<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>Vincristine sulphate
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 Vincristine sulphate
Uw kenmerk              : DGV/MBO/U-932542
Ons kenmerk             : U-1482/JR/pg/246-W11
Bijlagen                :1
Datum                   : 12 december 2007
Geachte minister,
Graag bied ik u hierbij het advies aan over de kankerverwekkendheid van vincristinesulfaat.
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>Vincristine sulphate
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. 2007/10OSH, 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. Vincristine sulphate; Evaluation of the carci-
nogenicity and genotoxicity. The Hague: Health Council of the Netherlands,
2007; publication no. 2007/10OSH.
all rights reserved
ISBN: 978-90-5549-674-7
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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 18
4   Mutagenicity and genotoxicity 21
4.1 In vitro assays 21
4.2 In vivo assays 22
    Contents                                    7
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<pre>5   Classification 25
5.1 Evaluation of data on carcinogenicity and genotoxicity 25
5.2 Recommendation for classification 26
    References 27
    Annexes 31
A   Request for advice 33
B   The committee 35
C   Comments on the public review draft 37
D   IARC Monograph 39
E   Mutagenicity and genotoxicity data 43
F   Carcinogenic classification of substances by the committee 47
G   Guideline 93/21/EEG of the European Union 49
8   Vincristine sulphate
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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 vincristinesul-
faat onder de loep. Vincristinesulfaat is een cytostatisch geneesmiddel dat wordt
gebruikt ter bestrijding van kanker.
De commissie meent dat vincristinesulfaat onvoldoende is onderzocht. Hoewel
de gegevens het niet toelaten de stof te classificeren als kankerverwekkend voor
de mens of als moet beschouwd worden als kankerverwekkend voor de mens, is
de commissie van mening dat waakzaamheid geboden is. De commissie advi-
seert daarom vinblastinesulfaat te classificeren als verdacht kankerverwekkend
voor de mens. Volgens de richtlijnen van de Europese Unie komt dit overeen met
een classificatie in categorie 3. Binnen deze categorie komt de situatie het meest
overeen met subcategorie b.
Samenvatting                                                                       9
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<pre>10 Vincristine sulphate</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 vincristine sul-
phate. Vincristine sulphate is an antineoplastic cytotoxic agent that is used in the
treatment of cancer.
The committee concludes that vincristine sulphate has been insufficiently inves-
tigated. While the available data do not warrant a classification as carcinogenic
to humans or as should be regarded as carcinogenic to humans, they indicate that
there is cause for concern for man. This recommendation corresponds to EU
classification in category 3. This situation is, furthermore, comparable with sub-
category b of this category.
Executive summary                                                                    11
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<pre>12 Vincristine sulphate</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 G). 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 F). This report contains
        the evaluation of the carcinogenicity of vincristine sulphate.
1.2     committee and procedure
        The evaluation is performed by the committee on Classifying Carcinogenic Sub-
        stances 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 IA van de Gevel and MI
        Willems, from the Department of Occupational Toxicology of the TNO Nutrition
        and Food Research, by contract with the Ministry of Social Affairs and Employ-
        ment.
        Scope                                                                               13
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<pre>         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
    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 sci-
    entific 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 genotox-
    icity of the substance in question. In the case of vincristine sulphate, 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  Vincristine sulphate
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<pre>Chapter 2
        General information
2.1     Identity and physico-chemical properties
        Vincristine sulphate is an antineoplastic cytotoxic agent that is mainly used to
        treat certain types of cancer, such as Hodgkin’s disease, non-Hodgkin’s lympho-
        mas, and cancer of the breast or testicles.1 It is also used in some non-malignant
        conditions, such as idiopathic thrombocytopenia. Occupational exposure may
        occur during manufacturing or packaging, or during the final preparation and
        administration to patients. Below is given the identity and some of its physico-
        chemical properties.
        Chemical name     : Vincaleukoblastine, 22-oxo-, sulfate (1:1) (salt)
        CAS registry no.  : 2068-78-2
        EINECS no.        : 218-190-0
        Synonyms          : Leurocristine sulfate (1:1) (salt); Des-Na-methyl-Na-formylvinblastine sul-
                            phate; LCR sulphate; leurocristine sulphate; leurocristine, sulphate (1:1)
                            (salt); VCR sulphate; NSC 67574, Oncovin; Onkovin; Vincristine; Vincrisul;
                            37231
        Description       : White to slightly yellow, odourless, very hygroscopic, amorphous or crystal-
                            line powder
        Occurrence        : Vincristine is a naturally occurring alkaloid, which has been isolated from
                            several members of the plant genus Catharanthus (formerly called Vinca), a
                            pantropical shrub.
        Molecular formula : C46H56N4O10 • H2SO4
        General information                                                                              15
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<pre>    Structure         :
    Molecular weight  : 923.0
    Melting point     : After recrystalization from absolute ethanol, 273-281°C
    Solubility        : Soluble in water (1 in 2), ethanol (1 in 600), chloroform (1 in 30) and metha-
                        nol; insoluble in diethyl ether
    Stability         : Sensitive to hydrolysis, oxidation and heat
2.2 IARC classification
    In 1981 and 1987, IARC concluded that there is no evidence of carcinogenicity
    in rats or mice on the basis of available data. The data from studies in man are
    inadequate to evaluate the carcinogenicity of vincristine sulphate in humans.
    Overall, there is no evidence currently available to indicate that vincristine sul-
    phate is carcinogenic to humans, but the compound has not been extensively
    investigated. As a consequence, IARC concluded that vincristine sulphate was
    not classifiable as to its carcinogenicity to humans (Group 3).1,2
16  Vincristine sulphate
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<pre>Chapter 3
        Carcinogenicity studies
3.1     Observations in humans
        In general, vincristine sulphate is given together with certain other types of
        agents, such as bleomycin, procarbazine, nitrogen mustard, prednisone, or meth-
        otrexate, in combination with ionizing radiation therapy.2 These agents and treat-
        ments have the potential to induce secondary cancers by themselves, and are as
        such suspected carcinogens. None of the published data on humans, which are
        available to the committee, concern vincristine sulphate application alone.
        Therefore, it is difficult to assess from observational data whether vincristine sul-
        phate is the only responsible agent that may have caused secondary cancers.
        Below is given a short evaluation of some of the data on combination therapy.
            Various case reports and epidemiological studies have been reported on
        patients with Hodgkins’ disease, who were treated with combined chemotherapy,
        including vincristine sulphate, and who developed acute nonlymphocytic leuke-
        mia as a secondary cancer.1.
            Coleman et al. (1977) performed a follow-up study on 680 patients with
        Hodgkins’ disease.1,3 No excess risk of developing acute nonlymphocytic leuke-
        mia was observed in the group of patients receiving combination therapy. How-
        ever, various other patient-cohort studies on patients with Hodgkins’ disease
        reported on excess risk of acute nonlymphocytic leukemia by combination ther-
        apy compared to other forms of therapy or control groups.1
        Carcinogenicity studies                                                               17
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<pre>        To assess an association between treatment of cancer patients and develop-
    ment of secondary cancers, a group of Italian investigators performed a series of
    randomized studies on more than 1,000 patients who have been cured of their
    Hodgkin’s disease, and who were given various different treatments to control
    cancer.4-6 One of these treatments concerned chemotherapy with MOPP (mixture
    of mechloretamine, vincristine sulphate, prodacarbazine, and prednisone), with
    or without radiotherapy. The median follow-up time was 10 years. Overall, the
    15-year actuarial risk of developing leukemia was: 2.8% for patients treated with
    MOPP alone; 0.3% for patients having radiotherapy alone; and, 5.4% for com-
    bined modality therapy.
        No data were available on occupational exposure to the agent.
3.2 Carcinogenicity studies in animals
    Weisburger (1977) used Sprague-Dawley rats and Swiss mice to study the possi-
    ble carcinogenicity of various agents, including vincristine sulphate.7 Animals
    (n=25/group/sex/species) were given intraperitoneal injections at maximum or
    half maximum tolerated doses of vincristine sulphate, three times per week for 6
    months. After those six months, animals were followed an additional year before
    the study was ended.
        In male and female rats receiving 0.06 or 0.12 mg/kg bw, the survival time
    ranged from 19 to 100% (males), and 100% (females), compared to the survival
    times of non-treated control animals. The number of tumour-bearing male ani-
    mals was 3/16. The types of tumours found were two pituitary tumours, and one
    testis tumour. None of these were malignant. The tumour incidence in treated
    males was lower than in controls (18% versus 34% in control animals (also
    tumours in pituitary gland and testis)). In female rats, twelve of them developed
    tumours (12/22), of which two had malignant tumours. The principal tumours
    were 10 breast tumours, 3 pituitary tumours, and one adrenal tumour. The tumour
    incidence in vincristine sulphate treated animals was comparable with that in
    controls (55% versus 58% in control animals (tumours in same organs as treated
    animals)).
        In male and female mice receiving 0.075-0.15 mg/kg bw, the survival time
    ranged from 33 to 55% (males), and 97 to 100% (females), compared to the sur-
    vival time in non-treated mice. Only one benign skin tumour was observed in
    one male mouse (1/15). In female mice, four animals developed tumours (4/13),
    of which 3 had malignant tumours (3 lung tumours, and one leukemia). Overall,
    the tumour incidences did not differ markedly from the 26% incidence seen in
    both male and female controls. In its monograph, IARC noted the incomplete
18  Vincristine sulphate
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<pre>reporting of certain items, such as on survival times, the amalgation of various
experimental groups and tumours types, as well as the lack of age-adjustment in
the analyses.1 For this reason a complete evaluation was not possible and no final
conclusion could be given. The committee agrees with the comments of IARC.
Carcinogenicity studies                                                            19
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<pre>20 Vincristine sulphate</pre>

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<pre>Chapter 4
        Mutagenicity and genotoxicity
        Vincristine sulphate is a Vinca alkaloid, which is known to bind to the microtubu-
        lar proteins of the mitotic spindle. This causes microtubule-destabilisation,
        which finally leads to mitotic arrest or cell death. It are these properties, which
        are related to the antineoplastic activity of the agent.8,9 The possible mutagenic
        and genotoxic properties are summarized in the next sections. The outcomes of
        the individual studies are given in annex E.
4.1     In vitro assays
        Vincristine sulphate did not induce reverse point mutations in Salmonella
        typhymurium strains TA98 or TA100.2 It did induce gene mutations in mouse
        lymphoma cells, but not in other commercial cell lines.2 No unscheduled DNA
        synthesis was observed in mammalian and human cells.2,10
        Regarding its aneugenic and clastogenic potential, the agent did not induce a
        clear increase in chromosomal aberrations in various mammalian, and in human
        peripheral blood lymphocytes.2,11 Vincristine sulphate induced sister chromatid
        exchanges in human lymphocytes in one study, but not in three other studies.2
            A few micronucleus assays were performed to study the induction of micro-
        nuclei by vincristine sulphate in various mammalian cell types, and in human
        peripheral blood lymphocytes.2,12-14 All scores were positive.
        Mutagenicity and genotoxicity                                                       21
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<pre>        More recently, Kopjar and Garaj-Vrhovac (2000) used human peripheral
    blood lymphocytes from one donor, to assess aneugenic and clastogenic potency
    of vincristine sulphate.15 Administration of 0.0875 μg vincristine/mL for up to 72
    hours resulted in DNA damage and chromosome aberrations (chromatid breaks
    and acentric fragments). Also polyploid cells with numerous chromosomes were
    observed.
        Furthermore, vincristine sulphate induced aneuploidy and cell transforma-
    tions in Syrian hamster embryo cells, and in human fibroblasts.16,17 However, no
    transformed cells were found in vincristine-treated C3H/10T½ cells.18
    Overall, based on the current knowledge, vincristine sulphate is considered an
    aneugen. This indicates that it induces numerical chromosomal changes rather
    than chromosome breakage.19 In somatic cells, aneuploidy is associated with the
    development of several cancers, although the exact mechanism of action of aneu-
    genic agents is not yet completely understood.
4.2 In vivo assays
    No data were available to the committee on the mutagenic potential of vincristine
    sulphate in humans and animals.
    In various animal studies, a single intraperitoneal injection of vincristine sulphate
    increased the frequencies of micronuclei in bone marrow cells, in a dose-related
    matter, compared to non-treated controls.20-24 The animals included mice, ham-
    sters and rats. Based on the crescent shape and large size of the micronuclei, Tin-
    well and Ashby (1991) considered vincristine sulphate aneugenic.24 In two
    separate animal studies, vincristine sulphate also increased the frequency of
    chromosomal aberrations.25,26.
        In two separate experiments, Sheu et al. (1990, 1992) treated male Chinese
    hamsters with vincristine sulphate to study aneuploidy.27,28 By counting chromo-
    somes in the metaphase, no aneuploidy (hyperploidy) was observed in bone mar-
    row cells. Also no increased frequency in hyperploidy was observed in
    spermatogonial and germ cells in the meiotic I phase, but it did increase the fre-
    quency in germ cells in the meiotic II phase.
    In Drosophila melanogaster flies the results were negative in the sex-linked
    recombination lethal test, but positive in the somatic mutation and recombination
    assay.2,29,30 For instance, Tiburi et al. (2002) used the wing somatic mutation and
    recombination test of Drosophila melanogaster, to test for genotoxicity of vinc-
22  Vincristine sulphate
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<pre>ristine sulphate.29 In marker-heterozygous flies, the agent statistically signifi-
cantly increased the frequencies of total spots, which were mainly related to
small single spots. These single spots can be produced by somatic point muta-
tions, chromosome aberrations, and/or by mitotic recombinations. In balancer-
heterozygous flies, in the highest exposure level, vincristine sulphate clearly
increased the total number of total spots. In this fly, the presence of spots can be
caused by somatic point mutations and/or chromosome aberrations, but not by
mitotic recombinations.
Mutagenicity and genotoxicity                                                        23
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<pre>24 Vincristine sulphate</pre>

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<pre>Chapter 5
        Classification
5.1     Evaluation of data on carcinogenicity and genotoxicity
        Human carcinogenicity data on vincristine sulphate is limited to patients, who
        underwent curative therapy to cure a primary cancer. No epidemiological studies
        or case reports were available in which vincristine sulphate was the only agent
        used to cure these patients.
            Currently, there is inadequate evidence that vincristine sulphate is carcino-
        genic to humans. Also, no clear evidence was found that vincristine sulphate is
        carcinogenic to animals, but the committee emphasizes that the number of ani-
        mal studies was limited, and that the reporting of the available studies was
        incomplete. Overall, the carcinogenic properties of vincristine sulphate have
        been insufficiently investigated.
            Vincristine sulphate did not induce gene mutations in bacteria or mammalian
        cells, but it did induce micronuclei and aneuploidy in various test systems in vivo
        and in vitro. Controversial findings were reported on chromosome aberrations
        and sister chromatid exchanges. In addition, based on the available genotoxicity
        data and the current understanding of the mechanism of action, the committee is
        of the opinion that vincristine sulphate is an aneugen.
        Classification                                                                      25
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<pre>5.2 Recommendation for classification
    The committee concludes that vincristine sulphate has been insufficiently inves-
    tigated. While the available data do not warrant a classification as carcinogenic
    to humans or as should be regarded as carcinogenic to humans, they indicate that
    there is cause for concern for man. This recommendation corresponds to EU
    classification in category 3. This situation is, furthermore, comparable with sub-
    category b of this category.
26  Vincristine sulphate
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<pre>  References
1 IARC. Vincristine sulphate. IARC Monogr Eval Carcinog Risk Chem Hum 1981; 26: 365-384.
2 IARC. Vincristine sulphate. IARC Monogr Eval Carcinog Risks Hum Suppl 1987; 26(Suppl.6): 563-
  565.
3 Coleman CN, Williams CJ, Flint A, Glatstein EJ, Rosenberg SA, Kaplan HS. Hematologic neoplasia
  in patients treated for Hodgkin's disease. N Engl J Med 1977; 297(23): 1249-1252.
4 Brusamolino E, Anselmo AP, Klersy C, Santoro M, Orlandi E, Pagnucco G et al. The risk of acute
  leukemia in patients treated for Hodgkin's disease is significantly higher aft [see bined modality
  programs than after chemotherapy alone and is correlated with the extent of radiotherapy and type
  and duration of chemotherapy: a case-control study. Haematologica 1998; 83(9): 812-823.
5 Brusamolino E, Baio A, Orlandi E, Arcaini L, Passamonti F, Griva V et al. Long-term events in adult
  patients with clinical stage IA-IIA nonbulky Hodgkin's lymphoma treated with four cycles of
  doxorubicin, bleomycin, vinblastine, and dacarbazine and adjuvant radiotherapy: a single-institution
  15-year follow-up. Clin Cancer Res 2006; 12(21): 6487-6493.
6 Valagussa P, Bonadonna G. Hodgkin's disease and the risk of acute leukemia in successfully treated
  patients. Haematologica 1998; 83(9): 769-770.
7 Weisburger EK. Bioassay program for carcinogenic hazards of cancer chemotherapeutic agents.
  Cancer 1977; 40(4 Suppl): 1935-1949.
8 Mukherjee AK, Basu S, Sarkar N, Ghosh AC. Advances in cancer therapy with plant based natural
  products. Curr Med Chem 2001; 8(12): 1467-1486.
9 Zhou XJ, Rahmani R. Preclinical and clinical pharmacology of vinca alkaloids. Drugs 1992; 44
  Suppl 4: 1-16.
  References                                                                                           27
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<pre>10 Benigni R, Calcagnile A, Dogliotti E, Falcone E, Giuliani A. DNA repair induction by cytostatic
   drugs in proliferating and quiescent MRC-5 cells. Teratog Carcinog Mutagen 1983; 3(6): 481-490.
11 Kucerová M, Polívková Z. Mutagenic effects of different mutagens on human chromosomes in vitro
   as detected by conventional and "harlequin" methods. Dev Toxicol Environ Sci 1977; 2: 319-325.
12 Darroudi F, Meijers CM, Hadjidekova V, Natarajan AT. Detection of aneugenic and clastogenic
   potential of X-rays, directly and indirectly acting chemicals in human hepatoma (Hep G2) and
   peripheral blood lymphocytes, using the micronucleus assay and fluorescent in situ hybridization
   with a DNA centromeric probe. Mutagenesis 1996; 11(5): 425-433.
13 Miyakoshi Y, Suzuki Y, Ooida M, Takahashi A, Tsukui M. Micronucleus test using cultured new born
   rat astrocytes. Ind Health 1999; 37(1): 95-102.
14 Vian L, Bichet N, Gouy D. The in vitro micronucleus test on isolated human lymphocytes. Mutat Res
   1993; 291(1): 93-102.
15 Kopjar N, Garaj-Vrhovac V. Application of cytogenetic endpoints and Comet assay on human
   lymphocytes treated with vincristine in vitro. Neoplasma 2000; 47(3): 162-167.
16 Natarajan AT, Duivenvoorden WC, Meijers M, Zwanenburg TS. Induction of mitotic aneuploidy
   using Chinese hamster primary embryonic cells. Test results of 10 chemicals. Mutat Res 1993;
   287(1): 47-56.
17 Tsutsui T, Suzuki N, Maizumi H, Barrett JC. Aneuploidy induction in human fibroblasts: comparison
   with results in Syrian hamster fibroblasts. Mutat Res 1990; 240(4): 241-249.
18 Benedict WF, Banerjee A, Gardner A, Jones PA. Induction of morphological transformation in mouse
   C3H/10T1/2 clone 8 cells and chromosomal damage in hamster A(T1)C1-3 cells by cancer
   chemotherapeutic agents. Cancer Res 1977; 37(7 Pt 1): 2202-2208.
19 Aardema MJ, Albertini S, Arni P, Henderson LM, Kirsch-Volders M, Mackay JM et al. Aneuploidy:
   a report of an ECETOC task force. Mutat Res 1998; 410(1): 3-79.
20 Grawe J, Abramsson-Zetterberg L, Eriksson L, Zetterberg G. The relationship between DNA content
   and centromere content in micronucleated mouse bone marrow erythrocytes analysed by flow
   cytometry and fluorescent in situ hybridization. Mutagenesis 1994; 9(1): 31-38.
21 Krishna G, Fiedler R, Theiss JC. Simultaneous evaluation of clastogenicity, aneugenicity and toxicity
   in the mouse micronucleus assay using immunofluorescence. Mutat Res 1992; 282(3): 159-167.
22 Krishna G, Urda G, Theiss JC. Comparative mouse micronucleus evaluation in bone marrow and
   spleen using immunofluorescence and Wright's Giemsa. Mutat Res 1994; 323(1-2): 11-20.
23 Shi XC, Krishna G, Ong TM. Induction of micronuclei in rat bone marrow by four model compounds.
   Teratog Carcinog Mutagen 1991; 11(5): 251-258.
24 Tinwell H, Ashby J. Micronucleus morphology as a means to distinguish aneugens and clastogens in
   the mouse bone marrow micronucleus assay. Mutagenesis 1991; 6(3): 193-198.
25 Choudhury RC, Das B, Misra S, Jagdale MB. Spermatogonial cytogenetic toxicity of vincristine and
   its transmission in the germline cells of Swiss mice. J Environ Pathol Toxicol Oncol 2002; 21(3):
   249-257.
28 Vincristine sulphate
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<pre>26 Sieber SM, Whang-Peng J, Botkin C, Knutsen T. Teratogenic and cytogenetic effects of some plant-
   derived antitumor agents (vincristine, colchicine, maytansine, VP-16-213 and VM-26) in mice.
   Teratology 1978; 18(1): 31-47.
27 Sheu CW, Lee JK, Arras CA, Jones RL, Lavappa KS. The feasibility of using Chinese hamsters as an
   animal model for aneuploidy. Environ Mol Mutagen 1990; 16(4): 320-323.
28 Sheu CW, Lee JK, Arras CA, Jones RL, Lavappa KS. Detection of vincristine-induced hyperploidy in
   meiotic II metaphases of male Chinese hamsters. Mutat Res 1992; 280(3): 181-186.
29 Tiburi M, Reguly ML, Schwartsmann G, Cunha KS, Lehmann M, Rodrigues de Andrade HH.
   Comparative genotoxic effect of vincristine, vinblastine, and vinorelbine in somatic cells of
   Drosophila melanogaster. Mutat Res 2002; 519(1-2): 141-149.
30 Vogel EW, Nivard MJ. Performance of 181 chemicals in a Drosophila assay predominantly
   monitoring interchromosomal mitotic recombination. Mutagenesis 1993; 8(1): 57-81.
31 Seino Y, Nagao M, Yahagi T, Hoshi A, Kawachi T, Sugimura T. Mutagenicity of several classes of
   antitumor agents to Salmonella typhimurium TA98, TA100, and TA92. Cancer Res 1978; 38(7):
   2148-2156.
32 Suter W, Brennand J, McMillan S, Fox M. Relative mutagenicity of antineoplastic drugs and other
   alkylating agents in V79 Chinese hamster cells, independence of cytotoxic and mutagenic responses.
   Mutat Res 1980; 73(1): 171-181.
33 Matheson D, Brusick D, Carrano R. Comparison of the relative mutagenic activity for eight
   antineoplastic drugs in the Ames Salmonella/microsome and TK+/- mouse lymphoma assays. Drug
   Chem Tox 1978; 1: 277-304.
34 Tsutsui T, Suzuki N, Maizumi H, Barrett JC. Vincristine sulfate-induced cell transformation, mitotic
   inhibition and aneuploidy in cultured Syrian hamster embryo cells. Carcinogenesis 1986; 7(1): 131-
   135.
35 Au WW, Johnston DA, Collie-Bruyere C, Hsu TC. Short-term cytogenetic assays of nine cancer
   chemotherapeutic drugs with metabolic activation. Environ Mutagen 1980; 2(4): 455-464.
36 Maier P, Schmid W. Ten model mutagens evaluated by the micronucleus test. Mutat Res 1976; 40(4):
   325-337.
37 Morgan WF, Crossen PE. Mitotic spindle inhibitors and sister-chromatid exchange in human
   chromosomes. Mutat Res 1980; 77(3): 283-286.
38 Raposa T. Sister chromatid exchange studies for monitoring DNA damage and repair capacity after
   cytostatics in vitro and in lymphocytes of leukaemic patients under cytostatic therapy. Mutat Res
   1978; 57(2): 241-251.
39 Banerjee A, Benedict WF. Production of sister chromatid exchanges by various cancer
   chemotherapeutic agents. Cancer Res 1979; 39(3): 797-799.
40 Todd GC, Gibson WR, Morton DM. Toxicology of vindesine (desacetyl vinblastine amide) in mice,
   rats, and dogs. J Toxicol Environ Health 1976; 1(5): 843-850.
41 Yamamoto KI, Kikuchi Y. Studies on micronuclei time response and on the effects of multiple
   treatments of mutagens on induction of micronuclei. Mutat Res 1981; 90(2): 163-173.
   References                                                                                           29
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<pre>42 Norppa H, Penttila M, Sorsa M, Hintikka EL, Ilus T. Mycotoxin T-2 of Fusarium tricinctum and
   chromosome changes in Chinese hamster bone marrow. Hereditas 1980; 93(2): 329-332.
30 Vincristine sulphate
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<pre>A Request for advice
B The committee
C Comments on the public review draft
D IARC Monograph
E Mutagenicity and genotoxicity data
F Carcinogenic classification of substances by the committee
G Guideline 93/31/EEG of the European Union
  Annexes
                                                             31
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<pre>32 Vincristine sulphate</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                                                                                        33
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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.
34  Vincristine sulphate
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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                                                               35
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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.
36  Vincristine sulphate
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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                                               37
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<pre>38 Vincristine sulphate</pre>

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<pre>Annex D
      IARC Monograph
      D.1        VOL.: 26 (1981) (p. 365)1
      Summary of Data Reported and Evaluation
      Experimental data
      Vincristine sulphate was tested in mice and rats by intraperitoneal injection. In
      these limited studies no evidence of carcinogenicity was found.
      Vincristine sulphate can induce teratogenic effects in several animal species, and
      it induced embryolethality at doses nontoxic to the mother. There is no evidence
      to suggest that this compound is mutagenic.
      Human data
      Vincristine sulphate has been used since the early 1960s for treatment of acute
      leukaemia in children, often in combination with other antineoplastic agents. It is
      also frequently a part of combination chemotherapeutic regimens for Hodgkin's
      disease, non-Hodgkin's lymphoma, chronic lymphocytic leukaemia and other
      adult neoplasms.
      IARC Monograph                                                                      39
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<pre>   The available data are insufficient to evaluate the teratogenicity of this drug in
   humans. No data were available on its mutagenic or chromosomal effects.
   Both case reports and epidemiological studies indicate that acute nonlympho-
   cytic leukaemia is produced in patients with Hodgkin's disease treated with com-
   bined therapeutic regimens which include vincristine sulphate, alkylating agents
   and procarbazine hydrochloride, often in conjunction with radiotherapy. No data
   were available on vincristine sulphate alone.
   Evaluation
   The available data in experimental animals were insufficient for evaluation.
   There is sufficient evidence for the carcinogenicity in humans of intensive che-
   motherapeutic regimens that include alkylating agents, vincristine sulphate, pro-
   carbazine hydrochloride and prednisone. There is inadequate evidence for the
   carcinogenicity of vincristine sulphate itself.
   On the basis of the available data, no conclusion could be drawn as to the carci-
   nogenicity of vincristine sulphate.
   D.2        Supplement 7: (1987) (p. 372)2
   CAS No.: 2068-78-2
   Chem. Abstr. Name: Vincaleukoblastine, 22-oxo, sulfate (1:1) (salt)
   A Evidence for carcinogenicity to humans (inadequate)
   No epidemiological study of vincristine sulphate as a single agent was available
   to the Working Group. Intensive combination chemotherapy with regimens
   including vincristine has been shown to result in increased risks for acute non-
   lymphatic leukaemia (ANLL). (See also the summary of data on MOPP and
   other combined chemotherapy including alkylating agents) Such combinations
   usually include procarbazine together with an alkylating agent such as nitrogen
   mustard (see p. 269), both of which are potent animal carcinogens, suggesting
   more plausible explanations for the association between combination chemother-
   apy and ANLL. In the presence of concurrent therapy with other putative carcin-
   ogens, including ionizing radiation and other potent drugs, occasional case
   reports of exposure to vincristine sulphate do not constitute evidence of carcino-
   genesis [ref: 1].
40  Vincristine sulphate
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<pre>  B Evidence for carcinogenicity to animals (inadequate)
  In limited studies in mice and rats, no evidence of carcinogenicity was found
  after intraperitoneal administration of vincristine sulphate [ref: 1].
  C Other relevant data
  No data were available on the genetic and related effects of vincristine sulphate
  in humans.
  Vincristine sulphate induced micronuclei in bone-marrow cells of mice and ham-
  sters treated in vivo. Conflicting results were obtained for induction of sister
  chromatid exchanges in human lymphocytes in vitro. It induced aneuploidy in
  and transformation of Syrian hamster embryo cells, but it did not transform
  mouse C3H 10T1/2 cells. It did not induce chromosomal aberrations, sister chro-
  matid exchanges or unscheduled DNA synthesis in rodent cells in vitro. It
  induced mutation in mouse lymphoma cells but not in other rodent cells. It did
  not induce sex-linked recessive lethal mutations in Drosophila and was not
  mutagenic to bacteria [ref: 2].
  Overall evaluation
  Vincristine sulphate is not classifiable as to its carcinogenicity to humans
  (Group 3).
  References
1 IARC Monographs, 26, 365-384, 1981
2 IARC Monographs, Suppl. 6, 563-565, 1987
  IARC Monograph                                                                    41
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<pre>42 Vincristine sulphate</pre>

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<pre>Annex         E
              Mutagenicity and genotoxicity data
Table E.1 Mutagenicity and genotoxicity of vincristine sulphate in in vitro test systems.
test system                               dose range        Result            remarks                  reference
                                                            - negative
                                                            + positive
Mutagenicity
Ames Salmonella assay; TA98 and           250 μg/mL         -                 No further details given Seino et al.
TA100; with and without metabolic acti-                                                                197831
vation
Hprt locus; Chinese hamster lung V79 0.3 μg/mL              -                 No further details given Suter et al.
cells; no metabolic activation                                                                         198032
TK locus ; mouse lymphoma L5178Y > 5 to 40 g/mL             +                 No further details given Matheson et
cells; with and without metabolic activa-                   (>1 g/mL)                                  al. 197833
tion
Other animal cells                        0.03 μg/mL        -                 No further details given Tsutsui et al.
                                                                                                       198634
Chromosome aberrations
Chinese hamster cells; with and without 10 μg/mL            -                 No further details given Au et al.
metabolic activation                                                                                   198035
Chinese hamster cells; no metabolic       0.1 μg/mL         -                 No further details given Maier et al.
activation                                                                                             197636
Syrian hamster cells; no metabolic acti- 0.03 μg/mL         -                 No further details given Tsutsui et al.
vation                                                                                                 198634
Transformed cells; no metabolic activa- 0.05 μg/mL          -                 No further details given Benedict et
tion                                                                                                   al. 197718
              Mutagenicity and genotoxicity data                                                                  43
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<pre>Human peripheral blood lymphocytes; 0.1 – 1,000 ng/mL -                    No further details given. The author Kučerová
no metabolic activation                                                    considers the increase in numbers of and Polívk-
                                                                           SCE/cell to be of doubtful if any sig- ová 197711
                                                                           nificance, since it concerned only
                                                                           one observation. In addition the num-
                                                                           ber of SCE/cell observed was in the
                                                                           range of the control limits of 8
                                                                           donors.
Human peripheral blood lymphocytes       Dose applied was +                Chromatid breaks and acentric frag- Kopjar and
from one donor                           0.0875 μg/mL for                  ments were observed. Authors also Garaj-Vrho-
                                         up to 72 hours; neg-              reported of DNA damage in the same vac 200015
                                         ative control was                 cells, as measured by Comet assay,
                                         included                          and of polyploidy.
Sister chromatid exchange
Human peripheral blood lymphocytes; 0.1 – 10 ng/mL            -            No further details given               Kučerová
no metabolic activation                                                                                           and Polívk-
                                                                                                                  ová 197711
Human lymphocytes; no metabolic acti- 1.0 μg/mL               -            No further details given               Morgan et
vation                                                                                                            al. 198037
Human lymphocytes; no metabolic acti- 0.1 μg/mL               +            No further details given               Raposa et al.
vation                                                                                                            197838
Transformed cells; no metabolic activa- 1.0 μg/mL             -            No further details given               Banerjee et
tion                                                                                                              al. 197939
Micronuclei
Human peripheral blood lymphocytes; 1 - 5 pM                  + (>3 pM)    Dose-related increase in micronucle- Vian et al.
no metabolic activation                                                    ated cells                             199314
Human peripheral blood lymphocytes 3 – 1.2 nM                 + (>3 nM)    Dose-related increase in micronucle- Darroudi et
(CB-FISH micronucleus assay) ; no met-                                     ated cells; 87-89% of micronuclei al. 199612
abolic activation                                                          centromere positive; 50% of con-
                                                                           trols were centromere positive
Primary rat astrocytes; no metabolic     8.5 – 135 nM         + (>8.5 nM)  Dose- and time-related increase        Miyakoshi et
activation                                                                                                        al. 199913
Aneuploidy
Syrian hamster embryo cells (chromo- 1-30 ng/mL               + (>3 ng/mL)                                        Tsutsui et al.
some counting in metaphases)                                                                                      198634
Human JHU-1 fibroblasts (chromosome 1 – 10 ng/mL              + (10 ng/mL)                                        Tsutsui et al.
counting in metaphases)                                                                                           198634
Chinese hamster embryo cells             0.005 – 0.50 μg/mL   +            Increases concern both anauploidy      Natarajan et
                                                                           and polyploidy                         al. 199316
Cell transformation
C3H/10T½ cells; no metabolic activa- 0.075 µg/mL              -            No further details given               Benedict et
tion                                                                                                              al. 197718
Syrian hamster embryo cells, clonal      1.0 ng/mL            +            No further details given               Tsutsui et
assay; no metabolic activation                                                                                    al.198634
Unscheduled DNA synthesis
Human fibroblast MRC-5 cells; no met- 0.0025 - 25 µg/mL       -                                                   Benigni et
abolic activation                                                                                                 al. 198310
Other animal cells; no metabolic activa- 0.10 µg/mL           -            No further details given               Tsutsui et
tion                                                                                                              al.198634
44             Vincristine sulphate
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<pre>Table E.2 Mutagenicity and genotoxicity of vincristine sulphate in in vivo test systems.
test system                         dose range             Result        remarks                                  reference
                                                           - negative
                                                           + positive
Mutagenicity
Sex-linked recessives lethal test, 15 μg/mL                -             No further details given                 Todd et al.
using Drosophila melanogaster                                                                                     198340
flies
Eye mosaic assay, using Drosophila 0 – 0.2 mM              +             The clone induction was relatively low,  Vogel et al.
melanogaster flies                                                       and was associated with indications of   199330
                                                                         reduced survival and cytotoxicity
Somatic mutation and recombina- 0 – 0.05 mM                +                                                      Tiburi et al.
tion test, using Drosophila melano-                                                                               200229
gaster flies
Chromosome aberrations
Pregnant mice, bone marrow and single intraperitoneal +                  Increase concerned numerical and struc-  Sieber et al.
embryonic tissue                    injection of                         tural chromosome aberrations             197826
                                    0.3 mg/kg bw, on day
                                    6 of pregnancy
Male Swiss mice, spermatogonia single intraperitoneal +                  Also a statistically significantly       Choudhury et
                                    injection of 0.25, 0.5               increased frequency of aberrant sper-    al. 200225
                                    or 1.0 mg/kg bw. Ani-                matogonial metaphases was observed.
                                    mals were followed
                                    for 24 hours, 4 weeks,
                                    and 8 weeks
Micronuclei (micronucleus test assays)
Mice, bone marrow                   0.05 mg/kg bw          +             No further details given                 Maier et al.
                                                                                                                  197636
Mice, bone marrow                   0.125 mg/kg bw         +             No further details given                 Yamamoto et
                                                                                                                  al. 198141
Hamsters                            0.2 mg/kg bw           +             No further details given                 Norppa et al.
                                                                                                                  198042
CBA mice, bone marrow               single intraperitoneal + (>0.025     Dose-related increase in micronucleated Tinwell and
                                    injection of 0.001 – mg/kg bw)       polychromatic erythrocytes. Micro-       Ashby 199124
                                    0.25 mg/kg bw                        scopic examination revealed an increased
                                                                         incidence of crescent-shaped and large-
                                                                         sized next to normal-sized micromuclei
CD-1 mice, bone marrow and          single intraperitoneal +             Dose-related increase in micronucleated Krishna et al.
spleen                              injection of 0.1-0.2                 polychromatic erythrocytes in bone mar- 1992,
                                    mg/kg bw                             row and spleen in both sexes. In males 199421,22
                                                                         87-89% of micronuclei K+, in females 75
                                                                         - 82%.
CBA mice, males bone marrow         Single intraperitoneal +             Percentage centromere positive: 76% in Grawe et al.
                                    injection of 0.125 mg/               treated animals versus 20% in controls 199420
                                    kg bw
Sprague-Dawley rats, males, bone Single intraperitoneal +                Dose- and time-related increase in micro-Shi et al.
marrow                              injection of 0.05-0.2                nucleated polychromatic erythrocytes     199223
                                    mg/kg bw
               Mutagenicity and genotoxicity data                                                                            45
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<pre>Aneuploidy
Chinese hamsters, bone marrow       single intraperitoneal -         No treatment-related increase in numbers Sheu et al.
(chromosome counting in             injection of 0.25-0.75           of chromosomes per metaphase was         199027
metaphase)                          mg/kg bw                         observed
Chinese hamsters, germ cells: sper- Single intraperitoneal + (hyper- Treatment-related increased frequency of Sheu et al.
matogonia, meiotic I and meiotic II injection of 0.25 –    ploidy)   hyperploid meiotic II metaphase cells at 199228
                                    0.75 mg/kg bw                    6, 24 and 48 h but not 72 and 96 h after
                                                                     treatment
46          Vincristine sulphate
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<pre>Annex         F
              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                                             47
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<pre>48 Vincristine sulphate</pre>

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<pre>Annex G
      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                                                             49
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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.
50  Vincristine sulphate
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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                                                                 51
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<pre>52 Vincristine sulphate</pre>

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