DNA
      was depressed in trichloroacetate-promoted liver tumours at 44 weeks and
      termination of treatment 1 week prior to sacrifice did not reverse this effect. An
      increased expression of c-jun and c-myc proto-oncogenes was observed when the
      5-methylcytosine levels in their respective promoter regions decreased (Tao et al.,
      2000a34) and administration of methionine 30 min after trichloroacetate inhibited
      expression of both proto-oncogenes (Tao et al., 2000b35). Increased cell
      replication rates and decreased methylation of the c-myc gene were first observed
      simultaneously in mice 72 h after the start of exposure to trichloroacetic acid.
      Trichloroacetic acid induced DNA hypomethylation by inducing DNA replication
      and preventing the methylation of the newly synthesized strands of DNA (Ge et
      al., 200136). The authors speculated that trichloroacetate depleted S-
      adenosylmethionine levels. Depressed levels of 5-methylcytosine were observed
      in the kidney and bladder as well as the liver.
           Li et al. (abstract)37 determined the methylation status the regulatory region of
      17 and 30 CpG sites in the tumour suppressor genes estrogen receptor (ER)-alpha
      and p16. DNA was isolated from mouse liver tumours induced by dichloroacetic
      acid and trichloroacetic acid, treated with bisulfite, PCR-amplified for the genes,
      and sequenced. The percentage of the CpG sites that were methylated in the ER-
      alpha gene was 3.9 ± 1.9% in normal liver tissue, while in dichloroacetic acid and
      trichloroacetic acid -promoted tumours the percentages was increased to 42.3 ±
      10.9% and 23.5 ± 7.9%, respectively. The extent to which the CpG sites in the p16
      gene were methylated in normal liver ranged from 0 to 1 site, while dichloroacetic
      acid and trichloroacetic acid -promoted tumours contained 2-3 methylated CpG
      sites. Hence, dichloroacetic acid and trichloroacetic acid increased the
      methylation of the two tumour suppressor genes in liver tumours.
      Mode of action                                                                         22
</pre>

====================================================================== Einde pagina 22 =================================================================

<br><br>====================================================================== Pagina 23 ======================================================================

<pre> hapter 5
        Classification
5.1     Evaluation and conclusion
        The Committee is of the opinion that there is insufficient evidence for the
        carcinogenicity of trichloroacetic acid in humans. The basis for this conclusion
        was the observation that in all human studies that analysed the risk for cancer, the
        investigated population was exposed to complex mixtures, i.e. disinfection by-
        products that are found in most chlorinated and chloraminated drinking-waters,
        and none of them specifically to trichloroacetic acid. Although some of these
        studies showed indications of increased relative risks for some tumours, i.e.
        melanoma and tumours of the urinary bladder, liver, colon and lung, no
        conclusion could be drawn with respect to the carcinogenicity in humans by
        trichloroacetic acid specifically. The Committee shares the opinion of IARC
        (2004)1 that the human data do not allow a conclusion on the carcinogenicity ot
        trichloroacetic acid. The Committee is aware that since this 2004 evaluation by
        IARC no additional human studies have appeared.
            With regard to the evidence from animal carcinogenicity data, the Committee
        observed increased incidences of hepatocellular adenomas and carcinomas in
        mice: in both male and female mice, after prolonged exposure to (neutralized)
        trichloroacetic acid via drinking-water. No increase in incidence of liver tumours
        or tumours at any other site was observed in a 2-year study with male rats. The
        Committee shares the opinion of IARC that trichloroacetic acid induces
        Classification                                                                       23
</pre>

====================================================================== Einde pagina 23 =================================================================

<br><br>====================================================================== Pagina 24 ======================================================================

<pre>peroxisome proliferation in the livers of mice at doses within the same range as
those that induce hepatic tumours.
    Peroxisome proliferators cause proliferation of peroxisomes and
hepatocarcinogenesis in rodent liver. This is mediated by peroxisome
proliferator-activated receptor-alpha (PPARalpha), a nuclear receptor protein
functioning as transcription factor.
    A number of other effects of trichloroacetic acid were observed: in in vitro
transfection experiments it was demonstrated that trichloroacetate directly
activates PPAR-alpha in primary cultures of hepatocytes from rats and mice, but
not from humans, whereas human hepatocytes co-transfected with mouse PPAR-
alpha did respond to trichloroacetate. This strongly suggested peroxisome
proliferation as underlying mechanism for liver tumour-induction in these rodent
species. Also, brief stimulation of cell division in the liver during the first days of
treatment, and depressed cell replication upon chronic treatment were observed.
The initial increase in cell proliferation correlated with a decreased methylation
of the promoter regions of the c-jun and c-myc proto-oncogenes coupled with an
increased expression of these genes.
    A small number of recent mechanistic animal studies have appeared. These
studies confirm the potential of trichloroacetic acid to induce peroxisome
proliferation in livers of mice and rats4,6. Smith et al.28,29 showed that induction
of peroxisome proliferation by trichloroacetic acid occurred in primary cultures
of hepatocytes from rats and mice but not in those from humans: the data
demonstrate that human hepatocytes are refractory to the induction of DNA
synthesis and apoptosis by trichloroacetic acid and that the PPAR-alpha is
required for the induction of DNA synthesis observed following trichloroacetic
acid exposure in B6C3F1 and 129/Sv wildtype mice. Accordingly, these data
support the notion that the induction of liver tumours in mice via this mechanism
is unlikely to be of relevance to human health.38
    A critical question is whether there is any role for direct DNA interactions of
trichloroacetic acid that could underly part of the liver tumours observed. From
the available in vitro data on mutagenicity and genotoxicity it is clear that
trichloroacetic acid is not a directly acting mutagenic chemical, either with or
without metabolic activation. The positive in vivo data apparently are due to
indirect effects induced by high doses of trichloroacetic acid and relate to its
peroxisome proliferating effects, e.g. the production of superoxide anions, lipid
peroxidation, DNA adducts by malondialdehyde, and DNA single-strand
breaks.1 As also demonstrated by the data of Smith et al.28,29, of Walgren et al.
(200026,27; cited in IARC, 20041), and the arguments indicated above, these
effects are not expected to occur in humans.
Classification                                                                          24
</pre>

====================================================================== Einde pagina 24 =================================================================

<br><br>====================================================================== Pagina 25 ======================================================================

<pre>5.2 Recommendation for classification
    The Committee is of the opinion that the available data are insufficient to
    evaluate the carcinogenic properties of trichloroacetic acid (category 3).*
    According to the classification system of the Health Council (see Annex F).
    Classification                                                              25
</pre>

====================================================================== Einde pagina 25 =================================================================

<br><br>====================================================================== Pagina 26 ======================================================================

<pre>References
IARC. Trichloroacetic Acid. IARC Monographs on the Evaluation of the Carcinogenic Risk of
Chemicals to Humans. 2004: Vol. 84: pp403-440.
IARC. Trichloroacetic Acid. IARC Monographs on the Evaluation of the Carcinogenic Risk of
Chemicals to Humans. 1995: Vol. 63: pp291-314.
Pereira MA. Carcinogenic activity of dichloroacetic acid and trichloroacetic acid in the liver of
female B6C3F1 mice. Fundam Appl Toxicol 1996; 31(2): 192-199.
DeAngelo AB, Daniel FB, Most BM, Olson GR. Failure of monochloroacetic acid and trichloroacetic
acid administered in the drinking water to produce liver cancer in male F344/N rats. J Toxicol
Environ Health 1997; 52(5): 425-445.
Bull RJ, Orner GA, Cheng RS, Stillwell L, Stauber AJ, Sasser LB et al. Contribution of
dichloroacetate and trichloroacetate to liver tumor induction in mice by trichloroethylene. Toxicol
Appl Pharmacol 2002; 182(1): 55-65.
DeAngelo AB, Daniel FB, Wong DM, George MH. The induction of hepatocellular neoplasia by
trichloroacetic acid administered in the drinking water of the male B6C3F1 mouse. J Toxicol Environ
Health A 2008; 71(16): 1056-1068.
Pereira MA, Phelps JB. Promotion by dichloroacetic acid and trichloroacetic acid of N-methyl-N-
nitrosourea-initiated cancer in the liver of female B6C3F1 mice. Cancer Lett 1996; 102(1-2):
133-141.
Pereira MA, Li K, Kramer PM. Promotion by mixtures of dichloroacetic acid and trichloroacetic acid
of N-methyl-N-nitrosourea-initiated cancer in the liver of female B6C3F1 mice. Cancer Lett 1997;
115(1): 15-23.
References                                                                                          26
</pre>

====================================================================== Einde pagina 26 =================================================================

<br><br>====================================================================== Pagina 27 ======================================================================

<pre>  Pereira MA, Kramer PM, Conran PB, Tao L. Effect of chloroform on dichloroacetic acid and
  trichloroacetic acid-induced hypomethylation and expression of the c-myc gene and on their
  promotion of liver and kidney tumors in mice. Carcinogenesis 2001; 22(9): 1511-1519.
0 Harrington-Brock K, Doerr CL, Moore MM. Mutagenicity of three disinfection by-products: di- and
  trichloroacetic acid and chloral hydrate in L5178Y/TK +/- (-) 3.7.2C mouse lymphoma cells. Mutat
  Res 1998; 413(3): 265-276.
1 Ferreira-Gonzalez A, DeAngelo AB, Nasim S, Garrett CT. Ras oncogene activation during
  hepatocarcinogenesis in B6C3F1 male mice by dichloroacetic and trichloroacetic acids.
  Carcinogenesis 1995; 16(3): 495-500.
2 Mackay JM, Fox V, Griffiths K, Fox DA, Howard CA, Coutts C et al. Trichloroacetic acid:
  investigation into the mechanism of chromosomal damage in the in vitro human lymphocyte
  cytogenetic assay and the mouse bone marrow micronucleus test. Carcinogenesis 1995; 16(5):
  1127-1133.
3 Bhunya SP, Behera BC. Relative genotoxicity of trichloroacetic acid (TCA) as revealed by different
  cytogenetic assays: bone marrow chromosome aberration, micronucleus and sperm-head abnormality
  in the mouse. Mutat Res 1987; 188(3): 215-221.
4 Giller S, Le CF, Erb F, Marzin D. Comparative genotoxicity of halogenated acetic acids found in
  drinking water. Mutagenesis 1997; 12(5): 321-328.
5 Bhunya SP, Jena GB. The evaluation of clastogenic potential of trichloroacetic acid (TCA) in chick in
  vivo test system. Mutat Res 1996; 367(4): 254-259.
6 DeMarini DM, Perry E, Shelton ML. Dichloroacetic acid and related compounds: induction of
  prophage in E. coli and mutagenicity and mutation spectra in Salmonella TA100. Mutagenesis 1994;
  9(5): 429-437.
7 Chang LW, Daniel FB, DeAngelo AB. Analysis of DNA strand breaks induced in rodent liver in
  vivo, hepatocytes in primary culture, and a human cell line by chlorinated acetic acids and
  chlorinated acetaldehydes. Environ Mol Mutagen 1992; 20(4): 277-288.
8 Plewa MJ, Kargalioglu Y, Vankerk D, Minear RA, Wagner ED. Mammalian cell cytotoxicity and
  genotoxicity analysis of drinking water disinfection by-products. Environ Mol Mutagen 2002; 40(2):
  134-142.
9 Stauber AJ, Bull RJ, Thrall BD. Dichloroacetate and trichloroacetate promote clonal expansion of
  anchorage-independent hepatocytes in vivo and in vitro. Toxicol Appl Pharmacol 1998; 150(2):
  287-294.
0 Beland FA. NTP technical report on the toxicity and metabolism studies of chloral hydrate (CAS No.
  302-17-0). Administered by gavage to F344/N rats and B6C3F1 mice. Toxic Rep Ser 1999;(59):
  1-E7.
1 Parrish JM, Austin EW, Stevens DK, Kinder DH, Bull RJ. Haloacetate-induced oxidative damage to
  DNA in the liver of male B6C3F1 mice. Toxicology 1996; 110(1-3): 103-111.
  References                                                                                            27
</pre>

====================================================================== Einde pagina 27 =================================================================

<br><br>====================================================================== Pagina 28 ======================================================================

<pre>2 Austin EW, Parrish JM, Kinder DH, Bull RJ. Lipid peroxidation and formation of 8-
  hydroxydeoxyguanosine from acute doses of halogenated acetic acids. Fundam Appl Toxicol 1996;
  31(1): 77-82.
3 Von Tungeln LS, Yi P, Bucci TJ, Samokyszyn VM, Chou MW, Kadlubar FF et al. Tumorigenicity of
  chloral hydrate, trichloroacetic acid, trichloroethanol, malondialdehyde, 4-hydroxy-2-nonenal,
  crotonaldehyde, and acrolein in the B6C3F(1) neonatal mouse. Cancer Lett 2002; 185(1): 13-19.
4 Zhou YC, Waxman DJ. Activation of peroxisome proliferator-activated receptors by chlorinated
  hydrocarbons and endogenous steroids. Environ Health Perspect 1998; 106 Suppl 4: 983-988.
5 Elcombe CR, Rose MS, Pratt IS. Biochemical, histological, and ultrastructural changes in rat and
  mouse liver following the administration of trichloroethylene: possible relevance to species
  differences in hepatocarcinogenicity. Toxicol Appl Pharmacol 1985; 79(3): 365-376.
6 Walgren JE, Kurtz DT, McMillan JM. Expression of PPAR(alpha) in human hepatocytes and
  activation by trichloroacetate and dichloroacetate. Res Commun Mol Pathol Pharmacol 2000a;
  108(1-2): 116-132.
7 Walgren JE, Kurtz DT, McMillan JM. The effect of the trichloroethylene metabolites trichloroacetate
  and dichloroacetate on peroxisome proliferation and DNA synthesis in cultured human hepatocytes.
  Cell Biol Toxicol 2000b; 16(4): 257-273.
8 Smith DJ, Kamendulis LM, Klaunig JE. Effect of Trichloroacetic acid in Male B6C3F1 Mouse
  Hepatocytes. Toxicologist 2004; 78(1-S): 223-224.
9 Smith DJ, Pu X, Kamendulis LM, Klaunig JE. Comparing effects of trichloroacetic acid on rodent
  and human hepatocytes: Role of peroxisome proliferator activated receptor alpha (PPARalpha).
  Toxicol Sci 2005; 84((1-S)): 155.
0 Walgren JL, Jollow DJ, McMillan JM. Induction of peroxisome proliferation in cultured hepatocytes
  by a series of halogenated acetates. Toxicology 2004; 197(3): 189-197.
1 Stauber AJ, Bull RJ. Differences in phenotype and cell replicative behavior of hepatic tumors
  induced by dichloroacetate (DCA) and trichloroacetate (TCA). Toxicol Appl Pharmacol 1997;
  144(2): 235-246.
2 DeAngelo AB, Daniel FB, Wong DM, George MH. The induction of hepatocellular neoplasia by
  trichloroacetic acid administered in the drinking water of the male B6C3F1 mouse. J Toxicol Environ
  Health A 2008; 71(16): 1056-1068.
3 Tao L, Kramer PM, Ge R, Pereira MA. Effect of dichloroacetic acid and trichloroacetic acid on DNA
  methylation in liver and tumors of female B6C3F1 mice. Toxicol Sci 1998; 43(2): 139-144.
4 Tao L, Yang S, Xie M, Kramer PM, Pereira MA. Hypomethylation and overexpression of c-jun and
  c-myc protooncogenes and increased DNA methyltransferase activity in dichloroacetic and
  trichloroacetic acid-promoted mouse liver tumors. Cancer Lett 2000; 158(2): 185-193.
5 Tao L, Yang S, Xie M, Kramer PM, Pereira MA. Effect of trichloroethylene and its metabolites,
  dichloroacetic acid and trichloroacetic acid, on the methylation and expression of c-Jun and c-Myc
  protooncogenes in mouse liver: prevention by methionine. Toxicol Sci 2000; 54(2): 399-407.
  References                                                                                          28
</pre>

====================================================================== Einde pagina 28 =================================================================

<br><br>====================================================================== Pagina 29 ======================================================================

<pre>6 Ge R, Yang S, Kramer PM, Tao L, Pereira MA. The effect of dichloroacetic acid and trichloroacetic
  acid on DNA methylation and cell proliferation in B6C3F1 mice. J Biochem Mol Toxicol 2001;
  15(2): 100-106.
7 Li L, Tao L, Kramer PM, Pereira MA. Dichloroacetic And Trichloroacetic Acid Induced Alteration
  In The Methylation Of Tumor Suppressor Genes And In The Acetylation Of Histone H3 In Mouse
  Liver And Tumors. Toxicologist 2004; 78(1-S): 224.
8 Peters JM CCaGFJ. Perixosome proliferator-acivated-alpha and liver cancer: Where do we stand?
  J Mol Med 2005; 83: 774-785.
9 Health Council of the Netherlands. Guideline to the classification of carcinogenic compounds. The
  Hague, The Netherlands: 2010: publication no. A10/07E.
  References                                                                                        29
</pre>

====================================================================== Einde pagina 29 =================================================================

<br><br>====================================================================== Pagina 30 ======================================================================

<pre>A Request for advice
B The Committee
C The submission letter
D Comments on the public review draft
E IARC Monograph
F Carcinogenic classification of substances by the Committee
  Annexes
                                                             30
</pre>

====================================================================== Einde pagina 30 =================================================================

<br><br>====================================================================== Pagina 31 ======================================================================

<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 governmen-
     tal 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                                                                                        31
</pre>

====================================================================== Einde pagina 31 =================================================================

<br><br>====================================================================== Pagina 32 ======================================================================

<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.
Request for advice                                                                                    32
</pre>

====================================================================== Einde pagina 32 =================================================================

<br><br>====================================================================== Pagina 33 ======================================================================

<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
     •  G.B. van der Voet, scientific secretary
        Health Council of the Netherlands, The Hague
     The Committee                                                              33
</pre>

====================================================================== Einde pagina 33 =================================================================

<br><br>====================================================================== Pagina 34 ======================================================================

<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.
The Committee                                                                      34
</pre>

====================================================================== Einde pagina 34 =================================================================

<br><br>====================================================================== Pagina 35 ======================================================================

<pre>nnex C
     The submission letter
     Subject            : Submission of the advisory report Trichloroacetic acid
     Our reference      : U-7411/BvdV/fs/246-B17
     Your Reference     : DGV/MBO/U-932342
     Enclosed           :1
     Date               : November 13, 2012
     Dear State Secretary,
     I hereby submit the advisory report on the effects of occupational exposure to
     Trichloroacetic acid.
     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 (DECOS). The advisory report
     has been assessed by the Health Council’s Standing Committee on Health and
     the Environment.
     The submission letter                                                          35
</pre>

====================================================================== Einde pagina 35 =================================================================

<br><br>====================================================================== Pagina 36 ======================================================================

<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
The submission letter                                                         36
</pre>

====================================================================== Einde pagina 36 =================================================================

<br><br>====================================================================== Pagina 37 ======================================================================

<pre>nnex D
     Comments on the public review draft
     A draft of the present report was released in June 2012 for public review. The fol-
     lowing organisations and persons have commented on the draft document:
     • National Institute for Occupational Safety and Health (NIOSH), Cincinnati,
        USA.
     Comments on the public review draft                                                 37
</pre>

====================================================================== Einde pagina 37 =================================================================

<br><br>====================================================================== Pagina 38 ======================================================================

<pre>nnex E
     IARC Monograph
     Volume 84, (excerpt from Trichloroacetic acid, pp. 403-440)
     Summary of Data Reported and Evaluation
     1          Exposure data
     Trichloroacetic acid is mainly used as a selective herbicide. It also finds use in
     the metal, plastics and textile industries and as an analytical reagent. It is used in
     the topical treatment of warts, cervical lesions and other dermatological
     conditions. Trichloroacetic acid is a major end metabolite of trichloroethylene
     and tetrachloroethylene. Wider exposure to trichloroacetic acid occurs at
     microgram-per-litre levels in drinking-water and swimming pools as a result of
     chlorination or chloramination.
     2          Human carcinogenicity data
     Several studies analysed risk with respect to one or more measures of exposure
     to complex mixtures of disinfection by-products that are found in most
     chlorinated and chloraminated drinking-water. No data specifically on
     trichloroacetic acid were available to the Working Group.
     IARC Monograph                                                                         38
</pre>

====================================================================== Einde pagina 38 =================================================================

<br><br>====================================================================== Pagina 39 ======================================================================

<pre>3           Animal carcinogenicity data
In four studies, neutralized trichloroacetic acid, when administered in the
drinking-water to female and/or male mice, increased the incidences of
hepatocellular adenomas and carcinomas. In a study in male rats, trichloroacetic
acid did not increase the incidence of liver tumours or tumours at any other site.
When administered in the drinking-water, trichloroacetic acid promoted the
induction of hepatocellular adenomas and/or carcinomas in carcinogen-initiated
male and female mice and of kidney tumours in male mice.
4           Other relevant data
The half-life of trichloroacetic acid, given orally or formed as a metabolite of
trichloroethylene or trichloroethanol, is longer in humans than in rodents.
Trichloroacetic acid may be reduced in vivo to dichloroacetic acid, but the
artefactual conversion of trichloroacetic acid to dichloroacetic acid hinders any
clear conclusions. A fraction of trichloroacetic acid is metabolized to carbon
dioxide.
Trichloroacetic acid induces peroxisome proliferation in the livers of mice at
doses within the same range as those that induce hepatic tumours. A brief
stimulation of cell division is observed in the liver during the first days of
treatment, but depressed cell replication results from chronic treatment. The
initial increase in cell proliferation was correlated with decreased methylation of
the promoter regions of the c-jun and c-myc proto-oncogenes and increased
expression of these genes.
Effects of trichloroacetic acid on reproduction and development in rats have been
reported, but were not confirmed in a subsequent study. In-vitro results suggest
that trichloroacetic acid can produce teratogenic effects at high doses.
In male mice, trichloroacetic acid modified neither the incidence of mutations in
exon 2 of H-ras in carcinomas, nor the mutational spectrum observed in tumours
that bore a mutation in exon 2. In female mice, 27% of tumours promoted by
trichloroacetic acid exhibited loss of heterozygosity at a minimum of two loci on
chromosome 6.
IARC Monograph                                                                      39
</pre>

====================================================================== Einde pagina 39 =================================================================

<br><br>====================================================================== Pagina 40 ======================================================================

<pre>In mouse liver in vivo, measurements of trichloroacetic acid-induced 8-
hydroxydeoxyguanosine DNA adducts gave different results depending on the
route of administration. Trichloroacetic acid induced abnormal sperm in mice in
vivo in one study and chromosomal aberrations in mouse and chicken bone
marrow in vivo. The results of in vivo studies in rodents on the induction of
DNA strand breaks and micronuclei were inconsistent. It induced the formation
of micronuclei in newt larvae in vivo.
In human cells in vitro, trichloroacetic acid did not induce chromosomal
aberrations or DNA strand breaks in single studies. In single studies on cultured
rodent cells, trichloroacetic acid was weakly mutagenic; no effect was observed
in a DNA strand-break assay or a single-cell gel assay. It also inhibited
intercellular communication in cultured rodent cells. Trichloroacetic acid caused
neither mutation in bacteria nor SOS repair.
5          Evaluation
There is inadequate evidence in humans for the carcinogenicity of trichloroacetic
acid.
There is limited evidence in experimental animals for the carcinogenicity of
trichloroacetic acid.
Overall evaluation
Trichloroacetic acid is not classifiable as to its carcinogenicity to humans (Group
3).
For definition of the italicized terms and definition of Groups, see Preamble
Evaluation.
Previous evaluation: Vol. 63 (1995)
Last updated: 29 September 2004.
IARC Monograph                                                                      40
</pre>

====================================================================== Einde pagina 40 =================================================================

<br><br>====================================================================== Pagina 41 ======================================================================

<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
                                                                                67/548/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 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: Health
 ouncil of the Netherlands, 2010; publication no. A10/07E.39
             Carcinogenic classification of substances by the Committee                                                41
</pre>

====================================================================== Einde pagina 41 =================================================================

<br><br>