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

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<pre>Gezondheidsraad                                          Vo o r z i t t e r
Health Council of the Netherlands
Aan de minister van Sociale Zaken en Werkgelegenheid
Onderwerp               : Aanbieding advies Chlorozotocin
Uw kenmerk              : DGV/MBO/U-932542
Ons kenmerk             : U-1478/JR/pg/246-S11
Bijlagen                :1
Datum                   : 12 december 2007
Geachte minister,
Graag bied ik u hierbij het advies aan over de kankerverwekkendheid van chlorozotocine.
Het 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.
     Het 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 voorgelegd aan de Commissie GBBS en vervolgens getoetst
door de Beraadsgroep Gezondheid en omgeving van de Gezondheidsraad.
Ik heb dit advies vandaag ter kennisname toegezonden aan de minister van Volksgezond-
heid, Welzijn en Sport en de minister van Volkshuisvesting, Ruimtelijke Ordening en
Milieubeheer.
Hoogachtend,
prof. dr. J.A. Knottnerus
Bezoekadres                                                                 Postadres
Parnassusplein 5                                                            Postbus 16052
2 5 11 V X D e n          Haag                                              2500 BB Den            Haag
Te l e f o o n ( 0 7 0 ) 3 4 0 6 6 3 1                                      Te l e f a x ( 0 7 0 ) 3 4 0 7 5 2 3
E - m a i l : j o l a n d a . r i j n k e l s @ g r. n l                    w w w. g r. n l
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<pre></pre>

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<pre>Chlorozotocin
Evaluation of the carcinogenicity and genotoxicity
Subcommittee on the classification of carcinogenic substances of the
Dutch Expert Committee on Occupational Standards,
a committee of the Health Council of the Netherlands
to:
the Minister of Social Affairs and Employment
No. 2007/07OSH, The Hague, December 12, 2007
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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...” (Section
22, Health Act).
     The Health Council receives most requests for advice from the Ministers of
Health, Welfare & Sport, Housing, Spatial Planning & the Environment, Social
Affairs & Employment, and Agriculture, Nature & Food Quality. 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 gov-
ernment 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. Chlorozotocin; Evaluation of the carcinoge-
nicity and genotoxicity. The Hague: Health Council of the Netherlands, 2007;
publication no. 2007/07OSH.
all rights reserved
ISBN: 978-90-5549-671-6
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<pre>    Contents
    Samenvatting 9
    Executive summary 11
1   Scope 13
1.1 Background 13
1.2 Committee and procedure 13
1.3 Data 14
2   General information 15
2.1 Identity and physico-chemical properties 15
2.2 IARC classification 16
3   Carcinogenicity studies 17
3.1 Observations in humans 17
3.2 Carcinogenicity studies in animals 17
4   Mutagenicity and genotoxicity 19
4.1 In vitro assays 19
4.2 In vivo assays 20
4.3 Carcinogenic mechanism 20
    Contents                                    7
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<pre>5   Classification 21
5.1 Evaluation of data on carcinogenicity and genotoxicity 21
5.2 Recommendation for classification 21
    References 23
A   Request for advice 27
B   The committee 29
C   Comments on the public review draft 31
D   IARC Monograph 33
E   Carcinogenic classification of substances by the committee 37
F   Guideline 93/21/EEG of the European Union 39
8   Chlorozotocin
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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 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 chlorozotocine
onder de loep. Chlorozotocine is een cytostaticum dat wordt gebruikt ter behan-
deling van kanker.
Op basis van de beschikbare gegevens leidt de commissie af dat chlorozotocine
beschouwd moet worden als kankerverwekkend voor de mens. Dit komt overeen
met een classificatie in categorie 2 volgens de richtlijnen van de Europese Unie.
De commissie concludeert verder dat chlorozotocine stochastisch genotoxisch is.
Samenvatting                                                                      9
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<pre>10 Chlorozotocin</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 sub-
stances to which workers are occupationally exposed. The evaluation is per-
formed by the subcommittee on Classifying Carcinogenic Substances of the
Dutch Expert Committee on Occupational Standards of the Health Council, here-
after called the committee. In this report, the committee evaluated chlorozotocin.
Chlorozotocin is a cytostatic agent that is used to treat cancer.
Based on the available information, the committee is of the opinion that chloro-
zotocin should be considered as carcinogenic to humans. This recommendation
corresponds to the EU classification in category 2. The committee concludes fur-
thermore that chlorozotocin acts by a stochastic genotoxic mechanism.
Executive summary                                                                  11
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<pre>12 Chlorozotocin</pre>

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<pre>Chapter 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 classifica-
        tion with reference to an EU-directive (see annex A and F). In addition to classi-
        fying 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 E). This report contains
        the evaluation of the carcinogenicity of chlorozotocin.
1.2     Committee and procedure
        The evaluation is performed by the subcommittee on Classifying Carcinogenic
        Substances of the Dutch Expert Committee on Occupational Standards of the
        Health Council, hereafter called the committee. The members of the committee
        are listed in annex B. The first draft was prepared by MI Willems, from the
        Department of Occupational Toxicology of the TNO Nutrition and Food
        Research, by contract with the Ministry of Social Affairs and Employment.
            In 2007 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 C. 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 commit-
    tees’ 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 commit-
    tee when these are considered most relevant in assessing the carcinogenicity and
    genotoxicity of the substance in question. In the case of chlorozotocin, such an
    IARC-monograph is available, of which the summary and conclusion of IARC is
    inserted in annex D.
         More recently published data were retrieved from the online databases Med-
    line, Toxline, Chemical Abstracts, and RTECS. The last updated online search
    was in March 2007. The new relevant data were included in this report.
14  Chlorozotocin
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<pre>Chapter 2
        General information
2.1     Identity and physico-chemical properties
        Chlorozotocin is a cytostatic agent that is used in the treatment of gastrointestinal
        cancers, cancer of the lungs, melanomas, and multiple myelomas.1,2 Occupational
        exposure may occur during manufacturing or packaging, or during the final prep-
        aration and administration to patients.
            Below is given the identity and some of its physical and chemical properties.
        Chemical name           : D-glucose, 2-({[(2-chloroethyl)nitrosoamino] carbonyl}amino)-2-
                                  deoxy-
        CAS registry number     : 54749-90-5
        IUPAC name              : Ethanamine-2-chloro-N,N-bis(2-chloroethyl)
                                  hydrochloride
        Synonyms                : D-Glucopyranose; 2-({[(2-chloroethyl)nitrosoamino] carbonyl}-
                                  amino)-2-deoxy-1-(2-chloroethyl)-1-nitroso-3-(D-glucos-2-yl)urea;
                                  DCNU; NSC-178248; Dome
        Description             : Ivory crystals. Chlorozotocin is synthesized by nitrosation of the urea
                                  derivative prepared from D-glucosamine and 2-chloroethylisocy-
                                  anante. Chlorozotocin is not known to occur naturally. Chlorozotocin
                                  is available as a lyophilized powder in vials containing 50 mg of the
                                  compound with 48 mg citric acid and sodium hydroxide to adjust the
                                  pH.
        Molecular formula       : C9H16ClN3O7
        General information                                                                               15
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<pre>    Structure                :
    Molecular weight         : 313.69
    Boiling point            : -
    Melting point            : 140-148 °C (decomposes)
    Vapour pressure          : -
    Solubility               : Soluble in water
    Stability                : Stable in solution at room temperature for 3 hours and at 2-8 °C for 24
                               hours; powder is stable for 24 months under refrigeration
    Partition coefficient    : 3 (octanol:water)
2.2 IARC classification
    In 1990, IARC concluded that there is sufficient evidence for the carcinogenicity
    of chlorozotocin in experimental animals, but that there were no carcinogenicity
    data available from studies in humans.1 Therefore, according to the IARC guide-
    lines, it classified chlorozotocin in Group 2A, which means that the agent is
    probably carcinogenic to humans. IARC also took notice of the possible geno-
    toxic effects of chlorozotocin.
16  Chlorozotocin
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<pre>Chapter 3
        Carcinogenicity studies
3.1     Observations in humans
        No data were available to evaluate the carcinogenicity of chlorozotocin in
        humans.
3.2     Carcinogenicity studies in animals
        Habs et al. (1979) reported on a carcinogenicity study on Sprague-Dawley rats,
        which received intraperitoneal injections of chlorozotocin at doses of 0.4 and 2
        mg/kg bw, once a week for more than 2 years.3 Also vehicle-control animals
        were included. The median survival time was lowest for the high-dose group,
        and highest for the control animals. The number of animals with tumours was
        significantly increased in the exposed groups compared to the control group: sar-
        comas and mesotheliomas of the peritoneal cavity, 13/20 and 16/20 (high dose,
        male and female, respectively), 14/20 and 10/20 (low dose, male and females,
        respectively), 0/20 and 1/20 (control, male and female, respectively). No other
        types of tumours were reported.
            In another study, thirty male Wistar rats were given ten intravenous injections
        of chlorozotocin at doses of 9.5, 19, and 38 mg/kg bw, one injection every six
        weeks. Also vehicle-control animals were included.1,4 The median survival time
        was lowest for the high-dose group, and highest for the control animals. The
        authors reported on increased numbers of animals with malignant tumours of the
        Carcinogenicity studies                                                             17
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<pre>   nervous system, lung, and forestomach in the exposed groups compared to the
   control animals: 4, 5 and 4% versus 1, 0, and 1% in low, mid, and high exposure
   group, respectively, versus controls. IARC noted the poor survival and limiting
   reporting.1
       The carcinogenic potency, expressed as TD50*, is estimated to be 0.0375.5 No
   other animal carcinogenicity studies were available to the committee.
*  TD50 is daily dose rate in mg/kg bw/day to induce tumours in half of test animals that would have remained
   tumour-free at zero dose.
18 Chlorozotocin
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<pre>Chapter 4
        Mutagenicity and genotoxicity
        The molecular mechanism by which chlorozotocin exerts its antineoplastic
        action is thought to be modification of DNA. However, this DNA modification
        may also contribute to its toxic and carcinogenic properties. The possible
        mutagenic and genotoxic properties are further reviewed in the next sections.
4.1     In vitro assays
        Chlorozotocin induced base-pair substitutions but not frame shift mutations in
        Salmonella typhimurium, in the presence and absence of an exogenous metabolic
        activation system.1 Also, it induced mutations at the hprt locus in V79 Chinese
        hamster cells.
            Chlorozotocin alkylated the DNA in mouse leukaemia L1210 cells. It also
        induced DNA strand breaks in L1210 and V79 Chinese hamster cells.1 In addi-
        tion, DNA interstrand cross-links were observed in mouse leukaemia L1210 and
        in human embryo cells.
            In mouse leukaemia L1210 cells, and in 9L rat brain tumour cells, chlorozo-
        tocin increased the frequencies of sister chromatid exchanges. It also induced
        mitotic gene conversion in Saccharomyces cerevisiae.1
        Mutagenicity and genotoxicity                                                   19
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<pre>4.2 In vivo assays
    Chlorozotocin induced sex-linked recessive lethal mutations in Drosophila mela-
    nogaster.1
         Furthermore, in the bone-marrow cells of Wistar rats, which were given a
    single intraperitoneal injection of 100 μmol chlorozotocin per kg bw, increased
    numbers of DNA strand breaks and interstrand cross links were observed.6
4.3 Carcinogenic mechanism
    Chlorozotocin is structurally related to other chloroethyl nitrosoureas, which are
    listed as ‘known human carcinogens’ or ‘should be regarded as a human carcino-
    gen’.1,2 These agents, including chlorozotocin, exert their carcinogenic effects
    through the formation of mono- and bifunctional alkylating agents.2 As such they
    are able to alkylate, and thus to damage, DNA.1,7-11
20  Chlorozotocin
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<pre>Chapter 5
        Classification
5.1     Evaluation of data on carcinogenicity and genotoxicity
        No data on the genotoxicity and carcinogenicity of chlorozotocin in humans
        were available, nor were any data available on inhalation exposure in animals.
        Overall, the availability of animal carcinogenicity studies is limited, but in one of
        the two available, chlorozotocin induced tumours throughout the body. These
        findings give sufficient evidence that exposure to chlorozotocin can result in can-
        cer development.
            Chlorozotocin is an alkylating agent, and as such has been shown to cause
        mutations and DNA-damage in in vitro and in vivo test systems. In addition,
        chlorozotocin causes mutations in cultured mammalian cells, as well in Droso-
        phila melanogaster. For this reason the committee considers chlorozotocin as a
        stochastic genotoxic carcinogen.
            The committee did not find indications that the observations in animals, and
        the proposed carcinogenic mechanism would not occur in humans.
5.2     Recommendation for classification
        The committee is of the opinion that chlorozotocin should be considered as carci-
        nogenic to humans. This recommendation corresponds to the EU classification in
        category 2. The committee concludes furthermore that chlorozotocin acts by sto-
        chastic genotoxic mechanism.
        Classification                                                                        21
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<pre>22 Chlorozotocin</pre>

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<pre>   References
1  IARC. Chlorozotocin. IARC Monogr Eval Carcinog Risks Hum 1990; 50: 65-75.
2  National Toxicology Program. Chlorozotocin. Report on Carcinogens, Eleventh Editions; US
   Department of Health and Human Services, Public Health Services, National Toxicology Program;
   2002. Internet: PM:15320326.
3  Habs M, Eisenbrand G, Schmahl D. Carcinogenic activity in Sprague-Dawley rats of 2-[3-(2-
   chloroethyl)-3-nitrosoureido]-D-gluco-pyranose (chlorozotocin). Cancer Lett 1979; 8(2): 133-137.
4  Eisenbrand G, Habs M. Chronic toxicity and carcinogenicity of cytostatic N-nitroso-(2-chloroethyl)
   ureas after repeated intravenous application to rats. Dev Toxicol Environ Sci 1980; 8: 273-278.
5  University of Berkeley. Carcinogenic Potency Database. University of Berkeley, California, USA,
   http://potency.berkeley.edu/; 2007.
6  Bedford P, Eisenbrand G. DNA damage and repair in the bone marrow of rats treated with four
   chloroethylnitrosoureas. Cancer Res 1984; 44(2): 514-518.
7  Berger M. Carcinogenicity of alkylating cytostatic drugs in animals. IARC Sci Publications 1986; 78:
   161-176.
8  Eisenbrand G, Muller N, Denkel E, Sterzel W. DNA adducts and DNA damage by antineoplastic and
   carcinogenic N-nitrosocompounds. J Cancer Res Clin Oncol 1986; 112(3): 196-204.
9  Lemoine A, Lucas C, Ings RM. Metabolism of the chloroethylnitrosoureas. Xenobiotica 1991; 21(6):
   775-791.
10 Vogel EW, Nivard MJ. Performance of 181 chemicals in a Drosophila assay predominantly
   monitoring interchromosomal mitotic recombination. Mutagenesis 1993; 8(1): 57-81.
   References                                                                                           23
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<pre>11 Vogel EW, Barbin A, Nivard MJ, Stack HF, Waters MD, Lohman PH. Heritable and cancer risks of
   exposures to anticancer drugs: inter-species comparisons of covalent deoxyribonucleic acid-binding
   agents. Mutat Res 1998; 400(1-2): 509-540.
24 Chlorozotocin
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<pre>A Request for advice
B The committee
C Comments on the public review draft
D IARC Monograph
E Carcinogenic classification of substances by the committee
F Guideline 93/21/EEG of the European Union
  Annexes
                                                             25
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<pre>26 Chlorozotocin</pre>

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<pre>Annex 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 fol-
      lows:
      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                                                                                        27
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<pre>       for advice. If possible this evaluation should lead to a health based recommended exposure limit,
       or, in the case of genotoxic carcinogens, a ‘exposure versus tumour incidence range’ and a calcu-
       lated 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 classifica-
       tion 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.
28 Chlorozotocin
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<pre>Annex B
      The committee
      •   G..J. Mulder, chairman
          emeritus professor of toxicology, Leiden University, Leiden
      •   P.J. Boogaard
          toxicologist, SHELL International BV, The Hague
      •   Ms. M.J.M. Nivard
          Molecular biologist and genetic toxicologist, Leiden University Medical
          Center, Leiden
      •   G.M.H. Swaen
          epidemiologist, Dow Chemicals NV, Terneuzen
      •   R.A. Woutersen
          toxicologic pathologist, TNO Nutrition and Food Research, Zeist
      •   A.A. van Zeeland
          professor of molecular radiation dosimetry and radiation mutagenesis,
          University Medical Center, Leiden
      •   E.J.J. van Zoelen
          professor of cell biology, Radboud University Nijmegen, Nijmegen
      •   J.M. Rijnkels, scientific secretary
          Health Council of the Netherlands, The Hague
      The committee consulted an additional expert, Prof dr G Mohn, working at
      Department of Radiation Genetics and Chemical Mutagenesis of the University
      of Leiden, with respect to the genotoxic data.
      The committee                                                               29
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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 Coun-
   cil Committee. Transparency regarding possible conflicts of interest is nonethe-
   less important, both for the President 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 mate-
   rial 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 establishment meeting the declarations issued are dis-
   cussed, so that all members of the Committee are aware of each other’s possible
   interests.
30 Chlorozotocin
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<pre>Annex C
      Comments on the public review draft
      A draft of the present report was released in 2007 for public review. The follow-
      ing organisations and persons have commented on the draft document:
      • G. Jonkers, Vereniging van Verf en Drukinktfabrikanten, the Netherlands;
      • E. González-Fernández, Ministerio de Trabajo y Asuntos Sociales, Spain;
      • R.D. Zumwalde, National Institute for Occupational Safety and Health, the
          USA.
      Comments on the public review draft                                               31
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<pre>32 Chlorozotocin</pre>

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<pre>Annex D
      IARC Monograph
      VOL: 50 (1990) (p. 65)1
      CAS No.: 54749-90-5
      Chem. Abstr. Name: D-Glucose, 2({[(2-chloroethyl)nitrosoamino]carbonyl}-
      amino)-2-deoxy-
      Summary of Data Reported and Evaluation
      Exposure data
      Chlorozotocin has been used as a cytostatic drug for the treatment of cancers at a
      variety of sites.
      Experimental carcinogenicity data
      Chlorozotocin was tested for carcinogenicity in single experiments in rats by
      intraperitoneal and intravenous injection. Intraperitoneal administration induced
      a high incidence of sarcomas and mesotheliomas in the peritoneal cavity in rats
      of each sex. The study by intravenous administration was inadequate for evalua-
      tion.
      IARC Monograph                                                                     33
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<pre>   Human carcinogenicity data
   No data were available to the Working Group.
   Other relevant data
   Chlorozotocin alkylates DNA and protein and causes DNA interstrand cross-
   links. In humans, it induces leukopenia and thrombocytopenia; in animals, it sup-
   presses the bone marrow and affects immune response.
   It is hepatotoxic in both humans and experimental animals.
   Chlorozotocin induced DNA damage in bone-marrow cells of rats in vivo. It
   induced DNA damage in human, mouse and Chinese hamster cells in vitro, sister
   chromatid exchange in mouse and rat cells and gene mutation in Chinese hamster
   cells. It induced sex-linked recessive lethal mutations in Drosophila and gene
   conversion in Saccharomyces cerevisiae. Chlorozotocin induced mutations in
   Salmonella typhimurium.
   Evaluation
   There is sufficient evidence for the carcinogenicity of chlorozotocin in experi-
   mental animals.
   No data were available from studies in humans on the carcinogenicity of chloro-
   zotocin.
   In making the overall evaluation, the Working Group also took note of the fol-
   lowing information. Chlorozotocin is an alkylating agent and is structurally
   related to other chloroethyl nitrosoureas, one of which, 1-(2-chloroethyl)-3-(4-
   methylcyclohexyl)-1-nitrosourea (methyl-CCNU), is carcinogenic to humans
   (Group 1) and two of which, bischloroethyl nitrosourea (BCNU) and 1-(2-chlo-
   roethyl)-3-cyclohexyl-1-nitrosourea (CCNU), are probably carcinogenic to
   humans (Group 2A) (IARC, 1987). Chlorozotocin has given consistently posi-
   tive results in a broad spectrum of assays for genetic and related effects, includ-
   ing those involving mammalian cells.
34 Chlorozotocin
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<pre>Overall evaluation
Chlorozotocin is probably carcinogenic to humans (Group 2A).
IARC Monograph                                               35
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<pre>Annex         E
              Carcinogenic classification of
              substances by the committee
The committee expresses its conclusions in the form of standard phrases:
Judgment of the committee                                                                       Comparable with EU class
This compound is known to be carcinogenic to humans                                             1
•   It is stochastic or non-stochastic genotoxic
•   It is non-genotoxic
•   Its potential genotoxicity has been insufficiently investigated. Therefore, it is unclear
    whether it is genotoxic
This compound should be regarded as carcinogenic to humans                                      2
•   It is stochastic or non-stochastic genotoxic
•   It is non-genotoxic
•   Its potential genotoxicity has been insufficiently investigated. Therefore, it is unclear
    whether it is genotoxic
This compound is a suspected human carcinogen.                                                  3
•   This compound has been extensively investigated. Although there is insufficient evidence    (A)
    for a carcinogenic effect to warrant a classification as ‘known to be carcinogenic to
    humans’ or as ‘should be regarded as carcinogenic to humans’, they indicate that there is
    cause for concern.
•   This compound has been insufficiently investigated. While the available data do not war-    (B)
    rant a classification as ‘known to be carcinogenic to humans’ or as ‘should be regarded as
    carcinogenic to humans’, they indicate that there is a cause for concern.
This compound cannot be classified                                                              not classifiable
•   There is a lack of carcinogenicity and genotoxicity data.
•   Its carcinogenicity is extensively investigated. The data indicate sufficient evidence sug-
    gesting lack of carcinogenicity.
              Carcinogenic classification of substances by the committee                                             37
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<pre>Annex F
      Guideline 93/21/EEG of the European
      Union
      4.2            Criteria for classification, indication of danger, choice of risk phrases
      4.2.1          Carcinogenic substances
      For the purpose of classification and labelling, and having regard to the current state of knowledge,
      such substances are divided into three categories:
      Category 1:
      Substances known to be carcinogenic to man.
      There is sufficient evidence to establish a causal association between human exposure to a substance
      and the development of cancer.
      Category 2:
      Substances which should be regarded as if they are carcinogenic to man.
      There is sufficient evidence to provide a strong presumption that human exposure to a substance may
      result in the development of cancer, generally on the basis of:
      •    appropriate long-term animal studies
      •    other relevant information.
      Guideline 93/21/EEG of the European Union                                                             39
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<pre>   Category 3:
   Substances which cause concern for man owing to possible carcinogenic effects but in
   respect of which the available information is not adequate for making a satisfactory assess-
   ment.
   There is some evidence from appropriate animal studies, but this is insufficient to place the substance
   in Category 2.
   4.2.1.1       The following symbols and specific risk phrases apply:
   Category 1 and 2:
   T; R45 May cause cancer
   However for substances and preparations which present a carcinogenic risk only when inhaled, for
   example, as dust, vapour or fumes, (other routes of exposure e.g. by swallowing or in contact with
   skin do not present any carcinogenic risk), the following symbol and specific risk phrase should be
   used:
   T; R49 May cause cancer by inhalation
   Category 3:
   Xn; R40 Possible risk of irreversible effects
   4.2.1.2       Comments regarding the categorisation of carcinogenic substances
   The placing of a substance into Category 1 is done on the basis of epidemiological data; placing into
   Categories 2 and 3 is based primarily on animal experiments.
   For classification as a Category 2 carcinogen either positive results in two animal species should be
   available or clear positive evidence in one species; together with supporting evidence such as geno-
   toxicity data, metabolic or biochemical studies, induction of benign tumours, structural relationship
   with other known carcinogens, or data from epidemiological studies suggesting an association.
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<pre>Category 3 actually comprises 2 sub-categories:
a    substances which are well investigated but for which the evidence of a tumour-inducing effect is
     insufficient for classification in Category 2. Additional experiments would not be expected to
     yield further relevant information with respect to classification.
b    substances which are insufficiently investigated. The available data are inadequate, but they
     raise concern for man. This classification is provisional; further experiments are necessary
     before a final decision can be made.
For a distinction between Categories 2 and 3 the arguments listed below are relevant which reduce
the significance of experimental tumour induction in view of possible human exposure. These argu-
ments, especially in combination, would lead in most cases to classification in Category 3, even
though tumours have been induced in animals:
•    carcinogenic effects only at very high levels exceeding the 'maximal tolerated dose'. The maxi-
     mal tolerated dose is characterized by toxic effects which, although not yet reducing lifespan, go
     along with physical changes such as about 10% retardation in weight gain;
•    appearance of tumours, especially at high dose levels, only in particular organs of certain species
     is known to be susceptible to a high spontaneous tumour formation;
•    appearance of tumours, only at the site of application, in very sensitive test systems (e.g. i.p. or
     s.c. application of certain locally active compounds);
•    if the particular target is not relevant to man;
•    lack of genotoxicity in short-term tests in vivo and in vitro;
•    existence of a secondary mechanism of action with the implication of a practical threshold above
     a certain dose level (e.g. hormonal effects on target organs or on mechanisms of physiological
     regulation, chronic stimulation of cell proliferation;
•    existence of a species - specific mechanism of tumour formation (e.g. by specific metabolic
     pathways) irrelevant for man.
For a distinction between Category 3 and no classification arguments are relevant which exclude a
concern for man:
•    a substance should not be classified in any of the categories if the mechanism of experimental
     tumour formation is clearly identified, with good evidence that this process cannot be extrapo-
     lated to man;
•    if the only available tumour data are liver tumours in certain sensitive strains of mice, without
     any other supplementary evidence, the substance may not be classified in any of the categories;
•    particular attention should be paid to cases where the only available tumour data are the occur-
     rence of neoplasms at sites and in strains where they are well known to occur spontaneously with
     a high incidence.
Guideline 93/21/EEG of the European Union                                                                 41
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