<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>Cyclosporin
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 Cyclosporin
Uw kenmerk              : DGV/MBO/U-932542
Ons kenmerk             : U-1479/JR/pg/246-T11
Bijlagen                :1
Datum                   : 12 december 2007
Geachte minister,
Graag bied ik u hierbij het advies aan over de kankerverwekkendheid van cyclosporine. Het
maakt deel uit van een uitgebreide reeks waarin kankerverwekkende stoffen worden geclas-
sificeerd 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>Cyclosporin
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. 2007/06OSH, 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. Cyclosporin; Evaluation of the carcinogenic-
ity and genotoxicity. The Hague: Health Council of the Netherlands, 2007; publi-
cation no. 2007/06OSH.
all rights reserved
ISBN: 978-90-5549-669-3
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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 21
4   Mutagenicity and genotoxicity 25
4.1 In vitro assays 25
4.2 In vivo assays 26
4.3 Carcinogenic mechanism 27
    Contents                                    7
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<pre>5   Classification 29
5.1 Evaluation of data on carcinogenicity and genotoxicity 29
5.2 Recommendation for classification 30
    References 31
    Annexes 35
A   Request for advice 37
B   The committee 39
C   Comments on the public review draft 41
D   IARC Monograph 43
E   Carcinogenic classification of substances by the committee 47
F   Guideline 93/21/EEG of the European Union 49
8   Cyclosporin
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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 cyclosporine
onder de loep. Cyclosporine verlaagt de immunologische afweer en wordt
gebruikt als immunosuppressieve therapie voor onder andere patiënten die een
orgaantransplantatie hebben ondergaan.
Op basis van de beschikbare gegevens leidt de commissie af dat cyclosporine
kankerverwekkend is voor de mens. Dit komt overeen met een classificatie in
categorie 1 volgens de richtlijnen van de Europese Unie. De commissie conclu-
deert verder dat cyclosporine niet genotoxisch is.
Samenvatting                                                                  9
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<pre>10 Cyclosporin</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 cyclosporin.
Cyclosporin is an immunosuppressive agent that is given to patients to prevent
graft-versus-host reactions after organ transplantation, and to control certain dis-
eases in which the immunological defense plays a role.
Based on the available information, the committee is of the opinion that
cyclosporin should be classified as known to be carcinogenic to humans. This
recommendation corresponds to the EU classification in category 1. The commit-
tee concludes furthermore that cyclosporin is not genotoxic.
Executive summary                                                                    11
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<pre>12 Cyclosporin</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 cyclosporin.
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 cyclosporin, 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  Cyclosporin
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<pre>Chapter 2
        General information
2.1     Identity and physico-chemical properties
        Cyclosporin is a fungal nonpolar oligopeptide, consisting of eleven amino acids,
        including a beta-hydroxy single unsaturated amino acid, which is isolated from
        the fungus Tolypocladium inflatum Grams1. It is the most commonly used immu-
        nosuppressive agent for organ transplantation.1 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 physical and chemical properties.
        Chemical name           : {R-[R*,R*-(E)]}-L-alanyl-N-methyl-L-leucyl-N-methyl-leucyl-N-
                                   methyl-L-valyl-3-hydroxy-N,4-dimethyl-L-2-amino-6-octenoyl-L-
                                   -aminobutyryl-N-methylglycyl-N-methyl-L-leucyl-L-valyl-N-
                                   methyl-L-leucyl)
        CAS registry number     : 79217-60-0 (cyclosporine A: 59865-13-3)
        Synonyms                : Cyclosporin A; ciclosporin, cyclosporin; OL-27-400; cyclo{[(E)-
                                   2S,3R,4R)-3-hydroxy-4-methyl-2-(methylamino)-6-octenoyl]-L-2-
                                   aminobutyryl-N-methylglycyl-N-methyl-L-leucyl-L-valyl-N-
                                   methyl-L-leucyl-L-alanyl-D-alanyl-N-methyl-L-leucyl-N-methyl-
                                   L-leucyl-N-methyl-L-valyl}; cyclo{[4-(E)-but-2-enyl-N,4-dime-
                                   thyl-L-threonyl]-L-homoalanyl(N-methyl-glycyl)(N-methyl-L-
                                   leucyl)-L-valyl(N-methyl-L-leucyl)-L-analyl-D-alanyl-(N-methyl-
                                   L-leucyl)(N-methyl-L-leucyl)(N-methyl-L-valyl); antibiotic S
                                   7481F1; Ramihyphin A; S 7481F1; Sandimmun; Sandimmune
        Description             : White prismatic crystals from acetone
        General information                                                                        15
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<pre>    Molecular weight        :  1202.64
    Boiling point           :  -
    Melting point           :  148-151 °C
    Vapour pressure         :  -
    Solubility              :  Cyclosporin is insoluble in water and n-hexane and very soluble in
                               all other organic solvents
    Stability               : Cyclosporin is stable in solution at temperature below 30 °C and is
                               sensitive to light, cold and oxidization
    Partition coefficient   : -
    Molecular formula       : C62 H111N11O12
    Structure               :
2.2 IARC classification
    In 1990, IARC concluded that there is sufficient evidence for the carcinogenicity
    of cyclosporin in humans, and that there is limited evidence for the carcinogenic-
    ity in experimental animals.1 Therefore, according to the IARC guidelines, it
    classified cyclosporin in Group 1, which means that cyclosporin is carcinogenic
    to humans.
16  Cyclosporin
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<pre>Chapter 3
        Carcinogenicity studies
3.1     Observations in humans
3.1.1   Case reports
        In 1990, IARC reported in its monograph of numerous case reports of neoplasms
        in patients, who received long-term cyclosporin therapy alone after organ trans-
        plantation.1 In general, the most reported neoplasms in these recipients were lym-
        phomas, leukaemias and skin cancer (Kaposi’s sarcomas). The lymphomas were
        predominantly found in the gastrointestinal tract.
            In his reviews, Ryffel concluded that the incidence of lymphoproliferative
        disorders and other malignant diseases in cyclosporin-immunosuppressed
        patients was higher than in the general population, but comparable to patients
        who were treated with other immunosuppressive agents, such as azathioprine and
        prednisone.2,3 Furthermore, data from the Collaborative Transplant Study have
        suggested that the combination therapy with cyclosporin and azathioprine was
        associated with an increased risk of non-Hodgkin's lymphoma.4
            Cases are still reported and not only concern organ transplant recipients, but
        also psoriatic patients who suffered skin cancer after photochemotherapy. In
        most of these cases, cyclosporin was the only given immunosuppressive agent in
        combination with UV-irradiation.5-11
            In a few case-reports regression of lymphomas were reported, following
        withdrawal of cyclosporin.12
        Carcinogenicity studies                                                            17
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<pre>           Overall, case reports do not allow a conclusion on the association between
      cyclosporin treatment and the induction of cancer in humans. No cases were
      reported on occupational inhalation or dermal exposure in healthy workers.
3.1.2 Epidemiological studies
      IARC reported on a number of cohort studies on organ transplant recipients.1
      Some of these cohorts concerned combined exposure with cyclosporin and other
      immunosuppressive agents or cytotoxic drugs, such as cyclophosphamide and
      azathioprine. Overall, IARC noted that there were a number of limitations in the
      cohort studies, such as: missing data on dose, survival and follow-up time; uncer-
      tainty whether cyclosporin was given in combination with other agents; and, the
      lack of comparison with the general population. From five cohort-studies, it was
      certain that only cyclosporin was given. From these studies, IARC estimated the
      upper limits for the expected values of Non-Hodgkin’s lymphoma in organ trans-
      plant recipients, which are shown in table 3.1. Although IARC made a lot of
      assumptions, the incidence of lymphomas was remarkably high.
      Table 3.1 Non-Hodgkin's lymphomas in organ transplant patients treated with cyclosporin (IARC
      1990).
      number of maximal follow-up Non-Hodgkin’s                             Reference
      patients      (years)         lymphomas
                                    expecteda         observed
      28            1.5             0.02               2                    Calne et al. (1979)13
      498           4               1.0               11                    Starzl et al. (1984)14
      67            1.5             0.05               0                    Bencini et al. (1986)15
      120           5 b
                                    0.3                0                    Sheil et al. (1987)16
      160           5               0.4                0                    Smith et al. (1989)17
      873 (total)                   1.8               13
      a    as estimated by the IARC
      b    mean as given in paper
      After the publication of the IARC monograph several other cohort studies were
      performed, of which a short description is given below.
      Organ transplant recipients
      Cockburn and Krupp reviewed the clinical situation of three different surveil-
      lance studies, concerning the occurrence of neoplasms in renal transplant recipi-
      ents treated with cyclosporin.18 Below only data of a post marketing surveillance
18    Cyclosporin
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<pre>study are given. The surveillance started in 1982, and in 1989 already 5,000
recipients were followed, which comprised 4,040 renal transplant recipients. The
overall incidence for both sexes of de novo neoplasms was 1.5%. The most fre-
quent types of neoplasms were skin cancer (Kaposi’s sarcomas and basal cell
carcinomas), but also some lymphomas were diagnosed, whereas no leukaemia
was found. The cyclosporin-treated recipients had a double risk for developing
neoplasms compared to the normal non-exposed population. The two-fold
increase in risk was comparable with the two- to four-fold increased risk that
recipients had, who received other immunosuppressive agents. The authors con-
cluded that the risk between the different types of immunosuppressive therapies
did not differ, but that the latency period appeared to be shorter in patients treated
with cyclosporin (about 10 months compared to about 3.5 years).
     Baildam et al. (1996), performed a prospective cohort study on 39 women
under the age 55 years, who had received a renal transplant at least 1 year ear-
lier.19 Of the 29 patients currently receiving cyclosporin or who had previously
done so, 13 had fibroadenomas in their breast. None of the patients who received
steroids or azathioprine developed fibroadenomas.
     Dantal et al. (1998) reported of a randomized clinical trial study on renal
transplant recipients who received a long-term combination therapy of
cyclosporin and azathioprine.20 The recipients were randomized one year after
transplantation in two groups. The first group consisted of 115 recipients who
received, after that first year, normal doses of cyclosporin (blood concentration,
150-250 ng/mL), the second group of 115 recipients received low doses of
cyclosporin (blood concentration, 75-125 ng/mL). Both groups were followed
for 66 months. A total of 60 recipients developed at least one cancer (normal-
versus low-dose group, 37 versus 23). The difference between the groups was
significant (p<0.034). Of the malignant tumours, 66% concerned skin cancer
(normal- versus low-dose group, 26 versus 17). Overall, the authors observed
that low-doses of cyclosporin reduced the number of malignant disorders, but
increased the number of organ rejections. So far, only indirect evidence sug-
gested that lowering the dose of cyclosporin could reduce cancer development.21
     More recently, Kessler et al. (2006) reported on increased standard incidence
rates (SIR) for various types of de novo cancer in a group of 488 renal transplant
recipients, who received dual or triple therapy with cyclosporin, prednisone and
azathioprine.22 The recipients were followed up to ten years after transplantation.
For all cancers the SIR was 2.2 (1.5-3.0) in males, and 3.0 (1.9-4.6) in females.
More specifically, in males there was a significant excess of cancer for native
kidneys, prostate cancer, and post-transplant lymphoproliferative disorder com-
pared with the general population in France; in females it concerned excesses of
Carcinogenicity studies                                                                19
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<pre>   cervical cancer, native kidneys, and post-transplant lymphoproliferative disorder.
   Also the incidence of nonmelanoma skin cancer was high, but the authors could
   not calculate SIRs for this type of cancer, due to a lack of data in the general pop-
   ulation.
   Psoriasis patients
   In a follow-up study, 1,223 patients with psoriasis who received cyclosporin
   were followed up for up to 60 months.23 Also a control group of patients with
   high blood pressure not receiving such a treatment, and considered to have a
   same risk of developing cancer as the general population, was included. Of the
   cyclosporin-treated patients, 28 developed cancer. Almost 50% concerned skin
   cancer. The relative risk of all cancers in cyclosporin-treated patients versus the
   control group was 5.6 (95% confidence interval, 3.9-8.0); skin cancer, 12.4 (95%
   CI, 7.3-21.2); solid tumours, 3.2 (95% CI, 1.8-5.6); and, lymphomas, 5.1 (95%
   CI, 1.2-21.2). The authors discussed the complexity of their study results, in that
   several bias may have influenced the outcome. Therefore, it is difficult to prove
   the causality between cyclosporin-treatment and cancer. At least the study shows
   a trend that cyclosporin may increase cancer risk in psoriasis patients.
       In 2004, Paul et al. reported on a prospective five-year cohort study to inves-
   tigate the association between the incidence of malignancies in severe psoriasis
   patients and the treatment with cyclosporin.24 A total of 1,252 patients from 10
   different European countries, and Canada, participated in the study. Incidence
   rates for malignancy were compared to the standardized incidence ratios in the
   general population. On average the patients received cyclosporin-therapy for 1.9
   years. In 3.8% of the patients malignancies were diagnosed, of which 49% had
   skin malignancies (mostly squamous cell carcinoma). The standardized inci-
   dence ratio was 2.1 as compared to the general population. The increased ratio
   was mainly ascribed to the higher incidence of skin malignancies; the overall
   incidence of nonskin malignancies was comparable with that of the general pop-
   ulation. The authors also noted that a longer treatment period increased the risk
   for tumour development, and that previous photochemotherapy (psoralen and
   ultraviolet A) and previous treatment with other immunosuppressive agents con-
   tributed to the overall risk.
       More recently, Behnam et al. (2005) performed a review of the literature in
   search for human data on dermatology patients and the adverse effects of
   cyclosporin.25 Between the period 1979 and 2003, the authors found 26 English
   articles that provided some relevant data, of which the study by Paul et al. (2004)
   gave the most relevant information.26 The reviewers commented that most scien-
20 Cyclosporin
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<pre>    tific papers had limitations, such as limitations in study design, and lack of com-
    parisons with the general population. The reviewers, however, did not make clear
    what their final conclusion was on the appearance of malignancies in dermatol-
    ogy patients.
    Other diseases
    Over a thousand patients with rheumatoid arthritis (RA) were treated with
    cyclosporin (maximum daily dose of 5 mg/kg bw).27 Seventeen of these patients
    developed a malignancy (skin cancer, 4 patients; B-cell lymphoma, 1 patient;
    solid tumours at various sites of the body, 12 patients). The time between first
    treatment and the development of the cancer varied greatly and ranged from 8
    days to 46 months. Further analyses revealed that the overall relative risk for
    developing cancer in RA patients is 1.4 (95% CI, 1.4-1.5), which was lower than
    for cyclosporin-treated RA patients (3.6; 95% CI, 2.2-5.8). However, the relative
    risk of cyclosporin-treated patients was comparable with the risk of RA patients
    receiving a different treatment.
    No observational studies were performed in an occupational setting in healthy
    workers, who produced or prepared cyclosporin.
3.2 Carcinogenicity studies in animals
    IARC reported on several long-term animal studies.1 No increased incidence of
    tumours was observed in OF1 mice (n=50/group/sex), which were given
    cyclosporin at a daily base in their diet (1, 4, or 16 mg/kg of diet) for 78 weeks,
    compared to non-treated animals. Also no increased incidence of tumours was
    observed in OFA rats (n=50/group/sex), which were fed cyclosporin-enriched
    food (0.5, 2 or 8 mg/kg of diet) for 95 weeks. In a screening assay based on the
    accelerated induction of leukaemia in a strain highly susceptible to develop this
    disease, 30 male AKR mice were fed cyclosporin at 150 mg/kg of diet for 17 up
    to 34 weeks. Not only the latency period was much shorter, but also the treated
    animals had a higher incidence of leukaemia than non-treated control animals.
         IARC also reported on animal studies in which cyclosporin treatment was
    combined with other treatments.1 Overall, cyclosporin enhanced the development
    of lymphomas induced in two strains of male mice by a single whole-body irradi-
    ation or by a single injection of N-methyl-N-nitrosourea. In grafted macaques,
    cyclosporin increased the incidence of lymphomas, a neoplasm that occurs
    extremely infrequently in this species of monkeys. When given in combination
    Carcinogenicity studies                                                             21
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<pre>   with various other immunosuppressive regimens, cyclosporin induced a substan-
   tial increase in the incidence of lymphomas when compared to immunosuppres-
   sive regimens without cyclosporin. The substance also enhanced the incidence of
   intestinal adenocarcinomas in male rats, which were pre-treated with the geno-
   toxic carcinogen N-methyl-N-nitrosourea.
   After the publication of the IARC-monograph a few other animal studies were
   performed. These studies all concerned the promotion of cancer by cyclosporin,
   or the accelerated cancer development in genetically modified animals, which
   were made susceptible for certain diseases.
        In 1993, Masuhara et al. reported a significant accelerated development of
   hepatocellular carcinomas in forty male F344 rats fed cyclosporin-enriched diet
   (amount in diet, 0.015%) for a maximum of 10 weeks, compared to a group of
   animals that did not receive cyclosporin.28 This food regimen was followed after
   an initiation and promotion period of seven weeks, in which the animals were
   given a single intraperitoneal injection of the genotoxic carcinogen diethylnitro-
   samine and a choline-deficient diet.
        In addition, a comparable animal study was performed by Yabu et al.
   (1991).29 In that study male F-344 rats were given diethylnitrosamine (DEN) by
   intraperitoneal injections. Depending on the DEN regimen animals were given
   cyclosporin-enriched diets (0.011% or 0.015%), starting one or seven weeks
   after the last injection. A further eight or 14/15 weeks later, all animals were
   euthanized, after which the livers were examined on the number and size of
   enzyme-altered foci (glutathion-S-transferase changes, placental form). From the
   results obtained, the authors concluded that cyclosporin enhanced the growth of
   carcinogen-induced preneoplastic foci. Whether the enhancement could be
   ascribed by inhibition of the regression of the foci, or by stimulation of their
   growth is unclear. Also the authors could not determine whether the preneoplas-
   tic lesions observed in the animals could be converted in hepatocellular carcino-
   mas by cyclosporine, because the study duration was too short for finding such
   an effect.
        More recently, Morton et al. reported on the cancer promotion of cyclosporin
   in Eker rats.30 These rats were genetically modified to study tuberous sclerosis,
   and are used as a model of dominantly inherited susceptibility to renal cell carci-
   nomas and mesenchymal tumours in other organs. Twenty to twenty-five male
   animals were given cyclosporin orally by gavage at a dose of 30 mg/kg bw/day,
   on a daily base for 4 or 6 months. Tumour development was compared to concur-
   rent untreated and vehicle controls. Both after 4 and 6 months, the cyclosporin-
   treated animals significantly showed more renal neoplasms (adenomas and carci-
22 Cyclosporin
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<pre>nomas) than control groups (4 months: 80 % versus 26.7% (olive oil), respec-
tively, p<0.0001; 6 months: 92% versus 55% (olive oil), respectively,
p<0.0001). The cyclosporin treatment could not last longer, because of the high
spontaneous renal neoplasia incidence at one year of age (almost 100%).
    Koehl et al. (2006) used p53 knock-out mice (p53-/-) to study de novo cancer
prevention by several classes of immunosuppressive agents (rapamycin, myco-
phenolate mofetil, and cyclosporin).31 Concerning cyclosporin, nine mice
received cyclosporin in their diet at a daily base for a total of 20 weeks (blood
levels of cyclosporin averaged 404±141 ng/mL). The treatment neither pro-
moted, nor inhibited, de novo cancer development, as compared to the non-
treated control group. However, the tumours found in cyclosporin-treated ani-
mals, showed a more systemic manifestation than in the tumours found in the
control group. The authors showed that rapamycin did reduce spontaneous de
novo cancer development in the knockout mice.
Carcinogenicity studies                                                           23
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<pre>24 Cyclosporin</pre>

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<pre>Chapter 4
        Mutagenicity and genotoxicity
4.1     In vitro assays
        Cyclosporin did not induce reverse mutations in tests with various Salmonella
        typhimurium strains up to a dose of 3,000 μg/plate, nor did it induce mutations in
        the hprt locus of Chinese hamster V79 cells, in the presence or absence of a met-
        abolic activation system.1,32
        Herman et al. (2001) reported of a dose-dependent reduction in DNA repair in
        peripheral blood mononuclear cells, which were incubated in vitro with various
        concentrations of cyclosporin (up to 50 μg/mL cell suspension).33 Cyclosporin
        was added to the cell cultures directly after stopping UV radiation to initiate
        DNA damage. The blood cells were obtained from healthy blood bank donors.
        Also in kidney transplant recipients, they found reduced DNA repair activity in
        blood cells in vivo during cyclosporin-treatment; however it is difficult to assess
        whether the reduction was due to cyclosporin alone, because the recipients
        received a combination therapy with azathioprine and prednisone.34,35
        Concerning clastogenic activity, no chromosomal aberrations were found in
        human peripheral blood lymphocytes obtained from healthy volunteers, with or
        without a metabolic activation system, at cyclosporin doses up to 300 μg/mL.36
        However, in another study, sister chromatid exchanges (SCE) were observed in
        the same type of cells, in the absence of an activation system (cyclosporin doses
        Mutagenicity and genotoxicity                                                       25
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<pre>    up to 4 μg/mL).37 Later the same authors repeated the test and noted again that
    cyclosporin induced sister chromatid exchanges, but to a lesser extent than other
    immunosuppressive agents.38 Zwanenburg (1988), who reviewed the results of
    this study, explained the lower SCE frequency by the low cell growth.39 Overall,
    it is difficult to associate clastogenicity found in human cells to cyclosporin
    exposure, because other yet unknown biological factors may have influenced the
    outcome.
4.2 In vivo assays
    Concerning mutagenicity, Fracasso et al. (1993) studied the urinary mutagenic
    activity in fifty kidney transplant recipients, using a liquid incubation assay with
    E. Coli WP2uvrA as the test strain.40 This assay detects base pair substitutions in
    the presence or absence of a metabolic activation system. The recipients were
    divided into three groups: i) recipients (n=10) receiving an oral dose of
    cyclosporin (4.5 mg/kg bw); ii) recipients (n=20) receiving an oral dose of aza-
    thioprine (1.26 mg/kg bw); and, iii) recipients (n=20) receiving a combined oral
    dose of cyclosporin (3.89 mg/kg bw) and azathioprine (1.15 mg/kg bw). No
    mutagenic activity was detected in the group treated with cyclosporin alone.
    Contrary to this finding, 85% of the urines of recipients receiving azathioprine
    alone, and 40% of the urines of recipients receiving the combined therapy
    showed high mutagenic activity.
         Using male CD-1 mice, Matter et al. (1982) could not demonstrate dominant
    lethal mutations after a single oral application of 100 and 1,000 mg cyclosporin/
    kg bw, nor did they observe unscheduled DNA synthesis.41 In another study,
    although the study reporting was limited, also no unscheduled DNA synthesis
    was observed in blood lymphocytes of kidney transplant recipients, who under-
    went immunosuppressive therapy with cyclosporin.1
         Ember and Kiss (1997) studied the expression of several oncogene and sup-
    pressor genes (c-myc, Ha-ras, p53) in CBA/Ca mice (n=6/group/sex), which
    received a single intraperitoneal dose of cyclosporin of 16 mg/kg bw, or cyclo-
    phosphamide (20 mg/kg bw).42 The dose of cyclosporin corresponded to the ther-
    apeutic doses given to humans. As measured after 2 and 6 days, and 1, 6 and 12
    months after the treatment, in cyclosporin-treated mice no significant induction
    of the gene expression was observed in the thymus, spleen, bone marrow, and
    mesenteric lymph nodes. On the contrary, in those organs, cyclophosphamide did
    increase the expression of all the measured genes.
26  Cyclosporin
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<pre>    A few studies were performed in organ transplant recipients to investigate cyto-
    genetic or clastogenic endpoints. For instance, in the blood lymphocytes of thirty
    renal transplant recipients, the frequencies of sister chromatid exchanges were
    tested before and 3 months after the start of a cyclosporin therapy. As a result of
    the therapy, the SCE frequencies increased significantly compared to the pre-
    treatment period (p<0.05).43
         IARC reported on a study, in which twenty-five kidney transplant recipients
    showed increased frequencies of chromosomal aberrations in their blood lym-
    phocytes during cyclosporin therapy.1 However, due to serious methodological
    shortcomings, the results of this study should be regarded as questionable.
         Matter et al. (1982) applied cyclosporin (up to 1,500 mg/kg bw) orally to
    CD-1 mice and Chinese hamsters (4 animals/group).44 However, no induction of
    micronucleated erythrocytes in bone marrow smears was observed. In addition,
    no chromosomal aberrations could be detected in Chinese hamsters (6 animals/
    group).
4.3 Carcinogenic mechanism
    Overall, the precise carcinogenic mechanism(s) of cyclosporin are not com-
    pletely understood by insufficient evidence. However, the weight of evidence
    from experimental data indicates a lack of genotoxicity. In addition, Rosenkranz
    and Klopman proposed that cyclosporin is devoid of mutagenicity, clastogenicity
    and DNA-modifying activity.45,46 They made this conclusion by using a knowl-
    edge-based structure-activity relational expert system, called CASE.
         Since cyclosporin is used as an immunosuppressive agent, and chemical
    immunosuppression carries the intrinsic risk of tumour growth, immunosuppres-
    sion was, until recently, the most likely explanation of the tumour stimulatory
    effect of cyclosporin.3,47,48. Also certain other immunosuppressive agents, such as
    azathioprine and cyclophosphamide, have been associated with increased risk of
    cancer.49,50
         As an immunosuppressive agent, cyclosporin affects both humoral and cellu-
    lar immune defence systems. The exact mechanism of suppression is not com-
    pletely understood, but it is clear that cyclosporin blocks the production of
    several lymphokines, such as interleukin-2, which are produced by T-lympho-
    cytes.1,51-53 This blockage prevents the stimulation of cells, which are involved in
    the regulation of immune responses. In addition, the impairment of immunologi-
    cal defence system may result in uncontrolled neoplastic cell growth after induc-
    tion by a viral infection or other causes.2,3,51,54,55
    Mutagenicity and genotoxicity                                                        27
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<pre>       In 2004, André et al. hypothesised that not only immunosuppressive proper-
   ties of cyclosporin stimulate tumour growth, but also its ability to facilitate accu-
   mulation of DNA mutations, to diminish the clearance of altered cells, and to
   transform cancer cells into aggressive cancer cells.56 They postulated their
   hypothesis after reviewing several scientific papers on the carcinogenic action
   mechanism of cyclosporin, which are published in the past years. In addition,
   five years earlier, Hojo et al. performed a series of in vitro experiments, and an in
   vivo animal study using immune-deficient SCID-beige mice, to study possible
   cancer progression of cyclosporin by mechanisms other than immunosuppres-
   sion.57 The investigators found that cyclosporin altered the characteristics of sev-
   eral cancerous cell lines in vitro and in vivo, into more aggressive and invasive
   cells. Furthermore, they found out that these effects were prevented by adding
   antibodies directed at transforming growth factor-β (TGF-β). The latter finding
   suggests that cyclosporin stimulates the production of TGF-β, a factor that not
   only stimulates cancer growth, but also suppresses immune responses.58,59 Also
   Masuhara et al. (1993) postulated that cyclosporin may promote tumour develop-
   ment by stimulating specific growth factors, but he did not find evidence for this
   in an experimental animal model.60
28 Cyclosporin
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<pre>Chapter 5
        Classification
5.1     Evaluation of data on carcinogenicity and genotoxicity
        Overall, data obtained from epidemiological studies on organ transplant recipi-
        ents and psoriasis patients, who received cyclosporin therapy, produced suffi-
        cient evidence that cyclosporin acts as a systemic carcinogen in humans. The
        types of cancer included lymphomas, leukaemias and skin cancer.
             Increased numbers of tumours were found in animals, in which cancer was
        initiated by genotoxic carcinogens or by irradiation, or in genetically modified
        animals, which are prone to cancer development. The types of cancer observed
        included lymphomas, leukaemia, intestinal adenocarcinomas, hepatocellular car-
        cinomas, and renal cancer. No increased cancer development was observed in
        two animal studies, using normal animals without pre-treatment to initiate can-
        cer.
             Overall, the exact carcinogenic mechanisms are not completely unravelled,
        but since mutagenicity and genotoxicity tests were negative, the committee is of
        the opinion that cyclosporin is not mutagenic nor genotoxic. There are, however,
        strong indications that cyclosporin stimulates and promotes tumour development
        by suppressing the immunological defence system by inhibiting lymphokine pro-
        duction. More recently, it is also suggested that cyclosporin may stimulate cancer
        development by stimulating specific growth factors that are directly involved in
        cancer cell growth.
        Classification                                                                     29
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<pre>5.2 Recommendation for classification
    Based in the available information, the committee is of the opinion that
    cyclosporin should be classified as known to be carcinogenic to humans. This
    recommendation corresponds to the EU classification in category 1. The commit-
    tee, furthermore, concludes that cyclosporin is not genotoxic.
30  Cyclosporin
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<pre>   References
1  IARC. Ciclosporin. IARC Monogr Eval Carcinog Risks Hum 1990; 50: 77-114.
2  Ryffel B, Mihatsch MJ, Fisher GL. Immunosuppression and cancer: the ciclosporin case. Drug Chem
   Toxicol 1992; 15(2): 95-115.
3  Ryffel B. The carcinogenicity of ciclosporin. Toxicology 1992; 73(1): 1-22.
4  Bernabeu M, Krupp P, Wiskott E. Long-term safety of cyclosporine in renal transplant recipients:
   worldwide experience. Transplant Proc 1993; 25(4 Suppl 3): 17-19.
5  Drahy G, Dion E, Faucher C, Bellin MF. [Renal lymphoma in a transplanted patient treated with
   cyclosporine]. J Urol (Paris) 1993; 99(1): 35-37.
6  Green C, Hawk JL. Cutaneous malignancy related to cyclosporin A therapy. Clin Exp Dermatol
   1993; 18(1): 30-31.
7  Masouye I, Salomon D, Saurat JH. B-cell lymphoma after cyclosporine for keratosis lichenoides
   chronica. Arch Dermatol 1993; 129(7): 914-915.
8  Piepkorn M, Kumasaka B, Krieger JN, Burmer GC. Development of human papillomavirus-
   associated Buschke-Lowenstein penile carcinoma during cyclosporine therapy for generalized
   pustular psoriasis. J Am Acad Dermatol 1993; 29(2 Pt 2): 321-325.
9  Swoboda A, Fabrizii V. Tonsillar carcinoma in a renal graft recipient treated with cyclosporine A.
   Clin Nephrol 1993; 39(5): 272-274.
10 van de Kerkhof PC, de Rooij MJ. Multiple squamous cell carcinomas in a psoriatic patient following
   high-dose photochemotherapy and cyclosporin treatment: response to long-term acitretin
   maintenance. Br J Dermatol 1997; 136(2): 275-278.
11 Weinstein SP, Orel SG, Collazzo L, Conant EF, Lawton TJ, Czerniecki B. Cyclosporin A-induced
   fibroadenomas of the breast: report of five cases. Radiology 2001; 220(2): 465-468.
   References                                                                                         31
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<pre>12 Bencini PL, Marchesi L, Cainelli T, Crosti C. Kaposi's sarcoma in kidney transplant recipients treated
   with cyclosporin. Br J Dermatol 1988; 118(5): 709-714.
13 Calne R, White D, Thiru S, Evans D, McMaster P, Dunn D et al. Cyclosporine in patients receiving
   renal allografts from cadaver donors. Lancet 1978; ii: 1323-1327.
14 Starzl TE, Nalesnik MA, Porter KA, Ho M, Iwatsuki S, Griffith BP et al . Reversibility of lymphomas
                                                                            l
   and lymphoproliferative lesions developing under cyclosporin-steroid therapy. Lancet 1984; 1(8377):
   583-587.
15 Bencini PL, Montagnino G, Sala F, De Vecchi A, Crosti C, Tarantino A. Cutaneous lesions in 67
   cyclosporin-treated renal transplant recipients. Dermatologica 1986; 172(1): 24-30.
16 Sheil AG, Flavel S, Disney AP, Mathew TH, Hall BM. Cancer incidence in renal transplant patients
   treated with azathioprine or cyclosporine. Transplant Proc 1987; 19(1 Pt 3): 2214-2216.
17 Smith JL, Wilkinson AH, Hunsicker LG, Tobacman J, Kapelanski DP, Johnson M et al. Increased
   frequency of posttransplant lymphomas in patients treated with cyclosporin, azathioprine, and
   prednisone. Transplant Proc 1989; 21(1 Pt 3): 3199-3200.
18 Cockburn IT, Krupp P. The risk of neoplasms in patients treated with cyclosporine A. J Autoimmun
   1989; 2(5): 723-731.
19 Baildam AD, Higgins RM, Hurley E, Furlong A, Walls J, Venning MC et al. Cyclosporin A and
   multiple fibroadenomas of the breast. Br J Surg 1996; 83(12): 1755-1757.
20 Dantal J, Hourmant M, Cantarovich D, Giral M, Blancho G, Dreno B et al. Effect of long-term
   immunosuppression in kidney-graft recipients on cancer incidence: randomised comparison of two
   cyclosporin regimens. Lancet 1998; 351(9103): 623-628.
21 Otley CC, Maragh SL. Reduction of immunosuppression for transplant-associated skin cancer:
   rationale and evidence of efficacy. Dermatol Surg 2005; 31(2): 163-168.
22 Kessler M, Jay N, Molle R, Guillemin F. Excess risk of cancer in renal transplant patients. Transpl Int
   2006; 19(11): 908-914.
23 Arellano F. Risk of cancer with cyclosporine in psoriasis. Int J Dermatol 1997; 36 Suppl 1: 15-17.
24 Paul CF, Ho VC, McGeown C, Christophers E, Schmidtmann B, Guillaume JC et al. Risk of
   malignancies in psoriasis patients treated with cyclosporine: a 5 y cohort study. J Invest Dermatol
   2003; 120(2): 211-216.
25 Behnam SM, Behnam SE, Koo JY. Review of cyclosporine immunosuppressive safety data in
   dermatology patients after two decades of use. J Drugs Dermatol 2005; 4(2): 189-194.
26 Paul CF, Ho VC, McGeown C, Christophers E, Schmidtmann B, Guillaume JC et al. Risk of
   malignancies in psoriasis patients treated with cyclosporine: a 5 y cohort study. J Invest Dermatol
   2003; 120(2): 211-216.
27 Arellano F, Krupp P. Malignancies in rheumatoid arthritis patients treated with cyclosporin A. Br J
   Rheumatol 1993; 32 Suppl 1: 72-75.
28 Masuhara M, Ogasawara H, Katyal SL, Nakamura T, Shinozuka H. Cyclosporine stimulates
   hepatocyte proliferation and accelerates development of hepatocellular carcinomas in rats.
   Carcinogenesis 1993; 14(8): 1579-1584.
32 Cyclosporin
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<pre>29 Yabu K, Warty VS, Shinozuka H. Cyclosporine enhances the growth of carcinogen-induced enzyme-
   altered foci in rat liver. Hepatology 1991; 13(2): 304-309.
30 Morton LD, Youssef AF, Lloyd E, Kiorpes AL, Goldsworthy TL, Fort FL. Evaluation of carcinogenic
   responses in the Eker rat following short-term exposure to selected nephrotoxins and carcinogens.
   Toxicol Pathol 2002; 30(5): 559-564.
31 Koehl GE, Gaumann A, Zuelke C, Hoehn A, Hofstaedter F, Schlitt HJ et al. Development of de novo
   cancer in p53 knock-out mice is dependent on the type of long-term immunosuppression used.
   Transplantation 2006; 82(6): 741-748.
32 Olshan AF, Mattison DR, Zwanenburg TS. International Commission for Protection Against
   Environmental Mutagens and Carcinogens. Cyclosporine A: review of genotoxicity and potential for
   adverse human reproductive and developmental effects. Report of a Working Group on the
   genotoxicity of cyclosporine A, August 18, 1993. Mutat Res 1994; 317(2): 163-173.
33 Herman M, Weinstein T, Korzets A, Chagnac A, Ori Y, Zevin D et al. Effect of cyclosporin A on
   DNA repair and cancer incidence in kidney transplant recipients. J Lab Clin Med 2001; 137(1): 14-
   20.
34 Herman M, Weinstein T, Korzets A, Chagnac A, Ori Y, Zevin D et al. Effect of cyclosporin A on
   DNA repair and cancer incidence in kidney transplant recipients. J Lab Clin Med 2001; 137(1): 14-
   20.
35 Weinstein T, Korzets A, Chagnac A, Ori Y, Herman M, Zevin D et al. Effect of immunosuppressive
   therapy on DNA repair and cancer incidence in renal transplant recipients. Transplant Proc 2000;
   32(4): 694-695.
36 Zwanenburg TS, Cordier A. No cyclosporin-induced chromosomal aberrations in human peripheral
   blood lymphocytes in vitro. Mutat Res 1994; 320(3): 217-221.
37 Yuzawa K, Kondo I, Fukao K, Iwasaki Y, Hamaguchi H. Mutagenicity of cyclosporine. Induction of
   sister chromatid exchange in human cells. Transplantation 1986; 42(1): 61-63.
38 Yuzawa K, Fukao K, Iwasaki Y, Hamaguchi H. Mutagenicity of cyclosporine against human cells.
   Transplant Proc 1987; 19(1 Pt 2): 1218-1220.
39 Zwanenburg TS, Suter W, Matter BE. Absence of genotoxic potential for cyclosporine in
   experimental systems. Transplant Proc 1988; 20(3 Suppl 3): 931-933.
40 Fracasso ME, Barba A, Tessari G, Gasperini S, Brunello F. Urinary mutagenic activity after different
   immunosuppressive protocols in renal transplant patients. Mutat Res 1993; 319(4): 279-283.
41 Matter BE, Donatsch P, Racine RR, Schmid B, Suter W. Genotoxicity evaluation of cyclosporin A, a
   new immunosuppressive agent. Mutat Res 1982; 105(4): 257-264.
42 Ember I, Kiss I. In vivo effects of cyclophosphamide on oncogene and suppressor gene expression in
   a "follow up" study. Anticancer Res 1997; 17(5A): 3593-3597.
43 Palanduz S, Sever MS, Ozturk S, Tascioglu C, Karan MA, Sonmez G et al. Genotoxic potential of
   cyclosporin A in patients with renal transplantation. Cell Biol Toxicol 1999; 15(1): 13-17.
44 Matter BE, Donatsch P, Racine RR, Schmid B, Suter W. Genotoxicity evaluation of cyclosporin A, a
   new immunosuppressive agent. Mutat Res 1982; 105(4): 257-264.
   References                                                                                           33
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<pre>45 Rosenkranz HS, Klopman G. A re-examination of the genotoxicity and carcinogenicity of
   azathioprine. Mutat Res 1991; 251(1): 157-161.
46 Rosenkranz HS, Klopman G. A structural analysis of the genotoxic and carcinogenic potentials of
   cyclosporin A. Mutagenesis 1992; 7(2): 115-118.
47 Arellano F, Krupp P. Malignancies in rheumatoid arthritis patients treated with cyclosporin A. Br J
   Rheumatol 1993; 32 Suppl 1: 72-75.
48 Olshan AF, Mattison DR, Zwanenburg TS. International Commission for Protection Against
   Environmental Mutagens and Carcinogens. Cyclosporine A: review of genotoxicity and potential for
   adverse human reproductive and developmental effects. Report of a Working Group on the
   genotoxicity of cyclosporine A, August 18, 1993. Mutat Res 1994; 317(2): 163-173.
49 Arellano F, Krupp P. Malignancies in rheumatoid arthritis patients treated with cyclosporin A. Br J
   Rheumatol 1993; 32 Suppl 1: 72-75.
50 Olshan AF, Mattison DR, Zwanenburg TS. International Commission for Protection Against
   Environmental Mutagens and Carcinogens. Cyclosporine A: review of genotoxicity and potential for
   adverse human reproductive and developmental effects. Report of a Working Group on the
   genotoxicity of cyclosporine A, August 18, 1993. Mutat Res 1994; 317(2): 163-173.
51 Bennett WM, Norman DJ. Action and toxicity of cyclosporine. Annu Rev Med 1986; 37: 215-224.
52 Bussiere JL, Mather GG, Exon JH. Effect of cyclosporine on 3-methylcholanthrene-induced
   carcinogenesis and immune responses in the rat. Immunobiology 1991; 182(3-4): 205-215.
53 Kahan BD. Cyclosporine: the agent and its actions. Transplant Proc 1985; 17(4 Suppl 1): 5-18.
54 Bussiere JL, Mather GG, Exon JH. Effect of cyclosporine on 3-methylcholanthrene-induced
   carcinogenesis and immune responses in the rat. Immunobiology 1991; 182(3-4): 205-215.
55 Kahan BD. Cyclosporine: the agent and its actions. Transplant Proc 1985; 17(4 Suppl 1): 5-18.
56 Andre N, Roquelaure B, Conrath J. Molecular effects of cyclosporine and oncogenesis: a new model.
   Med Hypotheses 2004; 63(4): 647-652.
57 Hojo M, Morimoto T, Maluccio M, Asano T, Morimoto K, Lagman M et al. Cyclosporine induces
   cancer progression by a cell-autonomous mechanism. Nature 1999; 397(6719): 530-534.
58 Hojo M, Morimoto T, Maluccio M, Asano T, Morimoto K, Lagman M et al. Cyclosporine induces
   cancer progression by a cell-autonomous mechanism. Nature 1999; 397(6719): 530-534.
59 Nabel GJ. A transformed view of cyclosporine. Nature 1999; 397(6719): 471-472.
60 Masuhara M, Ogasawara H, Katyal SL, Nakamura T, Shinozuka H. Cyclosporine stimulates
   hepatocyte proliferation and accelerates development of hepatocellular carcinomas in rats.
   Carcinogenesis 1993; 14(8): 1579-1584.
34 Cyclosporin
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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
                                                             35
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<pre>36 Cyclosporin</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                                                                                        37
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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.
38 Cyclosporin
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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                                                               39
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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.
40 Cyclosporin
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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                                               41
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<pre>42 Cyclosporin</pre>

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<pre>Annex D
      IARC Monograph
      VOL.: 50 (1990) (p. 77)1
      CAS No.: 79217-60-0 (cyclosporin A)
      CAS No.: 59865-13-3 (cyclosporine)
      Chem. Abstr. Name: R-[R*,R*-(E)]}-L-Cyclic(L-alanyl-D-alanyl-N-methyl-L-
      leucyl-N-methyl-leucyl-N-methyl-L-valyl-3-hydroxy-N,4-dimethyl-L-2-amino-
      6-octenoyl-L- -aminobutyryl-N-methylglycyl-N-methyl-L-leucyl-L-valyl-N-
      methyl-L-leucyl)
      Summary of Data Reported and Evaluation
      Exposure data
      Ciclosporin has been used as an immunosuppressive agent since the mid-1980s.
      Experimental carcinogenicity data
      Ciclosporin was tested for carcinogenicity by oral administration in two studies
      in mice and in one study in rats. In one study in mice, it accelerated the develop-
      ment of leukaemias; tumours were not induced in a chronic bioassay. In rats,
      negative results were obtained in a study with limited sensitivity.
      IARC Monograph                                                                      43
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<pre>   Ciclosporin enhanced the development of lymphomas induced in two strains of
   male mice by single whole-body irradiation or N-methyl-N-nitrosourea. In
   grafted macaques, ciclosporin increased the incidence of lymphomas, a neo-
   plasm that occurs extremely infrequently in this species of monkeys. When given
   in combination with various other immunosuppressive regimens, ciclosporin
   induced a substantial increase in the incidence of lymphomas when compared to
   immunosuppressive regimens excluding ciclosporin. This drug also enhanced the
   incidence of intestinal adenocarcinomas induced in male rats by N-methyl-N-
   nitrosourea.
   Human carcinogenicity data
   In case reports, both lymphomas and Kaposi's sarcoma have been associated fre-
   quently with exposure to ciclosporin. Four cohort studies recorded a high inci-
   dence of lymphoma in organ transplant recipients; in two of these, ciclosporin
   was given without azathioprine or cytotoxic drugs. In several cases, there has
   been well-documented regression of lymphoma following withdrawal of the
   drug.
   Other relevant data
   Ciclosporin induced dose-dependent changes in reproductive organ weights in
   male rats and caused sterility at high doses. Fetal mortality was observed in rats
   and rabbits when the drug was administered during the second half of gestation
   at maternally toxic doses. No other sign of embryo- or fetotoxicity was noted.
   Ciclosporin is rapidly absorbed and widely distributed in humans and in experi-
   mental animals. It is extensively metabolized by the cytochrome P450 system.
   Adverse effects include nephro- and hepatotoxicity. The compound is immuno-
   suppressive, resulting in tolerance to tissue grafts; its main effect is on the early
   proliferation of T-cells.
   In a single study, ciclosporin was reported to increase the incidence of chromo-
   somal aberrations in the lymphocytes of kidney transplant patients.
   Ciclosporin did not induce dominant lethal mutations in mice, chromosomal
   aberrations in the bone marrow of Chinese hamsters or micronuclei in the bone
   marrow of Chinese hamsters or mice in vivo. It induced sister chromatid
   exchange in human peripheral lymphocytes in vitro but did not induce gene
44 Cyclosporin
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<pre>mutations in Chinese hamster cells. Ciclosporin did not induce mutations in Sal-
monella typhimurium.
Evaluation
There is sufficient evidence for the carcinogenicity of ciclosporin in humans.
There is limited evidence for the carcinogenicity of ciclosporin in experimental
animals.
Overall evaluation
Ciclosporin is carcinogenic to humans (Group 1).
IARC Monograph                                                                   45
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<pre>46 Cyclosporin</pre>

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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                                             47
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<pre>48 Cyclosporin</pre>

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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                                                             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.
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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 Cyclosporin</pre>

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<br><br>